| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Departments of Molecular and Cell Biology [D. L., X. J., H. Z., L. W., Z. Z.], Immunology [Y. W.], and Research [P. B.], ImClone Systems Inc., New York, New York 10014
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Flt-1 and KDR have distinct functions in vascular development in embryos. Targeted deletion of genes encoding either receptor in mice is lethal to the embryo, demonstrating the physiological importance of the VEGF pathway in embryonic development (13 , 14) . KDR-deficient mice have impaired blood island formation and lack mature endothelial cells (13) , whereas Flt-1-null embryos fail to develop normal vasculature due to defective formation of vascular tubes, albeit with abundant endothelial cells (14) . On the other hand, inactivation of Flt-1 signal transduction by truncation of the tyrosine kinase domain did not impair mouse embryonic angiogenesis and embryo development, suggesting that signaling through the Flt-1 receptor is not essential for vasculature development in the embryo (15) . The biological responses of Flt-1 and KDR to VEGF in the adult also appear to be different. It is generally believed that KDR is the main VEGF signal transducer that results in endothelial cell proliferation, migration, differentiation, tube formation, increase of vascular permeability, and maintenance of vascular integrity (1, 2, 3) . Flt-1 possesses a much weaker kinase activity and is unable to generate a mitogenic response in endothelial cells when stimulated by VEGF, although it binds to VEGF with an affinity that is approximately 10-fold higher than that of KDR (1, 2, 3) . However, Flt-1 has been implicated in VEGF- and PlGF-induced migration of monocytes/macrophage and production of tissue factor (16 , 17) .
We previously produced a neutralizing antibody to KDR, IMC-1C11, and demonstrated that this antibody was able to effectively inhibit VEGF-stimulated receptor activation and mitogenesis of HUVECs (7 , 18) . We recently produced a monoclonal antibody to human Flt-1, FBK612.3 FBK612 binds specifically to Flt-1 and blocks the receptor from binding to both VEGF and PlGF. Here we constructed a recombinant bifunctional diabody, a bispecific divalent scFv dimer (19) , using the antibody variable domains from both IMC-1C11 and FBK612 as the building blocks. The bifunctional diabody binds to both KDR and Flt-1 and strongly blocks interaction between the receptors and their ligands, namely, VEGF and PlGF. Furthermore, we demonstrate that the bifunctional diabody is a more potent inhibitor of VEGF-stimulated proliferation of human endothelial cells and both VEGF- and PlGF-induced migration of human leukemia cells than either of its parent antibodies.
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
), was established at ImClone Systems Inc. (New York, NY) from a mouse immunized with a recombinant form of the receptor. The single-chain antibody directed against KDR, scFv p1C11, was isolated from a phage display library constructed from the splenocytes of a mouse immunized with KDR (7)
. Primary cultured HUVECs were obtained from Dr. S. Rafii (Cornell Medical Center, New York, NY) and maintained in EBM-2 medium (Clonetics, Walkersville, MD) at 37°C in 5% CO2. The soluble fusion protein KDR-AP was expressed in stably transfected NIH 3T3 cells and purified from cell culture supernatant by affinity chromatography using immobilized monoclonal antibody to AP as described previously (20)
. VEGF165 protein was expressed in baculovirus and purified following the procedures described previously (20)
. PlGF and Flt-1-Fc fusion proteins were purchased from R&D Systems (Minneapolis, MN). The leukemia cell lines HL60 and HEL were maintained in RPMI 1640 containing 10% FCS and grown at 37°C with 5% CO2.
Cloning of the VH and VL Genes of the Anti-Flt-1 Antibody, FBK612.
