Characterization of Site-Specific ScFv PEGylation for Tumor-Targeting

Bioconjugate Chem. 2005, 16, 113−121

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Characterization of Site-Specific ScFv PEGylation for Tumor-Targeting Pharmaceuticals Arutselvan Natarajan, Cheng-Yi Xiong, Huguette Albrecht, Gerald L. DeNardo, and Sally J. DeNardo* University of California Davis Medical Center, Sacramento, California 95816. Received August 2, 2004; Revised Manuscript Received November 24, 2004

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.

INTRODUCTION

Radiolabeled monoclonal antibodies (MAb) have proven to be effective therapy for some radiosensitive cancers (1, 2). Because of their large size, blood clearance and tumor uptake occur slowly, decreasing the tumor to marrow therapeutic index. Unless bone marrow support is provided, hematological toxicity precludes administration of sufficient radiopharmaceutical to deliver effective radiation to most solid tumors (3, 4). Recombinant production of selected single chain Fv antibody fragments (scFv) provides small molecules for new paradigms designed to enhance the therapeutic index of tumortargeted or pretargeted radionuclide therapy (5-7). With advances in antibody engineering and phage-display, antibody fragments can be generated that have a high degree of target specificity and a range of binding affinities (8, 9). * Corresponding author: Sally J. DeNardo, M.D., Radiodiagnosis and Therapy, Molecular Cancer Institute, University of California, Davis Medical Center, 1508 Alhambra Blvd., Rm. 3100, Sacramento, CA 95816. Telephone: 916-734-3787, Fax: 916-451-2857, E-mail: [email protected].

Pharmacokinetic studies with radiolabeled scFvs have shown that their small size leads to rapid blood and renal clearance and insufficient opportunity for tumor uptake (10, 11). These small antigen-binding proteins can also serve as modules in multivalent tumor-targeting constructs designed to enhance radioimaging and therapy (12, 13). Poly(ethylene glycol) (PEG) polymers have the characteristics necessary to serve as scaffolds for these modules. PEGylation, the covalent attachment of PEG to other molecules, has become a validated drug delivery method, and several PEGylated drugs have been approved by the FDA. Better stability, decreased proteolysis, enhanced solubility, and longer circulation and body retention of PEGylated pharmaceuticals have led to their enhanced efficacy (14, 15). PEGylation of biologically active drugs has historically been accomplished by covalent conjugation to available primary amines of lysine residues. Significant loss of target binding or biologic activity frequently resulted as a consequence of conformational change or steric hindrance (16, 17). Since small molecules such as scFv allow little room for error, an expression vector has been

10.1021/bc0498121 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/31/2004

114 Bioconjugate Chem., Vol. 16, No. 1, 2005

Natarajan et al. Table 1. List of Various PEG Molecules Used for Site-Specific PEGylation no.

functionalized PEGs

molecular weight (kDa)

1 2 3 4 5 6 7 8 9 10

PEG-(Mal)2 NHS-PEG-Mal PEG-Mal PEG-Mal PEG-Mal (PEG)2-Mal PEG-(OPSS)2 PEG-OPSS PEG-OPSS PEG-OPSS

2, 3.4 3.4 5 10 20 40 2 3.4 10 20

a Methoxy-PEG: PEG, Mal: maleimide, OPSS: o-pyridyl disulfide, NHS: hydroxysuccinimide, PEG-(Mal)2/(OPSS)2: PEG have been functionalized at both ends.

Figure 1. PEG maleimide (PEG-Mal) structures and the maleimide (Mal) thiol conjugation reaction with scFv-c (scFvSH): (a) Methoxy-PEG-Mal. (b) PEG-(Mal)2. (c) Branched methoxy-PEG-Mal. (d) Formation of thioether bond between maleimide and cysteine of scFv-c. (e) PEG-Mal structures electrophoresed on SDS-PAGE gel (4-12%) under reducing conditions and stained by barium iodide, demonstrating relative mobility prior to protein conjugation.

engineered for the addition of a free cysteine (c) near the carboxyl end of the scFv (scFv-c), thereby providing a specific site for thiol conjugation of scFv-c produced in the vector (18). To determine the general applicability of scFv-c PEGylation, we evaluated the relative conjugation efficiency of several scFv-c with PEG molecules varying in size, structure, and thiol reactive groups, including mono- and bifunctional PEG molecules in linear and branched formats (Figure 1, Table 1). Evidence of a relationship between PEG structure, size, and the efficiency of scFv-c PEGylation was observed. MATERIALS AND METHODS

