Purpose: New strategies that target selected molecular characteristics and result in an effective therapeutic index are needed for metastatic, hormone-refractory prostate cancer.

Experimental Design: A series of preclinical and clinical studies were designed to increase the therapeutic index of targeted radiation therapy for prostate cancer. 111In/90Y-monoclonal antibody (mAb), m170, which targets aberrant sugars on abnormal MUC1, was evaluated in androgen-independent prostate cancer patients to determine the maximum tolerated dose and efficacy of nonmyeloablative radioimmunotherapy and myeloablative combined modality radioimmunotherapy with paclitaxel. To enhance the tumor to liver therapeutic index, a cathepsin degradable mAb linkage (111In/90Y-peptide-m170) was used in the myeloablative combined modality radioimmunotherapy protocol. For tumor to marrow therapeutic index improvement in future studies, anti-MUC1 scFvs modules were developed for pretargeted radioimmunotherapy. Anti-MUC1 and anti-DOTA scFvs were conjugated to polyethylene glycol scaffolds tested on DU145 prostate cancer cells and prostate tissue arrays, along with mAbs against MUC1 epitopes.

Results: The nonmyeloablative maximum tolerated dose of 90Y-m170 was 0.74 GBq/m2 for patients with not more than 10% axial skeleton involvement. Metastatic prostate cancer was targeted in all 17 patients; mean radiation dose was 10.5 Gy/GBq and pain response occurred in 7 of 13 patients reporting pain. Myeloablative combined modality radioimmunotherapy with 0.4 GBq/m2 of 90Y-peptide-m170 and paclitaxel showed therapeutic effects in 4 of 6 patients and 30% less radiation to the liver per unit of activity. Neutropenia was dose limiting without marrow support and patient eligibility was a major limitation to dose escalation. Hypoglycosylated MUC1 epitopes were shown to be abundant in prostate cancer and to increase with disease grade. Anti-MUC1 scFvs binding to prostate cancer tissue and live cells were developed into di-scFv binding modules.

Conclusions: The therapeutic index enhancement for prostate radioimmunotherapy was achieved in clinical studies by the addition of cathepsin cleavable linkers to 90Y-conjugated mAbs and the use of paclitaxel. However, the need for marrow support in myeloablative combined modality radioimmunotherapy restricted eligible patients. Therefore, modular pretargeted radioimmunotherapy, aiming at improving the tumor to marrow therapeutic index, is being developed.

Although localized prostate cancer is curable, metastatic hormone-independent prostate cancer is usually fatal. Better understanding of this disease has yielded information useful in designing new therapeutic strategies (1, 2). Studies of epithelial cancer biology have provided insights into the relationship of abnormal glycoproteins and prostate cancer grade of relevance for tumor targeting (37).

Cancer-related aberrations in MUC1 epithelial mucin present unique epitopes that can provide targets for site-specific therapy for many epithelial cancers. Normal MUC1 is apically distributed on the surface of epithelial cells as a large, complex glycoprotein composed of a polypeptide core protected by long oligosaccharide side chains on its extracellular region (8). A 20-amino-acid sequence repeated in tandem (variable number of tandem repeats) constitutes the MUC1 core (9). In most epithelial cancers, expression of MUC1 is up-regulated, hypo- and aberrantly glycosylated, and its cell surface distribution is no longer apical. Monoclonal antibodies (mAb) against the MUC1 mucin in breast cancer have previously been investigated using a panel of mAbs and MUC1 mucin–related synthetic peptides and glycopeptides (10). Whereas MUC1 glycoproteins have been found in the blood of cancer patients and used as markers for disease (11), mAbs to MUC1 epitopes not shed in the blood have been used for radioimaging and radioimmunotherapy (1215). These mAbs include those shown to target the aberrantly glycosylated MUC1 peptide core, and also mAbs m170 and 155 produced against a synthetic asialo GM1 disaccharide but reactive to Thompson-Friedenreich–related sialyl-Tn antigen (16, 17); the Thompson-Friedenreich antigen has been recently confirmed as MUC1 associated (18).

For targeted radiation therapy, such as radioimmunotherapy, to have an effect on prostate cancer, the therapeutic index, defined as the therapeutic effect on cancer cells compared with toxic effects on the most sensitive normal tissues, must be increased (19, 20). This report spans nonmyeloablative radioimmunotherapy-related responses through combined modality radioimmunotherapy trials to new developments for future therapeutic index enhancement through novel pretargeted radioimmunotherapy. Both patient radioimmunotherapy trials in prostate cancer patients and immunohistochemistry using novel scFvs on prostate cancer tissue arrays show that hypoglycosylated MUC1 epitopes provide excellent targets for hormone-independent metastatic prostate cancer radioimmunotherapy. To reduce marrow toxicity, new pretargeted radioimmunotherapy is being developed based on the MUC1 scFv epitopes in prostate cancer and using new di-scFv modular pretargeting molecules.

Radioimmunotherapy and combined modality radioimmunotherapy

Patients. Twenty-six patients with androgen-independent progressive prostate cancer were entered on one of two radioimmunotherapy protocols, each requiring a radioimmunoconjugate quantitative imaging pharmacokinetic/dosimetry study, 1 week before radioimmunotherapy. The initial quantitative imaging pharmacokinetic/dosimetry study from each patient was used in the dosimetry analysis and radioimmunoconjugate comparison.

Radiopharmaceuticals.m170 monoclonal antibody. mAb m170 is a murine immunoglobulin G developed using a synthetic asialo GM1 terminal disaccharide immunogen related to the Thompson-Friedenreich antigen recently shown on MUC1 (1618, 21). mAb m170 (current good manufacturing practice grade) from Biomira, Inc. (Edmonton, Canada) was >95% monomeric immunoglobulin G by PAGE and met U.S. Food and Drug Administration guidelines.

Immunoconjugates. DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid) was conjugated to m170 as 2IT-BAD-m170 immunoconjugate and DOTA-peptide-m170 (DOTA-glycyl-glycyl-glycyl-m170) was prepared by methods previously described, radiolabeled with 111In for imaging or with 90Y for treatment (Fig. 1; refs. 2224). The resulting radioimmunoconjugates were 99% pure by molecular sieving chromatography. Immunoreactivity was comparable to the unmodified mAb (24); cellulose acetate electrophoresis and high-performance liquid chromatography indicated that more than 95% of the radiometal was associated with the radioimmunoconjugates.

Fig. 1.

Schematic illustrating the two mAb-chelate linkages studied: A, mAb-2IT-BAD, which was not cleavable with cathepsin B; B, mAb-peptide-DOTA in which DOTA is linked to the mAb via a tetra-peptide (GGGF) susceptible to cleavage by cathepsin B; B′, mAb after peptide cleavage by cathepsin (66).