The VH and VL genes of FBK612 were cloned by reverse transcription-PCR from mRNA isolated from the hybridoma cells, following the procedures of Bendig and Jones (21)
. Eleven 5' primers specifically designed to hybridize to the 5' ends of mouse antibody light chain leader sequences and one 3' primer that hybridizes to the 5' end of mouse light chain constant region were used to clone the VL gene. Twelve 5' primers specifically designed to hybridize to the 5' ends of mouse antibody heavy chain leader sequences and one 3' primer that hybridizes to the 5' end of mouse IgG1 heavy chain constant region were used to clone the VH gene (see Ref. 21
for the complete list of primer sequences). In total, 23 PCR reactions (11 for the VL gene and 12 for the VH gene) were carried out for each of the antibodies, following the protocols described by Bendig and Jones (21)
. All PCR-generated fragments with sizes between 400 and 500 bp were cloned into pCR 2.1 vector as described in the manufacturer’s instructions (TA Cloning Kit; Invitrogen, Carlsbad, CA), followed by transformation of Escherichia coli strain XL-1. At least five colonies containing each of the inserts as identified by PCR screening were picked and subjected to DNA sequencing.
Construction of the Anti-KDR x Anti-Flt-1 Diabody.
PCR fragments encoding the VL and VH genes of FBK612 were first used to assemble a scFv of the antibody, scFv 612, using overlapping PCR. In this scFv, the COOH terminus of FBK612 VH was linked to the NH2 terminus of FBK612 VL via a 15-amino acid linker [glycine-glycine-glycine-glycine-serine)3 or (GGGGS)3; Fig. 1A
]. The scFv 612-encoding gene was then cloned into vector pCANTAB 5E (Amersham Pharmacia Biotech, Piscataway, NJ) for expression of the soluble scFv protein. To construct the diabody, variable domains of scFv p1C11 and scFv 612 were used for PCR-directed assembly to create the expression plasmid pDAB-KF1 (Fig. 1A)
. Briefly, the following gene fragments of VL and VH domains of p1C11 and KBK612 were first generated by PCR: (a) VL domain of p1C11 followed by a segment encoding a 5-amino acid linker (GGGGS); (b) VH domain of FBK612 preceded by a segment encoding the GGGGS linker; (c) VL domain of FBK612 preceded by a segment encoding the E. coli heat stable enterotoxin II (stII) signal sequence (22)
followed by a segment encoding the GGGGS linker; and (d) VH domain of p1C11 preceded by a segment encoding the GGGGS linker. Cross-over scFvs, pLH-1C11–612 and pLH-612–1C11, were constructed by the annealing of PCR fragments p1C11 VL and FBK612 VH and FBK612 VL and p1C11 VH, respectively, followed by PCR amplification to introduce appropriate restriction sites for subsequent subcloning. pDAB-KF1, the expression plasmid for cosecretion of the two cross-over scFvs, was constructed by ligation of the SfiI/NheI and NheI/NotI fragments from pLH-1C11–612 and pLH-612–1C11, respectively, into vector pCANTAB 5E. All sequences encoding the cross-over scFv fragments were verified by DNA sequencing.
|
Dual Antigen Binding of the Diabody to KDR and Flt-1.
Two assays were carried out to determine the dual antigen binding capability of the diabody. First, a cross-linking assay was used to investigate whether the diabody is capable of binding both of its target antigens simultaneously. Briefly, the diabody or its parent scFv was first incubated in a 96-well Maxi-sorp microtiter plate (Nunc, Roskilde, Denmark) precoated with Flt-1-Fc fusion protein (1 µg/ml x 100 µl/well overnight at 4°C) at room temperature for 1 h. The plate was washed three times with PBS containing 0.1% Tween, followed by incubation with KDR-AP fusion protein at room temperature for an additional hour. The plate-bound KDR-AP was then quantified by the addition of AP substrate, p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, MO), followed by reading of the absorbance at 405 nm (19)
. In the second assay, the direct binding assay, various amounts of diabody or scFv were added to KDR- or Flt-1-coated 96-well plates and incubated at room temperature for 1 h, and then the plates were washed three times with PBS containing 0.1% Tween. The plates were then incubated at room temperature for 1 h with 100 µl of an anti-E tag antibody-HRP conjugate (Amersham Pharmacia Biotech). The plates were washed, peroxidase substrate was added, and the absorbance at 450 nm was read following the procedure described previously (19)
.
