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Originally published In Press as doi:10.1074/jbc.M513415200 on February 15, 2006

J. Biol. Chem., Vol. 281, Issue 16, 10706-10714, April 21, 2006
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Single Variable Domain-IgG Fusion

A NOVEL RECOMBINANT APPROACH TO Fc DOMAIN-CONTAINING BISPECIFIC ANTIBODIES*

Juqun Shen1, Marie Danielle Vil, Xenia Jimenez, Michelle Iacolina, Haifan Zhang, and Zhenping Zhu2

From the Department of Antibody Technology, ImClone Systems Inc., New York, New York 10014

Received for publication, December 16, 2005 , and in revised form, February 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Both laboratory and early clinical studies to date have demonstrated that bispecific antibodies (BsAb) may have potentially significant application in cancer therapy. The clinical development of BsAb as therapeutics has been hampered, however, by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods has been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. Here we describe a novel recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a single variable domain antibody to the N terminus of the light chain of a functional IgG antibody of different specificity. A model BsAb was constructed using a single variable domain antibody to mouse platelet-derived growth factor receptor {alpha} and a conventional IgG antibody to mouse vascular endothelial growth factor receptor 2. The BsAb was expressed in mammalian cells and purified to homogeneity by one-step protein A affinity chromatography. Furthermore, the BsAb retains the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. This design and expression of Fc domain-containing, IgG-like BsAb should be applicable to the construction of similar BsAb from antibodies recognizing any pair of antigens.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bispecific antibodies (BsAb)3 are Ig-based molecules that can simultaneously bind to two different epitopes on either the same or distinct antigens. Both laboratory and early clinical studies to date have demonstrated that BsAb may have significant potential application in cancer therapy (1–3). The primary use of BsAb has been to redirect cytotoxic immune effector cells for enhanced killing of tumor cells. In this context, one arm of the BsAb binds an antigen on the tumor cell, and the other binds a determinant expressed on effector cells, such as CD3, CD16, or CD64, which are expressed on T lymphocytes, natural killer cells, or other mononuclear cells (Refs. 4–6 and for review see Ref. 7). By cross-linking tumor and effector cells, the BsAb not only brings the effector cells within the proximity of the tumor cells but also simultaneously triggers their activation, leading to effective tumor cell killing. In addition, BsAb has also been used to enrich the tumor/normal tissue localization ratio of chemo- or radiotherapeutic agents. In this setting, one arm of the BsAb binds an antigen expressed on the cell targeted for destruction, and the other arm binds a chemotherapeutic drug, radioisotope, or toxin. The naked BsAb is administered first, and after sufficient time has passed for the BsAb to bind tumor cells and to clear from normal tissue, the cytotoxic molecule is delivered, with rapid accumulation in the tumor, because of its affinity for the tumor-bound BsAb (8–11). Recently, a novel concept has emerged, the development of BsAb that target simultaneously two tumor-associated antigens (e.g. growth factor receptors) for down-regulation of multiple cell proliferation/survival pathways, which provide enhanced antitumor activity (12–15).

A major obstacle in the general development of BsAb has been the difficulty of producing materials of sufficient quality and quantity for both preclinical and clinical studies. Initially, the main route to the production of BsAb was by co-expression of both the light chains (LC) and both the heavy chains (HC) of two parent antibodies of different specificities (antibody A and antibody B) in a single cell through either the hybrid hybridoma technique (16) or DNA co-transfection. Unfortunately, assuming that all four polypeptide chains are equally expressed and that there is no pairing preference between any particular LC and HC, in addition to the desired heterodimeric BsAb product (LCA-HCA plus LCB-HCB), there are also a large number of undesired products formed from the 10 molecules that result from the 16 permutations of LC and HC pairings. Consequently, the desired binding-competent BsAb are a minor product (in theory, an eighth of the total), and purification from the other products is very difficult. Another traditional method for BsAb production is chemical conjugation of two antibodies (or their fragments) of different specificities (17). The chemical modification process may inactivate the antibody or promote aggregation. As purification from undesired products remains difficult, the resulting low yield and poor quality BsAb makes this process, like the hybrid hybridoma and DNA co-transfection, unsuitable for the large scale production required for clinical development.

In recent years, a variety of recombinant methods has been developed for the efficient production of BsAb, especially for BsAb antibody fragments (1, 18, 19). On the other hand, the successful design and production of full-length IgG-like, i.e. Fc domain-containing, BsAb has been generally limited (20, 21). In this study, we describe a novel recombinant method for the production of an Fc domain-containing BsAb molecule by direct fusion of a single variable domain (sVD) antibody to the N terminus of the LC of a functional IgG antibody of a different specificity. The resulting BsAb molecule was expressed in mammalian cells and purified by protein A chromatography. Furthermore, the BsAb retained the binding specificities and the receptor neutralizing activities of both of the parent antibodies.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Proteins and Cells—Recombinant mouse platelet-derived growth factor receptor {alpha} (mPDGFR{alpha})-Fc, mouse vascular endothelial growth factor receptor 2 (mVEGFR2)-Fc fusion proteins, and human PDGF-AA were purchased from R & D Systems (Minneapolis, MN). VEGF protein was expressed in baculovirus and purified at ImClone Systems (New York) following the procedure described elsewhere (22). eEnd.1 cells, a murine endothelium line transformed by polyoma middle T oncogene, was a kind gift from Dr. M. Pepper of University Medical Center, Geneva, Switzerland. The control antibodies 1C11, an antibody directed against human VEGFR2, and C225, an antibody directed against human epidermal growth factor receptor, were produced at ImClone Systems (23, 24).