Fig. 1.

Schematic illustrating the two mAb-chelate linkages studied: A, mAb-2IT-BAD, which was not cleavable with cathepsin B; B, mAb-peptide-DOTA in which DOTA is linked to the mAb via a tetra-peptide (GGGF) susceptible to cleavage by cathepsin B; B′, mAb after peptide cleavage by cathepsin (66).

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Pharmacokinetics and radiation dosimetry. In the nonmyeloablative protocol, 17 patients were given 111In-m170 followed 1 week later by 90Y-m170. 111In-m170 (0.185 GBq, 6-8 mg) was injected i.v. followed by quantitative imaging over 7 days; serial blood and 24-hour urine samples were quantitated. 90Y doses were determined by activity per square meter (0.185-0.74 GBq/m2, 10-25 mg mAb) independent of the radiation dosimetry.

In the combined modality radioimmunotherapy myeloablative protocol, nine patients were imaged with 111In-peptide-m170 and six of these patients received radioimmunotherapy (0.4 GBq/m2, 10-15 mg). The initial patient cohort received radioimmunotherapy alone and the subsequent cohort received combined modality radioimmunotherapy as radioimmunotherapy followed by paclitaxel, 75 mg/m2, 48 hours after radioimmunotherapy. At this interval, as previously published, the majority of the radiation dose to marrow had been delivered, and the majority of the radiation to tumor had not yet occurred (25). Cyclosporine A was given 5 mg/kg orally every 12 hours to prevent human antimurine antibody binding (26). Pharmacokinetic data from imaging were obtained as previously described (20, 27, 28). The geometric-mean method was used to quantify activity in the organs and tumors clearly delineated on conjugate views, and photon attenuation was corrected using a transmission scan (29). The effective point source method was used to quantify activity in most tumors and kidneys (27).

Cumulated activity was converted to radiation dose for 90Y using the Medical Internal Radiation Dose formula, Medical Internal Radiation Dose S values, and reference man masses (30). The radiation dose from blood to marrow and marrow-to-marrow was calculated as previously described (23, 29, 31). Only tumors with a measured volume of 10 mL or greater were used for dosimetric calculations.

Statistical methods. Cumulated activity was determined as described above. To test for differences between the linkers, a Wilcoxon rank-sum test was used. All reported P values were two tailed.

Pretargeted radioimmunotherapy development

Immunohistochemistry on prostate tissues. Formalin-fixed tissue embedded in paraffin, obtained from the University of California at Davis Human Biological Specimen Repository and categorized in terms of pathology and Gleason grade (32), included a range of prostate cancers from prostatic intraepithelial neoplasia to Gleason grade 5 and benign/normal prostate tissues. The prostate tissues represented in the 197 array cores were 77 normal/benign (39%), 31 prostatic intraepithelial neoplasia and Gleason grade 1 to 2 (16%), and 89 Gleason grades 3 to 5 (45%).

Three well-characterized anti-MUC1 mAbs, with different epitope specificities, were chosen to evaluate the presentation of MUC1 on the prostate tissue array (Table 1); mAb m170 was not included because it does not react well with fixed tissues.

Table 1.

Characteristics of anti-MUC1 mAbs used to validate the suitability of aberrant MUC1 as a target for pretargeted radioimmunotherapy in prostate cancer

mAbCloneImmunogenCore epitope
BrE3 BrE3 HMFG TRP 
B27.29 B27.29 Human ascite mucin DTRPAP 
HMFG1 1.10.F3 Delipidated HMFG PDTR 
mAbCloneImmunogenCore epitope
BrE3 BrE3 HMFG TRP 
B27.29 B27.29 Human ascite mucin DTRPAP 
HMFG1 1.10.F3 Delipidated HMFG PDTR 

NOTE: Because their MUC1 reactivity has been well characterized (10, 37), these mAbs were used to quantitate and characterize MUC1 on prostate tissue.

BrE3 monoclonal antibody. In previous studies, this mAb stained 97% of metastatic breast cancer specimens with >75% positive cells (33). BrE3 binds to glycosylated and nonglycosylated forms of MUC1 with similar affinity and its expression has been reported to be associated with breast cancer patient survival (3437). Normal tissues were not targeted on imaging studies (12, 13). BrE3 mAb was obtained from Dr. R. Ceriani.

B27.29 monoclonal antibody. In prior studies, mAb B27.29 showed increased diffuse, cytoplasmic MUC1 expression on prostate cancer compared with normal prostate (6). Nuclear magnetic resonance studies of the binding of B27.29 to peptides with various glycosylation patterns indicate this mAb binds to the core epitope spanning the PDTRP sequence and a carbohydrate epitope (38). In a study on breast cancers, low B27.29 staining was associated with higher-grade tumors (39), consistent with recognition of hypoglycosylated epitopes of MUC1. mAb B27.29 was obtained from Rebiodiagnostics (Edmonton, Canada).

HMFG1 monoclonal antibody. HMFG staining in breast cancer was not associated with decreased survival (40, 41). Partial deglycosylation of MUC1 resulted in lower binding by HMFG1 compared with increased binding by B27.29 and BrE3 (36). HMFG1 was obtained from Novocastra (Newcastle upon Tyne, United Kingdom).

ScFv-c. Two scFv-c from our MUC1 scFv phage library were selected based on reaction with MUC-1 on DU145 prostate cancer cells and membranes.

Tissue microarray analysis. Immunohistochemistry was done as previously described (42), with the following mAb and scFv-c concentrations: BrE3 at 5 μg/mL; B27.29 at 0.5 μg/mL; HMFG1 were tissue culture supernatants diluted to 1:10; and scFv-c at 50 μg/mL. Scores were assigned based on three criteria: percent of cancer cells stained, apical and/or cytoplasmic staining, and intensity of staining. Cores without cancer cells (normal/benign) were scored to reflect the percentage of epithelial glandular cells stained. H&E staining of a separate array slide was used to group the scores by tissue status (normal/benign, prostatic intraepithelial neoplasia or Gleason grade 1-2, and Gleason grade 3-5). A positive score was given if >50% of tumor cells were positive with a minimum staining intensity of 1. The highest scores were analyzed for trend of increased staining and Gleason grade using SAS software (SAS version 8.0, SAS Institute, Inc., Cary, NC).

