Znt. J. Cancer: 61,557-566 (1995) Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancel 0 1995 Wiley-Liss, Inc.

QUANTITATIVE AND QUALITATIVE EFFECTS OF EXPERIMENTAL RADIOIMMUNOTHERAPY ON TUMOR VASCULAR PERMEABILITY Rosalyn D. BLUMENTHAL, Rina KASHI, Robert M. SHARKEY and David M. GOLDENBERG' Garden State Cancer Center at the Center for Molecular Medicine and Immunology, Newark, NJ 07103, USA.

Localization of radiolabeled antibodies in the perivascular space of tumors resulted in morphological changes in blood vessel structure and physiological changes in tumor vessel function. Vessel diameter decreased by day 14 and was associ- ated with a significant decline in vascular volume 0. Upon recovery of W, the basement membrane surrounding the endothelium had thickened. Tumor vascular permeability (VP) decreased within 7 days of treatment and remained suppressed throughout the 42-day observation period of our study. The decline in VP, which could be visualized by fluorescent micros- copy of FITC-dextran extravasation, was dose-related and could be quantified at doses as low as 800 cGy. The radioanti- body-induced 50-80% decline in tumor VP was observed in 3 human colonic xenografts (GW-39, LS I74T and MOSER). De- creases in VP have been observed for proteins ranging in size from 20 to I50 kDa. Similar effects on VP were noted when low protein doses (10-30 pg), which resulted in heterogeneous antibody distribution, were used or with high protein doses (400-750 pg), which resulted in more uniform penetration of antibody. If IB8Re or 90Y, radiometals with higher P-energy and longer path lengths, were substituted for l3Il, a similar decrease in VP was observed. The radioimmunotherapy (RAIT)-induced decrease in tumor VP resulted in a 90% decline in accretion of a second dose of radioantibody. The 10% of the second dose that was taken up by the tumor targeted many already non-viable tumor regions and some, but not all, viable tumor cell clusters. o 1995 Wiley-Liss, Inc.

Colorectal cancer is a prime candidate for antibody-directed radiotherapy (Wessels et al., 1989). Studies to compare the therapeutic potential of RAIT with standard chemotherapy in 8 human colorectal xenografts (lines with varying growth rate, antigen content and in vitro responsiveness to 5-FU) have revealed greater growth inhibition in 5 lines by radioimmuno- therapy and similar therapeutic efficacy with both treatment modalities in 3 lines (Blumenthal et al., 1994).

An antibody's ability to target tumors in vivo has been well documented (Goldenberg, 1993), and there are many reports demonstrating RAIT ability to control tumor growth (Bucheg- ger et al., 1990). Clinical results with a radioiodinated antibody have shown efficacy in the treatment of breast cancer (De- Nardo et al., 1991) and appear to hold considerable promise for the treatment of hematologic tumors (Goldenberg, 1993). Radioantibody therapy is also now viewed as a promising adjuvant approach for small primary sites or for treating micrometastatic disease. In our experience with experimental RAIT (Blumenthal et al., 1989a), minimal tumor burden can be treated with a single course of RAIT. As tumor burden increases, surviving populations arise from single treatments of RAIT. To eliminate these survivors, multiple cycles of RAIT are necessary. Several factors must be considered to optimize the timing of additional treatments: (i) the human anti-murine antibody (HAMA) response (Klein et al., 1986) if a murine antibody is being used, (ii) host toxicity (myelotoxicity; Welt et al., 1994) and (iii) tumor biology. This final factor, changes in tumor physiology as a result of damage from the first therapeu- tic dose, could have a profound impact on the uptake and cytotoxicity of subsequent treatments with either radiolabeled antibodies or other anti-tumor agents.

Numerous reports have demonstrated that radioantibody accretion is dependent on blood flow in and around tumor masses and on vascular permeability (VP) of tumor vessels (Blumenthal et al., 19896; Sands et al., 1988). In one study

tumor oxygenation, a measure of underlying tumor vascularity, correlated closely with antibody delivery (Gatenby et al., 1988); i.e., when mean p 0 2 fell below 16 mm Hg, tumors could not be imaged, even when the presence of antigen was confirmed. Two additional studies have employed vasoactive agents to dilate tumor vessels and increase tumor uptake and therapeu- tic efficacy of anti-tumor agents (Smyth et al., 1987). Other methods to stimulate tumor VP, such as hyperthermia and irradiation, have also been suggested to increase drug delivery.

Vascular changes in various tissues following high-dose-rate ionizing radiation are well documented (Law et al., 1978). Normal endothelial cells are among the most radiosensitive of the mesenchymal tissue structures (Fajardo and Berthrong, 1988). Histopathological changes following irradiation have been uncovered by angiographic studies (Saeki et al., 1971). Capillary injury occurs within 5 days of a single dose and results in loss of the microvasculature. By 6 months, new capillaries form (by budding from surviving cells) but are often shrouded by collagen and exhibit basement membrane thicken- ing. The mechanism responsible for this fibrosis remains to be elucidated completely but may involve the conversion of interstitial fibrinogen to fibrin by thrombokinase released from dying cells (Law et al., 1978) or as a result of release of growth factors such as fibroblast growth factor (FGF) (Witte et al., 1989). In arterial injury, concentric or eccentric narrowing of the lumen also occurs. Microscopic evidence for radiation- induced capillary injury in humans comes from observations on irradiated skin. Radiation vasculopathy is often found to be dose- and time-dependent. In one study brachytherapy with either lZ5I or 1921r resulted in ruptured vessels and stenosis (Fajardo and Berthrong, 1988). Song and Levitt (1971) demon- strated a transitional increase in the extravasation of plasma proteins in the Walker 256 carcinoma 2 days after irradiation with 200 cGy, or 1 day after irradiation with 3,000 cGy, without an increase in intravascular volume. They postulated the release of an endogenous mediator, rather than structural damage to blood vessels, as the mechanism of irradiation- induced vascular changes. Changes in energy metabolism and blood perfusion following X-irradiation in a mouse mammary adenocarcinoma have been documented (Tozer et al., 1987). The lactate/pyruvate ratio, a sensitive index of tumor oxygen- ation, decreased following irradiation with 2,000 cGy delivered as a single or fractionated dose, suggesting an enhancement in blood flow. In another study a double-isotope technique was used to study VP changes following higher radiation doses (1,800 and 2,500 cGy) in normal tissue (Evans et al., 1986). All tissues exhibited a biphasic response to radiation. Permeability was elevated 2- to 3-fold on the 1st and 2nd day after treatment in both shielded and unshielded tissue, which then declined to baseline values. The 2nd wave of enhanced permeability (4- to 5-fold above baseline levels only in unshielded tissues) was seen 14-20 days after irradiation. One investigation (Law et al., 1987) described similar biphasic increases in dye extravasation in rabbit skin after a 1,000 or 2,000 cGy exposure.