Materials. Methoxy-PEG-maleimide (PEG-Mal) of 3.4, 5, 20, and 40 kDa and a bifunctional maleimide-PEGMal (PEG-(Mal)2) of 3.4 kDa were purchased from Nektar Therapeutics (San Carlos, CA). PEG-Mal of 10 and 20 kDa, methoxy-PEG-o-pyridyl disulfide (OPSS) of 5, 10, and 20 kDa, and PEG-(Mal)2 of 2 kDa were obtained from

Sunbio PEG-Shop (Anyang City, S. Korea). These were stored under N2 atmosphere. Titrisol Iodine solution was obtained from EM Science (Gibbstown, NJ) and tris(2carboxyethyl)phosphine hydrochloride (TCEP) from Molecular Probes (Eugene, OR). The Micro BCA Protein Assay Reagent kit was obtained from Pierce Biotechnology (Rockford, IL) and the RPAS purification module from Amersham Biosciences Corp (Piscataway, NJ). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Production and Purification of scFv-c. Four MUC-1 positive scFv-c were selected from previously developed MUC-1 immune phage display scFv libraries (19, 20). MUC-1 is a high molecular weight glycoprotein molecule, composed of an extracellular domain with tandem repeats of 20 amino acids, a transmembrane region, and a cytoplasmic tail. In many epithelial cancers, the extracellular domain presents novel epitopes of shortened carbohydrate chains and exposed portions of the 20 amino acid tandem repeats, hence providing unique relatively tumor specific targets for antibodies (20). Anti MUC-1 scFv gene inserts were isolated by SfiI/NotI double digestion and ligated into the pCANTAB 5E Cys vector (18). Clones isolated from transformants after electroporation into E. coli HB2151 were sequenced to confirm the presence of the extra cysteine-specifying codon at the 3′ end of the scFv (scFv-c). In the scFv-c protein, cysteine (C) is transcribed from this vector codon, at the end of the linked Vh-Vl sequence, immediately after the amino acid sequence of the NotI insertion site (underlined) and before the E-TAG amino acid sequence (bold): (Vh-Vl...TKLELKRAAACGAPVPYPDPLEPRAA) (20). All the scFvs-c were produced in shaker flasks and purified as previously described (18, 20). Briefly, periplasmic extracts were prepared and affinity purified by anti E-Tag chromatography of the scFv-c proteins using the RPAS purification module and buffer-exchanged into phosphate-buffered saline (PBS) pH 7.4 (18, 20). The protein concentration (mg/mL) of each E-Tag purified scFv-c product was determined by using the micro BCA protein reagent kit. To ensure the scFv-c was in the monomeric form for conjugation, the scFv-S-S-scFv linkage from the unpaired thiol groups was prevented by addition of the reducing agent, TCEP, in 2-fold molar excess. As shown previously (18), nearly 100% reduction of that thiol group could be maintained in this manner with no evidence of effect on the two intramolecular disulfide bridges of the scFv-c. The advantage of TCEP as the reducing agent was stability over time in a broad pH range (5.0-8.0); however, increased signal in ELISA studies has been noted at these concentrations, so that samples with and without TCEP are evaluated.