ScFv phage display library construction and scFv. These procedures have been described previously (43). Briefly, mice (Harlan Sprague-Dawley, Indianapolis, IN) received an injection of MUC1-positive MCF-7/HBT 3477 (10:1) cell membrane lysate, followed by three immunizations with keyhole limpet hemocyanin-MUC1 synthetic peptide (80-mer corresponding to four variable number of tandem repeats) at 3-week intervals. The scFv library was constructed using the RPAS mouse scFv module (Amersham Biosciences Corp., Piscataway, NJ). Selection was done using three or more rounds of decreasing amounts (100, 50, and 10 nmol/L) of MUC1 peptide conjugated to biotinylated bovine serum albumin on magnetic streptavidin beads (Dynal, Inc., Lake Success, NY) and evaluated by ELISA on DU145 and MCF-7 live cells and cell membranes. Plasmids (pCANTAB 5E) from selected clones were isolated (Qiagen, Valencia, CA) and the scFv cDNA sequenced (DBS Automated DNA Sequencing Facility, University of California at Davis).

ScFv-c and di-scFv-c clones. ScFv-c, containing a free thiol from the addition of an unpaired cysteine, were obtained by cloning scFv fragments from the pCANTAB 5E into pCANTAB 5E-Cys vector (42). ScFv-c clones were recovered following electroporation of the HB2151 bacterial host.

Di-scFv-c clones were assembled as follows. Linker sequences carrying a 5′ NotI restriction site and coding for either (G4S)4 or (G4S)2C(G4S)2 were introduced in front of the VH domain of a scFv, cloned in pCANTAB 5E, by consecutive insertional mutagenesis using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA); the resulting clones were named LscFv. Final di-scFv-C constructs were assembled by ligation of the NotI insert of the LscFv clones into the unique NotI restriction site present at the 3′ end of the corresponding scFv cloned in pCANTAB 5E or pCANTAB 5E-Cys. Di-scFv-c clones, with the 5′ to 3′ orientation of the NotI insert, were selected by KpnI restriction mapping.

ScFv production and purification. ScFv, scFv-c, or di-scFv-c clones in HB2151 bacteria were grown overnight in cell culture plates or shaker flasks at 30°C with shaking at 200 rpm in 2× YT media (17 g/L bacto-tryptone, 10 g/L bacto-yeast extract, 5 g/L NaCl) containing 2.0% glucose and 100 μg/mL of ampicillin. After changing the medium to 2× YT + ampicillin, scFv expression was induced for 6 hours or overnight at 30°C by addition of isopropyl-β-d-thiogalactopyranoside (U.S. Biochemical, Cleveland, OH) to 1 mmol/L final concentration.

The various scFv products were purified as soluble proteins from bacterial periplasmic extracts by anti–E-Tag affinity chromatography (Amersham Biosciences) as described previously (42). Purified scFvs were buffer exchanged into PBS (pH 7.4) and concentrated using YM10 centripreps (Millipore, Bedford, MA). Purified scFvs were quantitated (Micro BCA protein kit, Pierce Chemical Co., Rockford, IL) and either used immediately or stored at 70°C.

SDS-PAGE analysis. Proteins were analyzed by SDS PAGE using 4% to 18% NuPage gradient gels (Invitrogen, Carlsbad, CA) under nonreducing or reducing (presence of β-mercaptoethanol in the loading buffer) conditions. After electrophoresis, proteins were visualized by Coomassie blue staining.

Polyethylene glycol-Maleimide and PEGylation of scFv or di-scFv. Methoxy-polyethylene glycol-Maleimide (PEG-Mal) of 5 kDa was purchased from Nektar Therapeutics (San Carlos, CA). Maleimide-PEG-Maleimide (PEG-(Mal) 2) of 2 kDa was obtained from Sunbio PEG-Shop (Anyang City, South Korea). PEGylation reactions were carried out at molar ratios of 5:1 (PEG/scFv) in 0.1 mol/L sodium phosphate buffer (pH 7.0), 2 mmol/L EDTA, with scFv-c or di-scFv-c at 1 to 2 mg/mL in the presence of 2-fold molar excess of the reducing agent Tris (2-carboxy-ethyl)-phosphin-HCl (Molecular Probes, Eugene, OR). The reaction was left to proceed at 37°C overnight under N2 atmosphere.

Radioimmunotherapy and combined modality radioimmunotherapy

Improved tumor/liver therapeutic index with peptide linkage. Metastatic prostate cancer was targeted in 25 of 26 patients studied with 111In-DOTA-peptide m170 or 111In-2IT-BAD-m170. The mean radiation dose to 39 bone and 18 nodal metastases was 10.5 Gy/GBq (range 2.8-25.1). Comparison of pharmacokinetic data and radiation doses for tumor and organs in both patient groups was similar except the mean cumulated activity, and therefore the mean liver radiation dose, for DOTA-peptide m170 was 30% less than that for 2IT-BAD-m170 (Fig. 2). This provided an enhanced therapeutic index of tumor to liver for these patients.

Fig. 2.

Effects of radiochelate mAb linkage on pharmacokinetics and dosimetry in patients with prostate cancer comparing: A, mean radiation doses and SD for m170-2IT-BAD (A), n = 17 patients, and m170-peptide-DOTA (B), n = 9 patients. P = 0.02 for kidney, P < 0.01 for liver and lung, P = 0.58 for blood, P = 0.31 for tumor, P = 0.68 for body. Liver radiation dose was significantly reduced by the peptide linkage. B, cumulated activities in tumor (gray bars) and liver (white bars) for m170-2IT-BAD with noncleavable linkage and m170-peptide-DOTA with cleavable linkage. The therapeutic index (black bars) for tumor to liver for 90Y is indicated on the right.

Fig. 2.

Effects of radiochelate mAb linkage on pharmacokinetics and dosimetry in patients with prostate cancer comparing: A, mean radiation doses and SD for m170-2IT-BAD (A), n = 17 patients, and m170-peptide-DOTA (B), n = 9 patients. P = 0.02 for kidney, P < 0.01 for liver and lung, P = 0.58 for blood, P = 0.31 for tumor, P = 0.68 for body. Liver radiation dose was significantly reduced by the peptide linkage. B, cumulated activities in tumor (gray bars) and liver (white bars) for m170-2IT-BAD with noncleavable linkage and m170-peptide-DOTA with cleavable linkage. The therapeutic index (black bars) for tumor to liver for 90Y is indicated on the right.

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Clinical response. In the nonmyeloablative trial, 7 of the 13 patients reporting pain had partial or complete resolution of pain, and the prostate-specific antigen velocity was interrupted temporarily in 7 of 17 (41%) patients after a single dose of 90Y-m170. Of six patients with prostate cancer that received a dose of 90Y-DOTA-peptide-m170 in the combined modality radioimmunotherapy trial, two of three also receiving paclitaxel showed a 50% decrease in serum prostate-specific antigen lasted <2 months and a sustained decrease in bone pain.