'To whom correspondence and reprint requests should be sent, at Center for Molecular Medicine and Immunology, 1 Bruce Street, Newark, NJ 07103, USA. Fax: (201) 982-7047.

Received: November 14,1994 and in revised form January 17,1995.

558 BLUMENTHAL ETAL.

In contrast to the wealth of information available on the effect of high-dose-rate external beam irradiation on tumor vascular function, the only information on the effect of low-dose-rate radiation (eg. , from radioantibody therapy) can be found in Blumenthal et a1. (1991b), where we demonstrated that (i) our antibody distributes into the perivascular space, (ii) a single dose of 1311-IgG affects blood flow rate, vascular volume (VV) and VP of tumor but not normal tissue (liver and lung) from days 7-14 post-treatment through day 42 and (iii) these changes in vascular function affect accretion of a 2nd dose of radioantibody. Our present report expands on those initial findings and gives results of studies to evaluate the effect of continuous low-dose radiation delivered over a 14-day period on tumor VP using 2 different protein doses of antibody ( < 30 pg and 400 pg), 5-7 doses of radiation (total dose = 200- 4,200 cGy), 2 sizes of tumor (0.2-0.4 g and 1.0-1.5 g), 3 different nuclides (1311, 90Y and IssRe) and 3 different colonic tumor lines (GW-39, LS174T and MOSER).

MATERIAL AND METHODS Animal model

Serially propagated GW-39 human colonic tumor xenografts (Goldenberg et al., 1966) were excised, passed through a 40-mesh screen and thoroughly rinsed with 0.9% sterile NaCl to yield the desired cell suspension. Subcutaneous tumors were initiated in nude mice with 0.2 ml of a 10% suspension. A 10% suspension gave rise to 0.2-g tumors in 10 days and 1.0-g tumors within 24 days. Female 6-8-week-old nulnu mice (Harlan Sprague-Dawley, Indianapolis, IN) were used for all studies. For some studies, MOSER, a poorly differentiated adenocarcinoma, and LS174T, a moderately differentiated adenocarcinoma, were employed.

Radioiodination NP-4 anti-CEA (Sharkey et al., 1993) and Mu-9 anti-CSAp

(Gold et al., 1990) IgG monoclonal antibodies (MAbs) were purified from mouse ascites using Protein A. Radioiodination was done by the chloramine-T method (McConahey and Dixon, 1966). Free radioiodine was separated from antibody- bound iodine by passage over a PD-10 column (Pharmacia, Piscataway, NJ) equilibrated with 0.04 M PBS (0.04 M phos- phate, 0.15 M NaCI, 0.02% NaN3),.pH 7.4, containing 1% human serum albumin (HSA). Routine quality assurance of each radiolabeled antibody was done to detect aggregation and free radioiodine by size exclusion HPLC using Zorbax GF-250 (Dupont, Wilmington, DE) columns; immunoreactivity was determined to be greater than 80% for each radiolabel on CEA or CSAp immunoadsorbent columns.

lRXRe labeling Vials containing 1 mg Mu-9 formulated for direct labeling

were reconstituted with 1-20 mCi of lXxRe freshly eluted from a 1RRW/ls8Re generator system in 1-20 ml of 0.9% sterile sodium chloride (Griffith et al., 1991). Elutions from the generator were constantly assayed for alumina breakthrough, Ix*W breakthrough, lXsRe yield, sterility and pyrogenicity. Radiolabeled Mu-9 was analyzed at 30 rnin and 2 hr post- reconstitution by HPLC, instant thin-layer chromatography (ITLC) in 2 solvents (one for detecting unbound lXxRe, the other for lxXRe colloid) and by affinity chromatography for immunoreactivity determination. The product contained < 2% free lxXRe and was 86% immunoreactive. The effects of higher specific activity radiolabeling were studied. Any effect due to radiolysis at high specific activity could be discerned at this point.

90Y labeling Two volumes of 2.0 M NH40Ac (pH 5.5 metal free) were

added to the 90Y vial (NEN, Wilmington, DE) and incubated at room temperature for 20 min. Chelated antibody was prepared according to established methods (Brechbiel et al.,

1986) using a 1-(p-isothiocyanatobenzyl) derivative of DTPA. An equal volume of chelated antibody and 0.5 M NH40Ac (pH 5.5) were mixed in a 1.5-ml acid-washed Eppendorf vial. Buffered 90Y was added to chelated protein in the vial at a ratio of 4 mCi/mg and incubated for 60 min. The reaction was stopped by adding 21.6 mM DTPA in 0.5 M NH40Ac to a final concentration of 10 mM, incubated at room temperature for 10 min. The vial was brought up to 1-ml volume by adding buffer (20 mM MES and 150 mM NaCI) and the contents pushed through an Avidchrom desalting cartridge (Bioprobe, Syra- cuse, NY) that had been equilibrated with 10 ml2.5% HSA, 20 mM MES and 150 mM NaCl (pH 6.0). The labeled antibody was eluted in t k first 2.4 ml, contained < 4% free 90Y and was 85% immunoreactive.

