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,
Departments of
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Biological Structure and
Medicine, University of Washington, Seattle, WA 98195;
Fred Hutchinson Cancer Research Center, Seattle, WA 98104; and
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Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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55
kDa. The CD20 binding properties of the four 1F5 scFv-Ig were studied
by ELISA and flow cytometry. The 1F5 scFv-Ig with the 5-aa linker (GS1)
demonstrated significantly superior binding to CD20-expressing target
cells, compared with the other scFv-Ig constructs. Scatchard analysis
of the radiolabeled monovalent GS1 scFv-Ig revealed a binding avidity
of 1.35 x 108 M-1 compared with an
avidity of 7.56 x 108 M-1 for the native
bivalent 1F5 Ab. These findings suggest that the GS1 scFv-Ig with a
short linker peptide of 5 aa is the best of the engineered
constructs for future studies. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The variable domains of Fv
fragments are not associated by covalent bonds, and consequently the
domains tend to dissociate at low protein concentrations (5). One of
the approaches to improve the stability of the Fv domain association
involves construction of single-chain Abs (scFvs) in which the two
variable domains are linked via a short flexible peptide (Fig. 1).
Previous studies indicate that the lengths and sequences of the linker
peptide can significantly affect the properties of scFvs (6, 7, 8, 9, 10, 11, 12, 13, 14, 15).
Peptide sequences from known protein structures have been used as
linkers, the lengths and conformations of which are compatible with
bridging the variable domains of scFvs without serious steric
interference. The most widely used linker designs have sequences
consisting primarily of stretches of glycine (G) and serine (S)
residues. These small, polar amino acids are optimal for the linker
peptides because hydrophilic amino acids allow hydrogen bonding to the
solvent, and glycines provide the necessary flexibility (6). These
properties prevent the penetration of the linker peptide into the
hydrophobic interface formed in the association of the domains. An
example of this type of linker is the (GGGGS)3 peptide
designed by Huston et al. (9).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PCR reactions were conducted in 100 µl volumes containing 20 µmol of each dNTP, 50–75 pmol primers, 1 ng template, and Taq polymerase (Stratagene, Torrey Pines, CA) in the polymerase buffer supplied by the manufacturer. PCR was conducted with 33 cycles of 30 s denaturation at 94°C, 1.5 min annealing at 45°C, and 1.5 min extension at 72°C. Amplified DNA was digested with restriction endonucleases BclI and SacI for VH and SalI and SacI for VL (Boehringer Mannheim, Indianapolis, IN). Digested and purified DNA was cloned into the PUC19 plasmid using those sites. DH5a Escherichia coli cells were transformed with the plasmid and DNA. Miniprep DNA was purified using Geneclean (Bio101, La Jolla, CA). DNA from three of four clones of each VH and VL were sequenced to determine the consensus sequence by automated sequencing using 32P-labeled dNTP.
Three-way ligation
Aliquots (5 µl) of miniprep DNA from PUC19 plasmids carrying VL and VH gene fragments were digested with a mixture of SacI and SalI restriction enzymes for VL and BclI and SacI for VH. The digests were incubated for 2 h at 37°C (56°C for BclI), and DNA fragments were separated by electrophoresis on minigels of 0.75% Seakem agarose (FMC Bioproducts, Rockland, ME). DNA bands were visualized by staining with ethidium bromide. DNA bands corresponding to VL and VH fragments were cut from gels and purified using Geneclean. DNA fragments encoding VH and VL were cloned into modified PUC19 at the same time. The PUC19 plasmid was modified as follows: the HindIII site at the 5' end of the stuffer region was used to insert a HindIII-SalI fragment containing the anti-L6 Ig light chain leader sequence for secretion of scFv-Ig, and the XbaI site at the 3' end of the stuffer region was used to insert the DNA sequence encoding the hinge-CH2-CH3 portion of human IgG1 in which hinge cysteines had been mutated to serines to favor the production of monomeric scFv-Ig. The cloned DNA fragment was then cut by XbaI and HindIII, purified by Geneclean, and inserted into the pCDM8 vector for expression.