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Site-Specific PEGylation

Estimation of Maleimide Reactivity as PEG-Mal. To estimate the degree of functional maleimide groups available for conjugation in the PEG reagents, functional group analysis of the various PEG-Mal was carried out by adding up to a 5 M excess of cysteine to each PEGMal in 0.1 M sodium phosphate, pH 7, with 1 mM EDTA (Table 1, nos. 1-6). Free cysteine was then measure by reaction with DNTB as previously published (21). The results indicated that maleimide functional groups in all the PEG-Mal reagents were greater than 90% reactive toward cysteine and thus thiol groups. PEGylation of scFv-c. The PEG to scFv molar ratio for conjugation reactions was initially studied with scFv-c as D5-c PEG-(Mal)2 (2 kDa) at 2, 5,10, 20, and 50-fold molar excess of PEG. Since no increase in scFv conjugation was demonstrated in ratios over 5:1, PEGylation studies of all 4 scFv-c with the PEG-Mal and PEG-OPSS reagents was performed at a molar ratio of 5:1 (PEG: scFv). The reaction was carried out in 0.1M sodium phosphate buffer, pH 7.0, 2 mM EDTA, at 1-2 mg/mL scFv-c and 2-fold molar excess TCEP, and incubated under N2 atm at 37 °C overnight (22, 23). PEGylation efficiency was analyzed using separation of scFv-c and PEG-scFv species with SDS-PAGE (Novex XCell II) in 4-12% Bis-Tris NuPAGE gel and MES running buffer, according to the manufacturer’s methods (17, 22, 24). Coomassie blue and barium iodide staining detected scFvs (free and conjugated) and PEG molecules, respectively, and comparisons were made to the molecular weight standards. Coomassie blue-stained proteins in the PAGE gels were digitally scanned for permanent images and quantitation of the bands. The gels were then rinsed with distilled water, and 5% barium chloride solution was added for 10 min and again rinsed and placed in 0.1 M Titrisol iodine solution for color development (17, 25) (Figure 2). PEGylation yields were calculated from quantitation of the initial scFv-c compared to the resulting scFv-c as PEG-scFv conjugates vs unconjugated scFv-c. The relative amounts of each species in each reaction were obtained by calculations of digital information from densitometry performed on the scanned gel images and protein standards (Personal Densitometer S1, model PDS1, Molecular Dynamics Inc., Sunnyvale, CA), as previously described (26). Western Blot Analysis of scFv-c E-Tag-Purified Products. The 4 scFv-c and separately BA1-c and the purified BA1-c PEG-(scFv)2 products were evaluated with anti-E Tag by Western blotting. Two hundred and fifty nanograms of purified scFv-c and PEG-(scFv)2 were subjected to SDS-PAGE, transferred onto a PVDF (polyvinylidine difluoride) membrane to which HRPconjugated anti-E Tag (Amersham Biosciences) was added. After washings, reactivity was detected with the Super Signal (Pierce, Rockford, IL) substrate and exposed to chemiluminescent-sensitive film. Specific attention was given to the 17 and 19 kDa products seen on PAGE but not on Western with anti-E-Tag (Figure 3). Protein N-Terminal Sequencing of the 17 and 19 kDa Fragments. After separation of the BA1-c protein forms by nonreducing SDS-PAGE, ∼9.5 and 19 µg of BA1-c nonreduced scFv fragments, respectively, were loaded into two lanes of a 4-12% NuPAge gel (Invitrogen), and the proteins were electroblotted onto a PVDF membrane (Biorad, Hercules, CA) for direct N-terminal sequencing by Edman’s degradation. This protein microsequencing was performed on an ABI Procise HT

Edman Sequencer System (Perkin-Elmer/Applied Biosystems, Foster City, CA) at the Molecular Structure Facility at University of CaliforniasDavis. Cell Lines and Cell Lysate Preparation. MUC-1 positive human breast adenocarcinoma MCF-7 cells (American Type Culture Collection (ATCC) Manassas, VA) were grown to 75% confluence in DMEM (Gibco, Invitrogen Corp., Carlsbad, CA) medium supplemented with 5% fetal calf serum (FCS) (Gibco, Invitrogen Corp.). MUC-1-positive DU145 human prostate cancer cells (ATTC) were grown to 100% confluence in RPMI-1640 medium (Gibco, Invitrogen Corp.) supplemented with 10% FCS. Purification of PEG Conjugates. PEG-(BA1)2 reaction mixture, as shown in the (Figure 3a, lane B) containing 250 µg of PEGylated protein, was purified by molecular sieving column chromatography on a 30 × 1.5 cm glass column (Bio-Rad, Hercules, CA) packed with sephadex G-75 (Sigma Chemicals, St. Louis, MO). The column was preconditioned in 100 mM sodium phosphate buffer pH 7.0 and operated at a flow rate of 0.3 mL/min. Fractions were collected (200 µL), and the PEG-(scFvc)2 (65 kDa) was eluted immediately after the void volume. ScFv-c (31kDa) and PEG-scFv-c (34kDa) eluted together at midcolumn volume. Immunoreactivity. ELISA was used to determine immunoreactivity of PEGylated scFv against breast (MCF-7) and prostate cancer (DU145) cell membranes. Pro-Bind ELISA assay plates (Becton Dickinson Lab Ware, Franklin Lakes, NJ) were coated with 100 µL of cell lysate at 1 mg/mL. The wells were dried by warming the plates at 37 °C. Nonspecific binding was blocked with 200 µl/well of PBS/0.1% Tween 20 (Sigma-Aldrich) containing 3.0% nonfat dry milk (Bio-Rad, Hercules, CA) for 1 h at 37 °C. E-Tag-purified scFv-c, the reaction mixture of scFv-c and PEGylated scFv, and the purified PEG(scFv-c)2 were each studied in triplicate on three occasions, using 1 µg of scFv-c/well, a total volume of 100 µL/ well (PBS/0.01% Tween 20/0.3% nonfat dry milk), and 1 h incubation at 37 °C. Following each incubation, the plates were washed six times with PBS containing 0.01% Tween 20. Anti-E-Tag-HRP MAb (100 µL) diluted to 1:4000 in PBS/0.1% Tween/0.3% nonfat milk solution was added and incubated in the same manner. Following the last wash step, the 2,2′-amino-bis-3-ethylbenzthiazoline6-sulfonic acid (ABTS) substrate (Sigma-Aldrich), containing 0.3% H2O2, was added. As soon as color had developed, the content of each well was transferred to a new plate. Plates were read at A405 nm (Dynex microplate reader, Chantilly, VA). RESULTS

