Genetic Engineering, Expression, and Activity of a Chimeric

Feb 16, 2008 - ArmaGen Technologies, Inc., Santa Monica, California 90401, and Department of Medicine, University of California Los Angeles, Los Angel...
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Bioconjugate Chem. 2008, 19, 731–739

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Genetic Engineering, Expression, and Activity of a Chimeric Monoclonal Antibody-Avidin Fusion Protein for Receptor-Mediated Delivery of Biotinylated Drugs in Humans Ruben J. Boado,† Yufeng Zhang,† Yun Zhang,† Chun-fang Xia,‡ Yuntao Wang,‡ and William M. Pardridge*,‡ ArmaGen Technologies, Inc., Santa Monica, California 90401, and Department of Medicine, University of California Los Angeles, Los Angeles, California 90024. Received November 2, 2007; Revised Manuscript Received January 7, 2008

The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.

INTRODUCTION Molecular Trojan horses are genetically engineered receptorspecific monoclonal antibodies (mAb) that undergo receptormediated transport through biological membranes, including the blood-brain barrier (BBB) in vivo (1). Chimeric and humanized mAb’s to the human insulin receptor (HIR) have been genetically engineered, expressed, and shown to have transport properties in vivo comparable to the original murine HIRMAb (2). The chimeric or humanized HIRMAb can target to brain, and other organs, recombinant proteins and monoclonal antibodies, and fusion proteins of the HIRMAb and recombinant neurotrophins, single chain Fv antibodies, and lysosomal enzymes have been produced and validated in vivo (3–5). However, the fusion protein technology cannot be used to deliver oligopeptides or short interfering RNA (siRNA), because fusion proteins of the HIRMAb and the oligopeptide or siRNA cannot be engineered. Avidin–biotin technology, in conjunction with the fusion protein technology, can enable the in vivo targeting of peptides and siRNAs. In this approach, a fusion protein of the HIRMAb and avidin is genetically engineered in parallel with the production of a monobiotinylated peptide or siRNA. Recent work shows that siRNAs can be monobiotinylated on either the 5′- or 3′-terminus of the sense strand, and conjugated to streptavidin (SA)-mAb delivery systems without loss of gene siliencing biological activity (6). In prior work (6), the targeting mAb and the recombinant SA were conjugated with a thioether bond, as described previously (7). However, for manu* Correspondence: Dr. William M. Pardridge, UCLA Warren Hall 13-164, 900 Veteran Ave., Los Angeles, CA 90024, Ph: (310) 8258858, Fax: (310) 206-5163, Email: [email protected]. † ArmaGen Technologies, Inc. ‡ University of California Los Angeles.

facturing of human therapeutics, what is needed is a eukaryotic expression system for the production of mAb-avidin fusion proteins. Avidin is preferred over SA, because avidin is less immunogenic in humans than is SA (8). Previous investigations have demonstrated the feasibility of genetically engineering mAb-avidin fusion proteins, using a murine mAb to the rat transferrin receptor (TfR) (9). However, the HIRMAb delivery system is more active than is the TfRMAb delivery system (1). The purpose of the present investigation is to examine the feasibility of the genetic engineering and eukaryotic expression of a fusion protein of avidin and the chimeric HIRMAb, which is designated HIRMAb-AV. The avidin monomer is fused to the carboxyl terminus of the HIRMAb heavy chain constant region (Figure 1). It is assumed that a HIRMAb-avidin (AV) fusion protein that is expressed in eukaryotic expression systems, such as Chinese hamster ovary (CHO) cells, would be fully saturated by the endogenous biotin present in the culture medium, and that this saturation of the fusion protein by the endogenous biotin would effectively eliminate the biotin binding activity of the fusion protein. Once biotin is bound to avidin, it does not readily dissociate off the protein. The dissociation constant (KD) of avidin binding of biotin is 10-15 M, and the dissociation t1/2 is 89 days (10). Therefore, the present studies examine the feasibility of production of mAb-avidin fusion proteins by CHO cells cultured in biotin-depleted medium.

MATERIALS AND METHODS Cloning of Avidin cDNA. Avidin cDNA (GenBank X05343) corresponding to amino acids Ala1-Glu128 of the mature chicken avidin protein was cloned by the polymerase chain reaction (PCR) using the oligodexoynucleotides (ODNs) described in Table 1 and an avidin plasmid DNA template. The avidin cDNA was cloned by PCR using 40 ng plasmid cDNA, 0.2 µM avidin

10.1021/bc7004076 CCC: $40.75  2008 American Chemical Society Published on Web 02/16/2008

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Figure 1. Structure of the fusion protein showing fusion of the amino terminus of the mature avidin monomer to the carboxyl terminus of the CH3 region of the heavy chain of the chimeric HIRMAb. The fusion protein is a bifunctional molecule with dual functions. The fusion protein binds the HIR, at the BBB, to mediate transport into the brain, and binds biotinylated pharmaceuticals. Table 1. Oligodeoxynucleotides Primers Used in the Cloning of Avdin cDNA 1) Avidin FWD: 5′-phosphate-CGTCTGCCAGAAAGTGCTCGCTGAC-3′ 2) Avidin REV: 5′-phosphate-TCACTCCTTCTGTGTGCGCAGGCGA-3′