Transient transfection in COS cells
COS cells were seeded at a density of 106 cells per
10-cm diameter culture dish 18–24 h before transfection. Plasmid DNA
was added (15 µg/dish) in a volume of 5 ml of serum-free DMEM
containing 0.1 mM chloroquine and 600 µg/ml DEAE dextran, and cells
were incubated for 3–3.5 h at 37°C. Transfected cells were then
briefly treated (2 ml) with 10% DMSO in PBS and incubated at 37°C
for 16–24 h in DMEM containing 10% FCS. The culture medium was
removed 24 h after transfection and replaced with serum-free DMEM
(10 ml/dish). Incubation was continued for 3 days at 37°C, and then
the spent medium was collected and fresh serum-free medium was added.
After an additional 3 days at 37°C, the spent medium was again
collected and cells were discarded. Culture supernatants were tested
for the presence of scFv-Ig proteins and for specific binding activity
of those proteins as described below.
Purification of 1F5 scFv-Ig
MC106/p3 clones, which contained host pCDM8 vectors and produced functional scFv-Ig, were initially identified by screening supernatants of small-scale preparations for binding to Ramos cells using FACS. Then, large-scale transfections were set up to generate sufficient quantities of fusion proteins for purification and experimental testing. Subsequently, 800 ml of serum-free supernatants were collected over a 6-day culture period. Cellular debris was removed by low-speed centrifugation, and clarified supernatants were applied to columns of immobilized protein A at a rate of 1 ml/min. The columns were rinsed with PBS and proteins were eluted with 0.1 M glycine/0.15 M NaCl, pH 2.7, neutralized immediately with 1.0 M Tris-HCl, pH 9.0, and then dialyzed against PBS at 4°C.
Detection of 1F5 scFv-Ig by Western blotting
1F5 scFv-Ig fusion proteins were immunoprecipitated from culture supernatants, and samples were electrophoresed on linear gradient (6–15%) acrylamide gels at 200 V for 4–5 h. Gels were blotted to nitrocellulose membranes using a Western semidry transfer apparatus (Ellard Instrumentation, Seattle, WA) at 20 V (3 mA/cm2) for 1 h. Blots were blocked with blocking buffer (2% nonfat milk plus 0.1% Tween in PBS) and then incubated with goat anti-human IgG (GAH) conjugated to alkaline phosphatase (Boehringer Mannheim) in blocking buffer. Blots were washed three times in blocking buffer and developed in Western Blue (Promega, Madison, WI).
Gel filtration analysis of 1F5 scFv-Ig
The m.w. of 1F5 scFv-Ig before and after purification with protein A were analyzed by gel filtration chromatography on a Waters 7.8 x 300 mm 300SW HPLC column (Waters, Milford, MA). Samples were chromatographed at a flow rate of 0.35 ml/min in 10 mM Tris-HCl, pH 7.5, 10 mM potassium phosphate, and 0.15 M NaCl. The protein standards used to determine the relative molecular masses of 1F5 scFv-Ig were as follows: blue dextran, 2 x 106 Da; ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa; BSA, 67 kDa; OVA, 43 kDa; chymotrypsinogen, 25 kDa; and RNase, 13.7 kDa.
Binding activity of 1F5 scFv-Ig detected by ELISA
A total of 105 Ramos cells/well were added to U-shaped 96-well microtiter plates (Falcon 3072, Becton Dickinson, Lincoln Park, NJ). Plates were blocked with PBS containing 5% BSA, and purified 1F5 scFv-Ig fusion protein or 1F5 was added. Following a 1-h incubation at 4°C and removal of unbound material, bound protein was detected with GAH or goat anti-mouse IgG Ab (GAM) labeled with HRP (American Qualex, La Miranda, CA) in blocking buffer for 45 min at 4°C. Unbound conjugates were removed by washing with PBS plus Tween 20, and o-phenylenediamine (Sigma, St. Louis, MO) in citrate buffer was added to the wells. The reaction was stopped with 2 M HCl, and the OD was measured at 490 nm with a Titertek multiwell plate reader (Titertek instruments, Huntsville, AL).
Binding activity of 1F5 scFv-Ig detected by flow cytometry
Ramos cells were incubated with COS supernatants/PBS, purified 1F5 scFv-Ig fusion proteins, or purified intact 1F5 Ab in staining medium (RPMI 1640 plus 5% FBS plus 0.1% sodium azide) for 45 min on ice. Cells were washed, and bound scFv-Ig fusion proteins were detected with GAH conjugated to FITC (Tago, Burlingame, CA) for 30 min on ice. Bound murine 1F5 Ab was detected using a FITC-labeled GAM (Tago). The cells were washed with ice-cold staining medium and fluorescence was analyzed using a flow cytometer (Becton Dickinson, Mountain View, CA).