VEGF/KDR, VEGF/Flt-1, and PlGF/Flt-1 Blocking Assays.
The assay was carried out following a previously described protocol (7
, 19)
. Briefly, various amounts of the diabody or scFv were mixed with a fixed amount of KDR-AP (100 ng) or Flt-1-Fc fusion protein (50 ng) and incubated at room temperature for 1 h. The mixture was then transferred to 96-well microtiter plates precoated with VEGF165 (200 ng/well) or PlGF (200 ng/well) and incubated at room temperature for an additional 2 h, and then the plates were washed five times with PBS. For KDR-AP assay, the substrate for AP was added, followed by reading of the absorbance at 405 nm to quantify the plate-bound KDR-AP molecules. For Flt-1-Fc assay, the plate was incubated with a mouse antihuman Fc-HRP conjugate to quantify the plate-bound Flt-1-Fc fusion. The IC50, i.e., the antibody concentration required for 50% inhibition of KDR or Flt-1 binding to VEGF or PlGF, was then calculated.
Binding Kinetics Analysis of the Diabody.
The binding kinetics of the diabody and its parent scFv to KDR and Flt-1 were measured using a BIAcore biosensor (Pharmacia Biosensor). KDR-AP or Flt-1-Fc fusion protein was immobilized onto a sensor chip, and soluble antibodies were injected at concentrations ranging from 1.5–100 nM. Sensorgrams were obtained at each concentration and evaluated using a the BIA Evaluation 2.0 program to determine the rate constants kon and koff. The affinity constant (Kd) was calculated from the ratio of rate constants koff:kon.
Antimitogenic Assay.
HUVECs (5 x 103 cells/well) were plated onto 96-well tissue culture plates (Wallach, Inc., Gaithersburg, MD) in 200 µl of EBM-2 medium without VEGF, basic fibroblast growth factor, or EGF and incubated at 37°C for 72 h. Various amounts of the antibodies were added to duplicate wells and preincubated at 37°C for 1 h, and then VEGF165 was added to a final concentration of 16 ng/ml. After 18 h of incubation, 0.25 µCi of [3H]thymidine (Amersham) was added to each well and incubated for an additional 4 h. The cells were washed once with PBS, trypsinized, and harvested onto a glass fiber filter (Printed Filtermat A; Wallach) with a cell harvester (Harvester 96; MACH III M; TOMTEC, Orange, CT). The membrane was washed three times with H2O and air-dried. Scintillation fluid was added, and DNA incorporated radioactivity was determined on a scintillation counter (Wallach Model 1450 Microbeta Liquid Scintillation Counter).
Leukemia Migration Assay.
HL60 and HEL cells were washed three times with serum-free plain RPMI 1640 and suspended in the medium at 1 x 106 cells/ml. Aliquots of the 100-µl cell suspension were added to either 3-µm-pore transwell inserts (for HL60 cells) or 8-µm-pore transwell inserts (for HEL cells; Costar; Corning Inc., Corning, NY) and incubated with the antibodies for 30 min at 37°C. The inserts were then placed into the wells of 24-well plates containing 0.5 ml of serum-free RPMI 1640 with or without VEGF165. The migration was carried out at 37°C in 5% CO2 for 16–18 h (HL60 cells) or 4 h (HEL cells). Migrated cells were collected from the lower compartments and counted with a Coulter counter (Model Z1; Coulter Electronics Ltd., Luton, United Kingdom).
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Construction and Expression of the Anti-KDR x Anti-Flt-1 Diabody.
