The prospects of radiolabeled antibodies in cancer detection and therapy remain promising. However, efforts to achieve cures, especially of solid tumors, with the systemic administration of radiolabeled monoclonal antibodies (MAbs) have met with limited success. Using genetic engineering techniques, MAbs have been tailored to improve the therapeutic index (tumor:normal tissue ratio) in clinical radioimmunotherapy. In the present study, we investigated the potential of tetravalent {[sc(Fv)2]2} and divalent[sc(Fv)2] single chain Fvs of MAb CC49 for therapy in athymic mice bearing s.c. LS-174T human colon carcinoma xenografts. Mice received 1000 μCi of 131I-labeled[sc(Fv)2]2 or 131I-labeled sc(Fv)2, either as a single injection on day 6 or as four injections (250 μCi each) on days 6, 7, 8, and 9; the day of tumor implantation was taken as day 0. The median survival for the control group was 26 days. Comparisons of single and fractionated therapeutic regimens showed median survival as 32 (P < 0.001)and 53 days (P < 0.0001), respectively for[sc(Fv)2]2 and 26 (P >0.5) and 38 days (P < 0.0001), respectively for sc(Fv)2 when compared with the control groups. The time for the quadrupling of tumor volume for single and fractionated therapeutic treatments were: 9.0 ± 0.8 and 21.1 ± 2.9 days respectively for sc(Fv)2; 16.6 ± 1.9 and 32.9 ± 2.7 days respectively for [sc(Fv)2]2; and 8.3 ±0.7 and 8.4 ± 0.6 days respectively for the control group. No 131I-labeled systemic toxicity was observed in any treatment groups. The results show that radioimmunotherapy delivery for sc(Fv)2 and [sc(Fv)2]2 in a fractionated schedule clearly presented a therapeutic advantage over single administration. The treatment group receiving tetravalent scFv showed a statistically significant prolonged survival with both single and fractionated administrations suggesting a promising prospect of this reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.

RIT3is a rapidly developing therapeutic modality for the treatment of a wide variety of carcinomas (1). Numerous antibody-radionuclide combinations have been evaluated in clinical studies (2, 3, 4, 5, 6, 7, 8, 9). RIT has yielded complete responses in hematological diseases like Hodgkin and non-Hodgkin lymphoma (10, 11); however, for solid tumors only a partial clinical response has been observed (12, 13, 14). Methods for improving the therapeutic index (tumor:normal tissue ratio) remain to be optimized for the development of more effective RIT for solid tumors (15).

Most of the radioimmunoconjugates studied to date involve the use of whole immunoglobulins, particularly IgG. Intact MAbs(Mr ∼150,000) remain in circulation for an extended time and can therefore increase the radiation dose delivered to normal tissues (16). Moreover, the poor tumor penetration of MAbs results in the localization of only 0.0001–0.01%ID/g of tumor (17). Many solid tumors are relatively radioresistant; therefore, RIT of such tumors requires high radiation doses, which can result in significant nonspecific uptake by normal tissue leading to toxicity, especially to the hematopoietic system (18). Because the size of the antibody-based molecule relative to the renal threshold for first-pass clearance is a major factor in determining the residence time of the radioimmunoconjugate in circulation, a range of IgG formats, like F(ab′)2, Fab′, or Fab, have been investigated for therapeutic potential at preclinical (19, 20, 21) and clinical levels (5, 22, 23). Decreasing the size of the antibody molecule increased both the degree of penetration and the clearance rate from the blood pool; however, these molecules were difficult to generate at a large scale with clinical-grade purity and did not provide a significant increment in quantitative tumor retention as an intact antibody (24).

The advent of molecular biology has enabled the designing and purification in ample abundance of small, high-affinity, multivalent antibody-based molecules as carriers for radionuclides (25). ScFvs are recombinant proteins composed of a VL amino acid sequence of an immunoglobulin tethered to a VH sequence by a designed peptide (26, 27). Compared with an intact antibody, scFvs can bind to a tumor cell in a more homogeneous distribution (28, 29). Such fragments lead to a higher tumor:normal tissue ratio,but the percentage of injected dose delivered to the tumor is usually poor because of their monovalent nature and faster removal from the circulatory system. Moreover, the high renal accretion of these small molecules can lead to severe nephrotoxicity at therapeutic doses (24). To increase the functional affinity of scFvs, the valency of scFvs has been increased by connecting them together by either noncovalent or covalent interactions (28, 29, 30). The divalent scFvs have shown improved avidity and efficacy for tumor targeting at preclinical levels (31, 32, 33, 34, 35, 36).

CC49 is a second-generation murine MAb showing high affinity for the tumor-associated glycoprotein 72 (37). CC49 MAb is under clinical trials for radiation-mediated therapy of ovarian, colorectal,breast, and prostate carcinoma (4, 38, 39, 40, 41). In the present study, we have investigated for the first time the therapeutic potential of divalent [sc(Fv)2] and tetravalent{[sc(Fv)2]2} CC49 scFvs under single and dose-fractionation schedules in athymic mice bearing human colon carcinoma xenografts. Fractionated therapeutic treatment was found to be clearly superior to single administration for both sc(Fv)2 and[sc(Fv)2]2. We believe that the multivalent CC49 scFvs hold potential toward the generation of optimum tumor-targeting reagents in radionuclide-mediated therapy.

Protein Expression and Purification.

For functional expression of the divalent and tetravalent CC49 scFvs,the construct(VL-linker-VH-linker-VL-linker-VH-His6)was cloned in the yeast shuttle vector, pPICZαA (Invitrogen,Carlsbad, CA) and transformed into competent Pichia pastoris KM71 cells(his4arg4aox1Δ::ARG4) as described earlier (36). Upon expression, 20–30% of divalent scFvs were found to associate as tetravalent and/or higher aggregated forms. ScFvs were purified by immobilized metal affinity chromatography using the chelating resin Ni2+-nitrilotriacetic acid Superflow (Qiagen Inc., Valencia, CA; Ref. 36). A Superdex 200 column (1.6 × 60 cm; Pharmacia Biotech., Piscataway, NJ) was used to separate divalent and tetravalent scFvs. Protein concentrations were determined by the method of Lowry et al.(42). CC49 IgG used for control studies was purified on Protein G Sepharose Fast Flow resin (Pharmacia Biotech.) and dialyzed into HEPES saline buffer [10 mm HEPES, 150 mm NaCl, (pH 7.4)].

Characterization of Purified Multivalent scFvs.

The purity of scFvs was assessed by SDS-PAGE and HPLC size exclusion chromatography. SDS-PAGE was performed according to the method of Laemmli (43) under reducing and nonreducing conditions. The gels were stained with Coomassie Blue R-250. HPLC analyses were done on TSK G2000SW and TSK G3000SW (Toso Haas, Tokyo, Japan)size-exclusion columns connected in series using 67 mmsodium phosphate buffer (pH 6.8), 100 mm KCl as the mobile phase. The columns were calibrated using the Gel Filtration Calibration Kit (Bio-Rad, Hercules, CA). The elution was monitored by an in-line UV detector at 280 nm.

The immunoreactivity of scFvs was analyzed by a solid phase competition ELISA using BSM (Sigma Chemical Co., St. Louis, MO) as the antigen (36). The binding affinities of sc(Fv)2,[sc(Fv)2]2, and CC49 IgG for BSM were determined by SPR measurements using an upgraded version of BIAcore 1000 (Pharmacia Biosensor, Uppsala, Sweden) as described previously (36, 44). The kinetic rate constants(kon and koff) were measured, and the equilibrium association constant (KA)and equilibrium dissociation constant(KD) were derived.

Radioiodination of scFvs.