Radioiodination of Abs and Scatchard analysis of binding
1F5 and 1F5 scFv-Ig were labeled with Na125I by the Iodogen method. Ab (100 µg/ml) was incubated with 0.5 mCi of Na125I in glass tubes coated with 10 µg of Iodogen (Pierce, Rockford, IL) for 5 min at room temperature. Free Na125I was removed by chromatography on a Pharmacia PD-10 column (Pharmacia, Piscataway, NJ). Eluted fractions containing 125I-labeled Ab were pooled and stored at 4°C.
Cell binding assays were performed in 1.5 ml microcentrifuge tubes.
Labeled Abs were serially diluted in tissue culture medium (RPMI 1640
plus 2% BSA plus 0.02% sodium azide) and 100 µl of each
concentration mixed with an equal volume of serial cell dilutions in
microcentrifuge tubes. A 100-µl aliquot of each Ab dilution was also
counted in a Beckman 
5500 counter (Beckman Instruments, Palo Alto,
CA) to determine the sp. act. of the labeled Abs and the total amount
of Ab added for each dilution. The immunoreactive fraction of each Ab
preparation was determined by Lineweaver-Burk analysis using a fixed
concentration of Ab (40 ng/ml) incubated with varying numbers of target
cells (107–105/well) as described by Lindmo
(16). Scatchard analysis was performed by incubating serial dilutions
of Ab (10 µg/ml to 10 ng/ml) with a fixed number of cells (2 x
105). Tubes were incubated at 37°C for 1 h. The
target cells were then washed three times by centrifugation, and bound
activity was counted. Scatchard plots were then used to determine
binding avidities as described by Trucco (17).
Flow cytometric analysis of apoptosis using propidium iodide (PI) staining
Flow cytometric analysis of cellular DNA was performed following PI staining according to the method of Fried et al. (18). Briefly, 106 Ramos cells were incubated with 1F5 scFv-Ig in the presence or the absence of F(ab')2 of a GAH (Jackson ImmunoResearch Laboratories, West Grove, PA), washed in PBS, and then gently resuspended in 0.5 ml of a hypotonic fluorochrome solution (50 µg/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100; all from Sigma). Samples were stored in the dark at 4°C until flow cytometric analysis of individual nuclei using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) could be performed. The percentage of cells that were apoptotic was measured as described by Nicoletti et al. (19). Briefly, cellular debris was excluded from analysis by raising the forward scatter threshold, and the DNA content of intact nuclei was recorded on a logarithmic scale. Apoptotic cell nuclei containing hypodiploid DNA were enumerated as a percentage of the total population.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four different sense primers for the VH domain of the
1F5 scFv-Ig were designed to construct four new 1F5 scFv-Ig-containing
multimers of the (GGGGS) linker peptide (Fig. 2). These primers contained sequences
encoding (GGGGS)3, (GGGGS)2, and
(GGGGS)1 and sequences joining VH and
VL directly. In addition, a SacI restriction
site was added to the amino terminus of all four primers and to the
carboxyl terminus of the antisense primer for amplifying the
VL sequence, which was used later in a three-way ligation
reaction to join the cohesive ends of sequences encoding VH
and VL.
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The newly designed primers were used to amplify DNA fragments
encoding the VH and VL regions of the new 1F5
scFv-Ig constructs by PCR, using plasmids containing DNA fragments
encoding the original VH and VL of 1F5 scFv as
a template. After being purified and sequenced, isolated VH
and VL DNA fragments were simultaneously inserted into the
polylinker region of the modified PUC19 plasmid flanked by a sequence
encoding a leader peptide at the N terminus and a human IgG1 domain
(hinge, CH2, and CH3) at the C terminus in a three-way ligation
reaction. A single open reading frame was created encoding, from N to C
terminus, leader peptide, VL, VH with different
linker peptides, and a derivative of the human IgG1 Fc domain. Fig. 3 shows the sequence of 1F5 scFvs with
varying lengths of linker peptides. These fragments were then
transferred to an expression vector (pCDM8) before transfecting into
COS cells for expression. 1F5 scFv-Ig fusion proteins were purified
from culture supernatants of transfected COS cells by affinity
chromatography on immobilized protein A. Yields of purified protein
were typically about 1.5 mg/L.