Bone and soft tissue metastases were targeted in all of the patients and no unexpected normal tissue uptake was observed. Cyclosporine A prevented human antimurine antibody binding in all of the patients despite multiple doses of m170 mAb. However, combined modality radioimmunotherapy (radioimmunotherapy plus paclitaxel) at this radioimmunotherapy dose level resulted in myelosuppression manifested by transient neutropenia (two patients with absolute neutrophil count < 500 for 1 and 6 days; ref. 44).

Pretargeted radioimmunotherapy development

Prostate cancer MUC1 targets: epitope selection for pretargeted radioimmunotherapy molecules. Immunohistochemistry done on the tissue array with three anti-MUC1 mAbs (BrE3, B27.29, and HMFG1) gave a positive MUC1 score if 50% or more of the cells were stained (Fig. 3). The profile of BrE3 showed a strong correlation between prostate cancer stage and increase in MUC1 staining (P < 0.001). Because BrE3 recognizes predominately a peptidic epitope within the variable number of tandem repeat region of MUC1, this result suggests that hypoglycosylated MUC1 is a good marker for aggressive prostate cancer.

Fig. 3.

Prostate tissue microarray. Prostate tissue cores (197) representative of various grades of disease were placed in a single array so that each slice contained the same groups of core tissues. Immunohistochemistry was done using three well-described mAbs, BrE3, B27.29, HMGFG1, and two newly derived scFv-c. Immunohistochemistry examples of normal/benign (a), prostatic intraepithelial neoplasia or Gleason grade 1 to 2 (b), and Gleason grade 3 to 5 (c) tissue cores are shown for each mAb and scFv-c. A, immunohistochemistry with anti-MUC1 mAbs. Dark staining, indicative of specific binding of each mAb with hypoglycosylated MUC1 protein, can be seen, particularly with higher-grade prostate cancer. B, immunohistochemistry with anti-MUC1 scFvs. Both scFv-c display specific binding but differ in staining patterns (see part C). C, summary analysis and comparison of aberrant MUC1 detection in prostate cancer tissue by anti-MUC1 mAbs and scFv-c. Cores with 50% or more of the prostate cells stained were considered positive for the epitope targeted by the mAb or scFv-c. Staining pattern comparisons show that the pattern of the E1 scFv is similar to that of BrE3 mAb, both demonstrating increased staining in higher-grade prostate cancer (P < 0.001). The staining patterns of the G1 scFv are similar to these of B27.29 mAb.

Fig. 3.

Prostate tissue microarray. Prostate tissue cores (197) representative of various grades of disease were placed in a single array so that each slice contained the same groups of core tissues. Immunohistochemistry was done using three well-described mAbs, BrE3, B27.29, HMGFG1, and two newly derived scFv-c. Immunohistochemistry examples of normal/benign (a), prostatic intraepithelial neoplasia or Gleason grade 1 to 2 (b), and Gleason grade 3 to 5 (c) tissue cores are shown for each mAb and scFv-c. A, immunohistochemistry with anti-MUC1 mAbs. Dark staining, indicative of specific binding of each mAb with hypoglycosylated MUC1 protein, can be seen, particularly with higher-grade prostate cancer. B, immunohistochemistry with anti-MUC1 scFvs. Both scFv-c display specific binding but differ in staining patterns (see part C). C, summary analysis and comparison of aberrant MUC1 detection in prostate cancer tissue by anti-MUC1 mAbs and scFv-c. Cores with 50% or more of the prostate cells stained were considered positive for the epitope targeted by the mAb or scFv-c. Staining pattern comparisons show that the pattern of the E1 scFv is similar to that of BrE3 mAb, both demonstrating increased staining in higher-grade prostate cancer (P < 0.001). The staining patterns of the G1 scFv are similar to these of B27.29 mAb.

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Anti-MUC1 scFv selection and production. ScFvs binding to biotinylated bovine serum albumin-MUC1 peptide were selected from anti-MUC1 phage display libraries (43, 45). In these libraries, the format of a full-length scFv is VH-(G4S)3linker-VL. Production yields and additional characteristics of selected scFvs are indicated in Table 2. Production yields vary significantly from one scFv to another although their nucleotide sequences share 70% or greater homology (45). As deduced from their isoelectric point values, these scFvs vary in net charge at pH 7.

Table 2.

Characteristics of selected anti-MUC1 scFvs and their scFv-c counterparts

ScFvSourceSelectionLength (aa)pIProduction yield (mg/L)
E1 BALB/c mice: scFv gene library Biotinylated BSA-MUC1 258 5.84 0.25 
E1-c Cloning Extra TGT codon 259 5.84 0.10 
G1 BALB/c mice: scFv gene library Biotinylated BSA-MUC1 259 5.37 0.49 
G1-c Cloning Extra TGT codon 260 5.37 0.21 
D5 NZB mice: scFv gene library Biotinylated BSA-MUC1 259 8.92 0.7 
D5-c Cloning Extra TGT codon 260 8.76 1.5 
ScFvSourceSelectionLength (aa)pIProduction yield (mg/L)
E1 BALB/c mice: scFv gene library Biotinylated BSA-MUC1 258 5.84 0.25 
E1-c Cloning Extra TGT codon 259 5.84 0.10 
G1 BALB/c mice: scFv gene library Biotinylated BSA-MUC1 259 5.37 0.49 
G1-c Cloning Extra TGT codon 260 5.37 0.21 
D5 NZB mice: scFv gene library Biotinylated BSA-MUC1 259 8.92 0.7 
D5-c Cloning Extra TGT codon 260 8.76 1.5 

Abbreviations: aa, amino acid; pI, isoelectric point; BSA, bovine serum albumin.

The MUC1 binding of two scFvs, as scFv-c, E1 and G1, was characterized by comparing their prostate tissue binding patterns with those of the three anti-MUC1 mAbs (Fig. 3). As shown, the tissue binding of the E1 scFv-c increases with prostate cancer stage, whereas that of the G1 scFv-c seems less discriminating (Fig. 4). Therefore, BrE-3 and E1 scFv-c have MUC1 epitopes which become more prominent with disease progression.

Fig. 4.

Schematic of vectors used for the expression of scFv and scFv-c. A scFv expressed from the pCANTAB 5E vector carries a COOH-terminal E-Tag (E) that can be used for affinity purification and immunodetection. When expressed from pCANTAB 5E-Cys, the same scFv contains an additional COOH-terminal cysteine (C), located just upstream of the E-Tag, providing a free thiol for site-specific conjugation.

Fig. 4.

Schematic of vectors used for the expression of scFv and scFv-c. A scFv expressed from the pCANTAB 5E vector carries a COOH-terminal E-Tag (E) that can be used for affinity purification and immunodetection. When expressed from pCANTAB 5E-Cys, the same scFv contains an additional COOH-terminal cysteine (C), located just upstream of the E-Tag, providing a free thiol for site-specific conjugation.

