Molecular Targets for Therapy (MTT)

Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity

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Abstract

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain ‘liquid’ tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC50 of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.

Introduction

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis.1,2,3 VEGF, the primary endothelial-specific mitogen, exerts its effects via two high-affinity tyrosine kinase receptor, VEGFR1 (or fms-like tyrosine kinase, Flt-1), and VEGFR2 (or kinase insert domain-containing receptor, KDR).1,2,3 Tumor inhibition has been achieved by using agents that either interrupt VEGF/VEGFR interaction and/or block the VEGF/VEGFR signal transduction pathway.4,5,6 In addition, emerging evidence suggest that angiogenesis may also play an important role in the growth of ‘liquid’ tumors such as leukemia and lymphomas.7,8 For example, several reports have demonstrated that there is increased angiogenesis in the bone marrow of patients with various hematological malignancies such as acute myeloid leukemia (AML) and multiple myeloma,9,10,11,12 along with increased expression of VEGFR.13,14 Also, leukemia patients with elevated plasma concentration of VEGF and/or cell surface expression of VEGFR are usually associated with disease progression and poor outcome.15,16,17 Studies on the molecular mechanisms underlying leukemia growth and propagation revealed that certain leukemia cells not only produce significant amounts of VEGF, but also express functional VEGFR on their cell surface, which results in the generation of an autocrine loop for leukemia cell growth.18,19,20,21 Taken together, these results suggest that antiangiogenic therapies that interfere with the VEGF/VEGFR pathway may represent novel approaches to effective treatment for certain leukemia.22,23,24 In fact, inhibition of leukemia growth has been achieved both in vitro and in vivo in animal models with the use of several different classes of ‘antiangiogenic’ agents, including anti-VEGF antibody,20 anti-KDR antibody18,25 and small molecular tyrosine kinase inhibitors.26,27

Monoclonal antibodies, owing to their high specificity towards a given target, represent a unique class of novel therapeutics as angiogenesis inhibitors.28,29 We previously produced a chimeric anti-KDR antibody, IMC-1C11, and demonstrated that this antibody is capable of blocking KDR/VEGF interaction and inhibiting VEGF-stimulated receptor activation and mitogenesis of human endothelial cells.30,31 In addition, IMC-1C11 inhibited VEGF-induced proliferation of human leukemia cells in vitro, and prolonged survival of nonobese diabetic-severe combined immune-deficient (NOD-SCID) mice inoculated with human leukemia cells.18 Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library.32 Compared to IMC-1C11, these human antibodies bind to KDR with higher affinity, and are more efficacious in inhibiting leukemia growth in the NOD-SCID mice model. We also observed a direct correlation between antigen-binding affinity of the anti-KDR antibodies and their biological activities both in vitro and in vivo.

Materials and methods

Proteins and cell lines

Primary-cultured human umbilical vein endothelial cells (HUVEC) were maintained in EBM-2 medium (Clonetics, Walkersville, MD, USA) at 37°C, 5% CO2. The soluble fusion proteins, KDR-alkaline phosphatase (AP) fusion, was expressed in stably transfected NIH 3T3 and purified from cell culture supernatants by affinity chromatography using immobilized monoclonal antibody to AP.33 VEGF165 protein was expressed in baculovirus and purified following the procedures described.33 Placenta growth factor (PlGF) was purchased from R & D Systems (Minneapolis, MN, USA). IMC-C225, a clinical grade chimeric antibody directed against human epidermal growth factor receptor (EGFR), and MAB612, a mouse antibody directed against human VEGFR1 (Flt-1), were produced at ImClone Systems Incorporated (New York, NY, USA). The leukemia cell lines, HEL and U937, were maintained in RPMI 1640 containing 10% fetal calf serum (FCS), whereas HL60 cells were maintained in RPMI 1640 containing 5% FCS.