forward and reverse ODN primers (Table 1), 0.2 mM deoxynucleosidetriphosphates, and 2.5 U PfuUltra DNA polymerase (Stratagene, San Diego, CA) in a 50 µL Pfu buffer (Stratagene). The amplification was performed in a Mastercycler temperature cycler (Eppendorf, Hamburg, Germany) with an initial denaturing step of 95 °C for 2 min followed by 30 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s and amplification at 72 °C for 1 min; followed by a final incubation at 72 °C for 10 min. PCR products were resolved in 0.8% agarose gel electrophoresis, and the expected major single band of ∼0.4 kb corresponding to the avidin cDNA was produced (Figure 2). Engineering of HIRMAb-Avidin Expression Vectors. The ODNs used for PCR are 5′-phosphorylated for direct insertion into the HpaI site of either the pHIRMAb-HC expression plasmid or the pHIRMAb tandem vector (TV) expression plasmid, respectively (Figure 3A,B), as described previously (3). The pHIRMAb-HC plasmid encodes the HC of the chimeric HIRMAb, and dual transfection of COS cells with this plasmid and a light chain (LC) expression plasmid, pHIRMAb-LC,

Figure 2. Ethidium bromide stain of agarose gel of avidin cDNA (lane 1), which was produced by PCR with avidin-specific ODN primers (Table 1). Lane 2: molecular weight DNA standards.

Boado et al.

Figure 3. Engineering of the expression plasmid encoding the HIRMAb-AV fusion protein for either COS cells (A) or CHO cells (B). The PCR generated avidin cDNA was ligated into the single HpaI site of either the universal pHIRMAb-HC plasmid (A) or the pHIRMAb tandem vector (B) to form the pHIRMAb-HC-Avidin vector or the pHIRMAb-AV TVrev, respectively. The pHIRMAb-HC plasmid encodes the heavy chain (HC) of the chimeric HIRMAb, and is 5′flanked by the CMV promoter and is 3′-flanked by the BGH polyA (pA) sequence. The universal pHIRMAb-HC tandem vector (TV) contains expression cassettes, in series, for the HC and LC of the chimeric HIRMAb, and contains the expression cassette for murine DHFR in either forward (for) or reverse (rev) orientation relative to the HC and LC cassettes. In the pHIRMAb-AV TV rev, the DHFR cDNA is 5′-flanked by the hepatitis B virus pA sequence, and is 3′flanked by the SV40 promoter.

allows for transient expression of either the chimeric HIRMAb, or a fusion protein, in COS cells. The pHIRMAb TV is a tandem vector (TV), which contains multiple genes, each within separate tandem expression cassettes, on a single piece of DNA, and includes the HC expression cassette, the LC expression cassette, and an expression cassette for the murine dihydrofolate reductase (DHFR) gene for amplification of cell lines with methotrexate (MTX). If the DHFR cassette is in the same forward (for) orientation as the HC and LC cassettes, then the TV is designated TVfor; if the DHFR cassette is in reverse (rev) orientation, then the TV is designated TVrev. The TV is engineered for permanent transfection of host cells, such as DG44 Chinese hamster ovary (CHO) cells. Placement of the HC fusion gene, the LC gene, and the genes encoding the selectable markers on a single piece of tandem vector DNA allows for isolation of a high expressing CHO line where the HC fusion gene and LC gene are equally expressed within the host cell. The selectable markers on the TV include the neomycin resistance gene (neo), for selection with G418, and the DHFR gene, for amplification of cell lines with MTX (Figure 3). The forward avidin PCR primer introduces “CG” nucleotides to maintain the open reading frame and to introduce a Ser-SerSer linker between the carboxyl terminus of the CH3 region of the HIRMAb HC and the amino terminus of the avidin, respectively. The fusion of the avidin monomer to the carboxyl terminus of each HC is depicted in Figure 1. This design sterically restricts the avidin to a dimeric configuration, which replicates the native conformation of avidin, which is a dimer of dimers (11). The avidin reverse PCR primer introduces a stop codon, “TGA”, immediately after the terminal Glu of the mature avidin protein. The HIRMAb HC and LC cDNA expression cassettes are driven by the cytomegalovirus (CMV) promoter and contain the bovine growth hormone (BGH) polyadenylation (pA) sequence (Figure 3). The DHFR expression cassette is under the influence of the SV40 promoter and the hepatitis B virus