Evaluation of scFv-c Preparations. PAGE gels of the ETAG-purified products of each scFv-c in TCEP are illustrated in Figures 2. The major band, approximately 30 kDa, corresponds to the monomeric form of the scFv-c for CC6-c (Figure 2a), AD3-c (Figure 2b), D5-c (Figure 2c), and BA1-c (Figure 2d). Lower molecular weight recombinant products of AD-3-c, D5-c, and BA1-c can be seen approximating 17 and 19 kDa. These were demonstrated to have no E-Tag by Western Blot analysis, as exemplified by BA1-c (Figure 3). Minor bands at approximately 52 kDa were detected only in the initial BA1-c (Figure 2d) and were not studied. PEGylation of scFv-c. PEGylation was studied using PEG with either of two thiol reacting groups, Mal or OPSS (16, 27, 28). Effective conjugations were only obtained with Mal as either PEG-Mal or PEG-(Mal)2. The


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Figure 2. PEG-scFv conjugates on SDS-PAGE gel (4-12%) under reducing conditions. (Left) (a-d) Stained with Coomassie blue for protein identification. ScFv-c in each series of conjugation reactions, a: CC6-c, b: Ad3-c, c: D5-c, and d: BA1-c. (Right) (e-h) Stained with Coomassie blue for protein followed by barium iodide for PEG, allowing both scFv-PEG and PEG identification, e: CC6-c, f: Ad3-c, g: D5-c, and h: BA1-c. Lanes A and C are the results from PEGylation with PEG-(Mal)2, 2 and 3.4 kDa; Lane B, NHS-PEG-Mal 3.4 kDa; and Lanes D-G PEG-Mal of 5, 10, 20, and (branched) 40 kDa. Note should be made that lanes D-G showed PEGylation with increasing PEG molecule size. The identification of PEG and scFv-PEG by barium iodide and Coomassie blue is shown in Figure 2e-h with three different colors: blue (scFv), green/gray (PEG-scFv), and brown (PEG), providing identification of various molecules to confirm the PEG-scFv conjugation. Lanes A to F (Figures a-d), with Coomassie blue staining, show gradual increase in molecular weight in accordance with the molecular weight of the PEG used in the conjugation.

PEG with OPSS was evaluated (5:1), PEG-OPSS/scFv, and resulted in less than 10% scFv conjugation. PEGylation with identical molar ratios of PEG-Mal to scFv-c (5:1) was much more effective with all scFvs (Figures2 and 4). The PEG-Mal conjugated 30-85% of the scFvs (Figure 4), and the percent of scFv that conjugated as bi-scFv on PEG-(Mal)2 ranged from 15 to 35% for both 2 and 3.4 kDa PEG-(Mal)2 polymers. ScFv BA1-c had the highest efficiency of bi-scFv-c conjugation to produce PEG- (scFv)2 (Figure 4). Using the hetero-bifunctional PEG (3.4 kDa) (Table 1; no. 2) containing one Mal and N-hydroxysuccinimide (NHS), the latter for amine reactions, correlated with no effective production of bifunctional PEG-(scFv)2 with CC6-c, AD3-c, or D5-c (Figure 2a,b,c; lane B), but BA1-c conjugated with the NHS group

resulting in 40% of that scFv-c as PEG(scFv)2, although the reactivity of NHS should be minimized at this pH (Figure 2d, lane B). With different sizes of PEG, the PEGylation of scFv-c as one scFv-c per PEG resulted with scFv-c conjugation efficiency ranging from 45% to 75% (2 kDa), 30 to75% (3.4 kDa), 25 to 85% (5 kDa), and 10 to 70% (40 kDa) of the original scFv-c protein (Figure 4, blue bar). With PEG-(Mal)2, 15 to 45% (2 kDa), and 15 to 40% (3.4 kDa), the initial scFv-c protein PEGylated as two scFv per PEG (PEG-(scFv)2) (Figure 4, green bar). Conjugation efficiency with PEG varied moderately among the four scFv with BA1-c > AD3-c = D5-c > CC6-c. The 10 and 20 kDa PEG-Mal PEGylated less than 5% of three out of the four scFv-c.