Generation of anti-KDR antibodies

The chimeric anti-KDR antibody, IMC-1C11 (IgG1, κ), was produced in a stably transfected CHO cell line as previously described.31 The human anti-KDR antibody, IMC-2C6 (IgG1, κ), was constructed from a Fab fragment (Hu-2C6 Fab), originally isolated from a large human antibody phage display library34 by immunopanning against KDR-AP following the method previously described.30,32 The other human anti-KDR antibody, IMC-1121 (IgG1, κ), was constructed similarly from a Fab fragment (Hu-1121 Fab). Hu-1121 Fab was isolated by immunopanning against KDR under extremely stringent conditions using a library constructed by shuffling the antibody heavy chain of Hu-2C6 Fab against the entire antibody light-chain repertoire obtained from the original large phage library (D Lu et al, manuscript in preparation). Both IMC-2C6 and IMC-1121 were produced in stably transfected NS0 cell lines grown under serum-free conditions, and were purified from batch cell culture using Protein A affinity chromatography. The purity of the antibody preparations were analyzed by SDS-PAGE, and the concentrations were determined by ELISA, using an anti-human Fc antibody as the capturing agent and an anti-human κ chain antibody–horseradish peroxidase (HRP) conjugate as the detection agent. A clinical grade antibody, IMC-C225, was used as the standard for calibration. The endotoxin level of each antibody preparations was examined to ensure the products were free of endotoxin contamination.

Quantitative KDR binding and blocking assay

In the direct binding assay, various amounts of antibodies were added to KDR-coated 96-well Maxi-sorp microtiter plates (Nunc, Roskilde, Denmark) and incubated at room temperature (RT) for 1 h, after which the plates were washed three times with PBS containing 0.1% Tween-20. The plates were then incubated at RT for 1 h with 100 μl of a rabbit anti-human IgG Fc-HRP conjugate (Jackson ImmunoResearch Laboratory Inc., West Grove, PA, USA). The plates were washed and developed following a procedure previously described.30,32. Two competitive assays were used to examine the efficacy of the anti-KDR antibodies in blocking KDR from binding to VEGF. In the first ELISA-based assay, various amounts of antibodies were mixed with a fixed amount of KDR-AP (100 ng) and incubated at RT for 1 h. The mixture were then transferred to 96-well microtiter plates precoated with VEGF165 (200 ng/well) and incubated at RT for an additional 2 h, after which the plates were washed 5 times and the substrate for alkaline phosphatase (AP) (p-nitrophenyl phosphate, Sigma) was added, followed by reading the absorbance at 405 nm to quantify the bound KDR-AP molecules.30,32 IC50, that is, the antibody concentration required for 50% inhibition of KDR binding to VEGF, was then calculated. In the second assay, a cell-based radioimmunoassay, various amounts of anti-KDR antibodies were mixed with a fixed amount (2 ng) of 125I-labeled VEGF165 (R & D Systems) and added to an 80–90% confluent monolayer of HUVEC grown in a 96-well microtiter plate. The plate was incubated at RT for 2 h, washed five times with cold PBS, and the amounts of radioactivity that bound to the endothelial cells were counted.

Antibody affinity determination by BIAcore analysis

The binding kinetics, that is, the association rate constant (kon) and the dissociation rate constant (koff), of the anti-KDR antibodies were measured using a BIAcore biosensor (Biacore, Inc., Uppsala, Sweden) following the procedure previously described.30,32 The affinity constant, kd was calculated from the ratio of rate constants koff/kon.

Endothelial cell mitogenesis assay

The HUVEC mitogenic assay was carried out following a protocol previously described.30,32 Briefly, various amounts of the anti-KDR antibodies were first preincubated with growth factor-starved HUVEC (5 × 103 cells/well) at 37°C for 1 h, after which VEGF165 was added to a final concentration of 16 ng/ml. After 18 h of incubation, 0.25 μCi of [3H]-TdR (Amersham) was added to each well and incubated for an additional 4 h. The cells were washed, harvested, and DNA incorporated radioactivity was determined on a scintillation counter (Wallach, Model 1450 Microbeta Liquid Scintillation Counter).

Expression of VEGF and VEGFR by leukemia cell lines

Expression of VEGF and KDR by human leukemia cell lines, HL60, HEL and U937, was first examined by RT-PCR following protocols previously described.18 The following primers were used to amplify VEGF, Flt-1, KDR and the internal control, α-actin: VEGF forward: 5′-IndexTermTCGGGCCTCCGAAACCATGA-3′, and reverse: 5′-IndexTermCCTGGTGAGAGATCTGGTTC-3′; Flt-1 forward: 5′-IndexTermTTTGTGATTTTGGCCTTGC-3′, and reverse, 5′-IndexTermCAGGCTCATGAACTTGAAAGC-3′, KDR forward: 5′-GTGACCAACATGGA GTCGTG-3′, and reverse, 5′-IndexTermCCAGAGATTCCATGCCACTT-3′, α-actin forward: 5′-IndexTermTCATGTTTGAGACCTTCAA-3′, and reverse, 5′-IndexTermGTCTTTGCGGATGTCCACG-3′. The PCR products were analyzed on a 1% agarose gel.