Avidin-Antibody Fusion Protein

pA sequence. The engineering of universal pHIRMAb-HC and the pHIRMAb TV vectors was performed by insertion of a single HpaI site at the end of the HIRMAb HC CH3 open reading frame (orf) by site directed mutagenesis (SDM), as described previously (3). Transient Expression of HIRMAb-Avidin Fusion Protein in COS Cells. COS cells were dual transfected with pHIRMAb-LC and pHIRMAb-HC-Avidin, or monotransfected with the pHIRMAb-AV tandem vector, using Lipofectamine 2000, with a ratio of 1:2.5, ug DNA/uL Lipofectamine. Following transfection, the cells were cultured in serum free VP-SFM (Invitrogen, Carlsbad, CA). The conditioned serum free medium was collected at 3 and 7 days. The fusion protein was purified by protein A affinity chromatography. Permanent Expression of HIRMAb-AV Fusion Protein in Stably Transfected CHO Cells. Serum free medium (SFM) adapted DG44 CHO cells (Invitrogen) were electroporated with 5 ug pHIRMAb-AV TVfor or pHIRMAb-AV TVrev, following linearization with PvuI, using a Gene Pulser Xcell electroporator (BioRad, Hercules, CA). 5 × 106 cells were electroporated with the DNA in 200 uL of phosphate buffered saline (PBS) and 0.2 cm cuvettes using a square wave and 160 V. Cells were suspended in CHO serum free medium (SFM) (Hyclone, Logan, UT) and plated in 4 × 96-well plates. Selection of stable transfectants began 2 days following electroporation with 0.54 mg/mL G418. Aliquots of supernatant were taken for human IgG ELISA when colonies of transfectants were evident, i.e., 21 days. Positive clones were isolated and cultured individually for further characterization. DG44 cells lack endogenous DHFR, and rely on nutrients, hypoxanthine and thymidine (HT) for endogenous folate synthesis. Transfected cells carrying the TV express the exogenous DHFR. Transfected cell lines were further selected by placement in HT-deficient medium. Lines with amplification around the transgene insertion site were selected by subjecting the cells to increasing concentrations of MTX, starting at 20 nM MTX. Following stabilization of the cell line at 80 nM MTX, high producing clones were isolated by limited dilution cloning (DC) at 1 cell per well; a total of 4000 wells were plated at each round of DC, and medium IgG was measured with a human IgG ELISA using a high volume microplate dispenser and a microplate washer (Biotek, Winooski, VT). SDS-PAGE and Western blotting. The purity of protein A purified fusion protein produced by COS or CHO cells was evaluated with a reducing 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoreactivity was tested with a primary rabbit antibody to chicken egg white avidin (Abcam, Cambridge, MA) or a primary goat antiserum against human IgG heavy and light chains (Vector Laboratories, Burlingame, CA). HIR Receptor Assay. The affinity of the fusion protein for the HIR extracellular domain (ECD) was determined with an ELISA using the lectin affinity purified HIR ECD. CHO cells permanently transfected with the HIR ECD were grown in serum free media (SFM), and the HIR ECD was purified with a wheat germ agglutinin affinity column, as previously described (12). The HIR ECD (0.2 ug/well) was plated on Nunc-Maxisorb 96 well dishes and the binding of the chimeric HIRMAb, the HIRMAb-AV fusion protein, or human IgG1 to the HIR ECD was detected with a biotinylated goat antihuman IgG (H+L) antibody (0.3 ug/well), and the ABC Elite detection system (Vector Laboratories). The concentration that caused 50% binding to the HIR ECD, the ED50, was determined by nonlinear regression analysis using the WinNonlin software. Biotinylated BSA ELISA. The qualitative binding of the HIRMAb-AV fusion protein to a biotinylated ligand was assessed with an ELISA using biotinylated bovine serum

Bioconjugate Chem., Vol. 19, No. 3, 2008 733 Table 2. Concentration of HIRMAb-AV in Medium Following Transient Expression in COS Cellsa plasmid

IgG at 3 days (µg/mL)

IgG at 7 days (µg/mL)

pHIRMAb HC-avidin + pHIRMAb LC pHIRMAb-AV TV-for pHIRMAb-AV TV-rev

4.7 ( 0.2 1.0 ( 0.2 6.2 ( 0.2

10.7 ( 2.2 4.7 ( 1.3 7.8 ( 0.3

a

Data are mean ( SE (n ) 3).

albumin (BSA, Sigma Chemical Co., St. Louis, MO) as the ligand. The biotinylated BSA was plated to a 96-well ELISA plate (0.4 ug/well). Following aspiration, washing with HBS buffer with 0.05% Tween-20, where HBS ) 0.01 M HEPES/ 0.15 M NaCl/pH ) 7.4, and blocking with 2% goat serum in HBS, the HIRMAb-AV fusion protein or the chimeric HIRMAb was added in concentrations ranging from 0 to 3000 ng/mL for 90 min at room termperature. Detection of binding used a alkaline phosphatase conjugate of a goat antihuman kappa light chain antibody and p-nitrophenylphosphate. [3H]-Biotin Binding Ultrafiltration Assay. The quantitative binding of biotin to the HIRMAb-AV fusion protein was determined with an ultrafiltration assay, as described previously (7). The assay used [3H]-biotin (Perkin-Elmer, Boston, MA), which had a specific activity of 50 uCi/nmol, and an UltrafreeMC filter unit (Millipore, Billerica, MA), which has a low protein binding regenerated cellulose filter with a 10 kDa molecular weight cutoff. A 100 uL volume of PBS (0.01 M Na2HPO4/0.15 M NaCl/pH ) 7.4) was added to a microfuge tube (MFT), which contained 0.1 uCi/mL of 3H-biotin (2 nM), various concentrations of unlabeled biotin (25–1000 nM), and a fixed concentration of either avidin (5 ug/mL, 80 nM) or the HIRMAb-AV fusion protein (15 ug/mL, 80 nM). After a 30 min incubation at room temperature, the solution was transferred to the Ultrafree-MC ultrafiltration unit, which was placed in a 1.5 mL MFT, and the unit was centrifuged at 10 000 g for 5 min at room temperature. The total biotin binding was determined from the total biotin concentration and the percent binding of the [3H]-biotin, which was determined by sampling the ultrafiltrate for 3H radioactivity using a Perkin-Elmer liquid scintillation counter. The number of biotin binding sites per protein was determined from the ratio of the bound biotin concentration relative to the concentration of the avidin or the HIRMAb-AV fusion protein. Equilibrium Dialysis of HIRMAb-AV Fusion Protein. The protein A affinity purified HIRMAb-AV fusion protein produced by transfected COS cells was fully saturated by the biotin in the VP-SFM tissue culture medium. The biotin concentration in this medium, 0.75 ug/mL (3100 nM), is in a 55-fold molar excess when the concentration of the HIRMAb-AV fusion protein is 10 ug/mL (Table 2). Therefore, the biotin binding sites on the expressed HIRMAb-AV fusion protein are fully saturated, which would prevent the use of the fusion protein for the drug delivery of biotinylated therapeutics. In an attempt to dissociate the biotin from the HIRMAb-AV fusion protein, the protein was subjected to extensive equilibrium dialysis, using an avidin sequestration technique. The HIRMAb-AV fusion protein was placed in a 1 mL Slide-A-Lyzer dialysis cassette with a 7 kDa molecular weight cutoff (Pierce Chemical Co., Rockford, IL), which was placed in 10 L of PBS, which contained 2 mg/L of avidin and 0.05% sodium azide, and dialysis was performed at 4 °C for 72 h, with a complete change of the 10L dialysis buffer at 36 h. The presence of high concentrations of avidin in the dialysis buffer was intended to sequester in the buffer any biotin that dissociated from the HIRMAb-AV fusion protein during the 72 h. The dialyzed HIRMAb-AV fusion protein was then recovered, and the binding of [3H]-biotin was measured.