Figure 3. (a) SDS-PAGE reducing gel (4-12%) of 2 kDa PEG-(Mal)2 conjugation reactions with two different scFvs, BA1-c and D5-c, using two different molar ratios of scFv/PEG: 5:1 (lanes A and C), and 1:5 (lanes B and D). It should be noted that bivalent PEG-(scFv)2 product yield was not enhanced by multifold increase in scFv-c to PEG. (b) SDS-PAGE gel and (c) Western blot analysis of BA1-c compared to the purified PEG-(BA1)2 product from the conjugation reaction mixtures seen in part b. The Western blot was performed using HRP-conjugated anti-E-TAG antibody. Bands at 17 and 19 kDa in parts a and b demonstrate no E-TAG binding on Western blot. Prior sequencing indicates that these represent an scFv fragment, with the protein sequence from the amino terminus of VH through the linker region.

Figure 4. Yields of scFv-c and with PEG-Mal conjugation. The percent scFv-c protein conjugated by PEG-Mal (conjugation yield) calculated from densitometry of the Coomassie bluestained PAGE gel images. Conjugation yield of scFv-c with PEG(Mal)2 and PEG-Mal are separately color-coded: green, PEG(scFv-c)2; blue, PEG-scFv-c; and orange, unconjugated scFv-c. The scFv-c protein percent yield as PEG-scFv conjugates are on the x-axis and PEG-Mal sizes are on the y-axis. * PEG-(Mal)2. Panel of scFvs used in these studies were CC6-c, AD3-c, D5-c, and BA1-c, respectively.

The comparison of conjugation reactions of PEG-Mal molecules consisting of various molecular weights ranging from 2 kDa to 40 kDa (Figure 1) demonstrated higher efficiency scFv PEGylation with smaller PEG molecules and lower with larger PEG molecules (Figure 4). The

percent yield of PEG-scFvs was 45 to 75, 30 to 75, 25 to 80, and 10 to 70% for 2, 3.4, 5, and 40 kDa PEGs, respectively. BA1 (scFv) had above 40% conjugation with all size of PEG-Mal. AD3 had an exceptionally high percent conjugation of 85% with 5 kDa PEG-Mal. However, unexpectedly 10 and 20 kDa linear PEG-Mal compounds did not react with CC6, D5, and AD3, but BA1 had greater than 75% conjugation. The 40 kDa PEGMal branched PEG (Figure 1) conjugated with all four scFv-c (Figures 2 and 4) (29). We speculate that this may be due to the influence of the location of the Mal in the branched polymer, as the 10 and 20 kDa were terminal on the linear molecule (29, 30). SDS-PAGE Analysis of PEG-scFv and PEG(scFv)2 Products. The SDS-PAGE of protein by Coomassie blue is shown in Figure 2a-d. The identification of PEG and scFv-PEG by barium iodide and Coomassie blue is shown in Figure 2e-h with three different colors: blue (scFv), gray (PEG-scFv), and brown (PEG), providing identification of various molecules to confirm the PEG-scFv conjugation. The SDS-PAGE gel (Figure 1e) of the PEG reagents stained with barium iodide showed decreased mobility of the PEG molecules of 2, 3.4, 5,10, 20, and 40 kDa with increased molecular size (25). Lanes A to E and F (Figures 2a-d), of Coomassie blue staining, demonstrated gradual increase in protein conjugate molecular weight (bands) in accordance with the molecular weight of the PEG (Figure 1) used in the conjugation. The typical conjugation yield of PEG-scFvs varied between 20 and 80%. In Figure 2a-d, lanes A and C, we have used PEG-(Mal)2 for the conjugation, which showed 15-45% PEG-(scFv)2. Figure 2e-h, lanes A and C, supported this, as the light green/gray color of these bands confirmed bifunctional PEGylation. Figure 2d, lane G, demonstrated substantial PEGylation product of BA1-c and the branched PEG-Mal 40 kDa (98 kDa), while Figure 2a (lane G) of the scFv (CC6) showed less than 10% branched PEG-Mal 40 kDa conjugation product. Lanes E and F for the 10 and 20 kDa linear PEG with the three scFvs (CC6, AD3, and D5) showed very faint bands for the PEGylation yield. Lane G of the Figure 2e-h did not demonstrate visible green color for PEGylation due to masking by the dark brown color by high concentrations of PEG. Data from these conjugations is presented in Figure 4.