Microautoradiography Tumors were removed at the desired time after injection of

the radiotracer and fmed in acetic acid/ethanol. Tumors were embedded in paraffin and 5-pm sections were cut. Sections were rehydrated and then dipped into an aqueous (1:l) dilution of Kodak NTB-2 emulsion. Slides were air-dried and exposed for 7-12 days at 4°C. Slides were processed using D-19 developer (2 min) and GBX fixer (5 min) at 15"-19"C and post-stained with Harris's hematoxylin.

Vascular measurements All vascular measurements were normalized to tumor size

and all studies were performed with size-matched untreated and RAIT-treated tumors. VV and VP were quantitated using an in vivo labeling method of RBCs. Animals were injected i.v. with 2.5 ng stannous chloride (Dupont/NEN, N. Billerica, MA), followed 30 rnin later by i.v. injection of 15 pCi 9 9 m T ~ 0 4 (Mallinckrodt, St. Louis, MO) and 2.5 pCi of '251-labeled non-specific protein (irrelevant antibody with a m.w. of 150 or 50 kDa (irrelevant Fab') or 20 kDa (lysozyme purchased from Sigma, St. Louis, MO). Animals were anesthetized 1 hr later with pentobarbital, and blood was collected by intra-cardiac puncture. Animals were killed by cervical dislocation, and tissues were removed, weighed and counted with a gamma scintillation counter. VV and VP were calculated using the following established formulas (Sands ef al., 1985):

VV = ml bloodig tissue = (99mTc/g t i s ~ u e ) / ( ~ ~ ~ T c / g blood)

VP = total plasma in tissue - intravacular plasma = [('251/g tumor)/(lZsI/g plasma)] - [VV*(I - HCT)]

HCT was determined in Pyrex disposable Wintrobe hemato- crit tubes using a 10-min spin of a minimum of 600 pl of whole blood. The plasma sample was then collected and counted for L2sI for the VP calculation. Masson trichrome staining of tumor vessels

Tissue sections were prepared in the same manner as for microautoradiography and then fmed in B0uin.s solution for 1 hr at 56"C, cooled and washed in running water until the yellow color disappeared. Slides were then stained in Weigert's iron hematoxylin for 10 rnin, Biebrich's scarlet-acid fuchsin for 2 min and 2.5% phosphomolybdic acid for 10-15 min, followed by 2.5% aniline blue for 5 min. After each staining step slides were rinsed in water and after the last wash treated with 1% glacial acetic acid, dehydrated, cleared in xylene and cover- slipped with Permount. This stain results in black nuclei, red cytoplasm and intercellular fibers and blue collagen. In con- trast to other cells, RBCs appear bright red due to the absence of nuclei. Therefore, the location of blood vessels could be identified with ease.

Fluorescent microscopy to assess vascular penneabiliy Tumor vascular permeability in our model system was

visualized using the FITC-dextran method (Dvorak et al., 1988). Untreated and radioantibody-treated (150 $3 l3II- Mu-9) mice bearing 0.8-1.0 g GW-39 S.C. tumors were injected

RAIT AND TUMOR VASCULAR PERMEABILITY 559

with 0.5 pMol FITC-dextran (70 kDa; Sigma). Mice were killed 24 hr later by cervical dislocation. The tumor was dissected free and fixed in 70:30 ethano1:formalin mixture overnight. Tissues were then dehydrated, cleared in xylene and embedded in paraffin. All work was done in aluminum foil- covered vials and beakers to prevent loss of fluorescence. Five-micron sections of tumor were cut and floated onto slides using pre-warmed 50% methanol. Sections were deparaf- finized, coverslipped and evaluated by fluorescence micros- COPY.

Radioantibody uptake The percentage of radioantibody uptake in tissues was

determined for tumors and normal tissues growing in nude mice. Mice were injected with 25 pCi of 1251-M~-9 at 14 or 28 days after a 150 pCi dose of 1311-M~-9 or in the absence of RAIT. Groups of 5 animals were killed 3 days after the '251-M~-9 dose. 1251 activity in the tissues (tumor, liver, lung and blood) was quantitated by gamma scintillation counting after 10.5 half-lives of I3lI (approx. 85 days) and recorded as the percent injected dose per gram (%ID/g). All data were corrected for physical decay of the isotope. All studies were done as comparisons of radioantibody-treated and size- matched untreated tumors from the same generation implant.

Statistics Differences in vascular permeability were evaluated by a

2-tailed t-test (comparison of 2 groups) or a 1-factor analysis of variance with a 2-tailed F-test (comparison of more than 2 groups). Both tests measure the probability that difference between treatment groups can occur by chance alone.