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Culture of CHO Cells During Biotin Starvation. The CHO cells producing the HIRMAb-AV fusion protein were cultured in HyQ SFM4CHO-utility medium from Hyclone (Logan, UT), which contains 0.194 ug/mL (800 nM) of biotin. The medium biotin is present in a 14-fold molar ratio when the medium HIRMAb-AV fusion protein is 10 mg/L, and it would not be possible to produce fusion protein with unoccupied biotin binding sites. Therefore, the CHO cells were subjected to biotin starvation over a 27 day period. On day 1, CHO cells were centrifuged and resuspended in 25 mL fresh biotin-free serum free medium (SFM), which was supplemented with 20 nM biotin, which is a 40-fold reduction from the usual medium biotin concentration. The cells were cultured in square plastic (polyethylene terephthalate G copolyester, PETG) bottles (Nalge Nunc International, Rochester, NY), on an Innova 200 orbital shaker (New Brunswick Scientific (Edison, NJ) at 120 rpm, which was placed inside a humidified tissue culture incubator at 37 °C, and the cells continued to divide. Fresh SFM with 20 nM biotin was added on days 2 and 4 until the volume of medium was 135 mL. On day 8, fresh medium was added to a total volume of 400 mL and the biotin concentration was reduced to 10 nM. Over the next 18 days, the volume of medium was maintained at 400 mL (135 mL in each of 3 × 500 mL plastic square bottles). During this time, 1500 mL of conditioned medium was harvested from the 3 bottles, and the bottles were replenished with 1500 mL fresh biotin free SFM, so that the medium biotin concentration was successively reduced to 3 nM at day 14, to 1 nM at day 18, to 0.3 nM at day 21, and the culture was terminated at day 26. HPLC Size Exclusion Chromatography. The presence of aggregates of the HIRMAb-AV fusion protein was evaluated with size exclusion chromatography (SEC) using a 7.8 mm × 30 cm (5 µm) G3000SW high pressure liquid chromatography (HPLC) column (Tosoh Bioscience LLC, Montgomeryville, PA), and a Perkin-Elmer Series 200 pump and UV/vis detector. The HIRMAb-AV fusion protein (100 ug), produced by CHO cells under conditions of biotin starvation, and purified by protein A affinity chromatography, was injected on to the column in a 200 uL volume of PBS, followed by elution in PBS at a flow rate of 0.5 mL/min with detection at 280 nm. The elution profile was recorded with a Servocorder model SR6253 chart recorder (Datamark Corp., Costa Mesa, CA), at 0.5 cm/min, and the chart paper was scanned with a UMAX PowerLook III scanner (Techville, Inc., Dallas, TX) into Adobe Photoshop. Native Polyacrylamide Gel Electrophoresis. The ability of the HIRMAb-AV fusion protein to migrate into a native polyacrylamide gel was evaluated with native PAGE, using a 15% Ready Gel Tris-HCl Gel (Biorad, Richmond, CA), which was composed of a 8.6 × 6.8 cm 15% resolving gel and a 4% stacking gel. The HIRMAb-AV fusion protein, the chimeric HIRMAb, or human IgG1 was prepared in sample buffer (3 mM histidine, 3 mM MES, pH ) 6.1, 30% glycerol), and 5 ug protein per lane was applied, where MES is 2-(N-morpholino)ethanesulfonic acid. The electrophoresis buffer was 30 mM histidine, 30 mM MES, pH ) 6.1, and electrophoresis was performed at 150 V for 4 h. Since the HIRMAb-AV fusion protein is cationic with a predicted isoelectric point (pI) of 9.02 (Results), the electrophoresis was performed with reversed electrode polarity. Following electrophoresis, the gel was stained with Coomasie blue, and photographed. Biotin Uptake Assay. Human renal 293 epithelial cells were obtained from the American type Culture Collection (Rockville, MD), and grown on collagen coated 24-well dishes in DMEM medium with 10% fetal bovine serum. Prior to the uptake assay, the medium was discarded, and 0.4 mL/well of 0.01 M Tris/ 0.15 M NaCl/pH ) 7.4 (TBS) was added, which contained 0.5

Boado et al.