Figure 5. Comparison of MUC-1 binding by ELISA of BA1-c and its PEGylated products on DU145 cell lysate. Two micrograms of protein from each product were used. (A) BA1-c, no TCEP (pink) and BA1-c, with 2-fold molar excess of TCEP (green). (B) PEG-BA1 products (unpurified), code corresponds to conjugation reaction mixture tested i.e., 0 ) BA1-c, 1 ) 2kDa PEG-(BA1)2, 2 ) 3.4 kDa PEG-(BA1)2, 3 ) 3.4 kDa PEG-BA1, 4 ) 5 kDa PEG-BA1, 5 ) 10 kDa PEG-BA1, 6 ) 20 kDa PEGBA1 7 ) 40 kDa PEG-BA1, and 8 ) negative scFv-PEG control. (C) BA1-c with TCEP (green); 2 kDa PEG-(BA1)2, unpurified (yellow); 2 kDa PEG-(BA1)2, purified (blue). These data demonstrate that binding was generally retained after PEGylation. Further, binding of purified bifunctional 2kDa PEG-(BA1)2 was increased over the original scFv-c with or without the TCEP addition. Since TCEP has demonstrated enhanced ELISA signal at the concentrations used; scFv-c with and without TCEP were controls in these binding studies. Reaction mixture samples had negligible TCEP because of buffer exchange/concentration and therefore no enhanced signal. The control BA1-c (pink), without TCEP, seen in A, represents a more appropriate comparison for PEG-scFv reaction mixture than the BA1-c with TCEP (green). PEGylated monofunctional scFv-c products demonstrated binding very similar to the initial BA1-c, and the binding of the bifunctional 2 kDa PEG-(BA1)2 seen in C (blue) was enhanced. Graphs were derived from mean absorbance values normalized to background (no scFv). Corresponding standard errors (SE) from three experiments with triplicates in each of the three experiments are shown. Mean background values and SE was 0.11 ((0.009) for DU145 cell lysate.

PAGE and Western Blot Analysis. Figure 3a-c show the conjugation reaction mixture and purified product of BA1-c conjugated as PEG-(scFv)2 analyzed by SDS-PAGE and anti E-Tag probing of the Western blot. SDS-PAGE of BA1-c show the two bands frequently seen, corresponding to 17 and 19 kDa, which did not appear with anti E-Tag probing of the Western blot, indicating that these two proteins have no E-Tag (Figure 3c). Immunoreactivity. ELISA studies of in vitro binding to tumor cell membrane lysate were performed on the various PEGylation products, with varied results, particularly noted in samples with low PEGylation yield. Since the most efficient PEGylation with the various PEG structures was demonstrated with BA1-c (Figures 2 and 4), providing comparable samples for study, all of the BA1-c unpurified PEGylation products and the purified bivalent PEG-(BA1-c)2 from conjugation with 2 kDa PEG(Mal)2 were selected for further study. ELISA results of these BA1-c PEGylation products seen on SDS-PAGE in Figure 2d demonstrated good binding with all univalent products and better binding with the bivalent PEG(BA1-c)2 (Figure 5). The purified PEG-(BA1-c)2, in PBS with no TCEP, demonstrated the best results compared to the BA1-c with or without TCEP (Figure 5). Results on MCF-7 were remarkably similar and therefore are not shown. DISCUSSION

Intact antibodies represent epitope specific, bivalent molecules that are used to specifically target a selected

