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Clinical Cancer Research Vol. 5, 2613-2619, September 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Radioimmunotherapy of Human Colon Cancer Xenografts Using a Dimeric Single-Chain Fv Antibody Construct1

Gabriela Pavlinkova2, Barbara J. M. Booth, Surinder K. Batra and David Colcher

Departments of Pathology and Microbiology [G. P., B. J. M. B.] and Biochemistry and Molecular Biology [S. K. B.], University of Nebraska Medical Center, Omaha, Nebraska 68198-3135; and Coulter Pharmaceuticals Inc., San Francisco, California 94080 [D. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Progress in the use of monoclonal antibodies (MAbs) for the treatment of solid tumors is limited by a number of factors, including poor penetration of the labeled IgG molecule into the tumors, their inability to reach the tumor in sufficient quantities without significant normal tissue toxicity, and the development of a human antimouse antibody response to the injected MAb. One possible way to alter the pharmacology of antibodies is via the use of smaller molecular weight antibody fragments called single-chain Fvs (scFvs). A divalent construct of MAb CC49, CC49 (scFv)2, composed of two noncovalently associated scFvs, was generated and shown to bind a tumor-associated antigen (TAG-72) epitope with a similar binding affinity to that of the murine IgG. The therapeutic potential of this construct after labeling with 131I was examined in athymic mice bearing established s.c. human colon carcinoma (LS-174T) xenografts. Treatment groups (n = 10) received a single dose of 131I-labeled CC49 (scFv)2 (500–2000 µCi) or 131I-labeled CC49 IgG (250 and 500 µCi). The group of mice treated with the lowest dose of 131I-(scFv)2 (500 µCi) showed statistically significant prolonged survival, compared with controls (P = 0.036). Complete tumor regression was observed in 20% of mice given 1500 µCi of labeled (scFv)2 and 30 and 60% of mice treated with 250 and 500 µCi of labeled IgG, respectively. In conclusion, the CC49 (scFv)2 construct provides a promising delivery vehicle for therapeutic applications.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
RIT3 is a promising therapeutic approach for the treatment of a wide variety of malignancies. The antitumor effect of RIT is based on the selective delivery of a sufficient radiation dose to tumors without significantly affecting normal tissues. Multiple clinical trials have shown the RIT treatment to be effective for some types of lymphomas and leukemias (1, 2, 3, 4, 5) . However, despite the promising preclinical study results, only modest responses have been documented in patients with solid tumors (6, 7, 8, 9, 10, 11) . The efficacy of RIT is affected by numerous factors, such as vascular and tumor permeability, development of human antimouse antibodies, heterogeneity of antigenic expression, the intrinsic radiosensitivity of the targeted tumor, radiation emissions, properties of the radionuclide and chemical stability of the radioimmunoconjugate, and normal tissue toxicity that is secondary to circulating radiolabeled antibody (12) .

The choice of antibody is another important factor in RIT. Several MAbs have been generated against tumor-associated antigens and have been used for radioimmunodetection of tumors. MAb CC49 IgG is one example of such an antibody. It reacts with a unique sialyl-Tn antigen expressed on a tumor-associated mucin, TAG-72. CC49 IgG exhibits high reactivity against tumor cells in most adenocarcinomas from colorectum, ovary, breast, stomach, and pancreas, but it shows little reactivity against normal tissues (13) . The preclinical therapeutic studies using radiolabeled CC49 IgG showed reduced tumor growth in 80–100% of tumor-bearing animals (14) . 131I-labeled MAb CC49 used in clinical trials demonstrated excellent tumor targeting. However, despite the promising preclinical results, patient trials with radiolabeled CC49 IgG failed to identify any clinically effective responses, even at the highest administered activities (300 mCi/m2; Ref. 9, 10, 11) .