uCi/mL (11 nM) of [3H]-biotin (Perkin-Elmer NET721) and either 0 or 5 ug/mL (26 nM) of the HIRMAb-AV fusion protein. After incubation for 2–120 min at 37 °C in a humidified chamber, the incubation was terminated by aspiration of the medium, washing of the wells with cold TBS, and the cell monolayer was solubilized in 0.5 mL/well of 1 N NaOH, followed by determination of 3H radioactivity in the cell lysate and the medium. The cell protein was measured by the bicinchoninic acid (BCA) assay. The 3H-biotin volume of distribution (VD) was determined from the ratio of DPM/mg protein divided by the DPM/uL medium. Confocal Microscopy. The 293 cells were plated on collagen coated 4-well microscopy slides (Biocoat, Becton Dickinson Labware). Prior to the incubation, the medium was aspirated and replaced with 0.5 mL/well of fresh DMEM, containing 100 nM fluorescein-biotin (Invitrogen B-1370), and either 0 or 60 ug/mL of the HIRMAb-AV fusion protein. The cells were incubated at 37 °C for either 3 or 24 h. Following incubation, the medium was aspirated, the wells were washed well with cold PBS and fixed in 100% methanol for 20 min at -20 °C. The fixative was removed, the cells were washed with cold PBS, the nuclei were stained by incubation in 0.3 ug/mL propidium iodide (Sigma) for 5 min, the wells were washed with PBS, and coverslipped with Vectashield (Vector Laboratories, Burlingame, CA). Confocal microscopy was performed with a Zeiss LSM 5 PASCAL confocal microscope with dual argon and helium/neon lasers equipped with Zeiss LSM software for image reconstruction.

RESULTS DNA sequencing of the expression cassette of the pHIRMAbAvidin heavy chain expression plasmid (Figure 3A) encompassed 2,874 nucleotides (nt), including a 714 CMV promoter, a 9 nt Kozak sequence (GCCGCCACC), a 1782 nt coding sequence, and a 369 nt BGH transcription termination sequence. The deduced amino acid sequence showed the heavy chain fusion protein was comprised of 593 amino acids (AA), including a 19 AA signal peptide, a 443 AA chimeric HIRMAb heavy chain, a 3 AA linker (Ser-Ser-Ser), and a 128 AA avidin monomer, which was 100% identical to the AA sequence of avidin, NP_990651 (GenBank). The avidin sequence included a single N-linked glycosylation sequence, and a single internal disulfide bond. The predicted molecular weight of the fusion protein heavy chain, without glycosylation, is 63,191 Da with a pI of 9.02. The pHIRMAb-Avidin expression cassette was used for transient transfection of COS cells as described below. The fusion protein heavy chain (HC) expression cassette, the chimeric HIRMAb light chain (LC) expression cassette, and the DHFR expression cassette were all incorporated into a single tandem vector (TV), designated pHIRMAb-AV TVrev, as shown in Figure 3B; the reverse (rev) designation indicates the orientation of the DHFR cassette was in reverse direction, relative to the HC or LC expression cassettes. DNA sequence analysis showed the 3 expression cassettes were composed of 6332 nt, in the following order: a 731 nt CMV promoter, a 9 nt Kozak sequence, a 705 nt LC open reading frame (orf), a 291 nt BGH sequence, a 23 nt spacer, a 714 CMV promoter, a 9 nt Kozak sequence, a 1782 nt HC orf, a 301 nt BGH sequence, a 940 nt hepatitis B virus (HBV) transcription termination sequence, a 564 nt murine DHFR orf, a 9 nt Kozak sequence, and a 254 nt SV40 promoter. The murine DHFR sequence was 100% identical to nt 857–1510 of NM_010049. The predicted molecular weight of the LC was 23 398 Da with a pI of 5.45. Dual transfection of COS cells with the pHIRMAb-HCAvidin plasmid and the chimeric HIRMAb LC expression plasmid, pHIRMAb-LC, resulted in medium levels of fusion protein of the HIRMAb-AV fusion protein of 4.7 ( 0.2 µg/mL

Avidin-Antibody Fusion Protein

Figure 4. Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of chimeric HIRMAb and the HIRMAb-AV fusion protein. The size of the light chain, 28 kDa, is identical for the chimeric HIRMAb and the fusion protein; the size of the heavy chain of fusion protein is about 16 kDa larger than the chimeric HIRMAb heavy chain, owing to the presence of the avidin monomer. The molecular weights shown for the standards are the actual molecular weights, whereas the molecular weights of the HIRMAb and HIRMAbAV fusion protein are estimated.