The scFv forms were labeled with either Na125I or Na131I using 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril (IodoGen; Pierce Chemical Co., Rockford, IL) as the oxidant (45). For therapeutic labeling, iodinations were carried out with 15–20 mCi of Na131I (NEN, Boston, MA)/mg of scFvs in 0.1 m sodium phosphate buffer (pH 7.2) with IodoGen (250μg/mg of protein) and at a protein concentration of 2–3 mg/ml. Unincorporated radioiodine was separated from the labeled protein by size exclusion chromatography using a Sephadex G25 column(Pharmacia, Piscataway, NJ). The specific activity of 125I- and 131I-labeled scFv molecules was about 3–9 mCi/mg. The radiochemical purity of all of the radiolabeled scFvs was ≥98% as confirmed by ITLC.

Characterization of Radiolabeled scFvs.

Radiolabeled proteins were analyzed on SDS-PAGE gels, and the radioactivity associated with the protein was measured using the ImageQuant software of the PhosphorImager (Molecular Dynamics,Sunnyvale, CA). Analytical size-exclusion HPLC was performed as described above. Fractions (1 ml) of the radiolabeled protein were collected, and the radioactivity was determined in a Packard Minaxi Auto-Gamma 5000 gamma counter (Meriden, CT). The immunoreactivity of radiolabeled CC49 scFv forms was assessed by RIA where BSM (surrogate for TAG-72 antigen) and BSA (negative control) were attached to a solid-phase matrix (Reacti-Gel HW-65F; Pierce; Ref. 36).

Animals and Tumor Model.

Female athymic mice (nu/nu; 4–6 weeks of age) were used for the in vivo studies (Charles River, Wilmington, MA). The human colon carcinoma LS-174T cell line (American Type Culture Collection, Rockville, MD) was implanted s.c. (4 ×106), and the mice were used 6 days (tumor volume, ∼250–300 mm3) after the injection of cells. Mice were kept in microisolator cages and fed ad libitum pathogen-free mouse diet and water. The procedures used were in accordance with the USPHS Guidelines for the Care and Use of Laboratory Animals and were also approved by the University of Nebraska Medical Center IACUC. Potassium iodide (0.001%) was given in the drinking water for 3 days before and terminated 3 days after the administration of the radioiodinated scFv. This blocked the thyroid radioiodine uptake that enabled the excretion of free iodine out from the animal body without contributing substantially to the whole animal dose or therapeutic dose to the tumor.

Biodistribution and Pharmacokinetics Studies.

Dual-label biodistribution studies were performed in LS-174T human xenograft-bearing mice after a simultaneous i.v. injection via the tail vein of 125I-labeled sc(Fv)2 (5 μCi) and 131I-labeled[sc(Fv)2]2 (2.5 μCi) or 125I-labeled CC49 IgG (5 μCi) and 131I-labeled[sc(Fv)2]2 (2.5 μCi) as described earlier (35, 36). For the whole-body retention studies, mice bearing the LS-174T xenografts (three/group) received injections via the tail vein with 1.5 μCi of radioiodinated scFvs. Each scFv was evaluated separately. The whole-body radioactivity was determined at various times after injection in a custom-built NaI crystal. The blood clearance studies were performed as described previously (35, 36).

Therapy Studies with 131I-labeled scFvs.

For the therapy studies, mice bearing established LS-174T tumors were used. The animals were randomized, the initial body weight was recorded, and tumor volume was measured by caliper. Two therapy regimens were used: (a) a single i.v. dose of 1000 μCi of 131I-labeled sc(Fv)2(n = 10) or[sc(Fv)2]2(n = 10) administered on day 6; and (b) four i.v. doses of 250 μCi of 131I-labeled sc(Fv)2 (n = 10) or[sc(Fv)2]2(n = 10) on days 6, 7, 8, and 9. For the control groups(n = 5), mice received injections i.v. with PBS (pH 7.4) either as a single administration on day 6 or as four injections on days 6, 7, 8, and 9. The tumor growth was monitored twice a week by measuring the tumor in two dimensions where volume = (length of short axis in mm)2 × (length of long axis in mm)/2, as described earlier (35). The body weight of mice was recorded twice a week. Mice were euthanized when the short axis of the tumor was ≥12 mm, tumor ulceration was detected, or the animals lost ≥20% of their original body weight.

Radiation Dosimetry.

The radiation-absorbed dose delivered to the tumors and normal organs such as kidneys and liver was calculated according to the Medical Internal Radiation Dose committee of the American Society of Nuclear Medicine (46, 47). For kidneys (average weight, ∼0.15 g), electron/β were only included in the mean absorbed dose. For all of the other organs and tumors, the self-absorbed dose was calculated. The mouse organ-cumulated activity (μCi × h) was calculated by integrating time-activity curves derived from the biodistribution data after the first injection of 131I-labeled scFv. The data were expressed in μCi/g and was not corrected for decay. It was assumed that radionuclide localizes immediately, i.e.,no lag time, in the organ of interest. It was further assumed that the effective half-lives do not change with each consecutive injection of the protein in the fractionated administration scheme. For calculation of the cumulative tumor radiation doses, the absorption phase times were Tabs = 2.2 h and 2.6 h,for dimer and tetramer, respectively. The tumor was assumed not to grow during the treatment period.

Statistical Analysis.

For comparing time with tumor quadrupling between various groups of mice, the data were fitted to estimate the slope of the growth curve. The Wilcoxon signed rank test was used to generate the two-tailed Ps. The survival fraction of each treatment group was evaluated according to the method of Kaplan and Meier. The survival curves were compared, and a Logrank test was used to generate the Ps using the GraphPad Prism, Version 2.01 (GraphPad Software Inc., San Diego, CA).

Characterization of Divalent and Tetravalent CC49 scFvs.

After purification, sc(Fv)2 and[sc(Fv)2]2 were ≥95%pure as indicated by SDS-PAGE and HPLC analyses. The binding parameters derived from the SPR studies were: kon= 9.1 × 104m−1s−1, koff = 8.9 ×10−4 s−1, and KA = 1.0 ×108m−1 for[sc(Fv)2]2; and kon = 2.2 ×104m−1s−1, koff = 8.0 ×10−4 s−1, and KA = 2.8 ×107m−1 for sc(Fv)2.

After radioiodination, the integrity of proteins stored at 4°C was ascertained daily by SDS-PAGE and HPLC for at least 4 days. On SDS-PAGE, both sc(Fv)2 and[sc(Fv)2]2 migrated as Mr 58,000 under nonreducing and reducing conditions and were found to be stable when stored at 4°C in 1% mouse serum. HPLC analysis indicated that ≥95% of the radioactivity was associated with the protein peak for both sc(Fv)2 and[sc(Fv)2]2 (Fig. 1). However, the amount of free 131I increased to 12% (72 h after labeling) and was corrected for therapeutic administrations. The immunoreactivity of radiolabeled scFvs by solid phase RIA was 85–95% (0.8–1.5% nonspecific binding). At 72 h after labeling, a decrease of ∼15 and 20% in the immunoreactivity was observed for sc(Fv)2 and[sc(Fv)2]2, respectively.

Pharmacokinetics and Biodistribution of Radioiodinated scFvs.

Blood clearance curves showed elimination half-lives of 80, 170, and 330 min for sc(Fv)2,[sc(Fv)2]2, and CC49 IgG. Whole-body clearance studies confirmed the rapid elimination of scFv fragments. At 48 h after administration, ∼95% of both radioiodinated scFvs cleared from the body, whereas the clearance was only 75% for CC49 IgG. The tumor localization of[sc(Fv)2]2 was 2-fold higher than sc(Fv)2 beginning at 4 h after injection (Fig. 2). Uptake at earlier time points (i.e. <4 h) was similar for all of the proteins. At 24 h after administration, the %ID/g in tumor was 10.5 ± 1.1 for[sc(Fv)2]2 and 5.1 ± 0.7 for sc(Fv)2. The maximum tumor uptake of[sc(Fv)2]2 occurred between 6 and 16 h. CC49 IgG showed 28.4 ± 1.7%ID/g in tumor at 24 h after administration. However, in blood CC49 IgG showed 12.5 ± 0.9%ID/g as compared with 0.3 ± 0.1 and 0.2 ± 0.1%ID/g for[sc(Fv)2]2 and sc(Fv)2, respectively. RIs (%ID/g of tumor divided by %ID/g of normal tissue) of the major organs were determined for CC49 IgG, sc(Fv)2, and[sc(Fv)2]2. For well-perfused organs such as the liver and the spleen, the tumor:liver and tumor:spleen ratios were 2.3:1 and 2.6:1 for CC49 IgG, 8.1:1 and 8.2:1 for sc(Fv)2, and 3.9:1 and 10.4:1 for[sc(Fv)2]2 at 24 h after administration.