RESULTS

Seven days after radioantibody injection, both 1311-NP-4 anti-CEA and l3lI-Mu-9 anti-CSAp could be found in the perivascular space of GW-39 human colonic xenografts (Fig. 1). The greater density of silver grains from Mu-9 (lower panel) reflects a higher tumor accretion and longer retention of that antibody compared with NP-4 (Blumenthal et al., 1991~). When tumor-bearing animals were treated with growth- inhibitory doses of "'1-Mu-9, tumor viability was markedly reduced and morphological changes in the vasculature were observed. Typical Masson trichrome-stained tumor sections from untreated and radioantibody-treated tumors (Fig. 2) demonstrated that localized radiation at the site of tumor vasculature resulted in a narrowing of vessel diameter on day 14 post-therapy (middle panel) and a significant fibrotic thickening in the basement membrane on day 35 post-therapy (bottom panel). These micro-anatomical changes were accom- panied by physiological alterations as seen in Figure 3. Four- teen days post-150 pCi I3lI antibody (approx. 4,200 cGy had been delivered over this entire period) there was a significant decrease in vascular volume from 76.3 5 7.1 pl/g in untreated mice to 15.9 ? 9.0 pl/g ( p < 0.001). Reductions in VV on days 7 and 21 were significant at t h e p < 0.005 level. By day 28, VV was fully recovered (66.4 f 4.2 pl/g). In the same study vascular permeability declined from 43.8 f 9.2 pl/g/hr in untreated mice to 15.7 f 12.1 pl/g/hr on day 7 post- radioantibody therapy and remained suppressed through day 42 ( p < 0.005 on days 7,14 and 42; p < 0.002 on days 21 and 35 a n d p < 0.001 on day 28). During this period of reduced vascular activity, tumors ceased to grow. The onset of VP suppression occurred earlier than the reduction in VV, but the change in VP was of much longer duration and may be permanent. W and VP of normal tissues, such as liver and lung, were approximately 4.5- to 8.5-fold higher than in tumor tissue. In contrast to the vascular changes observed in tumor, liver and lung exhibited some reduction in the average VV and VP in the first 14 days after treatment, as compared to the

FIGURE 1 - Microautoradiography demonstrating perivascular distribution of anti-CEA and anti-CSAp radioantibodies. Nude mice bearing GW-39 S.C. tumors were injected with 100 pCi 1311-labeled (a) NP-4 anti-CEA or (b) Mu-9 anti-CSAp and sacrificed 7 days later. Five micron sections of tumor were deparaffinized, rehydrated and placed in Kodak NTB2 emulsion for 5-10 days. Silver grains were tixed following Dektol develop- ment. b.v., blood vessel; arrows, silver grains: bar: 100 pm.

initial measurement made in these animals. However, the values never decreased to a level significantly lower than that seen in an age-matched, untreated control group of animals (Fig. 4). The 150 pCi dose of l3II-Mu-9 IgG (4,200 cGy tumor dose) resulted in 400 and 713 cGy doses to liver and lung, respectively.

A visual depiction of the effect of RAIT on tumor VP is shown in Figure 5. Seven or 14 days after a 4,200 cGy dose from I3lI-Mu-9, the movement of FITC-dextran (m.w. = 70 kDa) over a 24-hr period from the intravascular space to the intratumor space is virtually non-existent (Fig. 5c and d ) , compared with the permeability and intratumor diffusion in untreated tumors (Fig. 5a and b) .

In our next study suppression of VP was evaluated as a function of radiation dose and tumor size. Two different tumor sizes were included to evaluate the importance of (i) early and later stages of neovascularization and (ii) the effect of crossfire of deposited radiation. To achieve a similar cGy dose in both small and large tumors, the pCi dose had to be adjusted based on previous biodistribution studies (data not shown), since radioantibody uptake is inversely related to tumor mass (Hagan et al., 1986); eg., for an 800 cGy dose, either 10 or 26 pCi was given to small and large tumor-bearing mice, respec- tively. Size-matched sets of untreated tumors were included.

560

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a 60-

L 2 - - - 5 0 > - b 40- a 8 ’ 2 0 -

BLUMENTHAL ETAL.

100

T

1 0

T

0 10 20 30 40 50

Days Post Injection of Radioantibody

FIGURE 3 - Radiotracer measurement of tumor vascular volume and vascular permeability 1-42 days post-150 $3 (approx. 4,200 cGy) dose of 1311-Mu-9 IgG. Six mice with size-matched GW-39 tumors were used per time point and the mean 2 S.D. are recorded. Dotted lines represent the range of normal values for tumors from untreated mice.

1200

8 1000 Liver Lung - . -

8 200 -

FIGURE 2 - Masson trichrome-stained GW-39 tumor sections from (a) untreated mice, (b) 14 days or (c) 35 days post-150 pCi 1311-M~-9 (4,200 cGy) demonstrating RAIT-induced changes in vessel size and thickness of the basement membrane. Arrows pointing to normal blood vessel in (a), compressed blood vessels in (b) and fibrosis of vessel in (c); bar: 50 km.

f 3 400

.C 300

200 n

Figure 6 shows that the VP of small untreated tumors in the 3 0 L I range of 0.2-0.4 g (69.4 t 16.3 pl/g/hr) was greater than for large untreated tumors in the range of 1.0-1.5 g (43.8 t 9.6 pl/g/hr). When the pCi dose of 1311-labeled antibody was less than or equal to 400 cGy, there was no change in tumor VP. However, an 800 cGy dose resulted in a 44% reduction of VP in small tumors ( p < 0.001) and a 54% decrease in VP in large tumors ( p < 0.001). Doses of 2,000 and 4,200 cGy produced 60% and 72% decreases in VP in small tumors and 78% and 83% decreases in VP in large tumors.