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scFvs with short or absent linker peptides have been shown to have
a tendency to aggregate and form dimers or multimers (8). The
aggregation of 1F5 scFv-Ig was examined on Western blots probed with
alkaline phosphatase-conjugated GAH after proteins were separated by
reducing or nonreducing SDS-PAGE gels (Fig. 4). When reduced, all four 1F5 scFv-Ig
migrated at molecular masses of 55 kDa, the approximate size
expected for a scFv fused to hinge-CH2-CH3 of human IgG1. About
10–30% of 1F5 scFv-Ig appeared at molecular masses of 110 kDa when
run on nonreducing gels (Fig. 4B). These data indicated that
most of the 1F5 scFv-Ig were monomeric in SDS despite the different
lengths of their linker peptides. The size of native 1F5 scFv-Ig was
also examined by size-exclusion HPLC analysis before and after protein
A purification (Fig. 5). A single peak
corresponding in size to the scFv-Ig monomer was observed for all 1F5
scFv-Ig from both cell culture supernatants and purified proteins.
Binding assays (see below) using peak fractions indicated that the
monomers were capable of binding specifically to CD20-expressing cells
(data not shown). These results demonstrated that no significant
aggregation occurred with any of the 1F5 scFv-Ig when analyzed by HPLC.
The presence of a minor degree of dimer formation on nonreducing
SDS-PAGE gels was presumably due to association of the CH3 domains of
the Ig tail, as has been reported previously (20).
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The relative CD20 binding activities of these new 1F5 scFv-Ig
constructs were compared in flow cytometric and ELISA assays using
CD20-expressing Ramos cells and control Jurkat cells. In the former
assay, the binding activity was tested by adding 1F5 scFv-Ig constructs
to Ramos cells followed by staining with FITC-conjugated GAH as a
second step reagent and analyzing by flow cytometry. The results
indicated that 1F5 scFv-Ig bind specifically to CD20 expressed on Ramos
cells because nonspecific binding of 1F5 scFv-Ig was not found with
Jurkat cells, which do not express the target CD20 Ag (Fig. 6). The order of binding activity was
(GGGGS)1 > (GGGGS)2 > (GGGGS)0 >
(GGGGS)3 (Fig. 6). The specificity of binding was also
confirmed using a competitive binding assay employing unlabeled 1F5
scFv-Ig to block binding of fluorescein-labeled intact 1F5 (data not
shown). The mean fluorescence intensity of Ramos cells incubated with
FITC-1F5 declined from channel 31.02 in the absence of blocking 1F5
scFv-Ig to channel 18.64 in the presence of unlabeled 1F5 scFv-Ig. The
relative binding activities of the different 1F5 scFv-Ig were confirmed
in an ELISA assay, performed by adding 25 µl of various
concentrations (12.5–100 µg/ml) of 1F5 scFv-Ig to 105
Ramos cells, followed by the addition of a peroxidase-conjugated GAH
second Ab (Fig. 7). Thus, all of the
scFv-Ig constructs retained CD20 binding activity, but the binding
activities were strongly influenced by the length of the linker.
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The binding activity of the best 1F5 scFv-Ig construct (GS1) was
further compared with the binding of the intact anti-CD20 mAb 1F5
by ELISA, flow cytometry, and radiolabeled cell binding assays. As
shown in Fig. 8, the binding activity of
the monomeric 1F5 scFv-Ig was comparable, though slightly inferior to
that of the intact bivalent 1F5. Similar results were obtained using
flow cytometry (data not shown). A more quantitative analysis of
comparative binding was obtained by radiolabeling both the GS1 1F5
scFv-Ig construct and the intact 1F5 Ab and performing a standard
Scatchard binding analysis. Because soluble CD20 was not available, the
binding of 125I-labeled 1F5 scFv-Ig and intact 1F5 were
measured using CD20-bearing Ramos cells, and data were analyzed by
Scatchard analysis by the method of Trucco (17). Using this approach,
the binding avidity of the 1F5 scFv-Ig was estimated to be 1.35 x
108 M-1, whereas the binding avidity of the
bivalent 1F5 Ab was 7.56 x 108 M-1.