We further assayed VEGF production by the three leukemia cell lines cultured under either 10% FCS or serum-free conditions. The leukemia cells were collected, washed with plain RPMI 1640 medium and seeded in 24-well plates at a density of 5 × 104/ml, with or without the addition of 10% FCS. The cells were cultured at 37°C for 72 h, after which total numbers of cells were counted using a Coulter counter (Model Z1, Coulter Electronics Ltd., Luton, England) and the VEGF concentration in the supernatant was determined using an ELISA kit (Biosource International, Camarillo, CA, USA).

Leukemia cell migration assay

Leukemia cells were washed three times with serum-free plain RPMI 1640 medium and suspended in the medium at 1 × 106/ml. Aliquots of 100 μl cell suspension were added to either 3-μm-pore transwell inserts (for HL60 cells), or 8-μm-pore transwell inserts (for HEL and U937 cells) (Costar®, Corning Incorporated, Corning, NY, USA) 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, 5% CO2 for 16–18 h for HL60 cells, or for 4 h for HEL and U937 cells. Migrated cells were collected from the lower compartments and counted with a Coulter counter.

Leukemia growth in vivo in NOD-SCID mice

Age-(6 to 8-week-old) and sex-matched female NOD-SCID mice were used in all the experiments. The mice were irradiated with 3.5 Gy from a 137Cs gamma-ray source at a dose rate of 0.9 Gy/min and intravenously inoculated with 2 × 107 HL60 cells. At 3 days after tumor inoculation, groups of 7–9 mice were treated twice weekly with various doses of IMC-1C11, IMC-2C6 or IMC-1121 via intraperitoneal injection. Mice were observed daily for signs of toxicity and recorded for time of survival. For statistic analysis, the nonparametric one-tailed Mann–Whitney rank sum test was used.

Results

KDR binding and KDR/VEGF blocking by the anti-KDR antibodies

The antigen-binding efficiency of the anti-KDR antibodies was determined by ELISA on immobilized receptor. The antibodies bind to KDR in a dose-dependent manner, with IMC-1121 being the strongest binder (Figure 1a). All three antibodies also strongly blocked KDR from binding to its ligand, VEGF (Figure 1b). Unlike their varying binding efficiency, the three antibodies blocked KDR/VEGF interaction with similar potency. The IC50 is approximately 0.8–1.0 nM for all three antibodies. The control antibody, IMC-C225, an antibody directed against human EGFR, does not bind KDR, nor block KDR/VEGF interaction.

Figure 1
figure1

Binding to KDR and blocking KDR/VEGF interaction by anti-KDR antibodies. (a) Dose-dependent binding of the anti-KDR antibodies to immobilized KDR. Various amounts of antibodies were added to 96-well plates coated with KDR (1.0 μg/ml) and incubated at RT for 1 h, after which the plates were incubated with a mouse anti-human Fc-HRP conjugate. The plates were washed, peroxidase substrate was added, and A450 nm was read. (b) Inhibition of binding of KDR to immobilized VEGF by the anti-KDR antibodies. Various amounts of antibodies were incubated with a fixed amount of KDR-AP in solution at RT for 1 h, after which the mixtures were transferred to 96-well plates coated with VEGF. The amount of KDR-AP that bound to the immobilized VEGF was quantified by incubation of the plates with AP substrate and reading of A405 nm. Data points are the means±s.d. of triplicate determinations and are representative of at least three separate experiments.

The kinetics of binding between antibodies to KDR were determined by surface plasmon resonance on a BIAcore instrument (Table 1). As seen previously, IMC-1C11 binds to immobilized KDR with an affinity (Kd) of 0.27 nM, about five-fold higher than its Fab counterpart. IMC-2C6 possesses an affinity of 0.2 nM, which is 18-fold higher than that of the monovalent Hu-2C6 Fab, mainly because of an improvement in the off-rate. Affinity maturation of Hu-2C6 led to Hu-1121 Fab with a 33-fold improvement in binding affinity (from 3.6 to 0.11 nM). However, unlike Hu-2C6, converting Hu-1121 Fab into bivalent IgG, IMC-1121, only resulted in two-fold increase in overall binding avidity.