The difficulties in the clinical application of radiolabeled CC49 IgG and other radiolabeled MAbs are primarily caused by normal tissue toxicity, immunogenicity, and relatively poor penetration into tumor. The development of genetically engineered single-chain antibody fragments (scFvs) is one way to potentially overcome some of these limitations. The scFv molecule is comprised of the variable regions of the immunoglobulin heavy and light chains, which are covalently connected by a flexible peptide linker (15 , 16) . The scFvs showed very rapid blood clearance, excellent penetration into the tumor from the vasculature, reduced immunogenicity, and higher tumor:normal tissue ratios (RIs) than corresponding IgG, F(ab')2, or Fab' fragments in animal models (17, 18, 19, 20, 21) . Larson et al. (22) showed that CC49 scFv is safe, that tissue equilibration and clearance are fast, and that same-day imaging of the primary and metastatic tumors is feasible in patients with colorectal carcinoma. However, due to their small size and monovalency, scFvs clear the body too rapidly to allow for sufficient tumor uptake and retention for therapeutic applications.

Recently, we developed a new CC49 scFv construct that forms high-affinity stable noncovalent dimers [(scFv)2]. The CC49 (scFv)2 showed a rapid blood clearance rate, excellent RIs, lack of uptake in normal tissues, and better tumor targeting properties than corresponding CC49 scFv or Fab' (23) . Here, we present the therapeutic potential of the 131I-labeled CC49 (scFv)2 construct in an experimental tumor system and compare it with that of the intact IgG molecule.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Purification of MAb CC49 IgG.
MAb CC49 IgG was developed by the immunization of mice with purified TAG-72, as described previously (24 , 25) . CC49 IgG was purified from ascitic fluid that was obtained from pristane-primed BALB/c mice using Protein G chromatography (Pharmacia Biotech, Piscataway, NJ). The protein concentration was determined by the method of Lowry et al. (26) .

Expression and Purification of CC49 (scFv)2.
Construction and expression of CC49/205C scFv (VL-Linker-VH) has been described previously by Pavlinkova et al. (27) . The CC49 scFv protein was purified from the periplasmic fraction of a 1.0-liter Escherichia coli overnight culture by ion-exchange chromatography using a Mono-Q column (Pharmacia Biotech). Dimeric and monomeric scFv forms were separated by size-exclusion chromatography using a Superdex 75 column (Pharmacia Biotech).

Characterization of Purified CC49 Immunoglobulin Forms.
CC49 (scFv)2 and IgG binding activity was determined by a competition ELISA (27) using microtiter plate wells coated with BSM (28) . Purified proteins were analyzed by SDS-PAGE (29) and stained with Coomassie blue. The molecular weight of (scFv)2 was estimated by comparison to the relative mobility values of the gel filtration standard (Bio-Rad, Hercules, CA) using HPLC.

The binding kinetics of CC49 IgG and scFv dimer was measured by the BIAcore biosensor (Pharmacia Biosensor, Uppsala, Sweden), as described previously (23) . Briefly, BSM (positive control) or BSA (negative control; 200 µg/ml) was injected until a surface of 700 resonance units was realized. Binding analyses were performed in HBS buffer [10 mM HEPES (pH 7.4), 0.15 M NaCl, 3.4 µM EDTA, and 0.005% surfactant P20] at a flow rate of 30 µl/min at 25°C. The association constant (KA) and dissociation constant (KD) were evaluated using BIAevaluation Version 3.0 (Pharmacia Biosensor, Uppsala, Sweden), in which the experimental design correlated with the Langmuir 1:1 interaction model (30) .

Radioiodination of CC49 Immunoglobulin Forms.
The MAb CC49 IgG and the scFv form were labeled with Na125I or Na131I using Iodo-Gen (Pierce Chemical, Rockford, IL), as described by Colcher et al. (31) . The iodination protocol yielded specific activities of ~3–9 mCi/mg. The purity of each radiolabeled preparation was analyzed by gel filtration HPLC on tandem TSK G3000SW and TSK G2000SW columns (Toso Haas, Montgomeryville, PA). The immunoreactivity was determined using BSM- or BSA-coated beads (Reacti-Gel HW-65F; Pierce). The coated beads (0.5 ml) were washed with 1% BSA-0.1% Tween 20 in PBS and resuspended in 0.5 ml of binding buffer (1% BSA in PBS). The radiolabeled samples were added to each tube and incubated for 1 h at RT, the unbound radiolabeled protein was removed by washing, and the pellet was counted in a gamma scintillation counter.