The reduction in tumor vascular permeability by radioanti- body therapy had been observed in 2 other human colonic xenografts. Figure 7 illustrates that after a single injection of 150 pCi of 1311-NP-4 anti-CEA, GW-39 VP decreased from 43.8 2 9.2 pl/g/hr to 23.4 ? 5.0 ( p < 0.01) on day 14 and to 13.2 +- 6.1 ( p < 0.001) on day 28. RAIT reduced LS174T VP

0 10 20 30 40 0 10 20 30 40 50

Days Post Injection of Radioantibody

FIGURE 4 - Radiotracer measurement of liver and lung vascular volume and vascular permeability 1-42 days post-150 pCi dose of 1311-Mu-9 (400 and 713 cGy to liver and lung, respectively). Six mice with size-matched GW-39 tumors were used per time point and the mean ? S.D. are recorded. Dotted lines represent the range of normal values for liver and lung samples taken from untreated animals.

from 34.4 t 6.1 pl/g/hr to 21.6 t 3.0 on day 14 and to 19.1 t 4.2 on day 28 ( p < 0.001 at both times). MOSER VP was reduced from 43.7 t 4.3 pl/g/hr to 10.9 * 1.0 on day 14 and to 23.8 t 5.7 on day 28 ( p < 0.001). Partial recovery of MOSER

RAIT AND TUMOR VASCULAR PERMEABILITY 561

FIGURE 5 - Fluorescein-dextran trace of permeability from tumor vessels taken from 5 p sections of GW-39 tumors grown S.C. in nude mice (a) untreated tumor on day 7, (b) untreated tumor on day 14, (c) 4,200 cGy I3lI-Mu-9 IgG to tumor on day 7 and (d) 4,200 cGy 1311-M~-9 IgG to tumor on day 14. Animals were injected i.v. with FITC-dextran 24 hr before death. Small arrows (a,b) indicate FITC-detran that has diffused into the interstitial space. Thick arrows (c,d) indicate FITC-dextran that is limited to the vascular space after RAIT. Thin long arrows (c,d) indicate autoradiographic demonstration of radioantibody. Bar: 30 pm.

VP on day 28 may reflect the poor retention of antibody by this tumor.

The restricted distribution of radioantibodies could be made more homogeneous by administering 40- to 75-fold higher antibody protein doses without affecting total antibody accre- tion (Blumenthal et a[., 1991~; Ong and Mattes, 1989). By increasing antibody penetration away from the perivascular space, the direct radiation dose to the endothelial cells would theoretically decrease, at least for isotopes emitting short- range beta particles. Thus a comparison of VP when RAIT is administered under low- and high-protein conditions would provide information on whether the effect required direct exposure of the blood vessels to radiation. Figure 8 shows that the RAIT-induced suppression of VP occurs independently of the heterogeneity of intratumor penetration. When 800 cGy of either Mu-9 or NP-4 are given with low (10 kg) or high (400 kg NP-4 and 750 pg Mu-9) protein, VP exhibits a significant reduction ( p < 0.01 for both NP-4 groups and p < 0.02 for both Mu-9 groups), but there was no significant difference between VP at the low- and high-protein treatment groups. Another approach to evaluate whether vascular damage result- ing from RAIT requires direct exposure of the endothelium to radiation was to assess changes in tumor VP using antibodies labeled with nuclides other than I3lI with longer path lengths. The Em,, for lssRe and is approx. 3.6-fold higher than for I3lI and their respective path lengths are approximately 10-fold larger than the path for 1311 in tissue. Under low-protein conditions, when the labeled antibody was restricted to the perivascular space, much of the radiation from the radiometals might have been directed beyond the region of the blood vessel. If vascular damage was a direct effect of radiation

100

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0 1000 2000 3000 4000 5000

Dose Delivered to Tumor (cGy)

FIGURE 6 - Based on previous biodistribution data for small tumors (0.2-0.4 g initially) and lar e tumors (1.0-1.5 g at the time of dosing), defined WCi doses of IJfI-Mu-9 IgG were administered equal to desired radiation doses (200-4,200 cGy). Since small tumors accrete more radioantibody than larger tumors, a lower pCi amount was injected for the small tumor than the large tumor to achieve the same rad dose (e.g., for 800 cGy, either 10 or 26 pCi was injected into small or large tumor-bearing mice, respectively). Vascular permeability was quantited by radiotracer measurements as in Figures 3 and 4. Results represent mean k S.D. for 5 mice.

562 BLUMENTHAL ETAL.

9 0 -

60 , I

Tumor I Untreated T I I-131-Mu-9 IqG

I Untreated I 14-days post RAlT 0 28-days post RAlT

T I . = I 4 0 1

LS174T MOSER GW-39

Colonic Tumor Line

FIGURE 7 - Tumor vascular permeability in 3 CEA-expressing human colonic tumor xenografts: LS174T (moderately differenti- ated), MOSER (poorly differentiated) and GW-39 (signet-ring- cell carcinoma) at 14 and 28 days after a 150 pCi dose of l3lI-NP-4 IgG anti-CEA therapy. Each bar represents mean ? S.D. for 5 mice with size-matched tumors.

hitting the endothelium, then lssRe antibodies may not have resulted in the same vascular suppression as 13*1 antibodies. If the radiation effect was indirect, i x . , affecting neighboring tumor cells that then released a permeability-inhibiting factor or blocked the release of a permeability-stimulating factor, then all radioantibodies should have resulted in the same vascular damage. We evaluated the effect of 800 cGy of 1311-, lXKRe-, and 9oY-labeled Mu-9 on tumor VP and found that all 3 radioantibodies resulted in similar decreases in VP (Fig. 9). The untreated VP of 73.4 -+ 25.4 was reduced to 28.7 2 6.1 on day 14 and 25.9 k 6.5 on day 28 post-I3l1 ( p < 0.01). Tumor VP 14 days post-lsXRe was 24.2 f 11.5 and 16.1 f 7.1 on day 28 post-RAIT ( p < 0.01). On days 14 and 28 after 90Y therapy, tumor VP was measured as 23.9 k 3.6 and 14.0 ? 2.9 ( p < 0.01 on day 14 a n d p < 0.001 on day 28), respectively. Liver and lung VP were not affected by any of the 3 radioanti- bodies at either time point.