Therapeutic Study with Single-dose Administration of Radioiodinated scFvs.

The rate of tumor growth in groups receiving[sc(Fv)2]2 was statistically different from that in the control animals(P = 0.03) but not for sc(Fv)2(P > 0.05; Fig. 3). The group administered with[sc(Fv)2]2 showed tumor regression for 14–16 days as compared with the control group, which showed approximately a 6–8-fold increase in tumor volume (Fig. 3). The times for tumor-quadrupling volume were 9.0 ± 0.8 and 16.6 ± 1.9 days for sc(Fv)2 and[sc(Fv)2]2, respectively(Table 1). The time for tumor-quadrupling volume for the control group was 8.3 ± 0.7 days (Table 1). A comparison of survival curves with the control group for single dosing showed the median survival time as 26 (P > 0.5) and 32 days (P < 0.001)for the sc(Fv)2 and[sc(Fv)2]2, respectively(Fig. 4). No apparent signs of systemic radiotoxicity were detected. Animals did not lose >20% of their body weight with 1000 μCi as a single dose (Fig. 5). The cumulative radiation doses delivered to the tumor for bolus sc(Fv)2 and[sc(Fv)2]2 were 8.5 and 36.0 rads, respectively (Table 2). Compared with sc(Fv)2, the higher uptakes of[sc(Fv)2]2 in the liver and kidneys resulted in a 2–3-fold increase in the absorbed radiation dose.

Therapeutic Study with Dose Fractionation of Radioiodinated scFvs.

Mice receiving fractionated therapeutic doses exhibited a statistically significant difference in tumor growth for both sc(Fv)2 (P ≥ 0.05) and[sc(Fv)2]2(P = 0.03) from the untreated control group (Fig. 3). For both therapeutic regimens, tumors regressed for 24–28 days. During the same time, the control group showed approximately a >18-fold increase in tumor volume or was removed from the study (Fig. 3). The times for tumor-quadrupling volume were calculated as 21.1 ± 2.9 days for sc(Fv)2, 32.9 ± 2.7 days for[sc(Fv)2]2, and 8.4 ± 0.6 days for the control group (Table 1). Survival analysis showed a significant tumor growth inhibition for sc(Fv)2and [sc(Fv)2]2 with the median survival time as 38 (P < 0.0001) and 53 days(P < 0.0001), respectively, as compared with the control group (Fig. 4). Mice treated with fractionated doses showed a ≤8% loss in body weight, in contrast to the mice that received injections with a single dose of 1000 μCi, which showed a≥10% body weight loss from 20 day onward (Fig. 5). In tumors, the absorbed radiation doses for sc(Fv)2 and[sc(Fv)2]2 were 38.2 and 168.9 rads, respectively, for each protein and were 4-fold higher than doses after a single administration (Table 2). The cumulative activity in the liver and kidneys also increased but was well below the threshold levels.

RIT using MAbs against tumor-associated antigens has shown limited clinical success for the treatment of solid tumors (15)attributable mainly to the intrinsic characteristics of solid tumors such as poor radiosensitivity, heterogeneous expression of antigens,relatively poor tumor vasculature, elevated interstitial pressure, and tumor necrosis (48, 49). Achieving optimal adsorbed dose in solid tumors therefore requires the systemic delivery of high administered activities that result in incidental radiotoxicity to the bone marrow and organs involved in the catabolism of the radiopharmaceuticals such as the kidneys and the liver (12, 14). Some of the approaches that have been developed for increasing the therapeutic index of radiolabeled MAbs include: using high affinity antibodies (50); administering multiple doses of radiolabeled MAbs (51, 52, 53); predosing with an unlabeled antibody before the radiolabeled antibody (10, 54); using a “cocktail” of MAbs rather than a single radiolabeled antibody (55); and using combined modalities treatments (15).

Alternatively, recombinant antibody-based molecules with high affinity,functional avidity, and optimal size are being engineered and evaluated for the improved RIT of solid tumors (35, 56, 57, 58, 59). However, only a few studies have investigated the tumor localization of trivalent antigen-binding antibody constructs(Fab′)3(60, 61, 62) and tetravalent IgG dimers (63, 64) in xenografted mice.

We have previously compared the therapeutic efficacy of 131I-labeled noncovalent scFv dimers and intact IgG of MAb CC49 in athymic mice bearing human colon carcinoma xenografts. The maximum tolerated dose for IgG was 500 μCi and could be escalated to 1500 μCi with 131I-labeled sc(Fv)2. Even with the lowest dose of 131I-labeled sc(Fv)2, a statistically significant prolonged survival time was observed as compared with the control (P = 0.036; Ref. 35). For CC49 IgG, single administration of 250 or 500μCi resulted in complete tumor regression in 30 and 60% of mice,respectively (35). Reduced tumor growth in 80–100% of mice has been reported earlier with 131I-labeled CC49 IgG (50). Although IgG has shown promising preclinical results, the clinical performance of intact antibody for cure of solid tumors has been limited (38, 40, 41). This is primarily due to slower clearance of IgG from the blood pool that limits the maximum tolerated dose and dose escalation essential for the treatment of solid tumors.

A covalent dimeric CC49 scFv [sc(Fv)2] was subsequently constructed and characterized for in vitroantigen-binding affinity and in vivo tumor targeting (36). Upon expression, 20–30% of sc(Fv)2 was found to associate as tetravalent and/or higher molecular weight aggregated forms. The tetravalent scFv{[sc(Fv)2]2} revealed a 3–4-fold higher KA than sc(Fv)2 in SPR studies. Also,[sc(Fv)2]2 showed a 2–3-fold slower koff as compared with CC49 IgG, indicating a stronger association with the antigen because of a higher functional affinity of the molecule. The gain in avidity due to tetravalency, along with a lower molecular weight than intact IgG,suggests this recombinant scFv as a good candidate for tumor targeting.

In biodistribution studies,[sc(Fv)2]2 exhibited an approximately 2-fold increase in tumor localization over sc(Fv)2 following 4 h after administration. Although tumor uptake of CC49 IgG was significantly higher at later time points (16 h onwards), the nonspecific retention of radioactivity was found to be elevated in the major organs studied because of the long circulation half-life of radiolabeled IgG (65). The tumor:blood ratio for sc(Fv)2 and[sc(Fv)2]2 was detected as 1.5- and 15-fold higher, respectively, than CC49 IgG at 24 h after administration. Blood clearance studies showed the elimination half-life of sc(Fv)2 and[sc(Fv)2]2 as approximately 4- and 2-fold faster than intact CC49 IgG. The tumor:liver and tumor:spleen ratios for 131I-labeled sc(Fv)2 were found to be lower than the values observed for 131I-labeled sc(Fv)2earlier (35). The increased RI of noncovalent sc(Fv)2 resulted primarily because of its faster clearance from the blood than covalent sc(Fv)2(36). For[sc(Fv)2]2, the RI for tumor:liver ratio was found lower compared with that for sc(Fv)2 due to the faster clearance of the sc(Fv)2 from the blood. The liver might be the possible site for elimination as also evidenced by a lower kidney uptake of [sc(Fv)2]2(Mr 120,000). High tumor localization with low nonspecific retention suggests that these multivalent scFvs may be preferred choices for RIT of solid tumors.