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antigen for radiodiagnosis or therapy (31). If there is abundant antigen present, simultaneous or serial binding to two adjacent antigen molecules leads to increased “functional affinity” (avidity) and enhanced retention time on the target. Abundant antigen target on the malignant cell membrane is frequently found in cancer because of up-regulation of gene products. Univalent antibody fragments, such as Fab fragments or scFv, exhibit faster blood clearance and better tissue penetration and provide similar epitope-binding specificity and single valance affinity, but substantially lower avidity (32). The selection and production of target-specific scFv proteins is well established (23, 33). The characteristics of the molecular format in which they can be used most effectively for tumor targeting depends on the diagnostic or therapeutic purpose so that the required molecular formats and pharmacokinetics vary. Small molecules, with rapid tissue penetration and clearance, provide high target to nontarget (T/NT) imaging agents for PET; larger heterofunctional formats, having longer blood and malignant cell residence, provide pretargeting molecules for multistep radioisotope therapy. To increase avidity and to generate both homo- and heterofunctional molecules, Fab and scFv have been engineered previously into dimeric, trimeric, or tetrameric conjugates through the use of either chemical or recombinant technology (33-35). The work reported here addresses another more generally applicable approach to create multimeric scFv molecules for cancer targeting, by site-specific conjugation to functionalize PEG (18). PEGylation has demonstrated the ability to increase in vivo bioactivity of a number of peptides, including asparaginase, interferon alpha, recombinant interleukin2, tumor necrosis factor-alpha, cytokine conjugated toxin, and photodynamic agents such as chlorine polymers (29, 36-38). The ability to control blood clearance based upon the selection of molecular size for PEGyated peptides has been demonstrated (39, 40). Increasing the “effective” molecular size of PEGylated recombinant interlecukin-2 from 40 kDa to 208 kDa increased the slow phase of plasma clearance, by more than 8-fold (39). The FDA has approved a number of PEGylated proteins for imaging (41) and therapy (38, 42). Other PEGylated compounds, to prolong circulation time and reduce immunogenicity, are in clinical trials (43). In general, PEGylation of proteins, including radiolabeled proteins, results in increased molecular size, increased blood retention, and decreased renal glomerular filtration (44). However, uptake of PEGylated proteins into renal tubular cells and hepatocytes, compared to non-PEGylated proteins, has been variable: increased (45-47), decreased (48), or unchanged (49). Conceptually, tumor to normal tissue concentration ratios can be enhanced for targeted radionuclide therapy with PEGylation of small tumor binding modules, but this has yet to be realized (23). PEGylation also affords the opportunity to form multidentate versions of peptides and scFv through the use of branched or star PEG polymers, with the likelihood of increasing avidity and residence time on target sites (50). Studies of the effects of PEGylation on biodistribution of antibody fragments with linear PEG structures have shown that linear PEGylated fragments have prolonged circulation times and increased tumor uptake (36, 39, 45, 50-53). In most cases, PEGylation has been performed by random conjugation with any of the many lysine residues of the protein. In small molecules, such as scFv, an


increased fraction of available lysine is located in or near the binding site (17). The resultant PEGylated proteins tend to be heterogeneous, composed of molecules having PEG conjugated at different positions, thus creating variable binding affinities (17). To overcome this major problem, an approach wherein the recombinant scFv production vectors encode an unpaired cysteine near the C terminus of any inserted scFv for site-specific PEGylation was devised; scFv-c from the pCANTAB 5E Cys vector was used to study thiol PEGylation in this report (18). The advantage of employing sulfhydryl-directed moieties in scFv engineering is that the four disulfidebonded cysteines of the scFv framework needed for the structural configuration are completely buried by the molecular conformation, thus relatively protected (19). Unpaired cysteines are relatively unprotected and not naturally present, thus providing a unique site-specific target for conjugation of any scFv-c made in this manner (54). In the PEGylation of scFv for developing tumortargeting radiopharmaceuticals, efficient conjugation of thiol-reacting PEG to scFv-c is needed. Although the proof of principle of site-specific PEGylation has been previously published (18, 23), the general applicability and efficiencies of conjugation of various scFv-c with a panel of PEG molecules have not been demonstrated. Using four MUC-1 scFvs, having different target epitopes and sequences (20), the relative scFv-c thiol conjugation was evaluated with an array of functionalized PEG molecules. Focus was particularly made on linear uniand bifunctional maleimide PEG molecules of varying molecular weights. In light of these studies, at least 60% (60-90%) of all four scFv could be conjugated with maleimide, demonstrating that the added cysteine provided an available SH for site-specific conjugation, as shown in Figures 2 and 4, ScFv-PEG molecular size increased consistent with the size of PEG attached, as expected (Figure 2a-d). However, when the molecular weight of PEG-Mal increased above 5 kDa, PEGylation efficiency substantially decreased despite evidence of appropriate Mal reactivity on these larger PEG molecules. Steric hindrance of the Mal group by coiling of the linear PEG polymers may provide an explanation. With the 40 kDa branched polymer, better conjugation yield over the linear 10 and 20 kDa PEG may also be due to less steric hindrance of PEG on Mal interaction with the cysteine, since Mal is at the “v” branching point in this PEG configuration (Figure 1) (29). Bifunctional conjugation was demonstrated with 2 and 3.4 kDa PEG bimaleimide. Using the same 1:5 scFv/PEG ratio with the (2 kDa and 3.4 kDa) PEG-(Mal)2, 20, 30, 15, and 42% of the 4 scFv-c was conjugated as (scFv)2PEG, with 2-40% scFv-c left unconjugated. The efficiency of scFv-c reacting with Mal-PEG in all formats did seem to relate to the specific scFv-c, although three of four scFv-c demonstrated more complete conjugation with smaller and linear PEG-Mal molecules. Little or no divalent PEG-(scFv)2, however, with the NHS-PEG-Mal was expected since NHS should not be conjugating at the pH used. This was the case with three of four scFvs. However, BA1-c did conjugate 40% of the BA1-c as scFvPEG-scFv with NHS-PEG-Mal. This mixed bifunctional PEG formed a resultant PEG-(BA1)2 that did bind tumor membranes well on ELISA (Figure 5). An available reactive group near the C-terminus of this scFv-c may be responsible. The PEG-(scFv)2 demonstrated improved binding to the cell membrane antigen when compared to the monomeric scFv-c. The improved binding of S-S linked homodimers