All permeability studies thus far were performed with a 150-kDa protein tracer. In the next study, changes in VP of smaller molecules following RAIT were evaluated. Figure 10 illustrates that baseline VP increases from 43.8 ? 9.2 pl/g/hr to 423.1 f 106.9 kl/g/hr to 835.9 k 66.9 Fl/g/hr as the size of the macromolecule tracer decreases from 150 to 50 to 20 kDa. Tumor VP of the 50 and the 20 kDa proteins decreased in WIT-treated mice to 206.8 ? 117.3 pl/g/hr ( p < 0.02) and 406.3 f 76.0 kl/g/hr ( p < O.OOl), respectively. In the last set of studies, the consequences of RAIT-suppressed tumor VP were addressed. Since the first dose of radioantibody resulted in a 60-80% decline in tumor VP, the ability to retarget tumor with a second dose of radioantibody was quantified. The 2nd dose of lZsI Mu-Y IgG was administered 14 or 28 days after the first dose of 150 pCi of 1311-Mu-9 IgG. Accretion of this 2nd dose was examined 3 days later. Figure 11 shows that the %ID/g in tumor was only 6-10% (4.57 f 1.39 when the 2nd dose was given on day 14 and 2.79 -+ 0.77 when the 2nd dose was given on day 28) of uptake of the first dose (48.67 ? 23.55; p < 0.01). Blood clearance and liver and lung accretion of the 2nd dose were not significantly different from the first radioan- tibody dose. This type of quantitative assessment does not provide any indication of the intratumor distribution of the 2nd dose, i.e., whether the 2nd dose targets the residual viable

90

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T I Untreated I Low Protein E=;l High Protein

Mu-9 anti-CSAp NP-4 anti-CEA

FIGURE 8 - Vascular permeability 14 and 28 days after adminis- tration of 400 cGy I3lI-Mu-9 IgG to small GW-39 tumors under low protein conditions (10-30 pg; poor antibody penetration) and under high protein conditions (400 pg NP-4 and 750 pg Mu-9; better antibody penetration). Protein dose effects on antibody penetration were reported in Blumenthal et al. (1Y91a). Results represent mean 2 S.D. for 5 samples.

60 0 Y-90-Mu-9 IgG

30 - f a 0

Liver

.- = zoo ' d 250 1 T

3 3 Lung T T I 280

210

140

70

0

7

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Days Post Radioimmunotherapy

FTGURE 9 - Vascular permeability of tumor, liver and lung 14 and 28 days post-radioimmunotherapywith equal 800 cGy doses of I3II (26 pCi), 90Y (51 pCi) and ls8Re (95 pCi) Mu-9 IgG in large GW-39 tumors. The pCi amounts approx. equal to 800 cGy were determined in previous biodistribution studies in size-matched GW-39 tumors. Results represent mean 2 S.D. for 5 samples.

tumor regions. A 100-pCi dose of the lZ5I-Mu9 (2nd dose) was administered 28 days after the first dose of 150 pCi I3'I-Mu-9. Tumors were removed 3 days after the 2nd dose, and tumor sections were placed into emulsion approx. YO days (10.5 half-lives) after the first 1311-MuY dose decayed (Fig. 12). These autoradiographic results can be compared with the

RAIT AND TUMOR VASCULAR PERMEABILITY 563

1000

I Untreated T 1 EEG3 14-days post RAlT I

150 k 50 k 20 k

Size of Protein

FIGURE 10 - Vascular permeability of macromolecules of 3 sizes (a 150 kDa non-specific antibody, 50 kDa monovalent antibody fragment and a 20 kDa I sozyme) 14 days after radioimmuno- therapy with 4,200 rad 4 - M u - 9 IgG RAIT. Results represent mean -+ S.D. for 5 samples.

80

& P 6ol I I Untreated

14-days post RAlT D 28-days post RAlT

Tumor Liver Lung Blood

FIGURE 11 - Day 3 accretion of a 2nd dose of 12sI-labeled Mu-9 anti-CSAp intact antibod administered 14 and 28 days after the first 150 pCi dose of &-labeled Mu-9 anti-CSAp antibody. Results are corrected for crossover of I3II into the lZ5I window and for physical decay of both isotopes. Untreated and treated tumors express high levels of antigen that could theoretically be targeted. Results represent mean 2 S.D. of 5 samples.

FIGURE 12 - Microautoradiographic distribution of a 2nd 100 pCi dose of 12sI-labeled Mu-9 anti-CSAp administered 28 days after the first 150 pCi therapeutic dose of '311-Mu-9 IgG. The first nuclide was allowed to decay before slides were placed in emulsion. Due to the lower accretion of the 2nd dose, exposure took 6-8 weeks instead of 7-10 days before Dektol development could be done. Arrows illustrate (a ) targeting to viable regions, (b) targeting to non-viable regions and (c) viable cells with no antibody targeting (bar: 50 km).

results in Figure lb, which shows typical restricted distribution of the radioantibody in the perivascular space. Since tumor accretion of the 2nd dose was low, tissue sections remained in emulsion for approx. 42-56 days instead of the 5-7 days usually needed to produce good images. Developed slides demon- strated that some viable cells along the rim were targeted by the 2nd dose (Fig. l h ) , some non-viable regions were targeted by the 2nd dose (Fig. 1%) and some viable regions were not retargeted (Fig. 1 2 ) .