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Di-scFv. Soluble bivalent di-scFv were produced in E. coli by expression of scFv genes cloned in tandem into the expression vector. These bivalent scFvs are covalently linked. Furthermore, the di-scFv design offers additional options for the location of the extra cysteine. Di-scFv-c were produced as scFv-c-scFv or scFv-scFv-c (Fig. 5A). Di-scFv-c production yields were influenced by the presence and location of the extra cysteine; the overall trend was toward a decreased yield in comparison with di-scFvs without an unpaired cysteine. SDS-PAGE profiles of E-Tag–purified scFv-c and di-scFv-c are shown in Fig. 5B; under reducing conditions, apparent molecular weights for scFv-c and di-scFv-c are 31 and 52 kDa, respectively.

Fig. 5.

Di-scFv-c. A, schematic of vectors used for the expression of di-scFv-c. Di-scFv-c were expressed from scFvs cloned in tandem into either pCANTAB 5E (scFv-c-scFv) or pCANTAB 5E-Cys (scFv-scFv-c). In all cases, the scFv joining linker was the 20-amino-acid-long (G4S)4 sequence. In scFv-scFv-c, the extra-cysteine (C) is inserted at the COOH terminus on the vector backbone, whereas in scFv-c-scFv, the cysteine is inserted within the (G4S)4 linker joining the two scFvs. B, reducing SDS-PAGE profiles of scFv-c and di-scFv-c. After E-Tag chromatography purification from E. coli periplasmic extracts, scFv-c and di-scFv-c proteins were loaded onto a 4% to 12% gradient polyacrylamide gel for electrophoresis under reducing and denaturing conditions. Subsequently, proteins were visualized by Coomassie blue staining. Lane 1, D5-c scFv (10 μg of total proteins); lane 2, D5D5-c di-scFv (24 μg of total proteins); KD lane, MW standards (from top to bottom): 185, 98, 52, 31, 19, and 11 kDa. Arrows, full-length scFv-c (lane 1) and di-scFv-c (lane 2) proteins. In addition to the major full-length proteins, some degradation products are copurified by E-Tag affinity chromatography.

Fig. 5.

Di-scFv-c. A, schematic of vectors used for the expression of di-scFv-c. Di-scFv-c were expressed from scFvs cloned in tandem into either pCANTAB 5E (scFv-c-scFv) or pCANTAB 5E-Cys (scFv-scFv-c). In all cases, the scFv joining linker was the 20-amino-acid-long (G4S)4 sequence. In scFv-scFv-c, the extra-cysteine (C) is inserted at the COOH terminus on the vector backbone, whereas in scFv-c-scFv, the cysteine is inserted within the (G4S)4 linker joining the two scFvs. B, reducing SDS-PAGE profiles of scFv-c and di-scFv-c. After E-Tag chromatography purification from E. coli periplasmic extracts, scFv-c and di-scFv-c proteins were loaded onto a 4% to 12% gradient polyacrylamide gel for electrophoresis under reducing and denaturing conditions. Subsequently, proteins were visualized by Coomassie blue staining. Lane 1, D5-c scFv (10 μg of total proteins); lane 2, D5D5-c di-scFv (24 μg of total proteins); KD lane, MW standards (from top to bottom): 185, 98, 52, 31, 19, and 11 kDa. Arrows, full-length scFv-c (lane 1) and di-scFv-c (lane 2) proteins. In addition to the major full-length proteins, some degradation products are copurified by E-Tag affinity chromatography.

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ScFv site–specific conjugation to polyethylene glycol. For site-specific conjugation of scFv-c modules onto a functionalized PEG scaffold, the unpaired cysteine was used. This scFv-c site–specific conjugation to PEG was evaluated in respect to several variables: (a) size of the PEG; (b) nature of the group reactive with thiol; (c) number of functional groups per PEG molecule; and (d) configuration (linear or branched) of the PEG molecule. None of the combinations tested led to >89% of the scFv-c PEGylation, although di-scFv-c has resulted in >95% PEGylation with the scFv-c-scFv format. Linear PEG molecules of 2 and 5 kDa (Fig. 6A) showed the highest efficiencies for PEGylation. The conjugate of interest (Fig. 6B), di-scFv-PEG, has been shown to retain the immunoreactivity of the di-scFv. These site-specific conjugation studies to PEG-Mal indicate that the free thiol is accessible in both scFv-c and di-scFv-c configurations.

Fig. 6.

PEG-Maleimide scFv site–specific conjugation. A, schematic of a bifunctional 2 kDa PEG-Maleimide. B, PEG-(scFv-c)2 (MW: 60 kDa) obtained through site-specific conjugation of two scFv-c (D5-c) to a 2 kDa bifunctional PEG maleimide (A). The PEG-(scFv)2 molecule was purified from the PEGylation mixture by molecular sieving (30 × 1.5 cm column packed with Sephadex G75). Proteins were resolved by reducing SDS-PAGE (4-12% polyacrylamide gradient) and visualized by Coomassie blue staining. KD lane, MW standards (from top to bottom): 185, 98, 52, 31, 19, and 11 kDa; lane 1, D5-c (15 μg of total proteins); lane 2, purified PEG-(scFv)2 bioconjugate (15 μg). Arrows, full-length scFv-c (lane 1) and PEG-(scFv)2 (lane 2).

Fig. 6.

PEG-Maleimide scFv site–specific conjugation. A, schematic of a bifunctional 2 kDa PEG-Maleimide. B, PEG-(scFv-c)2 (MW: 60 kDa) obtained through site-specific conjugation of two scFv-c (D5-c) to a 2 kDa bifunctional PEG maleimide (A). The PEG-(scFv)2 molecule was purified from the PEGylation mixture by molecular sieving (30 × 1.5 cm column packed with Sephadex G75). Proteins were resolved by reducing SDS-PAGE (4-12% polyacrylamide gradient) and visualized by Coomassie blue staining. KD lane, MW standards (from top to bottom): 185, 98, 52, 31, 19, and 11 kDa; lane 1, D5-c (15 μg of total proteins); lane 2, purified PEG-(scFv)2 bioconjugate (15 μg). Arrows, full-length scFv-c (lane 1) and PEG-(scFv)2 (lane 2).

Close modal

Systemically given targeted radionuclide therapy is a modality uniquely suited to provide effective therapy for metastatic and androgen-independent prostate cancer, if the therapeutic index can be enhanced. The series of preclinical and clinical studies presented here were designed toward this goal by selecting the aspects of MUC1 targeted/pretargeted radionuclide therapy that can be developed and combined to achieve an effective therapeutic index.