Selection of Anti-mPDGFR{alpha} Antibodies from a Phage Display Fab Library—A large naive human Fab phage display library from the Dyax Corp. (containing 3.7 x 1010 clones) (25, 26) was used to select antibodies directed against mPDGFR{alpha}-Fc protein. Briefly, the library stock (100 µl) was grown to log phase in 20 ml of 2YTAG medium (2YT contains 100 µg/ml ampicillin and 2% glucose), rescued with M13K07 helper phage, and amplified overnight in 2YTAK medium (2YT contains 100 µg/ml ampicillin and 50 µg/ml kanamycin) at 30 °C. The phage preparation was precipitated in 4% polyethylene glycol, 0.5 M NaCl, and then resuspended in 1 ml of 3% fat-free milk/PBS containing 240 µg/ml of an unrelated human IgG and incubated at 37 °C for 1 h to block nonspecific binding and to deplete phages displaying Fab directed against human IgG Fc fragment. For each round of selection, mPDGFR{alpha}-Fc-coated Maxisorp Star tubes (Nunc, Rosklide, Denmark) were first blocked with 3% milk/PBS at 37 °C for 1 h and then incubated with the phage preparation at room temperature for 1 h. The tubes were washed 15 times with PBS containing 0.1% Tween 20 followed by 15 washes with PBS. The bound phage was eluted at room temperature for 10 min with 1 ml of a freshly prepared solution of 100 mM triethylamine (Sigma). The eluted phages were incubated with 10 ml of mid-log phase TG1 cells at 37 °C for 30 min stationary and 30 min shaking. The infected TG1 cells were pelleted, plated onto three 2YTAG plates, and incubated overnight at 30 °C. All of the colonies grown on the plates were scraped into 3–5 ml of 2YTA medium, mixed with glycerol (final concentration, 10%), aliquoted, and stored at –80 °C. For subsequent rounds of selection, the phage stock (100 µl) from the previous round of selection was amplified and used for selection followed the procedure described above on Maxisorp Star tubes coated with decreasing amounts of mPDGFR{alpha}-Fc (50, 10, and 2 µg/ml for the 1st, the 2nd, and the 3rd round of selections, respectively).

ELISA Screening for Antibodies with Binding and Blocking Activities—Individual TG1 clones recovered after each round of selection were randomly picked and grown at 37 °C in 96-well plates. To produce phage, the cells were rescued with M13K07 helper phage as described above. To produce soluble Fab, the cells were incubated in 2YTA medium containing 1 mM isopropyl 1-thio-beta-D-galactopyranoside (Sigma) at 30 °C overnight. For binding ELISA, 96-well microtiter plates (Nunc) were coated with mPDGFR{alpha}-Fc (50 µl at 1 µg/ml, 4 °C overnight). The amplified phage preparation or the cell culture supernatant containing soluble Fab was blocked with 1/6 volume of 18% milk/PBS at room temperature for 1 h and added to the mPDGFR{alpha}-Fc-coated plates. After incubation at room temperature for 1 h, the plates were washed three times with PBST. For phage ELISA, the plates were incubated with a mouse anti-M13 phage antibody horseradish peroxidase (HRP) conjugate (Amersham Biosciences); for Fab ELISA, the plates were incubated with a rabbit anti-human Fab antibody-HRP conjugate (Jackson ImmunoResearch, West Grove, PA). The plates were then washed three times; TMB peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added, and the absorbance at 450 nm was read using a microplate reader (Molecular Devices, Sunnyvale, CA).

For blocking ELISA, phage or Fab supernatant was mixed with a fixed amount of mPDGFR{alpha}-Fc (0.5 µg/ml) and incubated at room temperature for 30 min. The mixture was then transferred to 96-well plates precoated with PDGF-AA (0.5 µg/ml) and incubated at room temperature for 1 h. The plates were then incubated at room temperature for 1 h with a rabbit anti-human Fc antibody-HRP conjugate (Jackson ImmunoResearch) and developed as described above to quantify the amount of plate-bound mPDGFR{alpha}-Fc protein.

Identification of Anti-mVEGFR2 Antibody 2B4—Anti-mVEGFR2 antibody, 2B4, was isolated from the Fab phage library (Dyax Corp.) following the same procedure described above on Maxisorp Star tubes coated with mVEGFR2-Fc fusion protein. 2B4 binds specifically to mVEGFR2 and blocks efficiently mVEGFR2/VEGF interaction.4

Expression and Purification of Soluble Fab Fragments—Phagemids of the individual selected clones were used to transform a nonsuppressor Escherichia coli host HB2151. Expression of the Fab fragments in HB2151 and purification of the soluble Fab proteins from the periplasmic extracts of the E. coli were carried out as described previously (26). The Fab proteins were purified using protein G columns following the manufacturer's protocol (Amersham Biosciences). The purity and the molecular size of the purified proteins were analyzed by electrophoresis using a NuPAGETM 4–12% BisTris gel (Invitrogen) followed by visualization by staining with SimplyBlueTM SafeStain (Invitrogen).

Construction of Bispecific Anti-mPDGFR{alpha} x Anti-mVEGFR2 Antibody Based on the Single VH Domain Antibody 1F2—The BsAb was prepared using the single VH domain of the anti-mPDGFR{alpha} antibody, 1F2, and the variable domains (both VL and VH) from the conventional anti-mVEGFR2 antibody, 2B4, as the building blocks (Fig. 4). In this construct, 1F2 VH domain was linked on its C terminus via a 5-amino acid linker, Ala-Ser-Thr-Lys-Gly, to the N terminus of 2B4 LC. The resultant polypeptide, 1F2(VH)-2B4(VL)-CL, was then co-expressed with the 2B4 HC, 2B4(VH)-CH1-CH2-CH3, to form the bispecific molecule 1F2–2B4IgG. The BsAb construct was transiently expressed in COS-7 cells and purified using protein A affinity chromatography. The purity of the BsAb was assayed by SDS-PAGE analysis under both reducing and nonreducing conditions, and the protein concentrations were determined by ELISA, using an anti-human Fc antibody as the capturing agent and an anti-human {kappa} chain antibody-HRP conjugate as the detection agent. A clinical grade antibody, C225, an antibody directed against human epidermal growth factor receptor, was used as the standard for calibration.

Quantitative Receptor Binding and Blocking Assays—Two different assays were carried out to examine the binding specificity and efficiency of the BsAb. In the first assay, the cross-linking assay, the BsAb was tested for its capability in simultaneously binding two target receptors; the BsAb or the monospecific antibodies (5 nM) were first incubated with mVEGFR2 or mPDGFR-{alpha} (100 ng) in solution and then transferred to a microtiter plate coated with the second receptor, mPDGFR-{alpha} or mVEGFR2 (100 ng/well), followed by incubation with a biotin-labeled polyclonal antibody to the first receptor (the receptor in solution), mVEGFR2 or mPDGFR-{alpha}. The plate was then developed as described above after further incubation with a streptavidin-HRP conjugate. In the second assay, the direct binding assay, various amounts of purified antibodies were added to mPDGFR{alpha}-Fc- or mVEGFR2-Fc-coated plates (1 µg/ml) and incubated at room temperature for 1 h, after which the plates were washed three times with PBST. The plates were then incubated at room temperature for additional 1 h with a rabbit anti-human Fab antibody-HRP conjugate (Jackson ImmunoResearch). The plates were washed and developed. In the blocking assay, various amounts of antibodies were first mixed with a fixed amount of mPDGFR{alpha}-Fc or mVEGFR2-Fc (0.5 µg/ml) and incubated at room temperature for 30 min. The mixture was then transferred to 96-well plates precoated with rhPDGF-AA or VEGF (0.5 µg/ml) and incubated at room temperature for 1 h. After washing three times, the plates were incubated with an anti-human Fc antibody-HRP conjugate and developed as described above. IC50, the antibody concentration that yielded 50% blockade of mPDGFR{alpha} or mVEGFR2 from binding to its respective ligand, was determined.