We have performed a direct comparison of the radioimmunotherapeutic efficacy of dimeric and tetrameric CC49 scFvs under both single and fractionated regimens. The outcome of RIT strongly depends upon the initial size of the tumor because the uptake of radiolabeled antibody decreases exponentially with the tumor size thereby reducing the tumor dose (66). Impressive therapeutic results have been documented in animals with small or micrometastatic tumors (51, 67, 68) and in patients with small tumor burdens (13, 14). In the present study, only larger tumors were used to extrapolate the situation found in a clinical setting with higher tumor burdens. We have reported previously that the therapy with 131I-labeled noncovalent dimers gave a tumor regression lasting for ∼16 days at 1500-μCi radiation dose (35). The treatment with 1000 μCi of covalent dimer provided less effective therapy, probably because of the larger and more aggressive tumors (250–300 mm3 at approximately day 6). However, with tetravalent scFv, a single therapeutic treatment of 1000 μCi showed a 2-fold slower tumor volume-quadrupling time than the untreated group, with the median survival time of the group increased by ∼1.3-fold. No significant systemic toxicity was recorded for the single administration of 1000μCi attributable mainly to the fast blood clearance properties of multivalent scFvs (∼95% were cleared out in 48 h).

With radiolabeled IgGs, both a specific (targeting the tumor-associated antigen) and a nonspecific (emission properties of the radionuclide)components can be associated with tumor regression. However, the therapeutic advantage attained by the specific antigen binding is significant over the nonspecific cytotoxic properties of 131I (50). Therefore, in the present study, PBS (pH 7.4) was used as the negative control rather than an irrelevant scFv. Also, the reference control of CC49 IgG was not included because the maximum tolerated dose for 131I-labeled CC49 IgG was 500 μCi for single administration (35).

Fractionated therapy at medium dose levels has been suggested to provide effective RIT (51, 53, 69). Therefore, a dose-fractionation study was performed to directly compare the advantages of RIT involving dose fractionation over single administration. Fractionated doses were administered every 24 h as whole-body clearance studies demonstrated that >90% of the radioiodinated sc(Fv)2 and[sc(Fv)2]2 was cleared from the animal body. Also, at 24 h after administration the specific accumulation of scFvs in the tumors was detected. For both sc(Fv)2 and[sc(Fv)2]2, dose fractionation yielded slower tumor growth with the tumor-quadrupling time about 2.5- and 4-fold slower than the control group, respectively. In our study, the groups that received fractionated therapy of radioiodinated sc(Fv)2 and[sc(Fv)2]2 showed a statistically significant lengthening of the median survival time,which was ∼1.6- and ∼2.4-fold longer than in the untreated group. As a reference, IgG was not included in the dose-fractionation experiment because intact IgG remains in circulation for a much longer period. At 24 h, the %ID/g in blood was >10 for IgG and could have lead to potential bone-marrow toxicity upon multiple administrations. In previous dose-fractionation studies with IgG,usually the therapeutic regimen consisted of two or three administrations of 300 μCi of 131I-labeled B72.3/CC49 IgG at either a 7-day interval (51) or a 3-day interval (53).

Besides the molecular size and functional affinity of the antibody fragment, the therapeutic efficacy of a radioimmunoconjugate can depend on tumor vasculature, the total antibody protein dose, and the radiation dose administered (70, 71, 72). Moreover, for fractionated RIT, morphological and physiological changes in the tumor blood vessels occur after the first administration of the radiolabeled antibody. This can significantly alter the localization of the subsequent dose of antibody (73). Adams et al.(72) performed a study to compare the effect of dose escalation (50 μg to 1000 μg) and repeated i.v. administrations on the tumor localization of divalent scFvs. They demonstrate that a highly specific localization can be maintained with multiple i.v. bolus injections of divalent scFv administered 24 h apart. We believe that the increased therapeutic effect seen in the present study by fractionating 1000 μCi dosage to four doses of 250 μCi each, given 24 h apart, helps in overcoming the rapid clearance of the scFvs and in maintaining a better tumor localization. Nevertheless, the fractionated doses cannot be considered as additive because the irradiation-induced vascular changes can be significant for the tumor.

The tumor dosimetry showed: single administration of[sc(Fv)2]2 resulted in 4-fold higher radiation dose as compared with sc(Fv)2; the radiation dose with fractionated therapeutic scheduling of sc(Fv)2 was equivalent to single administration of[sc(Fv)2]2; and dose fractionation of[sc(Fv)2]2 further increased (5-fold) the tumor radiation absorbed dose. No radiation-related problems are anticipated on important target organs like the liver and kidneys based on the radiation dose calculations.

One of the major concerns of therapeutic strategies based on multiple dose scheduling is the development of human antimouse antibodies. ScFvs should have reduced immunogenicity because they do not contain CH2 and CH3 domains of intact immunoglobulins, or the CH1 or CL domains (responsible for antiallotype responses) found in Fab′ or F(ab′)2 fragments. We have recently developed a hu/muCC49 scFv containing the human subgroup IV germ-line VL and variable region of the murine CC49 heavy chain (74). In vivotumor-targeting studies showed similar biodistribution and pharmacokinetic properties of the shuffled and completely murine scFv (74) with possible implications in the reduction of human antimouse antibody responses in patients.

Although in the present study only a partial tumor regression occurred,a definitive advantage of multivalency and dose fractionation was noticed. There are increasing reports where either a partial or a complete ablation of xenografted solid tumors have been shown using RIT in conjunction with chemotherapy (75), radiotherapy (76, 77), or blood flow-modifying agents (78). Recently Behr et al.(79)demonstrated that α-emitters like 213Bi hold advantage over conventional low linear energy transfer radionuclides like 90Y in curing solid tumors with antitumor Fab′ fragments. Also, pretargeting RIT approaches that use either radiolabeled bivalent haptens with bispecific antibody (80) or biotin-streptavidin as receptor-ligand pair (81) have shown improved cure rates. Studies are under way to try further dose escalation and use of pretargeting approaches to improve the therapeutic efficacy of the multivalent CC49 scFvs.

In summary, we investigated the potential of the genetically engineered, multivalent scFvs of MAb CC49 as candidates for RIT of colon carcinoma. Multiple administrations of the radiolabeled modality showed higher tumor:normal tissue ratios in a shorter time without normal tissue toxicity, leading to a statistically significant reduction in tumor progression and prolonged median survival times. Subsequent studies may combine the advantages of multivalency and pharmacokinetic properties of divalent and tetravalent scFvs with dose-fractionation administration to determine an optimum therapeutic treatment and improved diagnosis of cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

Supported by grants from the United States Department of Energy (DE-FG02-95ER62024) and NIH (P5O CA72712 and RO1 CA78590).

                
3

The abbreviations used are: RIT,radioimmunotherapy; scFv, single chain Fv; sc(Fv)2,covalent divalent scFv; [sc(Fv)2]2,noncovalent tetravalent scFv; HPLC, high performance liquid chromatography; MAbs, monoclonal antibodies; SPR, surface plasmon resonance; BSM, bovine submaxillary gland mucin; %ID/g, % of injected dose/g; RI, radiolocalization index; VL, variable light chain; VH, variable heavy chain.

Fig. 1.

HPLC size-exclusion profiles of radioiodinated CC49 [sc(Fv)2]2 and sc(Fv)2. Samples were analyzed using TSK G2000SW and TSK G3000SW size-exclusion columns connected in series. The [sc(Fv)2]2(♦) and sc(Fv)2 (□) were eluted as single peaks of Mr ∼120,000 and 60,000, respectively.

Fig. 1.

HPLC size-exclusion profiles of radioiodinated CC49 [sc(Fv)2]2 and sc(Fv)2. Samples were analyzed using TSK G2000SW and TSK G3000SW size-exclusion columns connected in series. The [sc(Fv)2]2(♦) and sc(Fv)2 (□) were eluted as single peaks of Mr ∼120,000 and 60,000, respectively.

Close modal
Fig. 2.