Combined modality radioimmunotherapy using 90Y DOTA-peptide-m170 with paclitaxel increased efficacy in patients with advanced androgen-independent, metastatic prostate cancer over that previously reported for radioimmunotherapy alone (19). Further dose escalation is possible with the use of PBSC. However, PBSC harvest is only modestly successful in this older population, thus greatly limiting eligible patients. A pretargeting radioimmunotherapy strategy with novel molecules holds more promise for achieving the needed therapeutic index. Enhanced therapeutic index for pretargeted radioimmunotherapy has been shown in animal models (4649) and in patients (5052). Pretargeting using various streptavidin-biotin formats has been investigated (51, 53); although highly efficient for capture of the radionuclide, they raise more immunogenicity concerns than other approaches under study (47, 49, 54).

Because pretargeting molecules have to first effectively target the tumor, MUC1 pretargeting molecules presented herein are multivalent for the tumor epitope. Over the past decade, a number of bispecific mAb and mAb fragments have been generated and methods to increase their functional affinity have been explored (55, 56). To obtain stable multimers, we used di-scFv-c modules covalently attached to a PEG scaffold. PEG was chosen because PEGylation is a validated drug delivery method for extension of serum half-life (57, 58).

The selection of MUC1 as the targeting epitope for pretargeted radioimmunotherapy of metastatic prostate cancer was based on several criteria: (a) tumor targeting obtained in clinical trials with mAbs, such as m170 and BrE3 (13, 19, 20, 59, 60); (b) hypoglycosylated MUC1 provides novel tumor-specific epitopes (61); and (c) peptide mimics of the 20-amino-acid motif repeated in the MUC1 variable number of tandem repeat region promote immune responses in mice for development of anti-MUC1 scFvs (43).

In contrast to the wealth of available studies on MUC1 in breast cancer, knowledge of MUC1 in prostate cancer was limited. To validate MUC1 epitope targets and select suitable scFvs from our library for pretargeted radioimmunotherapy of prostate cancer, we studied the binding of three well-characterized anti-MUC1 mAbs using immunohistochemistry on a prostate tissue array (Table 1). BrE3 abundantly stained prostate cancer and showed increased staining with higher Gleason grades. Because BrE3 predominately recognizes a peptide epitope within the variable number of tandem repeat region of MUC1, this result suggests that hypoglycosylated MUC1 is a good marker for more aggressive prostate cancer. Of the scFv-c described, E1-c had a markedly similar staining profile to that of BrE3, therefore also correlating with higher grades of disease.

Multivalent, bispecific PEG-scFv molecules designed to target MUC1 and bind the chelate have been developed in the following formats: (anti-MUC1 scFv)2-PEG-anti-DOTA scFv or (anti-MUC1 scFv)2-PEG-(anti-DOTA scFv)2. In theory, such molecules can be assembled by scFv site–specific conjugation to a tri- or tetra-functionalized PEG. In practice, however, homogeneous tri- or tetra-functionalized PEG are not available and PEGylation of different scFvs to multifunctionalized PEG is not readily controlled. Therefore, we achieved tetravalency by conjugation of di-scFv-c to the PEG scaffold; di-scFv-c were produced in E. coli by expression of scFv cDNAs cloned in tandem into the expression vector. Not surprisingly, di-scFv-c production yields in E. coli were decreased in comparison with those of scFv and di-scFv without an unpaired cysteine (42, 62). Regardless of the presence of an unpaired cysteine, high production yields have been reported in yeast (63, 64). Site-specific conjugation studies of di-scFv-c to PEG-Mal indicate that the free thiol is accessible in these configurations with a scFv-c-scFv format providing >90% conjugation. PEGylation efficiency seems inversely proportional to the length of the PEG (65). The di-scFv-c module for site-specific and covalent attachment to a PEG scaffold provides flexibility in the use of various scFv units.

In summary, the research presented here reflects serial developments and target choices toward an increase of the therapeutic index for metastatic prostate cancer targeted therapy. A biodegradable linked radiometal-targeting mAb combined with paclitaxel for radiosensitization resulted in combined modality radioimmunotherapy with modest therapeutic efficacy for aggressive prostate cancer. Prostate cancer tissue immunohistochemistry revealed hypoglycosylated MUC-1 up-regulation corresponding to increased disease grade, and identified scFv-c molecules suitable for pretargeted radioimmunotherapy for prostate cancer. Implementation of pretargeted radioimmunotherapy with multivalent, bispecific molecules capable of targeting abnormal MUC1 and capturing subsequently administered radiochelates holds promise for further therapeutic index enhancement in prostate cancer radioimmunotherapy.

Grant support: National Cancer Institute grant CA47829, Department of Energy grant DE-FG01-00NE22944, and Department of Defense grant DAMD17-01-1-0177.

Presented at the Tenth Conference on Cancer Therapy with Antibodies and Immunoconjugates, October 21-23, 2004, Princeton, New Jersey.