In constructing the diabody, the COOH terminus of the p1C11 VL domain was first connected to the NH2 terminus of the FBK612 VH domain using a 5-amino acid linker, GGGGS, to restrict intrachain pairing of VL and VH. A second cross-over scFv was then constructed similarly by connecting the COOH terminus of the FBK612 VL domain to the NH2 terminus of the p1C11 VH domain using the same linker. The two cross-over scFv were then PCR amplified and subcloned into expression vector pCANTAB 5E to create the expression vector for the diabody, pDAB-KF1 (Fig. 1A)
. Each cross-over scFv is preceded by a different signal sequence, the gene III signal sequence and the E. coli stII signal sequence, respectively, to direct secretion to the periplasmic space of E. coli. A polypeptide tag, the E tag, was fused immediately to the COOH terminus of the second cross-over scFv for purification and detection purposes. The diabody was produced in E. coli strain HB2151 containing the expression vector grown in shake flasks. The diabody was released from the periplasmic space of E. coli by osmotic shock and purified with anti-E tag affinity chromatography.
The purified diabody was analyzed by SDS-PAGE. The two component cross-over scFvs were resolved under the electrophoretic conditions and gave rise to two major bands with mobility close to that anticipated (Fig. 1B)
; the lower band represents the first cross-over scFv (Mr 25,179.6), and the upper band correlates with the second cross-over scFv with E-tag (Mr 26,693.8; Fig. 1A
).
Dual Specificity of the Diabody.
We carried out a cross-linking assay to investigate whether the diabody is capable of simultaneously binding to both of its target antigens. In this assay, the diabody was first allowed to bind to immobilized Flt-1, which was followed by incubation with KDR-AP to test the capability of the Flt-1-bound diabody to capture the soluble KDR. As shown in Fig. 2A
, the diabody, but not the parent monospecific scFv, efficiently cross-linked the soluble KDR to the immobilized Flt-1, as demonstrated by the plate-bound AP activity.
|
. On the other hand, binding to Flt-1 of the diabody was slightly reduced, compared with that of the parent scFv 612 (Fig. 2C)
. As expected, the KDR-specific scFv p1C11 did not bind to Flt-1 (Fig. 2B)
, nor did the Flt-1-specific scFv 612 bind to KDR (Fig. 2C)
.
The binding kinetics of the diabody to KDR and Flt-1 were determined by surface plasmon resonance using a BIAcore instrument (Table 1)
. Consistent with the observations from ELISA (Fig. 2)
, the diabody binds to KDR with kinetics similar to those of its parent scFv p1C11 with a Kd of 1.4 nM. The binding affinity of the diabody to Flt-1 was moderately reduced compared with that of scFv 612, mainly due to a slower on-rate of the diabody (Table 1)
.
|
shows that the diabody blocks KDR from binding to immobilized VEGF, in a dose-dependent manner, as efficiently as scFv p1C11, with an IC50 of approximately 2 nM. The diabody also blocks Flt-1 from binding to VEGF with an IC50 of 
10 nM, which is about 10-fold less potent than the parent scFv 612 (Fig. 3B)
. Furthermore, the diabody blocks PlGF, a Flt-1-specific ligand, from binding to immobilized Flt-1 with an IC50 of approximately 8 nM, compared with that of 1.5 nM for scFv 612 (Fig. 3C)
. As expected, scFv p1C11 had no effects on Flt-1-VEGF and Flt-1-PlGF interaction, whereas scFv 612 had no effects on KDR-VEGF interaction.
|
. scFv p1C11 and scFv 612 effectively inhibited VEGF- and PlGF-induced cell migration (Fig. 4, B, C, E, and F)
. The two scFvs, however, showed a different efficacy pattern; scFv p1C11 is a stronger inhibitor of VEGF-induced cell migration, whereas scFv 612 is slightly more potent in inhibiting PlGF-induced cell migration. In contrast, the diabody is equally effective in blocking cell migration induced by VEGF and PlGF. Combination of both scFv p1C11 and scFv 612, either as a simple mixture or in the diabody format, demonstrated a more potent inhibitory effect than either scFv alone. It is noteworthy that neither scFv p1C11 nor scFv 612 alone was able to completely inhibit VEGF- or PlGF-induced cell migration, even at the highest antibody concentration tested (i.e., 200 nM). In contrast, combination of scFv p1C11 and scFv 612, either as a mixture or a diabody, completely abolished cell migration at an antibody concentration of 200 nM. A Fab fragment of C225, an antibody directed against EGF receptor, did not show significant inhibition of cell migration in this assay.
|
.