Biodistribution of radioiodinated CC49 IgG(▴), sc(Fv)2 (•), and[sc(Fv)2]2 (▪) in groups of athymic mice bearing LS-174T human colon carcinoma xenografts. The results are%ID/g of tissue ± SD; n = 6.

Fig. 2.

Biodistribution of radioiodinated CC49 IgG(▴), sc(Fv)2 (•), and[sc(Fv)2]2 (▪) in groups of athymic mice bearing LS-174T human colon carcinoma xenografts. The results are%ID/g of tissue ± SD; n = 6.

Close modal
Fig. 3.

The effect of administration of(A) a single dose of 1000 μCi and (B)four dose fractions of 250 μCi each of 131I-labeled sc(Fv)2 or [sc(Fv)2]2 on growth of LS-174T xenograft in athymic mice. Each linerepresents the relative increase in tumor volume in an individual mouse.

Fig. 3.

The effect of administration of(A) a single dose of 1000 μCi and (B)four dose fractions of 250 μCi each of 131I-labeled sc(Fv)2 or [sc(Fv)2]2 on growth of LS-174T xenograft in athymic mice. Each linerepresents the relative increase in tumor volume in an individual mouse.

Close modal
Table 1

Therapeutic efficacy of 131I-labeled CC49 sc(Fv)2 and [sc(Fv)2]2 on the growth of LS174-T human colon carcinoma xenografts

The time for tumor-quadrupling volume for each group was determined with single and/or fractionated radioimmunotherapeutic administrations. The average tumor size at day 6 for the group was used as the initial value for calculating the relative tumor quadrupling time.

Therapeutic reagentDose and schedulingMean time for quadrupling of tumor volume (days)
sc(Fv)2 1000 μCi at day 6 9.0 ± 0.8 
sc(Fv)2 250 μCi× 4 at days 6, 7, 8, and 9 21.1 ± 2.9 
[sc(Fv)2]2 1000 μCi at day 6 16.6 ± 1.9 
[sc(Fv)2]2 250 μCi× 4 at days 6, 7, 8, and 9 32.9 ± 2.7 
Control Single injection of 200 μl PBS at day 6 8.3 ± 0.7 
Control 200 μl PBS× 4 at days 6, 7, 8, and 9 8.4 ± 0.6 
Therapeutic reagentDose and schedulingMean time for quadrupling of tumor volume (days)
sc(Fv)2 1000 μCi at day 6 9.0 ± 0.8 
sc(Fv)2 250 μCi× 4 at days 6, 7, 8, and 9 21.1 ± 2.9 
[sc(Fv)2]2 1000 μCi at day 6 16.6 ± 1.9 
[sc(Fv)2]2 250 μCi× 4 at days 6, 7, 8, and 9 32.9 ± 2.7 
Control Single injection of 200 μl PBS at day 6 8.3 ± 0.7 
Control 200 μl PBS× 4 at days 6, 7, 8, and 9 8.4 ± 0.6 
Fig. 4.

Survival analysis of athymic mice bearing LS-174T human colon carcinoma xenografts versus time after a single administration of 1000 μCi of sc(Fv)2(▪) or [sc(Fv)2]2 (♦) or four injections of 250 μCi each of sc(Fv)2 (□) or[sc(Fv)2]2 (⋄). The control group (•)received injections with PBS (pH 7.4). Arrow represents the day of initiation of therapeutic regimen, i.e. day 6.

Fig. 4.

Survival analysis of athymic mice bearing LS-174T human colon carcinoma xenografts versus time after a single administration of 1000 μCi of sc(Fv)2(▪) or [sc(Fv)2]2 (♦) or four injections of 250 μCi each of sc(Fv)2 (□) or[sc(Fv)2]2 (⋄). The control group (•)received injections with PBS (pH 7.4). Arrow represents the day of initiation of therapeutic regimen, i.e. day 6.

Close modal
Fig. 5.

Toxicity of radioiodinated scFvs as monitored by the percentage change in body weight. Animals were given either a single administration of 1000 μCi of sc(Fv)2 (▪) or[sc(Fv)2]2 (♦) or four injections of 250μCi each of sc(Fv)2 (□) or[sc(Fv)2]2 (⋄). The control group (•)received PBS (pH 7.4). Arrow represents day 6.

Fig. 5.

Toxicity of radioiodinated scFvs as monitored by the percentage change in body weight. Animals were given either a single administration of 1000 μCi of sc(Fv)2 (▪) or[sc(Fv)2]2 (♦) or four injections of 250μCi each of sc(Fv)2 (□) or[sc(Fv)2]2 (⋄). The control group (•)received PBS (pH 7.4). Arrow represents day 6.

Close modal
Table 2

Radiation dose estimates of 131I-labeled CC49 scFvs in LS-174T xenograft-bearing mice

Therapeutic scheduleCumulative radiation doses (rads)
TumoraLiverKidneys
sc(Fv)2, 1000 μCi 8.5 0.9 1.3 
sc(Fv)2, 250 μCi× 4 38.2 3.7 5.3 
[sc(Fv)2]2, 1000 μCi 36.0 3.0 2.9 
[sc(Fv)2]2, 250 μCi× 4 168.9 12.2 11.5 
Therapeutic scheduleCumulative radiation doses (rads)
TumoraLiverKidneys
sc(Fv)2, 1000 μCi 8.5 0.9 1.3 
sc(Fv)2, 250 μCi× 4 38.2 3.7 5.3 
[sc(Fv)2]2, 1000 μCi 36.0 3.0 2.9 
[sc(Fv)2]2, 250 μCi× 4 168.9 12.2 11.5 
a

Tumor doses are for 1 g nodule.

We thank K. Devish, J. Jokerst, H. Conway, and Erik Moore for expert technical assistance. We acknowledge the Molecular Biology Core Lab for sequencing studies, the Molecular Interaction Facility for BIAcore studies, and Kristi L. W. Berger, communications specialist and editor, Eppley Institute, University of Nebraska Medical Center, for editorial assistance. The monovalent CC49 scFv construct was a generous gift from the National Cancer Institute Laboratory of Tumor Immunology and Biology and the Dow Chemical Company.