1
Linton DK, Hamdy FC. Early diagnosis and surgical management of prostate cancer.
Cancer Treat Rev
2003
;
29
:
151
–60.
2
Kasper S, Smith JJA. Genetically modified mice and their use in developing therapeutic strategies for prostate cancer.
J Urol
2004
;
172
:
12
–9.
3
Pandey P, Kharbanda S, Kufe D. Association of the DF3/MUC1 breast cancer antigen with Grb2 and the Sos/Ras exchange protein.
Cancer Res
1995
;
55
:
4000
–3.
4
Langner C, Ratschek M, Rehak P, Schips L, Zigeuner R. Expression of MUC1 (EMA) and E-cadherin in renal cell carcinoma: a systematic immunohistochemical analysis of 188 cases.
Mod Pathol
2004
;
17
:
180
–8.
5
Zhang S, Zhang HS, Reuter VE, Slovin SF, Scher HI, Livingston PO. Expression of potential target antigens for immunotherapy on primary and metastatic prostate cancers.
Clin Cancer Res
2001
;
4
:
295
–302.
6
Kirschenbaum A, Itzkowitz SH, Wang JP, Eliashvili M, Levine AC. MUC1 expression in prostate carcinoma: correlation with grade and stage.
Mol Urol
1999
;
3
:
163
–8.
7
O'Connor JC, Julian J, Lim SD, Carson DD. MUC1 expression in human prostate cancer cell lines and primiary tumors.
Prostate Cancer Prostatic Dis
2005
;
8
:
36
–44.
8
Brayman M, Thathiah A, Carson DD. MUC1: a multifunctional cell surface component of reproductive tissue epithelia.
Reprod Biol Endocrinol
2004
;
2
:
4
–12.
9
Gendler S, Taylor-Papadimitriou J, Duhig T, Rothbard J, Burchell J. A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats.
J Biol Chem
1988
;
263
:
12820
–3.
10
Price MR, Rye PD, Petrakou E, et al. Summary report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal antibodies against the MUC1 mucin.
Tumour Biol
1998
;
19
:
1
–20.
11
Devine PL, McGuckin MA, Quin RJ, Ward BG. Serum markers CASA and CA 15–3 in ovarian cancer: all MUC1 assays are not the same.
Tumour Biol
1994
;
15
:
337
–44.
12
Kramer EL, DeNardo SJ, Liebes L, et al. Radioimmunolocalization of breast carcinoma using BrE-3 monoclonal antibody: phase I study.
J Nucl Med
1993
;
34
:
1067
–74.
13
DeNardo SJ, Kramer EL, O'Donnell RT, et al. Radioimmunotherapy for breast cancer using indium-111/yttrium-90 BrE-3: results of a phase I clinical trial.
J Nucl Med
1997
;
38
:
1180
–5.
14
Epenetos AA, Munro AJ, Stewart S, et al. Antibody-guided irradiation of advanced ovarian cancer with intraperitoneally administered radiolabelled monoclonal antibodies.
J Clin Oncol
1987
;
5
:
1890
–9.
15
MacLean GD, McEwan A, Noujaim A, et al. Two novel monoclonal antibodies have potential for gynecologic cancer imaging. Antibody.
Immunoconj Radiophar
1991
;
4
:
297
–308.
16
Nabhan C, Tallman M, Riley M, et al. Phase I study of rituximab and Campath-1H in patients with relapsed or refractory chronic lymphocytic leukemia.
Blood
2001
;
98
:
365a
.
17
Longenecker BM, Willans DJ, MacLean GD, et al. Monoclonal antibodies and synthetic tumor-associated glycoconjugates in the study of the expression of Thomsen-Friedenreich-like and Tn-like antigens of human cancers [abstract].
J Natl Cancer Inst
1987
;
78
:
489
–96.
18
Burdick MD, Harris A, Reids CJ, Iwamural T, Hollingsworth MA. Oligosaccharides expressed on MUC1 produced by pancreatic and colon tumor cell lines.
J Biol Chem
1997
;
272
:
24198
–202.
19
O'Donnell RT, DeNardo SJ, Yuan A, et al. Radioimmunotherapy with 111In/90Y-2IT-BAD-m170 for metastatic prostate cancer.
Clin Cancer Res
2001
;
7
:
1561
–8.
20
DeNardo SJ, DeNardo GL, Yuan A, et al. Enhanced therapeutic index of radioimmunotherapy in prostate cancer patients: Comparison of radiation dosimetry for DOTA-peptide versus 2-IT-DOTA MAb linkage for RIT.
Clin Cancer Res
2003
;
9
:
3938
–44.
21
Springer GF. T and Tn, general carcinoma autoantigens.
Science
1984
;
224
:
1198
–206.
22
McCall MJ, Diril H, Meares CF. Simplified method for conjugating macrocyclic bifunctional chelating agents to antibodies via 2-iminothiolane.
Bioconjug Chem
1990
;
1
:
222
–6.
23
DeNardo GL, O'Donnell RT, Shen S, et al. Radiation dosimetry for 90Y-2IT-BAD-Lym-1 extrapolated from pharmacokinetics using 111In-2IT-BAD-Lym-1 in patients with non-Hodgkin's lymphoma.
J Nucl Med
2000
;
41
:
952
–8.
24
Kukis DL, DeNardo GL, DeNardo SJ, et al. Effect of the extent of chelate substitution on the immunoreactivity and biodistribution of 2IT-BAT-Lym-1 immunoconjugates.
Cancer Res
1995
;
55
:
878
–84.
25
DeNardo SJ, Richman CM, Kukis DL, et al. Synergistic therapy of breast cancer with Y-90-chimeric L6 and paclitaxel in the xenografted mouse model: development of a clinical protocol.
Anticancer Res
1998
;
18
:
4011
–8.
26
Richman CM, DeNardo SJ, O'Grady LF, DeNardo GL. Radioimmunotherapy for breast cancer using escalating fractionated doses of 131I-labeled chimeric L6 antibody with peripheral blood progenitor cell transfusions.
Cancer Res
1995
;
55
Suppl:
5916
–20.
27
DeNardo SJ, O'Grady LF, Macey DJ, et al. Quantitative imaging of mouse L-6 monoclonal antibody in breast cancer patients to develop a therapeutic strategy.
Int J Rad Appl Instrum [B]
1991
;
18
:
621
–31.
28
Erwin WD, Groch MW, Macey DJ, et al. A radioimmunoimaging and MIRD dosimetry treatment planning program for radioimmunotherapy.
Nucl Med Biol
1996
;
23
:
525
–32.
29
Shen S, DeNardo GL, DeNardo SJ, Yuan A, DeNardo DA, Lamborn KR. Reproducibility of operator processing for radiation dosimetry.
Nucl Med Biol
1997
;
24
:
77
–83.
30
Snyder WS, Ford MR, Warner GG, et al. “S” absorbed dose per unit cumulated activity for selected radionuclides and organs. MIRD Pamphlet No. 11. New York: Society of Nuclear Medicine; 1975. p. 82–3.
31
Dillman LT. Radionuclide decay schemes and nuclear parameters for use in radiation-dose estimation, part 2. MIRD Pamphlet No. 6. New York: Society of Nuclear Medicine; 1970. p. 5–32.
32
Gleason DF. The Prostate. Histologic grading and clinical staging of prostatic carcinoma. In: Tannenbaum M, editor. Urologic pathology. Philadelphia: Lea & Febinger; 1977. p. 171–97.
33
Howell LP, DeNardo SJ, Levy N, Lund J, DeNardo GL. Immunohistochemical staining of metastatic ductal carcinomas of the breast by monoclonal antibodies used in imaging and therapy: a comparative study.
Int J Biol Markers
1995
;
10
:
126
–35.
34