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
2000-fold reduced binding to Flt-1 is equally as potent as the wild-type VEGF in stimulating KDR activation and cell proliferation (26)
and in increasing vascular permeability and angiogenesis in vivo (27)
. The role of Flt-1 in tumor-associated angiogenesis, on the other hand, has remained controversial. Early studies demonstrated that Flt-1 is unable to generate a mitogenic response when stimulated by VEGF in both HUVECs and porcine aortic endothelial cells transfected with the receptor (24
, 25)
. A recent report, however, showed that tumor cells transfected with PlGF-2, a Flt-1-specific ligand, grew more slowly in Flt-1 kinase-deficient mice with less blood vessel formation, whereas VEGF-transfected tumor cells grew equally well in both wild-type and the Flt-1 kinase-deficient mice, implicating the involvement of Flt-1 in tumor-associated angiogenesis (28)
. In addition, increased levels of PlGF mRNA and VEGF-B mRNA, both Flt-1-specific ligands, have been detected in several human cancers, including melanoma, brain tumors, and carcinomas of kidney, lung, and colorectum (29
, 30)
. Taken together, these observations suggest that both KDR- and Flt-1-mediated signal transduction pathways may play a positive role under pathological conditions of angiogenesis. The importance of VEGF and its receptors in tumor angiogenesis suggests that blockade of this pathway by antibody therapy would be an effective therapeutic strategy for inhibiting angiogenesis and tumor growth (5 , 31) . We have produced an anti-KDR antibody, IMC-1C11, and demonstrated that this antibody is a potent inhibitor of VEGF-stimulated receptor activation and endothelial cell mitogenesis (7 , 18) . Furthermore, IMC-1C11 effectively inhibited high oxygen-induced vascular proliferative retinopathy in a newborn dog model4 and propagation of human leukemia in xenotransplanted NOD-SCID mice (32) . Recently, we produced a rat antibody directed against mouse Flt-1 and showed that the antibody has antiangiogenic and antitumor activity in xenograft models.5 An intriguing question that remained to be addressed was whether a dual blockade of both KDR and Flt-1 by a combination of antagonistic antibodies would result in an enhanced VEGF-inhibitory effect. Here we test the hypothesis by producing an anti-KDR x anti-Flt-1 bifunctional diabody recombinantly using the variable domains of both IMC-1C11 and FBK612, two monoclonal antibodies directed against human KDR and Flt-1, respectively, followed by a series of assays to determine the biological activities of the diabody.
As expected, the diabody retained its capacity to bind to both of its targets, Flt-1 and KDR. Furthermore, the diabody is capable of simultaneously binding to both Flt-1 and KDR and blocking the interaction between the receptors and their respective ligands, VEGF and PlGF. In the migration assay, both scFv p1C11 and scFv 612 inhibited VEGF-induced leukemia cell migration. This is consistent with the fact that both Flt-1 and KDR are involved in VEGF-induced cell migration, although the mechanisms by which the receptors respond to VEGF stimulation are different (33) . A recent report suggested that Flt-1 regulates cell migration by modulating actin reorganization, whereas KDR exert its effect by regulating cell adhesion via the assembly of vinculin in the focal adhesion plaque and tyrosine phosphorylation of focal adhesion kinase and paxillin (33) . It is interesting to note that the KDR-specific scFv p1C11 also inhibited PlGF-induced cell migration, although not as potently as Flt-1-specific scFv 612. On the other hand, the bifunctional diabody is an equally potent inhibitor of cell migration induced by both VEGF and PlGF. Combination of scFv p1C11 and scFv 612, either as a simple mixture or a diabody, is more efficacious than the individual parent antibodies in inhibiting cell migration. Taken together, these observations suggest that although cell migration-related cytoskeleton and adhesion molecules are regulated by distinct receptors, Flt-1 and KDR mediate the chemotactic effects of both VEGF and PlGF in a cooperative manner, not only through Flt-1-mediated actin reorganization and KDR-mediated vinculin assembly (both of which are required for cell migration) but also possibly through cross-talk between other components of the intracellular signal transduction pathways or, at the extracellular level, the formation of KDR/Flt-1 heterodimers.