Antibody Affinity Determination—The binding kinetics of various antibodies to their target receptors were measured using a BIAcore 3000 biosensor (BIAcore, Inc., Uppsala, Sweden). Briefly, mPDGFR{alpha} or mVEGFR2-Fc fusion protein was immobilized onto a sensor chip, and soluble antibodies were injected at concentrations ranging from 1.5 to 100 nM. Sensorgrams were obtained at each concentration and were evaluated using the program BIAevaluation 2.0. The affinity constant, Kd, was calculated from the ratio of dissociation rate (koff)/association rate (kon).

Receptor Phosphorylation Assay—eEnd.1 cells were first examined for expression of mVEGFR2 and mPDGFR{alpha} via FACS analysis. Briefly, the cells were incubated with 2B4 IgG, 1F2-CH/CL, or 1F2–2B4IgG (10 µg/ml) at 4 °C for 1 h, followed by incubation with a PE-labeled goat anti-human Fc antibody (Jackson ImmunoResearch) for an additional hour, and analysis on a Guava Easycyte System (Guava Technologies, Inc., Hayward, CA). In receptor phosphorylation assay, eEnd.1 cells were plated onto 6-cm dishes and grown to 70–80% confluence, after which the cells were washed twice in PBS and cultured overnight in serum-free medium. The cells were first incubated with various antibodies at 37 °C for 30 min, followed by stimulation with VEGF or PDGF-AA at 37 °C for 15 min. The cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Na3VO4, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin) for 1 h, followed by centrifugation of the lysate at 12,000 rpm for 10 min at 4 °C. The receptors were immunoprecipitated from the cell lysate supernatant by an anti-mP-DGFR{alpha} (eBioscience, San Diego) or an anti-mVEGFR2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), followed by the addition of 20 µl of protein A/G-Sepharose beads (Santa Cruz Biotechnology). The precipitated receptor proteins were resolved on a 4–12% NuPAGE BisTris gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane. Phospho-mVEGFR2 and phospho-mPDGFR{alpha} were detected on the blot using an anti-phosphotyrosine antibody-HRP conjugate (Santa Cruz Biotechnology). Total receptor proteins loaded on the gel were assayed with antibodies to mPDGFR{alpha} or mVEGFR2 (both from Santa Cruz Biotechnology).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Single VH Domain Antibodies to mPDGFR{alpha} from a Fab Phage Display Library—A large naive human Fab phage display library containing 3.7 x 1010 clones was used to select antibodies directed against mPDGFR{alpha}. A total of three rounds of selection were performed on immobilized mPDGFR{alpha}-Fc fusion protein in the presence of a high concentration of an unrelated human IgG in the solution in order to deplete phage clones reacting with the Fc fragment. After the 2nd and the 3rd round of selection, 190 clones were randomly picked from each round and tested for PDGFR{alpha} binding and blocking activities by both phage ELISA and soluble Fab ELISA. More than 77% clones picked after the 2nd round and 99% of clones after the 3rd round bound specifically to mPDGFR{alpha}, suggesting a high efficiency of the selection process. Blocking assay revealed that ~4.2% of binders also blocked mPDGFR{alpha} from binding to its ligand PDGF-AA. DNA sequencing analysis yielded eight distinct clones among the blockers. All these blockers, plus one nonblocker (clone 3F3), were selected for further study (Table 1). The VH and VL subgroup was assigned according to the classification of the Andrew CR Martin's Bioinformatics Group. No identical VHs or VLs were found among the nine clones, except for clones 1C10 and 1F2 (which share the same VH). Most interestingly, 4 of 8 blockers, clones 1C10, 1F2, 1F9, and 3G7, only possess a truncated LC. Clones 1F2 and 1F9 lack most of the VL domain because of an in-frame gene deletion but possess an intact CL domain. Clone 1C10 has a frameshift mutation within the complementarity determining region (CDR) 3 of VL resulting in reading frameshift and a stop codon that leads to deletion of the entire CL. Clone 3G7 has a stop codon at the 5' end of VL gene because of a frameshift mutation leading to the loss of the entire LC (Fig. 1). Both clone 1C10 and 3G7 Fab-like fragments expressed very poorly in E. coli because they lacked the CL domain.


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  		TABLE 1
Identification of anti-mPDGFR{alpha} antibodies from a Fab phage display library

 
The Fab fragments of six clones were expressed in E. coli, purified by affinity chromatography using protein G column, and analyzed by SDS-PAGE (Fig. 2). Under nonreducing conditions, Fabs with complete light and heavy chains, clones 1A11, 1E10, 3B2, and 3F3, yielded a single protein band with a molecular mass of ~50 kDa (Fig. 2A). On the other hand, Fab fragments of 1F2 and 1F9 gave a small band of ~37.5 kDa because of the lack of VL domain (Fig. 2B). Under reducing conditions, the conventional Fab fragment showed two bands, the LC and the VH-CH1 fragment, of about the same size (~25 kDa), which was not distinguishable under the electrophoresis condition (Fig. 2B). 1F2 and 1F9 Fabs yielded two bands that were resolved under the electrophoretic conditions as follows: the upper bands correlate with the normal size VH-CH1 fragment, and the lower bands represent the CL fragment only (~12. 5 kDa) (Fig. 2B).

mPDGFR{alpha} Binding and mPDGFR{alpha}/PDGF-AA Blocking by the Anti-mPDGFR{alpha} Antibodies—Purified Fab fragments from six clones were compared quantitatively in their binding efficiency to mPDGFR{alpha} and their potency in blocking mPDGFR{alpha}/PDGF-AA interaction. Clone 1F2 Fab, a single VH domain clone, is the most efficient binder to the receptor, followed by 1F9, another VH domain-only clone, and then the conventional Fab fragments, clones 1A11, 1E10, 3F3, and 3B2 (Fig. 3A). Clone 1F2 is also the most potent blocker to receptor/ligand interaction, with an IC50 of 12 nM, which is followed by clone 1F9 (IC50, 57 nM), 1E10 (IC50, 140 nM), 1A11 (IC50, 220 nM) (Fig. 3B). Clone 3B2, a weak blocker in phage-based ELISA, failed show any blocking activity as soluble Fab protein. The control clone, 3F3, binds well to the receptor but does not block receptor/ligand interaction.