Chan CM, Baratta FS, Ozzello L, Ceriani RL. Monoclonal antibody BrE-3 participation in a multivariate prognostic model for infiltrating ductal carcinoma of the breast.
Breast Cancer Res Treat
1994
;
30
:
243
–61.
35
Ceriani RL, Chan CM, Baratta FS, Ozzello L, DeRosa CM, Habif DV. Levels of expression of breast epithelial mucin detected by monoclonal antibody BrE-3 in breast-cancer prognosis.
Int J Cancer
1992
;
51
:
343
–54.
36
Ceriani RL, Peterson JA, Blank EW, Derek TA. Epitope expression on the breast epithelial mucin.
Breast Cancer Res Treat
1992
;
24
:
103
–13.
37
Peterson JA, Zava DT, Duwe AK, Blank EW, Battifora H, Ceriani RL. Biochemical and histological characterization of antigens preferentially expressed on the surface and cytoplasm of breast carcinoma cells identified by monoclonal antibodies against the human milk fat globule.
Hybridoma
1990
;
9
:
221
–35.
38
Grinstead JS, Koganty RR, Krantz MJ, Longenecker BM, Campbell P. Effect of glycosylation on MUC1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions.
Biochemistry
2002
;
41
:
9946
–61.
39
Rahn JJ, Dabbagh L, Pasdar M, Hugh JC. The importance of MUC1 cellular localization in patients with breast carcinoma.
Cancer
2001
;
91
:
1973
–82.
40
Parham DM, Slidders W, Robertson AJ. Quantitation of human milk fat globule (HMFG1) expression in breast carcinoma and its association with survival.
J Clin Pathol
1988
;
41
:
875
–9.
41
Wilkinson MJS, Howell A, Harris M, Taylor-Papadimitriou J, Swindell R. The prognostic significance of two epithelial membrane antigens expressed by human mammary carcinomas.
Int J Cancer
1984
;
33
:
299
–304.
42
Albrecht H, Burke PA, Natarajan A, et al. Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand.
Bioconjug Chem
2004
;
15
:
16
–26.
43
Winthrop MD, DeNardo SJ, DeNardo GL. Development of a hyperimmune anti-MUC-1 single chain antibody phage display library for targeting breast cancer.
Clin Cancer Res
1999
;
10
:
3088
–94.
44
Richman CM, DeNardo SJ, O'Donnell RT, et al. Combined modality radioimmunotherapy (RIT) in metastic prostate (PC) and in breast cancer (BC) using paclitaxel (PT) and a MUC-1 monoclonal antibody, m170, linked to yttrium-90 (Y-90): a phase I trail.
J Clin Oncol
2004
;
22
:
2554
.
45
Winthrop MD, DeNardo SJ, Albrecht H, et al. Selection and characterization of anti-MUC-1 scFvs intended for targeted therapy.
Clin Cancer Res
2003
;
9
:
3845
–53s.
46
Gautherot E, Bouhou J, Le Doussal JM, et al. Therapy for colon carcinoma xenografts with bispecific antibody-targeted, iodine-131-labeled bivalent hapten.
Cancer
1997
;
80
:
2618
–23.
47
Gautherot E, Rouvier E, Daniel L, et al. Pretargeted radioimmunotherapy of human colorectal xenografts with bispecific antibody and 131I-labeled bivalent hapten.
J Nucl Med
2000
;
41
:
480
–7.
48
Chang CH, Sharkey RM, Rossi EA, et al. Molecular advances in pretargeting radioimmunotherapy with bispecific antibodies.
Mol Cancer Ther
2002
;
1
:
553
–63.
49
Sharkey RM, Karacay H, Chang CH, McBride WJ, Horak ID, Goldenberg DM. Improved therapy of non-Hodgkin's lymphoma xenografts using radionuclides pretarted with a new anti-CD20 bispecific antibody. Leukemia 2005;1–6.
50
Weiden PL, Breitz HB, Press O, et al. Pretargeted radioimmunotherapy (PRIT) for treatment of non-Hodgkin's lymphoma (NHL): initial phase I/II study results.
Cancer Biother Radiopharm
2000
;
15
:
15
–29.
51
Paganelli G, Bartolomei M, Ferrari M, et al. Pre-targeted locoregional radioimmunotherapy with 90Y-biotin in glioma patients: phase I study and preliminary therapeutic results.
Cancer Biother Radiopharm
2001
;
16
:
227
–35.
52
Knox SJ, Goris ML, Tempero M, et al. Phase II trial of yttrium-90-DOTA-biotin pretargeted by NR-LU-10 antibody/streptavidin in patients with metastatic colon cancer.
Clin Cancer Res
2000
;
6
:
406
–14.
53
Axworthy DB, Reno JM, Hylarides MD, et al. Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity.
Proc Natl Acad Sci U S A
2000
;
97
:
1802
–7.
54
Kranenborg MHGC, Boerman OC, Oosterwijk-Wakka JC, De Weijert MCA, Corstens FHM, Oosterwijk E. Two-step radio-immunotargeting of renal-cell carcinoma xenografts in nude mice with anti-renal-cell-carcinoma X anti-DTPA bispecific monoclonal antibodies.
Int J Cancer
1998
;
75
:
74
–80.
55
Pluckthun A, Pack P. New protein engineering approches to multivalent and bispecific antibody fragments.
Immunotechnology
1997
;
3
:
83
–105.
56
Hudson PJ, Souriau C. Recombinant antibodies for cancer diagnosis and therapy.
Expert Opin Biol Ther
2001
;
1
:
845
–55.
57
Bailon P, Palleroni A, Schaffer CA, et al. Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon α-2a for the treatment of heptitis C.
Bioconjug Chem
2001
;
12
:
195
–202.
58
Harris JM, Chess RB. Effect of pegylation on pharmaceuticals.
Nat Rev Drug Discov
2003
;
2
:
214
–21.
59
Richman CM, DeNardo SJ, O'Donnell RT, et al. Dosimetry-based therapy in metastatic breast cancer patients using 90Y monoclonal antibodies 170H.82 with autologous stem cell support and cyclosporin A.
Clin Cancer Res
1999
;
5
:
3243
–8s.
60
DeNardo SJ, O'Donnell RT, Richman CM, et al. Comparison of In-111/Y-90-m170 pharmacokinetics and dosimetry in prostate cancer patients and breast cancer patients on cyclosporin A (CSA).
J Nucl Med
1999
;
40
:
217
–1.
61
McGucken MA, Walsh MD, Hohn BG, Ward BG, Wright RG. Prognostic significance of MUC1 epithelial expression in breast cancer.
Hum Pathol
1995
;
26
:
432
–9.
62
Schmiedl A, Breitling F, Winter CH, Queitsch I, Dubel S. Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody contructs expressed in E. coli.
J Immunol Methods
2000
;
242
:
101
–14.
63
FitzGerald K, Holliger P, Winter G, Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris.
Protein Eng
1997
;
10
:
1221
–5.
64
Yang K, Basu A, Wang M, et al. Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation.
Protein Eng
2003
;
16
:
761
–70.
65
Natarajan A, Xiong CY, Albrecht H, DeNardo GL, DeNardo SJ. Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals.
Bioconjug Chem
2005
;
16
:
113
–21.
66
DeNardo GL, Richman CM, Kroger LA, et al. Novel catabolizable 90Y/111In-DOTA-peptide-ChL6 radioimmunoconjugates for cancer therapy. In: Limouris G, editor. Radionuclides for mammary gland. Athens: Mediterra; 1997. p. 201–12.