Biodistribution Studies.
Tumors were grown in 4–6-week-old female athymic mice (nu/nu; Charles River, Wilmington, MA) after a s.c. injection of 4 x 106 human colon carcinoma cells (LS-174T; Ref. 32 ). Radiolabeled dimeric scFv and IgG (5 µCi of 125I-labeled MAb and 2.5 µCi of 131I-labeled MAb) were coinjected i.v. into tumor-bearing animals ~10 days after injection of the cells. At specific times, mice (groups of six) were euthanized and dissected, and the major organs were weighed and counted in a gamma scintillation counter. The percentage ID per gram of tissue (expressed as % ID/g) was calculated. Pharmacokinetic studies were conducted by obtaining blood samples (5 µl) via tail bleeds at various times after the i.v. injection of 10 µCi of the radioiodinated CC49 forms. On average, six mice per group are presented. The whole-body retention of radiolabeled forms was also determined. Mice bearing the LS-174T xenograft (three mice per group) were injected via the tail vein with 1.5 µCi of radiolabeled CC49 forms and counted using a custom-built NaI crystal at various times after injection.

Biodistribution and pharmacokinetic data were analyzed by using Graphpad Prism, Version 2.01 (GraphPad Software Inc., San Diego, CA). To compare changes in % ID/g over time between IgG and (scFv)2, we performed statistical analyses using two-way ANOVAs. When a significant interaction was detected, the differences between IgG and (scFv)2 at each time point were further compared using the t test for unpaired data (significance assigned at the P < 0.05 level). The clearance of the (scFv)2 was very rapid, so single-phase exponential decay curve fit was used for pharmacokinetic analyses. The data for F(ab')2 and IgG were analyzed using a two-phase exponential decay curve fit.

Tumor Therapy Studies.
In all RIT experiments, female athymic mice (nu/nu), obtained from Charles River (Wilmington, MA) at 4–6 weeks of age, were injected s.c. on the back with 4 x 106 human colon carcinoma cells (LS-174T; Ref. 32 ). Tumor growth was determined by caliper measurement of the long and short axis for each tumor and the tumor volume was calculated as follows:

Eight days post-tumor implantation, the mice were distributed into groups (n = 10) to give similar tumor size distributions in all treatment groups. One group was injected with PBS (control group) or treated i.v. with a single dose of radioiodinated CC49 IgG or CC49 (scFv)2. Body weight and tumor size were monitored twice a week. Animals were observed until the animals died, the tumors exceeded 10% of the total body weight, the tumors began to ulcerate through the skin, or the animals lost >20% of their original weight, at which time the animals were removed from the group and killed. The survival fraction of each treatment group was evaluated according to the method of Kaplan and Meier. The survival curves were compared, and Ps were generated using the log-rank test. GraphPad Prism, Version 2.01 (GraphPad Software Inc.) was used for these analyses.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
We have developed a new recombinant fragment-dimeric CC49 scFv [(scFv)2]. We hypothesized that the characteristics of CC49 (scFv)2 may satisfy the requirements for successful RIT. CC49 scFv noncovalent dimer combines a small size for fast elimination from circulation with a reduction of normal tissue toxicity and better penetration into tumor due to smaller size, compared with intact IgG.

Biochemical Characterization and Immunoreactivity of CC49 (scFv)2.
The CC49/205C (scFv)2 was secreted as soluble, active protein using the pRW83 expression vector and purified by ion-exchange chromatography. SDS-PAGE analysis of the purified (scFv)2 showed >90% purity. The molecular weight of the (scFv)2 (Mr ~60,000) was estimated using size-exclusion HPLC by comparison to the relative mobility values of the standards. The association constants were determined by surface plasmon resonance (BIAcore) for CC49 IgG (KA = 1.14 x108) and (scFv)2 (KA = 4.46 x 107; Ref. 23 ).