and 10.7 ( 2.2 µg/mL at 3 and 7 days after transfection, respectively. These levels of medium IgG exceed those obtained following transient transfection of COS cells with the chimeric HIRMAb HC and LC genes. COS cells were also lipofected with the CHO cell tandem vectors, pHIRMAb-AV TVfor and pHIRMAb-AV TVrev, which are outlined in Figure 3B. The medium concentrations of the HIRMAb-AV fusion protein with the pHIRMAb-AV TV rev were comparable to that found with dual transfection with the separate HC and LC expression plasmids (Table 2). Lower levels of transient expression were observed with the pHIRMAb AV TVfor (Table 2). The HIRMAb-AV fusion protein secreted to the serum free medium by transfected COS cells was purified with protein A affinity chromatography. The fusion protein was purified to homogeneity on reduced SDS-PAGE (Figure 4). The HIRMAbAV fusion protein and the chimeric HIRMAb share the same light chain (LC) (Figure 4), whereas the size of the heavy chain (HC) of the HIRMAb-AV fusion protein is approximately 15 kDa larger than the size of the HC of the chimeric HIRMAb (Figure 4). Western blotting of the purified HIRMAb-AV fusion protein and chimeric HIRMAb was performed with primary antibodies to either human IgG (Figure 5A) or to avidin (Figure 5B). The antihuman IgG antibody reacts with both the HC and the LC of the HIRMAb-AV fusion protein and the chimeric HIRMAb, but not with avidin (AV), and the size of the immunoreactive HC is about 15 kDa larger for the HIRMAbAV HC as compared to the HIRMAb HC (Figure 5A). The antiavidin antibody reacts with both avidin and the HC of the HIRMAb-AV fusion protein, but not with LC of the HIRMAbAV fusion protein or chimeric HIRMAb, and the size of the immunoreactive HC is about 15 kDa larger for the HIRMAbAV HC as compared to the HIRMAb HC (Figure 5B). The affinity of the HIRMAb-AV fusion protein for the HIR ECD was determined with a binding assay, in parallel with measurements of the binding of the chimeric HIRMAb and human IgG1 (Figure 6). The concentration of chimeric HIRMAb that produced 50% saturation of binding, the ED50, was 0.50 ( 0.06 nM, and the ED50 of the COS-cell derived HIRMAb-AV fusion protein was 1.13 ( 0.20 nM; human IgG1 did not bind to the HIR ECD (Figure 6). An ELISA using biotinylated BSA as the ligand is outlined in Figure 7A. The HIRMAb-AV fusion protein, but not the

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Figure 5. (A) Western blot with antihuman IgG primary antibody. The size of the heavy chain of the fusion protein and the chimeric HIRMAb is 68 kDa and 52 kDa, respectively, and the size of the light chain for either the fusion protein or the chimeric HIRMAb is 28 kDa. (B) Western blot with antiavidin antibody, which reacts with either fusion protein or avidin, but not with chimeric HIRMAb. MW standards (STDS) are shown on the right side. The molecular weights (MW) shown for the standards (STDS) are the actual molecular weights, whereas the molecular weights of the HIRMAb and HIRMAb-AV fusion protein are estimated.

Figure 6. Saturable binding of the chimeric HIRMAb and the HIRMAbAV fusion protein to the HIR ECD is demonstrated over a range of 0–3000 ng/mL. There is no binding to the HIR by human IgG1. The concentration that produces 50% binding, the ED50, was computed by nonlinear regression analysis with units of ng/ml, and converted to nM, based on the molecular weight of the HIRMAb, 150 000 Da, and the molecular weight of the HIRMAb-AV fusion protedin, 185 000 Da.

chimeric HIRMAb bound to the biotinylated BSA over a concentration range of 0–3000 ng/mL (Figure 7B). This ELISA provides a qualitative index of HIRMAb-AV binding to biotinylated substrate, but does not provide a measure of the number of biotin binding sites on the HIRMAb-AV fusion protein. The latter is determined with a quantitative biotin binding assay using a [3H]-biotin ultrafiltration method (Methods). As shown in Figure 8, 80 nM avidin bound 250 nM biotin, which indicates there are 3 available biotin binding sites per avidin tetramer. However, in this assay, no measureable binding of [3H]-biotin was observed for the COS-derived HIRMAbAV fusion protein. This was attributed to near complete saturation of the available biotin binding sites on the HIRMAbAV fusion protein by the endogenous biotin in the tissue culture medium. The medium biotin concentration was 3.1 uM (Methods), as opposed to the concentration of HIRMAb-AV fusion protein, which ranged from 26 nM to 59 nM (Table 2). The HIRMAb-AV fusion protein was subjected to extensive dialysis using an avidin sequestration technique (Methods). However, there were still no measureable biotin binding sites on the fusion protein, as determined with the ultrafiltration assay. These observations suggested that it would not be possible to produce the HIRMAb-AV fusion protein in eukaryotic expression

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Boado et al. Table 3. Expression of HIRMAb-AV by CHO Cells under Conditions of Biotin Depletion in Serum Free Mediuma day of culture

total cells (×106)

viable cells (%)

biotin (nM)

HIRMAb-Av/ biotin ratio

8 14 18 21 24 27

2.7 1.8 2.6 2.0 0.8 1.0

94 93 83 69 70 33

10 3 1 0.3 0.3 0.3

2.4 3.9 11 41 233 332

a Cells were grown in a shake flask on an orbital shaker in serum free medium. The medium biotin was reduced from 800 nM to 20 nM on day 1, and was reduced from 20 nM to 10 nM on day 8.

Figure 7. (A) Outline of biotinylated bovine serum albumin (BSA) ELISA. Binding of the HIRMAb-AV fusion protein to the biotinylated BSA is detected with a peroxidase conjugate of a goat antihuman (GAH) IgG. (B) Binding of the HIRMAb-AV fusion protein, but not the chimeric HIRMAb, to biotinylated BSA is demonstrated over a concentration range of 0–3000 ng/mL.