DISCUSSION

This report is an extension of our previous work (Blumen- that et al., 19916), which describes the effect of radiolabeled

antibody therapy on human colonic tumor xenograft vascular morphology and VP. RAIT induces a thickening of the basement membrane similar to the documented fibrosis that occurs in response to high-dose-rate irradiation (Fajardo and Berthrong, 1988). The decline in VP has been monitored both qualitatively by FITC-dextran extravasation (Dvorak et a/., 1988) and quantitatively (Blumenthal et al., 1991b) by measur- ing the amount of non-specific radiolabeled protein found within the tumor 1 hr after i.v. administration (Sands et al., 1985) and was shown to be a specific effect found in tumor tissue. VP of liver and lung was unaffected since the radiation dose to these normal tissues was < 800 cCy, the threshold dose needed to see a change in VP in tumors. The effect is first

564 BLUMENTHAL ETAL

noted 7 days after RAIT, several days after tumor metabolic activity begins to decline (data not shown), and continues for the 42-day period of the study. It remains unknown whether recovery of baseline tumor VP is achieved at some later time. It will be interesting to determine whether the time frame for VP recovery is similar to that for tumor growth restoration (approx. 8-10 weeks after therapy).

In general, interstitial fluid pressure rises as tumors increase in size (Jain and Baxter, 1988). Therefore, baseline tumor VP is higher for smaller tumors than for larger tumors. Suppres- sion of VP in GW-39 tumors by RAIT was dose-related. The first significant effect was noted at an 800 cGy dose and became more severe as the total dose increased. At matched doses, the RAIT-induced decrease in VP was not statistically different between larger and smaller tumors, suggesting that the effect is not strongly influenced by the stage of neovascularization or the amount of crossfire of deposited radiation.

Suppression of tumor VP in response to a 150 pCi dose of RAIT was first observed in the GW-39 signet-ring carcinoma (approx. 1,800 cGy) and also in 2 other colonic tumors, the LS174T moderately differentiated adenocarcinorna (approx. 1,700 cGy) and MOSER, a poorly differentiated adenocarci- noma (approx. 800 cGy).

To understand the mechanism responsible for the RAIT- induced VP suppression, studies were designed to determine whether the effect was mediated by direct radiotoxicity to endothelial cells or indirect effects on the endothelium via a tumoricidal response. The first study evaluated changes in tumor VP following radioantibody therapy administered under low- and high-antibody permeating conditions. Autoradio- graphic studies have shown that antibody penetration into solid tumors is restricted to the perivascular space (Ong and Mattes, 1989), and mathematical modeling has suggested that tumor penetration could be enhanced by administration of higher protein doses (Fujimori et al., 1989). Studies in human tumor xenografts now support this theoretical model. If the protein dose is increased above 500-750 pg (normal dose is 10-30 pg) in our animal model, a more uniform distribution of radioantibody is apparent as early as 1 day after administration without any change in total antibody accretion (Blumenthal et al., 1991~) . The magnitude of reduction in VP in response to 800 cGy of I3lI-Mu-9 or I3lI-NP-4 IgG was similar under low- and high-antibody penetrating conditions. A dose of 800 cGy was selected for these studies so that at high-protein doses the rad dose remaining in the perivascular space would be less than the threshold dose of 800 cGy. These findings suggest that an indirect mechanism, possibly involving the release of a permeability-inhibiting substance or the reduced release of a permeability-enhancing substance, may be operational, a con- cept similar to one previously postulated to explain the effect of external radiation on permeability (Song and Levitt, 1971).

An alternative way to assess whether endothelial cells are indirectly affected by RAIT is to monitor changes in tumor VP following radioantibody therapy with different nuclides of therapeutic promise, which deposit different amounts of radia- tion in the immediate region of the blood vessels. lsxRe has an Em, that is 3.5-fold higher (2.12 MeV) than the Em,, for l3II (0.61 MeV). Its path length is approximately 12-fold larger (11.1 mm) than the path for l3lI in tissue (2.4 mm). Its half-life is only 17 hr compared with a 193 hr tl,? for I3lI. Similarly, another high-energy p-emitter with a longer half-life (tl/? = 2.67 days), with energy comparable to IR8Re (Em,, = 2.27 MeV) and a slightly longer path length (11.9 mm), was evaluated (Howell et al., 1989). We postulated that the restricted intratumor distribution of antibodies would result in much of the radiation from lxxRe and being directed beyond the region of the blood vessel. If vascular damage is a direct effect of radiation hitting the endothelium, then lsxRe and 90Y antibodies may not result in the same vascular

suppression as I3lI antibodies. If the radiation effect is indirect, then all 3 radionuclides should result in the same vascular damage. Therefore, the similar permeability-suppressive ef- fects of all 3 nuclides lends further support to the hypothesis that the effect of radioimmunotherapy is indirect.

VP is a function of the size of molecule permeating the vessel; i.e., as the size of the molecule decreases, the pl/g/hr found within the tumor increases as a result of increased thermal motion and ease of movement through the trans- endothelial pores. All of our initial permeability studies demonstrated a 5 0 4 0 % reduction in VP to a 150-kDa protein. In subsequent studies, we have shown that VP of smaller macromolecules (20-50 kDa) are also reduced by approx. 50%. This suggests that tumor accretion after a 2nd dose (m.w. 20-150 kDa) would be reduced. Indeed, we observed a 90% decline in uptake of a 2nd dose of radioantibody administered 14 or 28 days after the first dose, even though surviving tumor regions contain significant amounts of antigen (Blumenthal et al., 1991d). The 10% of the 2nd dose that did target was not restricted to viable cell clusters. This suggests that there may be limited therapeutic advantage to redosing with additional radioantibody during the first 42 days after RAIT.