1
DeNardo S. J., Kroger L. A., DeNardo G. L. A new era for radiolabeled antibodies in cancer?.
Curr. Opin. Immunol.
,
11
:
563
-569,  
1999
.
2
Cheung N. K., Kushner B. H., Yeh S. D. J., Larson S. M. 3F8 monoclonal antibody treatment of patients with stage 4 neuroblastoma: a Phase II study.
Int. J. Oncol.
,
12
:
1299
-1306,  
1998
.
3
Matthews D. C., Appelbaum F. R., Eary J. F., Fisher D. R., Durack L. D., Hui T. E., Martin P. J., Mitchell D., Press O. W., Storb R., Bernstein I. D. Phase I study of (131)I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome.
Blood
,
94
:
1237
-1247,  
1999
.
4
Meredith R. F., Khazaeli M. B., Macey D. J., Grizzle W. E., Mayo M., Schlom J., Russell C. D., LoBuglio A. F. Phase II study of interferon-enhanced 131I-labeled high affinity CC49 monoclonal antibody therapy in patients with metastatic prostate cancer.
Clin. Cancer Res.
,
5(Suppl.)
:
3254s
-3258s,  
1999
.
5
Juweid M. E., Hajjar G., Stein R., Sharkey R. M., Herskovic T., Swayne L. C., Suleiman S., Pereira M., Rubin A. D., Goldenberg D. M. Initial experience with high-dose radioimmunotherapy of metastatic medullary thyroid cancer using 131I-MN-14 F(ab)2 anti-carcinoembryonic antigen MAb and AHSCR.
J. Nucl. Med.
,
41
:
93
-103,  
2000
.
6
Foran J. M., Rohatiner A. Z., Cunningham D., Popescu R. A., Solal-Celigny P., Ghielmini M., Coiffier B., Johnson P. W., Gisselbrecht C., Reyes F., Radford J. A., Bessell E. M., Souleau B., Benzohra A., Lister T. A. European Phase II study of rituximab (chimeric anti-CD20 monoclonal antibody) for patients with newly diagnosed mantle-cell lymphoma and previously treated mantle-cell lymphoma, immunocytoma, and small B-cell lymphocytic lymphoma.
J. Clin. Oncol.
,
18
:
317
-324,  
2000
.
7
Akabani G., Cokgor I., Coleman R. E., Gonzalez Trotter D., Wong T. Z., Friedman H. S., Friedman A. H., Garcia-Turner A., Herndon J. E., DeLong D., McLendon R. E., Zhao X. G., Pegram C. N., Provenzale J. M., Bigner D. D., Zalutsky M. R. Dosimetry and dose-response relationships in newly diagnosed patients with malignant gliomas treated with iodine-131-labeled anti-tenascin monoclonal antibody 81C6 therapy.
Int. J. Radiat. Oncol. Biol. Phys.
,
46
:
947
-958,  
2000
.
8
Vose J. M., Wahl R. L., Saleh M., Rohatiner A. Z., Knox S. J., Radford J. A., Zelenetz A. D., Tidmarsh G. F., Stagg R. J., Kaminski M. S. Multicenter Phase II study of iodine-131 tositumomab for chemotherapy-relapsed/refractory low-grade and transformed low-grade B-cell non-Hodgkin’s lymphomas.
J. Clin. Oncol.
,
18
:
1316
-1323,  
2000
.
9
Winzelberg G. G., Grossman S. J., Rizk S., Joyce J. M., Hill J. B., Atkinson D. P., Sudina K., Anderson K., McElwain D., Jones A. M. Indium-111 monoclonal antibody B72.3 scintigraphy in colorectal cancer. Correlation with computed tomography, surgery, histopathology, immunohistology, and human immune response.
Cancer (Phila.)
,
69
:
1656
-1663,  
1992
.
10
Kaminski M. S., Zasadny K. R., Francis I. R., Milik A. W., Ross C. W., Moon S. D., Crawford S. M., Burgess J. M., Petry N. A., Butchko G. M., et al. Radioimmunotherapy of B-cell lymphoma with [131I]anti-B1 (anti-CD20) antibody.
N. Engl. J. Med.
,
329
:
459
-465,  
1993
.
11
DeNardo S. J., DeNardo G. L., Kukis D. L., Shen S., Kroger L. A., DeNardo D. A., Goldstein D. S., Mirick G. R., Salako Q., Mausner L. F., Srivastava S. C., Meares C. F. 67Cu-2IT-BAT-Lym-1 pharmacokinetics, radiation dosimetry, toxicity and tumor regression in patients with lymphoma.
J. Nucl. Med.
,
40
:
302
-310,  
1999
.
12
Crippa F., Bolis G., Seregni E., Gavoni N., Scarfone G., Ferraris C., Buraggi G. L., Bombardieri E. Single-dose intraperitoneal radioimmunotherapy with the murine monoclonal antibody I-131 MOv18: clinical results in patients with minimal residual disease of ovarian cancer.
Eur. J. Cancer
,
5
:
686
-690,  
1995
.
13
Juweid M., Sharkey R. M., Behr T. M., Swayne L. C., Dunn R., Ying Z., Siegel J. A., Hansen H. J., Goldenberg D. M. Clinical evaluation of tumor targeting with the anticarcinoembryonic antigen murine monoclonal antibody fragment, MN-14 F(ab)2.
Cancer (Phila.)
,
78
:
157
-168,  
1996
.
14
Behr T. M., Sharkey R. M., Juweid M. E., Dunn R. M., Vagg R. C., Ying Z., Zhang C. H., Swayne L. C., Vardi Y., Siegel J. A., Goldenberg D. M. Phase I/II clinical radioimmunotherapy with an iodine-131-labeled anti-carcinoembryonic antigen murine monoclonal antibody IgG.
J. Nucl. Med.
,
38
:
858
-870,  
1997
.
15
DeNardo G. L., O’Donnell R. T., Kroger L. A., Richman C. M., Goldstein D. S., Shen S., DeNardo S. J. Strategies for developing effective radioimmunotherapy for solid tumors.
Clin. Cancer Res.
,
5(Suppl.)
:
3219s
-3223s,  
1999
.
16
Reilly R. M., Sandhu J., Alvarez-Diez T. M., Gallinger S., Kirsh J., Stern H. Problems of delivery of monoclonal antibodies. Pharmaceutical and pharmacokinetic solutions.
Clin. Pharmacokinet.
,
28
:
126
-142,  
1995
.
17
Esteban J. M., Colcher D., Sugarbaker P., Carrasquillo J. A., Bryant G., Thor A., Reynolds J. C., Larson S. M., Schlom J. Quantitative and qualitative aspects of radiolocalization in colon cancer patients of intravenously administered MAb B72.3.
Int. J. Cancer
,
39
:
50
-59,  
1987
.
18
Buchsbaum D. J., Langmuir V. K., Wessels B. W. Experimental radioimmunotherapy.
Med. Phys.
,
20
:
551
-567,  
1993
.
19
Stein R., Blumenthal R., Sharkey R. M., Goldenberg D. M. Comparative biodistribution and radioimmunotherapy of monoclonal antibody RS7 and its F(ab′)2 in nude mice bearing human tumor xenografts.
Cancer (Phila.)
,
73
:
816
-823,  
1994
.
20
Buchegger F., Mach J. P., Folli S., Delaloye B., Bischof-Delaloye A., Pelegrin A. Higher efficiency of 131I-labeled anti-carcinoembryonic antigen-monoclonal antibody F(ab′)2 as compared to intact antibodies in radioimmunotherapy of established human colon carcinoma grafted in nude mice.
Recent Results Cancer Res.
,
141
:
19
-35,  
1996
.
21
Behr T. M., Memtsoudis S., Sharkey R. M., Blumenthal R. D., Dunn R. M., Gratz S., Wieland E., Nebendahl K., Schmidberger H., Goldenberg D. M., Becker W. Experimental studies on the role of antibody fragments in cancer radio-immunotherapy: influence of radiation dose and dose rate on toxicity and anti-tumor efficacy.
Int. J. Cancer
,
77
:
787
-795,  
1998
.
22
Becker W. S., Behr T. M., Cumme F., Rossler W., Wendler J., Kern P. M., Gramatzki M., Kalden J. R., Goldenberg D. M., Wolf F. G. 67Ga citrate versus 99mTc-labeled LL2-Fab′ (anti-CD22) fragments in the staging of B-cell non-Hodgkin’s lymphoma.
Cancer Res.
,
55(Suppl.)
:
5771s
-5773s,  
1995
.
23
Gulec S. A., Serafini A. N., Moffat F. L., Vargas-Cuba R. D., Sfakianakis G. N., Franceschi D., Crichton V. Z., Subramanian R., Klein J. L., De Jager R. L. Radioimmunoscintigraphy of colorectal carcinoma using technetium-99m-labeled, totally human monoclonal antibody 88BV59H21–2.