Figure 1
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  		FIGURE 1.
Deduced amino acid sequences of the VH and the VL domains of the anti-mPDGFR{alpha} antibodies and the anti-mVEGFR2 antibody 2B4. All the CDRs (shown in boldface) are defined according to Kabat's definition (27). {wedge}, frameshift mutation due to a single nucleotide insertion in the CDR3 region, leading to a shift in reading frame. *, stop codon due to frameshift mutations. @...@, almost the entire VL was deleted due to an in-frame deletion.

 


Figure 2
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  		FIGURE 2.
Expression and purification of the anti-mPDGFR{alpha} Fab fragments. The Fab fragments were expressed in E. coli host HB2151 cells, purified by affinity protein G chromatography, and analyzed by SDS-PAGE. A, conventional Fab fragments with complete LC and Fd domain, including clones 1A11, 1E10, 3B3, and 3F3, show a single protein band of ~50 kDa. B, 1F2 and 1F9 Fab fragments show a single band of ~37.5 kDa under nonreducing conditions (–DTT), because of the deletion of VL domain. Under reducing conditions (+DTT), both 1F2 and 1F9 gave rise to two protein bands of 25 (the HC) and 12.5 kDa (the CL domain only), whereas the conventional Fab yielded two bands of ~25 kDa each (the LC and the Fd domains), which were not resolved under the electrophoresis conditions. Also shown on the left are the positions of molecular mass standards (Std) in kDa.

 


Figure 3
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  		FIGURE 3.
Receptor binding and blocking activities of the purified anti-PDGFR{alpha} Fab fragments. A, quantitative binding ELISA of the purified Fab to immobilized mPDGFR{alpha}. Various amounts of Fab were incubated in 96-well plates precoated with mPDGFR{alpha}-Fc fusion protein at room temperature for 1 h, followed by incubation with a rabbit anti-human-{kappa} antibody HRP conjugate for an additional hour. The plates were then incubated with a peroxidase substrate, followed by reading of the absorbance at 450 nm. B, inhibition of binding of mPDGFR{alpha} to immobilized PDGF-AA by the purified Fab protein. Various amounts of Fab were incubated with a fixed amount of mPDGFR{alpha}-Fc in solution for 30 min. The mixtures were transferred to 96-well plates coated with PDGF-AA and incubated for 1 h. After washing, the plates were incubated with an anti-human-Fc antibody-HRP conjugate, followed by development of the plates as described above. Data shown represent the mean ± S.D. of duplicate samples.

 
The binding kinetics of various anti-mPDGFR{alpha} clones were determined by surface plasmon resonance on a BIAcore instrument. Consistent with ELISA binding assay, 1F2 showed the highest binding affinity with a Kd of 0.42 ± 0.21 nM (kon, 9.2 ± 4.2 x 105 M–1 s–1, koff, 3.1 ± 0.76 x 10–4 s–1), which is followed by clone 1F9 (Kd, 2.1 ± 0.75 nM) and clone 1E10 (Kd, 12.2 ± 2.9 nM). When clone 1F2 was converted into a divalent Fab form (VH-CH1/VH-CL), the affinity was further increased to 0.08 nM, representing a 5-fold enhancement over the monovalent 1F2 Fab. As a comparison, the divalent IgG form of clone 1E10 has a binding affinity of 0.81 ± 0.34 nM (data not shown).

Identification of the Anti-mVEGFR2 Antibody 2B4—The anti-mVEGFR2 antibody, 2B4, was isolated from the Fab phage library (Dyax Corp.) by selecting on Maxisorp Star tubes coated with mVDGFR2-Fc fusion protein (Fig. 1). Fab fragment of 2B4 binds specifically to mVEGFR2 and blocks mVEGFR2/VEGF interaction. The binding affinity of 2B4 Fab and IgG to mVEGFR2 was 6.7 ± 3.0 and 0.39 ± 0.1 nM, respectively, as determined by BIAcore analysis. 2B4 IgG blocks mVEGFR2/VEGF interaction with an IC50 value of ~3.5 nM.


Figure 4
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  		FIGURE 4.
A, schematic representation of the sVD-IgG fusion BsAb molecules 1F2–2B4IgG. In this format, 1F2 VH was fused to the N terminus of 2B4 VL via a 5-amino acid linker to form 1F2VH-2B4 LC, which was then co-expressed with the intact HC of 2B4. The bivalent monospecific 1F2-CH/CL and 2B4 IgG were also shown. B, expression and purification of the various antibody molecules in mammalian cells. The antibodies were transiently expressed in mammalian cells, purified by protein A affinity chromatography, and analyzed by SDS-PAGE under both nonreducing (–DTT) and reducing (+DTT) conditions. Also shown on the left are the positions of molecular mass standards (Std) in kDa.

 
Construction and Expression of sVD Antibody-based BsAb—The BsAb construct was prepared using the variable domains from the single VH domain antibody 1F2 and the conventional antibody, 2B4 (Fig. 4A). 1F2 VH was first fused to the N terminus of 2B4 VL via a 5-amino acid linker to form 1F2VH-2B4 LC, which was then co-expressed with the intact HC of 2B4 (Fig. 4A). As a control, a bivalent 1F2 molecule, 1F2-CH/CL, was also created by co-expression the 1F2VH-CH1-CH2-CH3 fusion along with the CL domain (Fig. 4A). All the antibody molecules were transiently expressed in COS-7 cells and purified from the supernatant of cell cultures by affinity chromatography using protein A columns.

The purified antibodies were analyzed by SDS-PAGE under both nonreducing and reducing conditions. When assayed under nonreducing conditions, the BsAb showed a single protein band of ~175 kDa, whereas the divalent 1F2-CH/CL demonstrated a single band at ~125 kDa, compared with that of 150 kDa of the monospecific 2B4 IgG (Fig. 4B). The two polypeptides of the antibodies were resolved under reducing conditions. Whereas 2B4 IgG yielded two bands of 50 (the HC) and 25 kDa (the LC), 1F2-CH/CL gave two species of 50 (the HC) and 12.5 kDa (the CL domain). As expected, 1F2–2B4IgG gave rise to two bands of 50 (the HC) and 37.5 kDa (the 1F2VH-2B4 LC fusion).