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The functional activities of 1F5 scFv-Ig were subsequently
assessed by measuring the capability of these constructs to induce
apoptosis of Ramos cells after cross-linking CD20 with 1F5 scFv-Ig plus
GAH. Our group (21) and others (22) have previously demonstrated that
intact anti-CD20 mAbs reproducibly induce apoptosis of
malignant human B cells if the CD20 molecule on mAb-coated cells is
cross-linked using GAM or FcR-bearing accessory cells. To investigate
whether 1F5 scFv can induce apoptosis in a similar fashion, we treated
Ramos cells with 1F5 scFv-Ig in the presence and absence of GAH and
then stained cells with PI followed by flow cytometry analysis. As
depicted in Fig. 9, 1F5 scFv-Ig could
induce a small amount of apoptosis in Ramos cells when used alone
(p < 0.05), but was more effective at inducing
apoptosis when further cross-linked by a secondary GAH Ab
(p < 0.01), similar to our observations with
intact murine 1F5.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1F5 is one of a panel of mAbs that have been produced recognizing the CD20 pan B cell Ag (34), and it was the first anti-CD20 mAb tested in lymphoma patients (26). Anti-CD20 mAbs are attractive for immunotherapy of B cell lymphomas because 1) anti-CD20 Abs do not internalize after cell surface binding, 2) anti-CD20 Abs are not shed from the cell surface, 3) CD20 is expressed at a high density on >95% of all B cell lymphomas, and 4) the antigenic density of CD20 is relatively homogenous from one tumor cell to another (35). To further improve the promising therapeutic effects of anti-CD20 mAbs by enhancing penetration into tumor sites, smaller fragments of these mAbs have been suggested to replace intact mAbs (27, 36, 37). However, preliminary efforts to generate 1F5 F(ab')2 enzymatically have been laborious and yields have been disappointing (our unpublished observations). Fab of these mAbs have been produced but their monovalent binding and lower avidities have impeded clinical applications (27, 36, 37). These disadvantages have induced us to pursue genetic approaches to synthesize new Ab derivatives. Ultimately, these efforts are focused on making these special Abs smaller, while retaining bivalent binding properties to make their affinities comparable to intact mAbs.
Recombinant fragments with two binding sites have been made in several ways, for example, by chemical cross-linking of the hinge cysteine residues (38) or by including a C-terminal peptide that promotes dimerization (39, 40). More recently, single-chain Abs have been reported by many research groups to have the tendency to form dimers and higher molecular multimers when the linker peptides are short, especially shorter than 15 aa residues (7, 15, 33). The explanation for this phenomenon is that short linkers presumably constrain the folding and appropriate association of VH and VL located on the same polypeptide, which expose hydrophobic residues normally concealed by the VH-VL interface, therefore increasing the likelihood of intermolecular associations and resulting in multimerization.
In our study, both Western blotting and HPLC data indicated that most of the functionally active 1F5 scFv-Ig were monomeric despite the differences in their linker peptide lengths. This was not expected because most of the other scFv produced with short linker peptides tend to form dimers or multimers (11, 33). A possible explanation for our findings is that the Ig tail may limit the aggregation of 1F5 scFv-Ig. Although a small amount of dimerization was found on nonreducing SDS-PAGE gels, this was probably due to intermolecular associations via the Ig CH3 domain as has been reported previously for scFv-Ig constructs (20).
One potential explanation for the apparent increase in the CD20 binding
activity of the 1F5 scFv-Ig constructs synthesized with shorter linker
peptides may be that they formed dimers or multimers in culture,
leading to an increase in their binding valency. However, this
hypothesis is apparently not consistent with our Western blot and HPLC
experiments demonstrating <30% dimerization. A possible explanation
for this discrepancy is that dimerization may have occurred after
binding of monomeric scFv-Ig to the CD20 Ag on the cell surface due to
local concentration effects promoting the formation of dimers and
higher m.w. forms as has been reported for other scFvs (7, 15). This
type of multimerization has been reported to be most likely for scFv
recognizing Ags present at high density on a solid phase such as cells
(7, 15). In support of this hypothesis, our experiments show that the
slope of 1F5 scFv-Ig GS1 binding in our ELISA assay exhibits a
cooperative binding effect in contrast to the slope of the 1F5 scFv-Ig
GS3 (Fig. 7
). An alternative explanation for the superior binding of
the short linker construct GS1 may be that the 5-aa linker caused
subtle conformational changes leading to a slightly altered binding
pocket, causing a better "fit" with the CD20 Ag.