Previous reports have demonstrated that KDR-mediated but not Flt-1-mediated signal transduction is primarily responsible for VEGF-stimulated DNA synthesis in HUVECs (7 , 18 , 24, 25, 26, 27) . Our results, which show that only the anti-KDR scFv p1C11 and not the anti-Flt-1 scFv 612 strongly inhibited VEGF-stimulated DNA synthesis, further confirmed the roles of each receptor in endothelial cell mitogenesis. It is noteworthy that, as seen in the migration assay, the bifunctional diabody is also a more potent inhibitor than either of its parent antibodies in neutralizing VEGF-stimulated endothelial cell mitogenesis. Kanno et al. (33) recently reported that a combination of anti-KDR and anti-Flt-1 antibodies was more effective in inhibiting both VEGF-stimulated phosphorylation of KDR and Flt-1 and DNA synthesis in HUVECs. VEGF induces the expression of Ets-1, a transcription factor that regulates angiogenesis by inducing expression of MMP-1, MMP-3, MMP-9, urokinase-type plasminogen activator, integrin ß3, and Flt-1 in endothelial cells (34, 35, 36) . An antibody to KDR, but not the antibody to Flt-1, inhibited VEGF-induced expression of Ets-1. However, the anti-Flt-1 antibody could significantly enhance the inhibitory effect of the anti-KDR antibody (33) . These observations suggest that KDR homodimer and KDR/Flt-1 heterodimer, but not the Flt-1 homodimer, are responsible for VEGF-mediated signal transduction that results in gene induction and DNA synthesis. Taken together, it appears that the enhanced effect of the anti-KDR x anti-Flt-1 bifunctional diabody on VEGF-stimulated DNA synthesis is likely due to a dual inhibition of both KDR homodimer and KDR/Flt-1 heterodimer-mediated signal transduction pathways.
Apart from VEGF and PlGF, several other growth factors related to VEGF have now been identified: (a) VEGF-B; (b) VEGF-C; (c) VEGF-D; and (d) VEGF-E (5) . VEGF-B, like PlGF, binds exclusively to Flt-1. VEGF-E is specific for KDR, whereas VEGF-C and VEGF-D can bind to KDR and another receptor, VEGFR-3 (Flt-4), which is expressed predominantly on lymphatic endothelium (for reviews, see Refs. 2 , 3 , and 5 ). In addition to their respective specific receptors, these ligands may form heterodimers that bind differentially to various receptor homo- or heterodimers and signal through different pathways. In theory, antibodies to an individual growth factor such as VEGF would only neutralize the angiogenic activity of the specific single ligand. In contrast, antagonistic antibodies to a VEGFR will block not only the angiogenic activity of VEGF but also that of other growth factors exerting their angiogenic effects via the receptor. For example, an anti-KDR antibody will potentially block angiogenic activity of VEGF, VEGF-C, VEGF-D, and VEGF-E, whereas an antibody to Flt-1 will inhibit the activity of VEGF, PlGF, and VEGF-B. Here we produced a bifunctional diabody that blocks both KDR and Flt-1, and we demonstrated that this diabody is a more potent inhibitor than the individual parent antibodies in neutralizing the chemotactic and mitogenic activities of both VEGF and PlGF. Our results suggest that dual receptor blockade with the bifunctional diabody is a more efficient approach to inhibiting VEGF-stimulated angiogenesis. Taken together, these results lend strong support for further evaluation of the bifunctional diabody as an antiangiogenic agent.