Dual Specificities of sVD Antibody-based BsAb—A number of assays were used to confirm the BsAb molecules were capable of binding to both mPDGFR{alpha} and mVEGFR2. In the first assay, the cross-linking assay, we examined whether the BsAb could bind to both its targets simultaneously. The antibodies were first incubated with a receptor in solution and transferred to a 96-well plate coated with the second receptor. The plate was then incubated with a biotin-labeled antibody to the receptor in solution, followed by a streptavidin-HRP. As shown in Fig. 5, A and B, only the BsAb, but not the parent monospecific 2B4 IgG and 1F2-CH/CL, was able to cross-link the two target receptors.

In the second assay, the BsAb were compared with their monospecific counterparts in antigen-binding efficiency. Various amounts of antibodies were added to 96-well plates coated with mPDGFR{alpha} or mVEGFR2 and assayed for their efficiency in binding to the receptors. Although 1F2-CH/CL and 2B4 IgG bound only to their respective targets, the BsAb reacted to both immobilized mPDGFR{alpha} and mVEGFR2 (Fig. 5, C and D). The ED50 values, i.e. the antibody concentrations that yield 50% of maximum binding, to mPDGFR{alpha} were 0.11 and 0.83 nM for 1F2-CH/CL and 1F2–2B4IgG, respectively, and to mVEGFR2 were 0.19 and 4.8 nM for 2B4 IgG and 1F2–2B4IgG, respectively. On BIAcore analysis, the BsAb binds to both mPDGFR{alpha} and mVEGFR2 with an affinity that is higher than that of its monovalent, monospecific counterparts, 1F2 and 2B4 Fab (Table 2). On the other hand, the binding affinity of the BsAb is ~6-fold lower in mPDGFR{alpha} binding and 4-fold lower in mVEGFR2 binding, compared with its respective monospecific bivalent molecules 1F2-CH/CL and 2B4 IgG (Table 2). When examined on binding to cell surface-expressed receptors on eEnd.1 cells (the cells that express both mPDGFR{alpha} and mVEGFR2), the BsAb demonstrated, however, higher efficiency than either of the parent monospecific bivalent antibodies (Fig. 5E). The mean fluorescence intensities on the cells were 15.7, 31, and 36.9 for 1F2-CH/CL (for mPDGFR{alpha} binding), 2B4 IgG (for mVEGFR2 binding), and 1F2–2B4IgG (for both mPDGFR{alpha} and mVEGFR2 binding), respectively.


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  		TABLE 2
Binding kinetics of various antibody molecules to immobilized mPDGFR{alpha} and mVEGFR2 receptor proteins

 


Figure 5
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  		FIGURE 5.
Dual binding activity of the bispecific 1F2–2B4IgG. In the cross-linking assay (A and B), the BsAb or the monospecific antibodies were first incubated with mPDGFR-{alpha} (A) or mVEGFR2 (B) in solution and then transferred to a microtiter plate coated with the second receptor, mVEGFR2 (A) and mPDGFR-{alpha} (B), followed by incubation with a biotin-labeled polyclonal antibody to the first receptor (the receptor in solution), mPDGFR-{alpha} or mVEGFR2. The plate was developed after further incubation with a streptavidin-HRP conjugate. 1C11, a control antibody directed against human VEGFR2. In the direct binding assay (C and D), various amounts of antibodies were first incubated in a 96-well plate precoated with mPDGFR{alpha}-Fc (C) or VEGFR2-Fc (D) fusion protein, followed by incubation with an anti-human-{kappa} antibody HRP conjugate. Data shown represent the mean ± S.D. of duplicate samples. E, FACS analysis of the bispecific 1F2–2B4IgG for binding to eEnd.1 cells. The cells were incubated with 2B4 IgG, 1F2-CH/CL, or 1F2–2B4IgG at 4 °C for 1 h, followed by incubation with a PE-labeled goat anti-human Fc antibody for an additional hour and analysis on a Guava Easycyte System. Anti-KLH, a control antibody directed against keyhole limpet hemocyanin (KLH). Numbers in parentheses represent the mean fluorescence intensity (MFI).

 


Figure 6
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  		FIGURE 6.
Dual blocking assays of the bispecific 1F2–2B4IgG. Various amounts of antibodies were incubated with a fixed amount of mPDGFR{alpha}-Fc (A) or VEGFR2-Fc (B) fusion protein in solution for 30 min. The mixtures were then transferred to plates precoated with PDGF-AA (A) or VEGF (B) and incubated for 1 h. The amounts of plate-bound receptors were then quantified by incubation with an anti-human-Fc antibody-HRP conjugate. Data shown represent the mean ± S.D. of duplicate samples.

 
Fig. 6 shows that the BsAb inhibit both mPDGFR{alpha} and mVEGFR2 from binding to their respective ligand, PDGF-AA and VEGF. The IC50 of the BsAb for blocking mPDGFR{alpha}/PDGF-AA is ~24 nM, compared with that of 2.7 nM for 1F2-CH/CL. On the other hand, the IC50 values for blocking the mVEGFR2/VEGF interaction are ~13.5 and 3.5 nM for 1F2–2B4IgG and 2B4 IgG, respectively (Fig. 6). As expected, 2B4 IgG had no effects on mPDGFR{alpha}/PDGF-AA interaction, nor did 1F2-CH/CL have any effects on mVEGFR2/VEGF interaction.

The BsAb Inhibits Ligand-induced Activation of Both mVEGFR2 and mPDGFR{alpha}—The biological activity of the BsAb was investigated using eEnd.1 cells, a murine endothelial cell line that expresses both mPDGFR{alpha} and mVEGFR2 receptors. As shown in Fig. 7, the BsAb inhibited both PDGF- and VEGF-stimulated phosphorylation of mPDGFR{alpha} and mVEGFR2 receptors, whereas its monospecific parent antibodies only blocked the activation of a single receptor stimulated by its cognate ligand. As a control, C225, the antibody to epidermal growth factor receptor, did not have any effect on ligand-stimulated activation of either receptor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we describe an efficient approach for the production of an Fc domain-containing, IgG-like, BsAb by recombinant fusion of an sVD antibody to the N terminus of an intact IgG antibody of different specificity. The BsAb is functionally expressed in mammalian cells and is capable of binding both of its target antigens. Furthermore, the BsAb retained the activities of both parent antibodies in blocking receptor/ligand interaction and in neutralizing ligand-induced biological activities. We believe that this approach of BsAb production is applicable to antibodies recognizing any pair of antigens. By using the sVD antibody as the building blocks, it is also possible to produce an IgG-like antibody molecule that is tetravalent (but monospecific) to the same antigen.