The purified CC49 (scFv)2 and IgG were radiolabeled with Na125I and analyzed for protein purity using SDS-PAGE and size-exclusion HPLC (Fig. 1)Citation . The immunoreactivity was evaluated using beads coated with BSM, which contains the epitope recognized by MAb CC49 on the human tumor-associated antigen (TAG-72), as a positive control or with BSA as a negative control. The (scFv)2 showed 88–94% binding to BSM, compared with intact IgG (87–90%), with <5% nonspecific binding. Radiolabeled samples of the CC49 (scFv)2 were stable for at least 35 days when they were stored at 4°C, as tested by HPLC and the binding assay. Radiolabeled (scFv)2 was also analyzed to determine its stability in serum. Unfortunately, blood clearance of CC49 scFv dimer is fast, and its concentration in blood is not sufficient to perform direct blood HPLC analysis. Therefore, we have designed conditions similar to in vivo conditions, when radiolabeled CC49 scFv dimer was incubated in murine serum at 37°C, and most importantly, the concentrations of radiolabeled CC49 (scFv)2 were appropriate for analysis using HPLC for extended time period (4 days). The labeled samples mixed with murine serum or 1% BSA were taken at various times and analyzed by HPLC. The CC49 (scFv)2 maintained its full integrity throughout the testing period (data not shown).



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  		Fig. 1. HPLC size-exclusion profiles of the radiolabeled CC49 IgG and (scFv)2. After radiolabeling, 125I-IgG and 125I-(scFv)2 were analyzed using TSK G3000SW and TSK G2000SW size-exclusion columns connected in series. The IgG (•) and the (scFv)2 ({blacktriangleup}) were eluted as single peaks of Mr ~150,000 and Mr ~60,000, respectively.

 
Pharmacokinetics and Tumor Targeting of the CC49 (scFv)2.
The blood clearance rate of radiolabeled CC49 (scFv)2 was compared with the blood clearance of the intact IgG and the divalent fragment [F(ab')2] in pharmacokinetic studies. As seen in Fig. 2Citation , the (scFv)2 (Mr ~60,000) blood clearance was faster than F(ab')2 (Mr ~110,000) or IgG (Mr ~150,000) with 50% cleared from the blood pool in 50, 400, and 1200 min, respectively. Whole-body clearance analysis also displayed a rapid (scFv)2 clearance, suggesting that CC49 noncovalent dimers were not being retained in the extravascular space or in any specific organ. The relative rates of clearance observed in the whole body experiments were similar to those observed with the blood clearance (data not shown).



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  		Fig. 2. CC49 IgG, F(ab')2, and (scFv)2 were injected into athymic mice (six per group) bearing LS-174T colon carcinoma xenografts, and blood samples were obtained at various times. IgG ({blacktriangleup}), F(ab')2 ({triangleup}), and (scFv)2 ({diamond}).

 
The antigen-binding ability of the CC49 (scFv)2 in vivo and its efficiency to target human colon carcinoma xenografts (LS-174T) were compared with CC49 IgG in biodistribution studies. At various times after injection, blood, tumor, and normal organs were analyzed to determine the amount of each radionuclide retained per gram of tissue. The (scFv)2 % ID/g levels in tissues are generally lower than those of the IgG, due to a more rapid blood and whole-body clearance (Table 1)Citation . The differences in the relative rates of blood clearance in the biodistribution studies were statistically significant (P < 0.001). As expected, (scFv)2 uptake in the kidneys was 5 times higher than uptake of IgG at 30 min (P < 0.001) but 4 times lower that reported for the kidney uptake for similarly size Fab' fragments (23) . However, the level of renal uptake detected with (scFv)2 was similar to the levels found in the other major organs by 4 h postadministration.


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  		Table 1 Comparative biodistribution studies with CC49 IgG and (scFv)2a

 
A lower level of tumor uptake (10.87% ID/g) was observed with in vivo tumor targeting with (scFv)2, as compared with IgG (23.12% ID/g) at 6 h postadministration (P < 0.001). However, the CC49 dimeric scFv appears to remain intact in vivo, as evidenced by the relatively high levels of tumor uptake up to 120 h. The CC49 IgG showed noticeably higher nonspecific uptake in the liver, spleen, blood, and kidneys, compared with the (scFv)2 construct after 24 h postadministration. Extremely high RIs (RI = ratio of the % ID/g in tumor to the % ID/g in normal tissue) were obtained with (scFv)2 with a tumor:blood (P < 0.001), tumor:liver (P = 0.01), and tumor:spleen (P = 0.02) ratios of 80.3, 25.5, and 31.2:1, respectively, compared with 3.4, 6.1, and 9.1:1 for IgG at 24 h.