Cancer Res.
,
55(Suppl.)
:
5774s
-5776s,  
1995
.
24
Behr T. M., Goldenberg D. M. Improved prospects for cancer therapy with radiolabeled antibody fragments and peptides?.
J. Nucl. Med.
,
37
:
834
-836,  
1996
.
25
Hudson P. J. Recombinant antibody constructs in cancer therapy.
Curr. Opin. Immunol.
,
11
:
548
-557,  
1999
.
26
Bird R. E., Hardman K. D., Jacobson J. W., Johnson S., Kaufman B. M., Lee S. M., Lee T., Pope S. H., Riordan G. S., Whitlow M. Single-chain antigen-binding proteins.
Science (Washington DC)
,
242
:
423
-426,  
1988
.
27
Huston J. S., McCartney J., Tai M. S., Mottola-Hartshorn C., Jin D., Warren F., Keck P., Oppermann H. Medical applications of single-chain antibodies.
Int. Rev. Immunol.
,
10
:
195
-217,  
1993
.
28
Colcher D., Bird R., Roselli M., Hardman K. D., Johnson S., Pope S., Dodd S. W., Pantoliano M. W., Milenic D. E., Schlom J. In vivo tumor targeting of a recombinant single-chain antigen-binding protein.
J. Natl. Cancer Inst. (Bethesda)
,
82
:
1191
-1197,  
1990
.
29
Yokota T., Milenic D. E., Whitlow M., Schlom J. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms.
Cancer Res.
,
52
:
3402
-3408,  
1992
.
30
Pluckthun A., Pack P. New protein engineering approaches to multivalent and bispecific antibody fragments.
Immunotechnology
,
3
:
83
-105,  
1997
.
31
Adams G. P., McCartney J. E., Tai M. S., Oppermann H., Huston J. S., Stafford W. F. d., Bookman M. A., Fand I., Houston L. L., Weiner L. M. Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single-chain Fv.
Cancer Res.
,
53
:
4026
-4034,  
1993
.
32
Wu A. M., Chen W., Raubitschek A., Williams L. E., Neumaier M., Fischer R., Hu S. Z., Odom-Maryon T., Wong J. Y., Shively J. E. Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers.
Immunotechnology (Amsterdam)
,
2
:
21
-36,  
1996
.
33
Adams G. P., Schier R., McCall A. M., Crawford R. S., Wolf E. J., Weiner L. M., Marks J. D. Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu.
Br. J. Cancer
,
77
:
1405
-1412,  
1998
.
34
Beresford G. W., Pavlinkova G., Booth B. J., Batra S. K., Colcher D. Binding characteristics and tumor targeting of a covalently linked divalent CC49 single-chain antibody.
Int. J. Cancer
,
81
:
911
-917,  
1999
.
35
Pavlinkova G., Booth B. J., Batra S. K., Colcher D. Radioimmunotherapy of human colon cancer xenografts using a dimeric single-chain Fv antibody construct.
Clin. Cancer Res.
,
5
:
2613
-2619,  
1999
.
36
Goel A., Beresford G. W., Colcher D., Pavlinkova G., Booth B. J. M., Baranowska-Kortylewicz J., Batra S. K. Divalent form of CC49 single-chain antibody constructs in Pichia pastoris: expression, purification and characterization.
J. Biochem. (Tokyo)
,
127
:
829
-836,  
2000
.
37
Colcher D., Minelli M. F., Roselli M., Muraro R., Simpson-Milenic D., Schlom J. Radioimmunolocalization of human carcinoma xenografts with B72.3 second generation monoclonal antibodies.
Cancer Res.
,
48
:
4597
-4603,  
1988
.
38
Murray J. L., Macey D. J., Kasi L. P., Rieger P., Cunningham J., Bhadkamkar V., Zhang H. Z., Schlom J., Rosenblum M. G., Podoloff D. A. Phase II radioimmunotherapy trial with 131I-CC49 in colorectal cancer.
Cancer (Phila.)
,
73
:
1057
-1066,  
1994
.
39
Murray J. L., Macey D. J., Grant E. J., Rosenblum M. G., Kasi L. P., Zhang H. Z., Katz R. L., Riger P. T., LeBherz D., Bhadkamkar V., et al Enhanced TAG-72 expression and tumor uptake of radiolabeled monoclonal antibody CC49 in metastatic breast cancer patients following α-interferon treatment.
Cancer Res.
,
55(Suppl.)
:
5925s
-5928s,  
1995
.
40
Divgi C. R., Scott A. M., Dantis L., Capitelli P., Siler K., Hilton S., Finn R. D., Kemeny N., Kelsen D., Kostakoglu L., et al. Phase I radioimmunotherapy trial with iodine-131-CC49 in metastatic colon carcinoma.
J. Nucl. Med.
,
36
:
586
-592,  
1995
.
41
Tempero M., Leichner P., Dalrymple G., Harrison K., Augustine S., Schlam J., Anderson J., Wisecarver J., Colcher D. High-dose therapy with iodine-131-labeled monoclonal antibody CC49 in patients with gastrointestinal cancers: a Phase I trial.
J. Clin. Oncol.
,
15
:
1518
-1528,  
1997
.
42
Lowry O., Rosenbrough N. J., Farr A. L., Randall R. J. Protein measurement by Folin phenol.
J. Biol. Chem.
,
193
:
265
-275,  
1951
.
43
Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (Lond.)
,
227
:
680
-685,  
1970
.
44
Colcher D., Pavlinkova G., Beresford G., Booth B. J., Batra S. K. Single chain antibodies in pancreatic cancer.
Ann. NY Acad. Sci.
,
880
:
263
-280,  
1999
.
45
Colcher D., Zalutsky M., Kaplan W., Kufe D., Austin F., Schlom J. Radiolocalization of human mammary tumors in athymic mice by a monoclonal antibody.
Cancer Res.
,
43
:
736
-742,  
1983
.
46
Stabin M. G. MIRDOSE: personal computer software for internal dose assessment in nuclear medicine.
J. Nucl. Med.
,
37
:
538
-546,  
1996
.
47
Howell, R. W., Wessels, B. W., Loevinger, R., Watson, E. E., Bolch, W. E., Brill, A. B., Charkes, N. D., Fisher, D. R., Hays, M. T., Robertson, J. S., Siegel, J. A., and Thomas, S. R. The MIRD perspective 1999. Medical internal radiation dose committee. J. Nucl. Med. 40: 3S–10S, 1999.
48
Jain R. K., Baxter L. T. Mechanisms of heterogeneous distribution of monoclonal antibodies and other macromolecules in tumors: significance of elevated interstitial pressure.
Cancer Res.
,
48
:
7022
-7032,  
1988
.
49
Jain R. K. Determinants of tumor blood flow: a review.
Cancer Res.
,
48
:
2641
-2658,  
1988
.
50
Schlom J., Eggensperger D., Colcher D., Molinolo A., Houchens D., Miller L. S., Hinkle G., Siler K. Therapeutic advantage of high-affinity anticarcinoma radioimmunoconjugates.
Cancer Res.
,
52
:
1067
-1072,  
1992
.
51
Schlom J., Molinolo A., Simpson J. F., Siler K., Roselli M., Hinkle G., Houchens D. P., Colcher D. Advantage of dose fractionation in monoclonal antibody-targeted radioimmunotherapy.
J. Natl. Cancer Inst. (Bethesda)
,
82
:
763
-771,  
1990
.
52
Meredith R. F., Khazaeli M. B., Liu T., Plott G., Wheeler R. H., Russell C., Colcher D., Schlom J., Shochat D., LoBuglio A. F. Dose fractionation of radiolabeled antibodies in patients with metastatic colon cancer.
J. Nucl. Med.
,
33
:
1648
-1653,  
1992
.
53
Buchsbaum D., Khazaeli M. B., Liu T., Bright S., Richardson K., Jones M., Meredith R. Fractionated radioimmunotherapy of human colon carcinoma xenografts with 131I-labeled monoclonal antibody CC49.
Cancer Res.
,
55(Suppl.)
:
5881s
-5887s,  
1995
.
54
Buchsbaum D. J., Wahl R. L., Glenn S. D., Normolle D. P., Kaminski M. S. Improved delivery of radiolabeled anti-B1 monoclonal antibody to Raji lymphoma xenografts by predosing with unlabeled anti-B1 monoclonal antibody.
Cancer Res.