Figure 8. The binding of [3H]-biotin to either avidin or the HIRMAbAV fusion protein produced in biotin-starved CHO cells is measured with an ultrafiltration assay. The concentration of avidin or the HIRMAb-AV fusion protein in the assay is 80 nM.

systems that retained functional biotin binding sites, unless the host cell line was cultivated in biotin-depleted medium under conditions of biotin starvation over several weeks. This required the availability of a permanently transfected host cell line, such as Chinese hamster ovary (CHO) cells. Following permanent transfection of DG44 CHO cells with linearized pHIRMAbAV TVrev (Figure 3B), a high producing CHO cell line was isolated following methotrexate amplification and dilutional cloning. The levels of human IgG in the medium of the transfected CHO line grown in serum free medium in shake flasks at a density of (1–2) × 106 cells/mL exceeded 40 mg/L following one round of dilutional cloning. The CHO cells were propagated in square plastic bottles on an orbital shaker, and the medium biotin concentration was reduced from 20 nM to 0.3 nM over a 27 day period (Table 3). During this time, the HIRMAb-AV fusion protein was secreted to the medium and the molar ratio of HIRMAb-AV fusion protein/biotin increased to >300 (Table 3). The HIRMAb-AV fusion protein produced by the biotinstarved CHO cells was purified by protein A affinity chromatography. The protein eluted as a single homogeneous peak on SEC HPLC, and migrated with a molecular weight of ap-

proximately 400 kDa (Figure 9A). The fusion protein migrated as a single homogeneous product in native PAGE gels, and migrated faster than the chimeric HIRMAb or human IgG1, which is consistent with the higher cationic charge of the fusion protein as compared to the HIRMAb alone (Figure 9B). The affinity of the CHO derived HIRMAb-AV fusion protein for the HIR ECD was measured, and the ED50 of HIRMAbAV fusion proteing binding was 0.58 ( 0.09 nM, which was not significantly different from the ED50 for the binding of the chimeric HIRMAb, 0.66 ( 0.14 nM. The HIRMAb-AV fusion protein produced by the biotin-starved CHO cells quantitatively bound [3H]-biotin, and a 80 nM concentration of fusion protein bound 80 nM biotin (Figure 8), indicating 1 of the 2 biotin binding sites on the fusion protein was unoccupied. The uptake of 3H-biotin by human renal 293 cells was minimal over a 2 h incubation period at 37C (Figure 10). However, in the presence of the HIRMAb-AV fusion protein, the cell uptake of the 3H-biotin increased over 15-fold, and the biotin VD exceeded 100 uL/mg protein at 120 min of incubation (Figure 10). Confocal microscopy showed no measureable uptake of fluorescein-biotin at 3 or 24 h of incubation in the absence of the HIRMAb-AV fusion protein (Figure 11A). However, in the presence of the HIRMAb-AV fusion protein, there was penetration of the fluorescein-biotin into the intracellular compartment, which was visible in intracellular endosomes, at 3 h (Figure 11B) and 24 h (Figure 11C,D) of incubation.

DISCUSSION The results of this study are consistent with the following conclusions. First, a fusion gene has been genetically engineered, which encodes a fusion protein wherein the amino terminus of avidin is fused to the carboxyl terminus of the CH3 region of the HC of the chimeric HIRMAb (Figure 1). Second, following expression in either COS or CHO cells, the HC of the fusion protein is 15 kDa larger than the HC of the chimeric HIRMAb (Figure 4), and the fusion protein HC reacts with antibodies to both human IgG and avidin (Figure 5). Third, the fusion protein is a bifunctional molecule, and binds both the HIR ECD (Figure 6), and binds biotinylated ligands (Figure 7). Fourth, the fusion protein can be produced in CHO cells under conditions of biotin starvation of periods of at least 27 days (Table 3), and the fusion protein retains unoccupied biotin binding sites (Figure 8). Fifth, the HIRMAb-AV fusion protein produced by CHO cells under conditions of biotin starvation is highly stable, does not form aggregates (Figure 9B), and elutes as a monomeric species on HPLC SEC (Figure 9A). Sixth, the HIRMAb-AV fusion protein enhances the cell uptake of biotin (Figure 10) or biotinylated ligands, such as fluorescein-biotin (Figure 11). The design of the HIRMAb-AV fusion protein places the 15 kDa avidin monomer at the carboxyl terminus of the heavy chain of the chimeric HIRMAb (Figure 1). This places the avidin moiety in a configuration that favors a parallel dimer conformation, which replicates the parallel association of 2 avidin or

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Figure 9. (A) SEC HPLC of the HIRMAb-AV fusion protein produced by biotin-starved CHO cells. The migration of 4 molecular size markers is shown by the solid circles, and in left-to-right order include, blue-dextran-2000 (2 million Da), ferritin (440 kDa), aldolase (158 kDa), and RNase A (14 kDa). (B) Native polyacrylamide gel electrophoresis (PAGE) of the HIRMAb-AV fusion protein, the chimeric HIRMAb, and human IgG1.