Therapeutic Efficacy of the Radiolabeled CC49 (scFv)2.
We have compared the therapeutic potential of radioiodinated CC49 (scFv)2 and IgG in two sets of RIT studies with athymic mice bearing established s.c. LS-174T tumors. Radiolabeled MAb administered dose titration and the size of the established s.c. LS-174T tumors were the variables in these studies. In the first study, mice with large s.c. LS-174T tumors received a single injection of 500, 750, or 1000 µCi of 131I-CC49 (scFv)2. In the second study, animals with smaller established s.c. tumors received a single injection of 1000, 1500, or 2000 µCi of 131I-CC49 (scFv)2 by i.v. injection. Each study also involved groups of mice receiving radiolabeled MAb CC49 IgG at doses of 250, 500 µCi for comparison as well as a control group. Survival analysis (Fig. 3)Citation showed significant tumor growth inhibition for all treated animal groups, as compared with controls. Even the lowest (scFv)2 administered dose of 500 µCi resulted in a statistically significant prolonged survival of treated mice when compared with untreated mice (P = 0.036), with median survival times of 27 and 18 days, respectively. Groups of mice treated with 750 and 1000 µCi of 131I-CC49 (scFv)2 demonstrated significant prolonged survival, compared with the control group (P < 0.001). However, complete tumor regression was observed only with higher levels of (scFv)2 (1500 µCi; 2 of 10) and with 250 µCi (3 of 10) and 500 µCi (6 of 10) of CC49 IgG (Fig. 3)Citation . An administered dose of 1500 µCi of 131I-labeled (scFv)2 was the maximum tolerated single dose.



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  		Fig. 3. Survival analysis of athymic mice with established s.c. LS-174T tumors versus time. Animals were treated with a single injection of 131I-labeled CC49 IgG or 131I-labeled CC49 (scFv)2 or left untreated (Control). The administered dose for IgG was 250 or 500 µCi and the dose for radiolabeled (scFv)2 was 500, 750, 1000, or 1500 µCi.

 
As expected, the initial size of tumors was another important factor influencing the efficacy of RIT. In the second study using athymic mice with smaller s.c. LS-174T tumors, the median survival was higher for treated groups when compared with the first study. Both study groups, the group with larger tumors (initial volume = 305 ± 64 mm3) and the group with smaller tumors (initial volume = 176 ± 31 mm3), were treated with 1000 µCi of (scFv)2 and 250 µCi and 500 µCi of IgG. Differences in the survival of mice treated with 1000 µCi of (scFv)2 were noted. Mice bearing smaller tumors had significantly prolonged survival (P = 0.02), compared with the group with larger tumors: median survival times of 44 and 25 days, respectively. Fifty days after RIT, 40% of treated animals were still alive in this group, whereas none of the treated mice survived in the group with larger tumors (Fig. 3)Citation . Similarly, treatment with radiolabeled IgG provided less effective therapy for larger established tumors. The median survival times of mice treated with 250 µCi IgG were 74 days in the first study and 130 days in the second group bearing smaller tumors. The growth rate of tumors was evaluated for each group. In both studies, the mice with established s.c. LS-174T tumors showed dose-dependent tumor growth inhibition curves, as compared with untreated controls. The mean values for each group are presented in Fig. 4Citation . The growth rate of tumors in the group with large tumor was very rapid, with mean volume quadrupling times of <7 days for controls, 15 days for mice treated with 1000 µCi of (scFv)2 and 19 days for mice treated with 250 µCi of IgG (Fig. 5)Citation . In contrast, the growth rate of tumors in the group with small tumor showed growth delay, with mean volume quadrupling times of 27 and 34 days for mice treated with 1000 and 1500 µCi of (scFv)2, respectively, and 62 days for mice treated with 250 µCi of IgG. The mean volume quadrupling time of the untreated control tumors was <8 days for the group with small tumors. The best growth inhibition was observed in mice treated with 500 µCi of IgG, in which the tumor volume never quadrupled.