Significant advancements have been made in the engineering and production of small BsAb fragments, e.g. diabody, minibody, and Fab-scFv fusion protein (see Refs. 28–31 and for reviews see Refs. 18, 19, and 32). These BsAb fragments may possess some advantages over the full-length IgG-like molecules for certain clinical applications, such as for tumor radio-imaging and targeting, because of its better tissue penetration and faster clearance from the circulation. On the other hand, IgG-like BsAb may prove to be better choices over smaller BsAb fragments for other in vivo applications, specifically for oncology indications, by providing the Fc domain that not only confers long serum half-life but also supports secondary immune function, such as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. Unlike their fragment counterparts, engineering and production of recombinant IgG-like BsAb has been, however, rather technically challenging, and the success in the field has been much limited. A number of approaches have been exploited in the past several years for efficient expression of Fc domain-containing BsAb molecules in mammalian cells (for reviews see Refs. 20 and 21). For example, production of homogeneous full-length IgG-like BsAb has been achieved by the so-called "knobs-into-holes" engineering for efficient Ig CH3 domain heterodimerization plus the use of a common LC shared by two antibodies of different specificities (33–35). Although functional IgG-like BsAb were formed with high yield (>95%), an obvious drawback of this method is that the inclusion of multiple mutations in the CH3 domains might pose an immunogenic risk in a therapeutic setting. Furthermore, it requires the identification of antibodies with common LC, which is rather infrequent, particularly for high affinity antibodies. Alternatively, IgG-like BsAb have also been produced by linking two single chain Fv (scFv) of different specificities onto both the N-terminal of the CL and the first constant heavy (CH1) chains (the "(scFv)4-IgG") (14, 36), or by fusing a scFv to the C terminus of the HC of a full-length IgG molecule of different specificity (37). A drawback of these formats is their low expression levels in mammalian cells, probably due to both its large size (~200 kDa) and structural complexity. Recently, by fusing a bispecific diabody to the Fc domain of an IgG, we constructed a tetravalent BsAb, the "Di-diabody," that could be efficiently expressed in mammalian cells (13). The Di-diabody demonstrated potent dual targeting activity both in vitro and in vivo but showed significant instability in mice because of the lack of covalent linkage between the two polypeptide chains constituting the bispecific molecule, and the antibody lost greater than 50% of binding activity at 72 h post-administration.5 The approach we described here, by directly fusing an sVD antibody to the N terminus of a full-length IgG antibody of a different specificity, resulted in the production of a tetravalent (i.e. two binding sites to each target), Fc domain-containing BsAb that could be efficiently expressed in mammalian cells and be purified to homogeneity with a single-step affinity chromatography. The binding affinities of the BsAb to both mPDGFR{alpha} and mVEGFR2 were significantly (2–4-fold) higher than those of its mono-specific, monovalent Fab counterparts (Table 2), demonstrating the benefit of bivalency antibody/antigen interaction within one BsAb molecule. In addition, although the BsAb showed lower binding activity (and affinity) to immobilized receptors (in both ELISA and BIAcore analysis; see Fig. 5, C and D, and Table 2) compared with its monospecific bivalent parent antibodies, the molecule was more efficient in interacting with cells that express both mPDGFR{alpha} and mVEGFR2 (Fig. 5E), clearly a result of dual receptor-targeting. Because the sVD antibody was genetically linked to the IgG, the BsAb should possess a good stability for in vivo indications. Finally, like in the Di-diabody format (13), the intact natural Fc domain is expected to retain full capability in mediating immune effector functions, including both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity.


Figure 7
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  		FIGURE 7.
Inhibition of PDGF-(A) and VEGF (B)-stimulated receptor phosphorylation by the bispecific 1F2–2B4IgG. eEnd.1 cells were first incubated with various antibodies at 37 °C for 30 min, followed by stimulation with VEGF or PDGF at 37 °C for 15 min. After cell lysis, the receptors were immunoprecipitated from the cell lysate supernatant by incubation with an anti-mPDGFR{alpha} or an anti-mVEGFR2 antibody, followed by protein A/G-Sepharose beads. The precipitated receptor proteins were resolved on a 4–12% NuPAGE BisTris gel and transferred to a polyvinylidene difluoride membrane. Phospho-mVEGFR2 and phospho-mPDGFR{alpha} were detected on the blot using an anti-phosphotyrosine antibody-HRP conjugate. Total receptor proteins loaded on the gel were assayed with antibodies to mPDGFR{alpha} or mVEGFR2. C225, a control antibody directed against human epidermal growth factor receptor.

 
The prototype sVD antibody we utilized in this study, clone 1F2, was derived from a large Fab phage display library via immunopanning on immobilized mPDGFR{alpha}. Identification of sVD (VL or VH only) antibodies from both the Fab and scFv phage display libraries has been, although infrequent, reported previously (38).4,5 Recent progress in antibody scaffold engineering has led to the production of a number of sVD antibody libraries, including those from the scaffolds of camelid VhH (39–41), human VH or VL (42, 43), lipocalins (44), shark V-NAR (45, 46), protein A IgG-binding Z domain (47), and fibronectin type III domain (48) (for reviews see Refs. 49–51). With the availability of these libraries, sVD antibodies to various antigens can be readily identified. These sVD antibodies represent excellent building blocks for the construction of bi- or multispecific antibody molecules in both fragment and, especially, the IgG-like formats (for review see Refs. 49–51). For example, in addition to the sVD-IgG fusion BsAb described in this report, it is also possible to construct a BsAb that is theoretically identical to a normal IgG molecule by linking a VL sVD and a VH sVD to the N terminus of the CL and the CH1, respectively. In theory, the sVD can be paired in various combinations including VH/VL, VL/VL, and VH/VH, for the construction of both fragment and IgG-like BsAb molecules. It is plausible, however, that the noncognate VL and VH pair may interact with each other in an "unwanted" or "undesirable" fashion, leading not only to a disruption of molecular folding but also to an interference (thus loss) of antigen binding activity of individual sVD in the final product.4,5 To ensure the production of an active BsAb molecule, it is therefore imperative to identify the appropriate sVD pair by testing carefully the effect of interaction between the selected sVD antibodies.