A major reservation to the substitution of scFv constructs for intact mAbs in immunotherapeutic approaches involves the possible loss of anti-tumor effect or functions including complement fixation, Ab-dependent cellular cytotoxicity, and induction of apoptosis. Although the first two of these functions are dependent on the physical presence of the Fc portion of the Ig molecule, apoptosis induction is triggered by binding to the target Ag and might be preserved with scFv constructs. Our results indicate that this may be the case with the 1F5 scFv described in this manuscript, though optimal induction of apoptosis required amplification by cross-linking the scFv with a GAH secondary reagent. Because the human IgG1 tail used in our scFv-Ig constructs can bind Fc receptors, this apoptotic effect may be augmented in vivo by cross-linking mediated by FcR-bearing accessory cells. We conclude that the 5-aa GS1 1F5 scFv construct merits further investigation for targeted therapy of CD20-expressing B cell malignancies including applications involving radionuclide conjugates and pretargeting strategies using scFv-streptavidin conjugates in combination with biotin-90Y secondary reagents. In addition, further structural modifications are underway to generate dimeric scFv, which may prove superior to the present constructs.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daming Shan, University of Washington Medical Center, Division of Medical Oncology, Box 356043, Seattle, WA 98195-6043. E-mail address:
3 Current address: Xcyte Therapies, 2203 Airport Way South, Suite 300, Seattle, WA 98134.
4 Abbreviations used in this paper: VH, variable domain of heavy chain; VL, variable domains of light chain; scFvs, single-chain Abs; scFv-Ig, single-chain Abs and Ig fusion protein; G, glycine; S, serine; GS0, VH domain with 0-aa linker peptides; GS1, VH domain with 5-aa linker peptides; GS2, VH domain with 10-aa linker peptides; and GS3, VH domain with 15-aa linker peptides; GAM, goat anti-mouse IgG Ab; GAH, goat anti-human IgG Ab; PI, propidium iodide.
Received for publication October 9, 1998. Accepted for publication March 10, 1999.
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receptor-positive tumor xenografts. Cancer Res. 55:5323.This article has been cited by other articles:
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  J. M. Pagel, Y. Lin, N. Hedin, A. Pantelias, D. Axworthy, D. Stone, D. K. Hamlin, D. S. Wilbur, and O. W. Press Comparison of a tetravalent single-chain antibody-streptavidin fusion protein and an antibody-streptavidin chemical conjugate for pretargeted anti-CD20 radioimmunotherapy of B-cell lymphomas Blood, July 1, 2006; 108(1): 328 - 336. [Abstract] [Full Text] [PDF]
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 A. M. Wu, G. J. Tan, M. A. Sherman, P. Clarke, T. Olafsen, S. J. Forman, and A. A. Raubitschek Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange Protein Eng. Des. Sel., December 1, 2001; 14(12): 1025 - 1033. [Abstract] [Full Text] [PDF]
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 J. Schultz, Y. Lin, J. Sanderson, Y. Zuo, D. Stone, R. Mallett, S. Wilbert, and D. Axworthy A Tetravalent Single-chain Antibody-Streptavidin Fusion Protein for Pretargeted Lymphoma Therapy Cancer Res., December 1, 2000; 60(23): 6663 - 6669. [Abstract] [Full Text]
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signal peptide. Four
versions of the (linker plus 1F5 VH) domain were generated
by PCR using different sense primers. The primers differed in the
number of (gly4ser)n repeats immediately
following the SacI site and just before the
VH coding region. The antisense primer contained a
BclI site for fusion to Ig tails or STOP cassettes. Four
scFvs were generated by ligation of the VL domain to one of
the four VH domains and were designated GS0, GS1, GS2, and
GS3 depending on the number of (gly4ser) repeats contained
in the peptide linker between VL and VH.