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  		Fig. 4. Growth kinetics of LS-174T tumors [large tumors (A), initial volume = 305 ± 64 mm3; and small tumors (B), initial volume = 176 ± 31 mm3] after treatment with 131I-labeled CC49 IgG or CC49 (scFv)2 and no therapy (Control). Data points, means for 10 animals in each group; bars, SE. The IgG administered dose was 250 or 500 µCi, and the administered dose of radiolabeled (scFv)2 was 500, 750, 1000, or 1500 µCi.

 


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  		Fig. 5. Therapeutic efficacy of 131I-labeled CC49 (scFv)2 and IgG treatment on the growth of established LS174-T xenografts represented as a mean volume quadrupling time. Columns, means for the groups of mice with large tumors ({blacksquare}) and small tumors (); bars, SE. Results with treatment of (scFv)2 at 500, 750, or 1000 µCi (n = 10) and 250 µCi of IgG (n = 9) in the large tumor group or 1000 (n = 10) or 1500 (n = 9) µCi of (scFv)2 and 250 µCi of IgG (n = 9) in the small tumor group were analyzed.

 
The selection of radionuclide is a critical factor to consider for RIT. The kidney was the dose-limiting organ in a preclinical therapy study with radiometal (90Y)-labeled Fab fragments (33) . However, radiation nephrotoxicity was not a problem in the therapeutic applications of radioiodinated antibody fragments, but the dose-limiting organ was the red marrow in a study in which Behr et al. (34) reported excellent therapeutic antitumor efficacy using 131I-labeled anti-CEA Fab. In this study, the Fab fragments were more efficient at controlling human colon cancer xenograft growth than the respective IgG. Treatment-related toxicity of 131I radiolabeled CC49 (scFv)2 and IgG was monitored by weight loss (Fig. 6)Citation . One of 10 animals died from a single injection in the group injected with 1500 µCi of 131I-CC49 (scFv)2 at 41 days posttreatment and in 500 µCi of 131I-CC49 IgG group at 44 days posttreatment. Treatment with 2000 µCi of 131I-CC49 (scFv)2 yielded 100% lethality at 26–59 days.



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  		Fig. 6. Relationship between dosage of radiolabeled antibody and the change in body weight. 131I-labeled CC49 (scFv)2 (at 500, 750, 1000, 1500, or 2000 µCi) and IgG (250 or 500 µCi) were injected into athymic mice (10 per group) bearing s.c. LS-174T large (A) and small (B) tumors. Data points, average body weights during first 60 days posttreatment.

 
In conclusion, we showed that the radiolabeled CC49 (scFv)2 construct is a promising candidate for therapeutic applications. The single administration of 1500 µCi of 131I-CC49 (scFv)2 resulted in complete tumor regression in ~20% of treated mice and significant improvement in survival times. This was the maximal tolerated dose for a single-treatment regimen. Future studies for RIT with the CC49 scFv dimer should include dose fractionation of the administered radiolabeled construct. Several investigators have reported that dose fractionation can be less toxic and more efficacious than a single dose (35, 36, 37) . The biological efficacy of time-dose fractionation depends on the total dose that can be administered, the dose rate, the biological half-life of the antibody, the physical half-life of the radionuclide, and the fractionation schedule (38) .


   ACKNOWLEDGMENTS
 
We thank Heather Conway, Kay Devish, and Jodi Halgren for their expert technical assistance and the Monoclonal Antibody Core Facility and Molecular Interaction Laboratory for their expertise. The CC49 scFv construct was a generous gift from the National Cancer Institute Laboratory of Tumor Immunology (Dr. Jeffrey Schlom) and the Dow Chemical Company.


   FOOTNOTES
 
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 These studies were supported by United States Department of Energy Grant DE-FG02-95ER62024 and were conducted in the J. Bruce Henrikson Cancer Research Laboratories. Back

2 To whom requests for reprints should be addressed, at Department of Pathology and Microbiology, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, NE 68198-3135. Phone: (402) 559-2976; Fax: (402) 559-8112; E-mail: gpavlink{at}unmc.edu Back

3 The abbreviations used are: RIT, radioimmunotherapy; MAb, monoclonal antibody; scFv, single-chain Fv; RI, radiolocalization index; BSM, bovine submaxillary mucin; HPLC, high-performance liquid chromatography; ID, injected dose. Back

Received 2/ 5/99; revised 5/19/99; accepted 5/21/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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