Unlike the effect of high-dose-rate external irradiation that causes a short-term increase in tumor VP (Song and Levitt, 1971; Evans et a/., 1986), low-dose-rate, exponentially decay- ing, continuous radiation from RAIT results in decreases in tumor VP. High-dose-rate radiation is known to result in an increase in mean pOz and a decrease in the hypoxic cell fraction shortly after treatment (Koutcher et al., 1992), which would be expected to increase radiation sensitivity during that window. Radiation therapy also decreases tumor interstitial pressure (Roh et al., 1991), which would affect the uptake and distribution of follow-up therapy. It remains to be determined what effect radioantibody therapy has on tumor pOz, pH and interstitial pressure, major physiological factors influencing tumor responsiveness to further chemo- and radiation therapy. Radioimmunotherapy is not the only therapeutic modality that affects the vasculature. Studies on photodynamic therapy- induced tumor destruction have revealed a microvascular shutdown effect resulting in tumor death from oxygen starva- tion (ischemic necrosis). Using laser Doppler measurements, maximal decreases in blood flow were observed within 5 min of illumination (Wieman et al., 1990). Therapy with biological response modifiers also influences tumor vasculature (Sidky and Borden, 1987). T N F has been reported to inhibit capillary growth by direct cytostatic and cytotoxic actions to microvascu- lar endothelial cells, and IFN-aA and IFN-p both inhibit tumor-induced angiogenesis by modulating the signal for new blood vessel formation. This latter effect has been shown to be independent of the anti-proliferative effect of IFN. Regulation of tumor blood vessel structure and function is an active area of investigation (Folkman, 1986). The biochemical mechanism responsible for the likely indirect VP suppression by RAIT remains to be determined. The possibility that the regulation of release of a vasoactive factor is involved is intriguing. Many human tumors synthesize and secrete high levels of a 34-42- kDa glycoprotein called vascular endothelial growth factor/ vascular permeability factor (VEGF/VPF), which both is mitogenic for endothelial cells and stimulates microvascular permeability (Collins et al., 1993). The biology of this multi- functional factor and its role in making tumor microvascula- ture hyperpermeable has been reviewed (Kim et a!., 1992). Two high-affinity VPF/VEGF receptors, both tyrosine ki- nases, have been identified on tumor endothelial cells, which result in activation of phospholipase C and induction of transient increases in intracellular Ca2+ (deVries et al., 1992). The presence of VEGF/VPF in both tumor cells and immedi- ately adjacent endothelium has been demonstrated by intense and specific immunohistochemical staining of VPF. mRNA can be found only in tumor cells as determined by in situ

RAIT AND TUMOR VASCULAR PERMEABILITY 565

hybridization (Dvorak et al., 1991). VPF-stained vessels are hyperpermeable to macromolecules, as judged by their capac- ity to accumulate circulating colloidal carbon. Vessels that are more than 0.5 mm from VPF+ tumor cells could not become hyperpermeable. Tumors that secrete more VPF exhibit higher in vivo vessel permeability and greater accumulation of drugs such as photosensitizers (Roberts and Hagan, 1993). The absence or reduced expression of VPF might be expected to reduce drug accumulation by tumors and plasma extravasa- tion, which is necessary for angiogenesis and tumor stroma formation. It has been demonstrated that inhibition of VEGF by a MAb specific for VEGF reduces angiogenesis and suppresses tumor growth in vivo (Kim et al., 1993).

Another factor that may play a role in RAIT-induced suppression of tumor vessel permeability is nitric oxide (NO). It has been shown that solid tumor VP is inhibited by N O scavengers and by N O synthase inhibitors (Maeda et al., 1994). A similar finding has been reported for coronary vessels (Yuan et al., 1992). In vivo, the amount of NO may be mediated by the amount of NO synthase available. Therefore, if cytotoxic radiation from RAIT decreases enzyme synthesis, NO would decrease and VP would be expected to decline. Alternatively, enzyme activity might be inhibited by radiation-induced super- oxide production (Rengasamy and Johns, 1993). Another possibility is that NO is produced but inactivated by reaction with superoxide radicals (Henryet al., 1991). The possible role of NO in mediating the RAIT effect needs to be explored.

While it is tempting to view the changes in tumor vascular permeability following RAIT as a function of a change in the structure of the vascular endothelium (e.g., endothelial junc- tions or vesicular transport), one must also consider the role of several other variables, including the concentration of material in plasma and tumor, which regulates the diffusion coefficient, and the fluid pressure in plasma and tumor, which regulates hydraulic conductivity, i.e., convection. Variation in any of

them would change VP. We have preliminary evidence that blood flow rate to tumor decreases in response to RAIT (Blumenthal et al., 1991b), which would decrease the convec- tive force. Others have shown that interstitial fluid pressure can decrease in response to radiotherapy (Roh et al., 1991); however, this change would result in an increase in tumor VP. Further work is needed to elucidate the mechanism respon- sible for RAIT-induced changes in VP.

In summary, our report shows that (i) radioantibodies distribute in the perivascular space in tumors; (ii) vessel diameter decreases and then increases on days 14 and 28 after RAIT, respectively, in comparison to their appearance in untreated tumors; (iii) tumor VP is reduced by > 50% within 7-14 days after RAIT and does not show real signs of recovery for at least 42 days, while normal tissues remain unaffected; (iv) the RAIT-induced suppression in VP has been found in 3 colonic tumor xenografts of varying histopathology; (v) VP to molecules as large as immunoglobulins (150 kDa) and as small as lysozyme (20 kDa) is disrupted; (vl, 13’1-, 90Y and lssRe- labeled antibody (with differing tIl2, Em, and path length) administered at an equal total cGy dose to tumor suppress VP similarly; (vii) the suppression of VP is similar, independent of the protein dose (degree of penetration); (viii) the suppression of VP is greater as the cGy dose increases and (iw) the RAIT-induced suppression of VP may explain the decline in accretion of a 2nd dose of radioantibody 14 or 28 days after the first dose, even though antigen remains expressed.

ACKNOWLEDGEMENTS

We thank Mr. M. Pryzbylowski for antibody purification, Mr. P. Andrews for radioiodination and quality assurance and Mr. D. Varga for lssRe and 90Y labeling. This work was supported in part by USPHS grants CA60764 and CA39841 from the NIH.

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