An apparent concern associated with the sVD-IgG fusion BsAb format described here is that the direct fusion of a polypeptide (such as a sVD) to the N terminus of an IgG may affect the overall folding as well as the antigen binding efficiency of the latter molecule. The observation that the antigen-binding affinity of 1F2–2B4IgG to mVEGFR2 is ~4-fold lower than that of the monospecific 2B4 IgG appears in support of this notion. An obvious alternative to address this issue is to link the sVD to the C terminus of the HC of the IgG to form an IgG-sVD fusion. In fact, in a separate study, we linked the 1F2 VH onto the C terminus of the HC of an IgG antibody directed against the mPDGFRbeta antibody 1B3. The resultant BsAb, 1B3IgG-1F2 fusion, retained full antigen binding efficiency to both mPDGFRbeta and mPDGFR{alpha}.4 Taken together, our sVD/IgG fusion format, either as sVD-IgG (N-terminal fusion) or IgG-sVD (C-terminal fusion), provides a novel and simple recombinant approach to the production of Fc domain-containing, IgG-like BsAb.


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

1 To whom correspondence and reprint requests may be addressed: ImClone Systems Inc., 180 Varick St., New York, NY 10014. Tel.: 212-645-1405; Fax: 212-645-2054; E-mail: juqun.shen{at}imclone.com. 2 To whom correspondence and reprint requests may be addressed. Tel.: 212-645-1405; Fax: 212-645-2054; E-mail: zhenping.zhu{at}imclone.com.

3 The abbreviations used are: BsAb, bispecific antibody; LC, light chain; HC, heavy chain; VL, variable light chain; VH, variable heavy light; sVD, single variable domain; PDGF, platelet-derived growth factor; mPDGFR{alpha}, mouse PDGF receptor {alpha}; VEGF, vascular endothelial growth factor; mVEGFR2, mouse VEGF receptor 2; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ELISA, enzyme-linked immunosorbent assay; DTT, dithiothreitol; FACS, fluorescence-activated cell sorter; CL, constant light. Back