(di-scFv) and PEG-(scFv)2 over the univalent scFv-c or scFv would also be expected because of the increase in avidity with bivalent binding (18, 55-57). However, selected PEG-linked scFv bioconjugates are expected to be superior in vivo tumor targeting agents compared to di-scFv or diabody forms since (i) PEGylated proteins have shown enhanced stability in the circulation in vivo; (ii) increased molecular size should result in decreased rapid kidney clearance, as the threshold for first pass elimination is approximately 65 kDa (58); (iii) the addition of PEG is likely to decrease proteolysis at the tumor site (42). In summary, 30-40% of the scFv-c protein from E-TAGpurified recombinant products was converted to malignant cell membrane binding PEG-(scFv)2 in three of the four scFv-c studied. In these three studied, 50-90% could be conjugated as univalent PEG-scFv, with comparable malignant cell binding before and after PEGylation. These results provide further evidence that novel constructs for imaging and therapy can be developed from scFv-c modules through site-specific PEGylation. ACKNOWLEDGMENT

U.S. Department of Energy Grant DE-FG0100NE22944, National Cancer Institute Grant PO1 CA47829, and Department of Defense Grant DAMD1701-0177 supported this work. We thank Trevor Peterson for excellent technical assistance to producing scFv for this work under a Society of Nuclear Medicine Extension and Research fellowship. LITERATURE CITED (1) DeNardo, S. J., DeNardo, G. L., O’Grady, L. F., Macey, D. J., Mills, S. L., Epstein, A. L., Peng, J.-S., and McGahan, J. P. (1987) Treatment of a patient with B cell lymphoma by I-131 Lym-1 monoclonal antibodies. Int. J. Biol. Markers 2, 49-53. (2) Juweid, M. E., Stadtmauer, E., Hajjar, G., Sharkey, R. M., Suleiman, S., Luger, S., Swayne, L. C., Alavi, A., and Goldenberg, D. M. (1999) Pharmacokinetics, dosimetry and initial therapeutic results with 131I- and 111In-/90Y-labeled humanized LL2 anti-CD22 monoclonal antibody (MAb) in patients with relapsed/refractory non-Hodgkin’s lymphoma (NHL). Clin. Cancer Res. 5, 3292s-3303s. (3) DeNardo, S. J., DeNardo, G. L., Yuan, A., Richman, C. M., O’Donnell, R. T., Lara, P. N., Kukis, D. L., Natarajan, A., Lamborn, K. R., Jacobs, F., and Siantar, C. L. (2003) Enhanced therapeutic index of radioimmunotherapy (RIT) in prostate cancer patients: comparison of radiation dosimetry for 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)-peptide versus 2IT-DOTA monoclonal antibody linkage for RIT. Clin. Cancer Res. 9, 3938s-3944s. (4) Jain, R. K. (1990) Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors. Cancer Res. 50, 814s-819s. (5) Winthrop, M. D., DeNardo, S. J., and DeNardo, G. L. (1999) Development of a hyper immune anti-MUC-1 single chain antibody fragments phage display library for targeting breast cancer. Clin. Cancer Res. 5, 3088s-3094s. (6) DeNardo, S. J., DeNardo, G. L., DeNardo, D. G., Xiong, C. Y., Shi, X. B., Winthrop, M. D., Kroger, L. A., and Carter, P. (1999) Antibody phage libraries for the next generation of tumor targeting radioimmunotherapeutics. Clin. Cancer Res. 10, 3213-3218. (7) Winter, G., Griffiths, A. D., Hawkins, R. E., and Hoogenboom, H. R. (1994) Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433-455. (8) Carter, P. (2001) Improving the efficacy of antibody-based cancer therapies. Nat. Rev. Cancer 1, 118-129. (9) Maynard, J., and Georgiou, G. (2000) Antibody engineering. Annual Review of Biomed. Eng. 2, 339-376.

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BC0498121

Characterization of Site-Specific ScFv PEGylation for Tumor-Targeting

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