Figure 10. Volume of distribution (VD) of [3H]-biotin in human 293 cells at 2 to 120 min of incubation at 37 °C after comixture of the labeled biotin with either PBS or the HIRMAb-AV fusion protein. Data points are mean ( SE (n ) 4 dishes per time point).

streptavidin monomers to form a dimer (11). Placement of the avidin monomer at the carboxyl terminus of the heavy chain, as opposed to the amino terminus eliminates interference of antibody binding to the HIR. Avidin is a dimer of dimers (11), and the HIRMAb-AV fusion protein may also form a tetrameric structure formed from the association of 2 antiparallel fusion proteins. The evidence for this is the SEC HPLC, which shows the HIRMAb-AV fusion protein migrates with a molecular weight of about 400 kDa, which is twice the expected molecular weight of the glycosylated HIRMAb-AV fusion protein. Avidin has the maximal affinity for biotin when the protein is assembled in the tetrameric configuration. The fact that bound biotin could not be dissociated off the HIRMAbAV fusion protein despite 72 h of dialysis against a 10 000fold increase in volume with high concentrations of avidin in the dialysate (Methods) is evidence for the very high affinity of the HIRMAb-AV fusion protein for biotin. The HIRMAb-AV fusion protein is secreted in high amounts in transfected COS cells (Table 2). The size of the heavy chain (HC) of the fusion protein is about 15 kDa larger than the HC of the chimeric HIRMAb, based on either SDS-PAGE (Figure 4) or Western blotting with an antihuman IgG antibody (Figure 5A). An antiavidin antibody reacts with the fusion protein HC, or with avidin, but not with the HC of the HIRMAb, or the light chain (LC) of either the HIRMAb or the HIRMAb-AV fusion protein (Figure 5B). The COS cell-derived HIRMAbAV fusion protein binds with high affinity to the HIR, albeit with about 50% reduced affinity as compared to the chimeric HIRMAb (Figure 6). However, the affinity of the CHO-derived HIRMAb-AV fusion protein for the HIR is identical to that of

the chimeric HIRMAb (Results), and this may be due to differential glycosylation in CHO and COS cells. The HIRMAb-AV fusion protein produced by COS cells is fully saturated with biotin, owing to the 50-fold molar excess of biotin over the fusion protein (Results). Nevertheless, the fusion protein reacts with biotinylated BSA in an ELISA (Figure 7). This reactivity is most likely due to the presence of a minor fraction of unoccupied biotin binding sites, perhaps 40 mg/L of human IgG in the serum free conditioned medium in shake flasks at a cell density of (1–2) × 106 cells/mL. The HIRMAb-AV fusion protein produced by CHO cells under conditions of biotin starvation bound 3H-biotin, and accelerated biotin uptake into human epithelial cells with a >15-fold increase in VD of the biotin at 2 h of incubation (Figure 10). The HIRMAb-AV fusion protein facilitated cell uptake of fluorescein-biotin, as demonstrated by confocal microscopy (Figure 11). There was no measureable cell uptake of fluoresceinbiotin alone (Figure 11A). However, following attachment of the fluorescein-biotin to the HIRMAb-AV fusion protein, there was enhanced cell uptake into the intracellular endosomal compartment at 3 and 24 h of incubation (Figure 11B,C,D). The expression of avidin, or avidin fusion proteins, in CHO cells has not been previously described, although avidin has been expressed in baculovirus, yeast, and bacterial expression systems (14). In order to produce fusion protein with unoccupied biotin binding sites, it was necessary to develop a culture methodology whereby the CHO cells were grown under conditions of biotin depletion. As shown in Table 3, the cells could be maintained for 4 weeks in very low biotin medium,

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Figure 11. Confocal microscopy of human 293 cells following the addition to the medium of fluorescein-biotin comixed in either PBS (A) or the HIRMAb-AV fusion protein (B,C,D). The cells were incubated for either 3 h (B) or 24 h (A,C,D), and were photographed with either a 40× (B) or 100× (A,C,D) objective. The nuclei are stained red by propidium iodide.

and the HIRMAb-AV fusion protein was continuously produced under these conditions. The biotin ultrafiltration assay demonstrated that there was 1 biotin binding site per antibody molecule (Figure 8). There are 2 predicted biotin binding sites per fusion protein (Figure 1). Therefore, these data show that the HIRMAbAV fusion protein can be produced in CHO cells under conditions of biotin starvation, to enable production of fusion protein with high affinity biotin binding properties. In summary, these studies describes the genetic engineering, expression, and validation of a drug delivery system that is active in humans, and is designed to deliver monobiotinylated pharmaceuticals, including siRNA (6), across biological membranes via the insulin receptor. The engineering of the HIRMAb-AV fusion protein represents the merger of molecular Trojan horse technology and avidin–biotin technology in drug targeting. Owing to the extremely high affinity of avidin binding of biotin (KD ) 10-15 M, dissociation t1/2 ) 89 days), the bond between the Trojan horse HIRMAb and the biotinylated therapeutic is extremely stable in vivo. The avidin–biotin bond does not spontaneously dissociate in vivo, and is disrupted only via the action of tissue enzymatic reactions, particularly in liver and kidney (15). Owing to the rapid binding of biotinylated drugs to avidin, the present approach enables “2-vial pharmaceutics,” where the HIRMAb-AV fusion protein is formulated in the first vial, and the monobiotinylated therapeutic is formulated in a second vial, and the 2 vials are mixed prior to systemic administration. The concern with avidin fusion proteins is immunogenicity of the chicken avidin in humans, and a human antiavidin response (HAAR). However, avidin has been administered

to humans in 5–10 mg doses intravenously without immunologic reactions (8, 16, 17). The human hypo-responsiveness to avidin may arise from oral antigen feeding (18). Avidin is abundant in egg whites, and virtually all members of Western societies have consumed dietary eggs, which is a form of oral avidin antigen feeding that could induce immune tolerance in humans to avidin. Support for this hypothesis is the finding that humans have serum antibodies to avidin, and that the titer is lower in subjects on egg-free diets (19). Therefore, an avidin fusion protein may be administered to humans without immunological reactions.

ACKNOWLEDGMENT Winnie Tai and Phuong Tram provided expert technical assistance.

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