,
52
:
637
-642,  
1992
.
55
Ceriani R. L., Blank E. W., Peterson J. A. Experimental immunotherapy of human breast carcinomas implanted in nude mice with a mixture of monoclonal antibodies against human milk fat globule components.
Cancer Res.
,
47
:
532
-540,  
1987
.
56
Sharkey R. M., Blumenthal R. D., Hansen H. J., Goldenberg D. M. Biological considerations for radioimmunotherapy.
Cancer Res.
,
50(Suppl.)
:
964s
-969s,  
1990
.
57
Schlom J., Milenic D. E., Roselli M., Colcher D., Bird R., Johnson S., Hardman K. D., Guadagni F., Greiner J. W. New concepts in monoclonal antibody based radioimmunodiagnosis and radioimmunotherapy of carcinoma.
Int. J. Radiat. Appl. Instrum. Part B
,
18
:
425
-435,  
1991
.
58
Pedley R. B., Boden J. A., Boden R., Dale R., Begent R. H. Comparative radioimmunotherapy using intact or F(ab′)2 fragments of 131I anti-CEA antibody in a colonic xenograft model.
Br. J. Cancer
,
68
:
69
-73,  
1993
.
59
Quadri S. M., Lai J., Mohammadpour H., Vriesendorp H. M., Williams J. R. Assessment of radiolabeled stabilized F(ab′)2 fragments of monoclonal antiferritin in nude mouse model.
J. Nucl. Med.
,
34
:
2152
-2159,  
1993
.
60
Schott M. E., Frazier K. A., Pollock D. K., Verbanac K. M. Preparation, characterization, and in vivo biodistribution properties of synthetically cross-linked multivalent antitumor antibody fragments.
Bioconjug. Chem.
,
4
:
153
-165,  
1993
.
61
King D. J., Turner A., Farnsworth A. P., Adair J. R., Owens R. J., Pedley R. B., Baldock D., Proudfoot K. A., Lawson A. D., Beeley N. R., et al Improved tumor targeting with chemically cross-linked recombinant antibody fragments.
Cancer Res.
,
54
:
6176
-6185,  
1994
.
62
Werlen R. C., Lankinen M., Offord R. E., Schubiger P. A., Smith A., Rose K. Preparation of a trivalent antigen-binding construct using polyoxime chemistry: improved biodistribution and potential for therapeutic application.
Cancer Res.
,
56
:
809
-815,  
1996
.
63
Wolff E. A., Esselstyn J., Maloney G., Raff H. V. Human monoclonal antibody homodimers.
Effect of valency on in vitro and in vivo antibacterial activity. J. Immunol.
,
148
:
2469
-2474,  
1992
.
64
Wolff E. A., Schreiber G. J., Cosand W. L., Raff H. V. Monoclonal antibody homodimers: enhanced antitumor activity in nude mice.
Cancer Res.
,
53
:
2560
-2565,  
1993
.
65
Pavlinkova G., Beresford G. W., Booth B. J., Batra S. K., Colcher D. Pharmacokinetics and biodistribution of engineered single-chain antibody constructs of MAb CC49 in colon carcinoma xenografts.
J. Nucl. Med.
,
40
:
1536
-1546,  
1999
.
66
Behr T. M., Goldenberg D. M., Becker W. S. Radioimmunotherapy of solid tumors: a review “of mice and men”.
Hybridoma
,
16
:
101
-107,  
1997
.
67
Blumenthal R. D., Sharkey R. M., Haywood L., Natale A. M., Wong G. Y., Siegel J. A., Kennel S. J., Goldenberg D. M. Targeted therapy of athymic mice bearing GW-39 human colonic cancer micrometastases with 131I-labeled monoclonal antibodies.
Cancer Res.
,
52
:
6036
-6044,  
1992
.
68
Blumenthal R. D., Sharkey R. M., Natale A. M., Kashi R., Wong G., Goldenberg D. M. Comparison of equitoxic radioimmunotherapy and chemotherapy in the treatment of human colonic cancer xenografts.
Cancer Res.
,
54
:
142
-151,  
1994
.
69
Buchsbaum D. J., Khazaeli M. B., Mayo M. S., Roberson P. L. Comparison of multiple bolus and continuous injections of 131I-labeled CC49 for therapy in a colon cancer xenograft model.
Clin. Cancer Res.
,
5(Suppl.)
:
3153s
-3159s,  
1999
.
70
Thomas G. D., Chappell M. J., Dykes P. W., Ramsden D. B., Godfrey K. R., Ellis J. R., Bradwell A. R. Effect of dose, molecular size, affinity, and protein binding on tumor uptake of antibody or ligand: a biomathematical model.
Cancer Res.
,
49
:
3290
-3296,  
1989
.
71
Boerman O. C., Sharkey R. M., Wong G. Y., Blumenthal R. D., Aninipot R. L., Goldenberg D. M. Influence of antibody protein dose on therapeutic efficacy of radioiodinated antibodies in nude mice bearing GW-39 human tumor.
Cancer Immunol. Immunother.
,
35
:
127
-134,  
1992
.
72
Adams G. P., McCartney J. E., Wolf E. J., Eisenberg J., Tai M. S., Huston J. S., Stafford W. F., III, Bookman M. A., Houston L. L., Weiner L. M. Optimization of in vivo tumor targeting in SCID mice with divalent forms of 741F8 anti-c-erbB-2 single-chain Fv: effects of dose escalation and repeated i.v. administration. Cancer Immunol.
Immunother.
,
40
:
299
-306,  
1995
.
73
Blumenthal R. D., Kashi R., Sharkey R. M., Goldenberg D. M. Quantitative and qualitative effects of experimental radioimmunotherapy on tumor vascular permeability.
Int. J. Cancer
,
61
:
557
-566,  
1995
.
74
Pavlinkova G., Colcher D., Booth B. J. M. B., Goel A., Batra S. K. Pharmacokinetics and biodistribution of a light-chain shuffled CC49 single-chain Fv antibody construct.
Cancer Immunol. Immunother.
,
49
:
267
-275,  
2000
.
75
Chalandon Y., Mach J. P., Pelegrin A., Folli S., Buchegger F. Combined radioimmunotherapy and chemotherapy of human colon carcinoma grafted in nude mice, advantages and limitations.
Anticancer Res.
,
12
:
1131
-1139,  
1992
.
76
Buchegger F., Rojas A., Delaloye A. B., Vogel C. A., Mirimanoff R. O., Coucke P., Sun L. Q., Raimondi S., Denekamp J., Pelgrin A., et al Combined radioimmunotherapy and radiotherapy of human colon carcinoma grafted in nude mice.
Cancer Res.
,
55
:
83
-89,  
1995
.
77
Vogel C. A., Galmiche M. C., Buchegger F. Radioimmunotherapy and fractionated radiotherapy of human colon cancer liver metastases in nude mice.
Cancer Res.
,
57
:
447
-453,  
1997
.
78
Pedley R. B., Boden J. A., Boden R., Boxer G. M., Flynn A. A., Keep P. A., Begent R. H. Ablation of colorectal xenografts with combined radioimmunotherapy and tumor blood flow-modifying agents.
Cancer Res.
,
56
:
3293
-3300,  
1996
.
79
Behr T. M., Behe M., Stabin M. G., Wehrmann E., Apostolidis C., Molinet R., Strutz F., Fayyazi A., Wieland E., Gratz S., Koch L., Goldenberg D. M., Becker W. High-linear energy transfer (LET) α versus low-LET β emitters in radioimmunotherapy of solid tumors: therapeutic efficacy and dose-limiting toxicity of 213Bi- versus 90Y-labeled CO17–1A Fab′ fragments in a human colonic cancer model.
Cancer Res.
,
59
:
2635
-2643,  
1999
.
80
Gautherot E., Rouvier E., Daniel L., Loucif E., Bouhou J., Manetti C., Martin M., Le Doussal J. M., Barbet J. Pretargeted radioimmunotherapy of human colorectal xenografts with bispecific antibody and 131I-labeled bivalent hapten.
J. Nucl. Med.
,
41
:
480
-487,  
2000
.
81
Axworthy D. B., Reno J. M., Hylarides M. D., Mallett R. W., Theodore L. J., Gustavson L. M., Su F., Hobson L. J., Beaumier P. L., Fritzberg A. R. Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity.
Proc. Natl. Acad. Sci. USA
,
97
:
1802
-1807,  
2000
.