4 J. Shen, M. D. Vil, X. Jimenez, M. Iacolina, H. Zhang, and Z. Zhu, unpublished results. Back

5 D. Lu and Z. Zhu, unpublished result. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kontermann, R. E. (2005) Acta Pharm. Sin. 26, 1–9
  2. Kipriyanov, S. M., and Le Gall, F. (2004) Curr. Opin. Drug Discovery Dev. 7, 233–242[Medline] [Order article via Infotrieve]
  3. Lum, L. G., and Davol, P. A. (2005) Cancer Chemother. Biol. Response Modif. Annu. 22, 273–291
  4. Xiong, D., Xu, Y., Liu, H., Peng, H., Shao, X., Lai, Z., Fan, D., Yang, M., Han, J., Xie, Y., Yang, C., and Zhu, Z. (2002) Cancer Lett. 177, 29–39[CrossRef][Medline] [Order article via Infotrieve]
  5. Kipriyanov, S. M., Cochlovius, B., Schäfer, H. J., Moldenhauer, G., Bähre, A., Le Gall, F., Knackmuss, S., and Little, M. (2002) J. Immunol. 169, 137–144[Abstract/Free Full Text]
  6. James, N. D., Atherton, P. J., Jones, J., Howie, A. J., Tchekmedyian, S., and Curnow, R. T. (2001) Br. J. Cancer 85, 152–156[CrossRef][Medline] [Order article via Infotrieve]
  7. Baeuerle, P. A., Kufer, P., and Lutterbuse, R. (2003) Curr. Opin. Mol. Ther. 5, 413–419[Medline] [Order article via Infotrieve]
  8. Corvalan, J. R., Smith, W., and Gore, V. A. (1988) Int. J. Cancer 2, (suppl.) 22–25
  9. Ford, C. H., Osborne, P. A., Rego, B. G., and Mathew, A. (2001) Int. J. Cancer 92, 851–855[CrossRef][Medline] [Order article via Infotrieve]
  10. Ferrini, S., Sforzini, S., and Canevari, S. (2001) Methods Mol. Biol. 166, 177–192[Medline] [Order article via Infotrieve]
  11. Chang, C. H., Sharkey, R. M., Rossi, E. A., Karacay, H., McBride, W., Hansen, H. J., Chatal, J.-F., Barbet, J., and Goldenberg, D. M. (2002) Mol. Cancer Ther. 1, 553–563[Abstract/Free Full Text]
  12. Lu, D., Jimenez, X., Zhang, H., Wu, Y., Bohlen, P., Witte, L., and Zhu, Z. (2001) Cancer Res. 61, 7002–7008[Abstract/Free Full Text]
  13. Lu, D., Zhang, H., Koo, H., Tonra, J., Balderes, P., Prewett, M., Corcoran, E., Mangalampalli, V., Bassi, R., Anselma, D., Patel, D., Kang, X., Ludwig, D. L., Hicklin, D. J., Bohlen, P., Witte, L., and Zhu, Z. (2005) J. Biol. Chem. 280, 19665–19672[Abstract/Free Full Text]
  14. Lu, D., Zhang, H., Ludwig, D., Persaud, A., Jimenez, X., Burtrum, D., Balderes, P. M., Bohlen, P., Witte, L., and Zhu, Z. (2004) J. Biol. Chem. 279, 2856–2865[Abstract/Free Full Text]
  15. Jimenez, X., Lu, D., Brennan, L., Persaud, K., Liu, M., Miao, H., Witte, L., and Zhu, Z. (2005) Mol. Cancer Ther. 4, 427–434[Abstract/Free Full Text]
  16. Milstein, C., and Cuello, A. C. (1983) Nature 305, 537–540[CrossRef][Medline] [Order article via Infotrieve]
  17. Brennan, M., Davison, P. F., and Paulus, H. (1985) Science 229, 81–83[Abstract/Free Full Text]
  18. Carter, P., Ridgway, J., and Zhu, Z. (1995) J. Hematother. 4, 463–470[Medline] [Order article via Infotrieve]
  19. Todorovska, A., Roovers, R. C., Dolezal, O., Kortt, A. A., Hoogenboom, H. R., and Hudson, P. (2001) J. Immunol. Methods 248, 47–66[CrossRef][Medline] [Order article via Infotrieve]
  20. Carter, P. (2001) J. Immunol. Methods 248, 7–15[CrossRef][Medline] [Order article via Infotrieve]
  21. Marvin, J., and Zhu, Z. (2005) Acta Pharm. Sin. 26, 649–658[CrossRef]
  22. Lu, D., Kussie, P., Pytowski, B., Persaud, K., Bohlen, P., Witte, L., and Zhu, Z. (2000) J. Biol. Chem. 275, 14321–14330[Abstract/Free Full Text]
  23. Zhu, Z., Rockwell, P., Lu, D., Kotanides, H., Pytowski, B., Hicklin, D. J., Bohlen, P., and Witte, L. (1998) Cancer Res. 58, 3209–3214[Abstract/Free Full Text]
  24. Zhu, Z., Lu, D., Kotanides, H., Santiago, A., Jimenez, X., Simcox, T., Hicklin, D. J., Bohlen, P., and Witte, L. (1999) Cancer Lett. 136, 203–213[CrossRef][Medline] [Order article via Infotrieve]
  25. Hoet, R. M., Cohen, E. H., Kent, R. B., Rookey, K., Schoonbroodt, S., Hogan, S., Rem, L., Frans, N., Daukandt, M., Pieters, H., van Hegelsom, R., Neer, N. C., Nastri, H. G., Rondon, I. J., Leeds, J. A., Hufton, S. E., Huang, L., Kashin, I., Devlin, M., Kuang, G., Steukers, M., Viswanathan, M., Nixon, A. E., Sexton, D. J., Hoogenboom, H. R., and Ladner, R. C. (2005) Nat. Biotechnol. 23, 344–348[CrossRef][Medline] [Order article via Infotrieve]
  26. Lu, D., Jimenez, X., Zhang, H., Bohlen, P., Witte, L., and Zhu, Z. (2002) Int. J. Cancer 97, 393–399[CrossRef][Medline] [Order article via Infotrieve]
  27. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th Ed., National Institutes of Health, Bethesda
  28. Holliger, P., Prospero, T., and Winter, G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6444–6448[Abstract/Free Full Text]
  29. Zhu, Z., Zapata, G., Shalaby, R., Snedecor, B., Chen, H., and Carter, P. (1996) Bio/Technology (N.Y.) 14, 192–196
  30. Hu, S., Shively, L., Raubitschek, A., Sherman, M., Williams, L. E., Wong, J. Y., Shively, J. E., and Wu, A. M. (1996) Cancer Res. 56, 3055–3061[Abstract/Free Full Text]
  31. Lu, D., Jimenez, X., Zhang, H., Bohlen, P., Witte, L., and Zhu, Z. (2002) J. Immunol. Methods 267, 213–226[CrossRef][Medline] [Order article via Infotrieve]
  32. Wu, A. M., and Yazaki, P. J. (2000) Q. J. Nucl. Med. 44, 268–283[Medline] [Order article via Infotrieve]
  33. Ridgway, J. B., Presta, L. G., and Carter, P. (1996) Protein Eng. 9, 617–621[Abstract/Free Full Text]
  34. Atwell, S., Ridgway, J. B., Wells, J. A., and Carter, P. (1997) J. Mol. Biol. 270, 26–35[CrossRef][Medline] [Order article via Infotrieve]
  35. Merchant, A. M., Zhu, Z., Yuan, J. Q., Goddard, A., Adams, C. W., Presta, L. G., and Carter, P. (1998) Nat. Biotechnol. 16, 677–681[CrossRef][Medline] [Order article via Infotrieve]
  36. Zuo, Z., Jimenez, X., Witte, L., and Zhu, Z. (2000) Protein Eng. 13, 361–367[Abstract/Free Full Text]
  37. Coloma, M. J., and Morrison, S. L. (1997) Nat. Biotechnol. 15, 159–163[CrossRef][Medline] [Order article via Infotrieve]
  38. Cai, X., and Garen, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6280–6285[Abstract/Free Full Text]
  39. Muyldermans, S., Atarhouch, T., Saldanha, J., Barbosa, J. A., and Hamers, R. (1994) Protein Eng. 7, 1129–1135[Abstract/Free Full Text]
  40. Desmyter, A., Decanniere, K., Muyldermans, S., and Wyns, L. (2001) J. Biol. Chem. 276, 26285–26290[Abstract/Free Full Text]
  41. Tanha, J., Xu, P. P., Chen, Z., Ni, F., Kaplan, H., Narang, S. A., and MacKenzie, C. R. (2001) J. Biol. Chem. 276, 24774–24780[Abstract/Free Full Text]
  42. Jespers, L., Schon, O., Famm, K., and Winter, G. (2004) Nat. Biotechnol. 22, 1161–1165[CrossRef][Medline] [Order article via Infotrieve]
  43. Jespers, L., Schon, O., James, L. C., Veprintsev, D., and Winter, G. (2004) J. Mol. Biol. 337, 893–903[CrossRef][Medline] [Order article via Infotrieve]
  44. Schlehuber, S., and Skerra, A. (2005) Drug Discov. Today 10, 23–33[CrossRef][Medline] [Order article via Infotrieve]
  45. Nuttall, S. D., Krishnan, U. V., Doughty, L., Pearson, K., Ryan, M. T., Hoogenraad, N. J., Hattarki, M., Carmichael, J. A., Irving, R. A., and Hudson, P. J. (2004) Proteins 55, 187–197[CrossRef][Medline] [Order article via Infotrieve]
  46. Stanfield, R. L., Dooley, H., Flajnik, M. F., and Wilson, I. A. (2004) Science 305, 1770–1773[Abstract/Free Full Text]
  47. Ronnmark, J., Gronlund, H., Uhlen, M., and Nygren, P. A. (2002) Eur. J. Biochem. 269, 2647–2655[Medline] [Order article via Infotrieve]
  48. Parker, M. H., Chen, Y., Danehy, F., Dufu, K., Ekstrom, J., Getmanova, E., Gokemeijer, J., Xu, L., and Lipovsek, D. (2005) Protein Eng. Des. Sel. 18, 435–444[Abstract/Free Full Text]
  49. Holt, L. J., Herring, C., Jespers, L. S., Woolven, B. P., and Tomlinson, I. M. (2003) Trends Biotechnol. 21, 484–490[CrossRef][Medline] [Order article via Infotrieve]
  50. Binz, H. K., and Pluckthun, A. (2005) Curr. Opin. Biotechnol. 16, 459–469[CrossRef][Medline] [Order article via Infotrieve]
  51. Holliger, P., and Hudson, P. J. (2005) Nat. Biotechnol. 23, 1126–1136[CrossRef][Medline] [Order article via Infotrieve]

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