|
FOOTNOTES |
|---|
1 To whom requests for reprints should be addressed, at Department of Molecular and Cell Biology, ImClone Systems Inc., 180 Varick Street, New York, NY 10014. Phone: (646) 638-5190; Fax: (212) 645-2054; E-mail: zhenping{at}imclone.com 
2 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; KDR, kinase insert domain-containing receptor (or VEGFR-2); Flt-1, fms-like tyrosine kinase receptor (or VEGFR-1); PlGF, placenta growth factor; HUVEC, human umbilical vein endothelial cell; ScFv, single chain Fv; AP, alkaline phosphatase; VL, variable domain of antibody light chain; VH, variable domain of antibody heavy chain; HRP, horseradish peroxidase; EGF, epidermal growth factor; MMP, matrix metalloproteinase.
3 Y. Wu and D. Hicklin, unpublished data.
4 G. Lutty, D. S. McLeod, L. Witte, Z. Zhu, unpublished data.
5 Y. Wu and D. Hicklin, unpublished data.
Received 4/24/01. Accepted 8/13/01.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C.-Y. Xiong, A. Natarajan, X.-B. Shi, G. L. Denardo, and S. J. Denardo Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding Protein Eng. Des. Sel., August 1, 2006; 19(8): 359 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Shen, M. D. Vil, X. Jimenez, M. Iacolina, H. Zhang, and Z. Zhu Single Variable Domain-IgG Fusion: A NOVEL RECOMBINANT APPROACH TO Fc DOMAIN-CONTAINING BISPECIFIC ANTIBODIES J. Biol. Chem., April 21, 2006; 281(16): 10706 - 10714. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Jimenez, D. Lu, L. Brennan, K. Persaud, M. Liu, H. Miao, L. Witte, and Z. Zhu A recombinant, fully human, bispecific antibody neutralizes the biological activities mediated by both vascular endothelial growth factor receptors 2 and 3 Mol. Cancer Ther., March 1, 2005; 4(3): 427 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hoeben, B. Landuyt, M. S. Highley, H. Wildiers, A. T. Van Oosterom, and E. A. De Bruijn Vascular Endothelial Growth Factor and Angiogenesis Pharmacol. Rev., December 1, 2004; 56(4): 549 - 580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Lu, H. Zhang, D. Ludwig, A. Persaud, X. Jimenez, D. Burtrum, P. Balderes, M. Liu, P. Bohlen, L. Witte, et al. Simultaneous Blockade of Both the Epidermal Growth Factor Receptor and the Insulin-like Growth Factor Receptor Signaling Pathways in Cancer Cells with a Fully Human Recombinant Bispecific Antibody J. Biol. Chem., January 23, 2004; 279(4): 2856 - 2865. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Mac Gabhann and A. S. Popel Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H153 - H164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Jiang and J. R. Couchman Perlecan and Tumor Angiogenesis J. Histochem. Cytochem., November 1, 2003; 51(11): 1393 - 1410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Rakic, V. Lambert, L. Devy, A. Luttun, P. Carmeliet, C. Claes, L. Nguyen, J.-M. Foidart, A. Noel, and C. Munaut Placental Growth Factor, a Member of the VEGF Family, Contributes to the Development of Choroidal Neovascularization Invest. Ophthalmol. Vis. Sci., July 1, 2003; 44(7): 3186 - 3193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Hashiguchi, T. Nakashima, A. Nitani, T. Yoshihara, K. Yoshinaga, Y. Ito, Y. Maeda, and K. Sugimura Human Fc{varepsilon}RI{alpha}-Specific Human Single-Chain Fv (scFv) Antibody with Antagonistic Activity toward IgE/Fc{varepsilon}RI{alpha}-Binding J. Biochem., January 1, 2003; 133(1): 43 - 49. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