Table 1 Binding kinetics of anti-KDR antibodies

Inhibition of VEGF-stimulated mitogenesis of HUVEC

The three anti-KDR antibodies were examined for their efficacy in blocking VEGF from binding to cell-surface-expressed KDR and in neutralizing VEGF-stimulated mitogenesis of human endothelial cells. As shown in Figure 2a, all three antibodies compete efficiently with radiolabeled VEGF for binding to HUVEC. The antibodies also blocked VEGF-stimulated HUVEC mitogenesis in a dose-dependent manner (Figure 2b). IMC-1121, the antibody with the highest affinity, is the most efficacious inhibitor with an ED50, that is, the concentration that results in 50% of inhibition of [3H]-TdR incorporation, of 0.7 nM, in comparison to that of 1.5 nM for both IMC-1C11 and IMC-2C6. It is noteworthy that at higher antibody concentrations (>2.5 nM), however, IMC-2C6 consistently yielded lower inhibition than did both IMC-1121 and IMC-1C11 (Figure 2b).

Figure 2
figure2

(a) Competitive blocking of radiolabeled VEGF from binding to endothelium surface-expressed KDR with anti-KDR antibodies. Various amounts of anti-KDR antibodies were mixed with 2 ng of 125I-labeled VEGF165 and added to an 80–90% confluent monolayer of HUVEC grown in a 96-well microtiter plate. The plate was incubated at RT for 2 h, washed with cold PBS, and the amounts of radioactivity that bound to the endothelial cells were counted. Data represent the means±s.d. of triplicate determinations. (b) Inhibition of VEGF-induced HUVEC mitogenesis by the human anti-KDR antibodies. Various amounts of anti-KDR antibodies were added to growth factor-straved HUVEC and incubated at 37°C for 1 h, after which VEGF was added to the wells to a final concentration of 16 ng/ml. After 18 h of incubation, 0.25 μCi of [3H]-TdR was added to each well and incubated for an additional 4 h. The cells were harvested and DNA incorporated radioactivity was determined with a scintillation counter. Controls included HUVEC incubated with plain medium in the presence or absence of VEGF. Data represent the means of duplicate determinations and are representative of at least three separate experiments.

Expression of VEGF and KDR by leukemia cells

We examined VEGF and KDR expression, by RT-PCR, in three myeloid leukemia cell lines including HL60 (promyelocytic), HEL (megakaryocytic) and U937 (histiocytic). As shown in Figure 3, all three cell lines are positive for VEGF, and HL60 and HEL, but not U937, are also positive for KDR expression. The three cell lines are also positive for Flt-1 expression as detected by RT-PCT (not shown). The leukemia cells secrete significant amount of VEGF when culture in vitro (Figure 3). It is noteworthy that both HL60 and U937 cells produced more VEGF under serum-starving conditions.

Figure 3
figure3

Expression of KDR and VEGF by human leukemia cell lines. (Top panel) cDNA was synthesized from total RNA extracted from serum-starved leukemia cells. PCR was carried out according to conditions described in Materials and methods. Lane 1, molecular weight standards. From top to bottom: 1000, 850, 650, 500 and 400 bp, respectively; lane 2, negative control (no cDNA added); lane 3, HL60 cells; lane 4, HEL cells; lane 5, U937 cells; lane 6, HUVEC. (Bottom panel) Secretion of VEGF by human leukemia cells cultured in 10% FCS or serum-free conditions. Data shown are mean±s.e. of two independent experiments of triplicate determinations.

Inhibition of VEGF-induced leukemia cell migration

As seen previously, all three leukemia cell lines, HL60, HEL and U937, migrate in response to the chemotactic activity of VEGF (Figure 4). Incubation with anti-KDR antibodies inhibited, in a dose-dependent manner, VEGF-induced migration of HL60 and HEL cells (Figure 4a and b), but had no effect on migration of U937 cells that do not express KDR (Figure 4c). The VEGF-induced migration of U937 cells was, however, efficiently inhibited by an anti-human Flt-1 antibody, MAB 612 (Figure 4c). There is no significant difference in the potency between the three anti-KDR antibodies in inhibiting VEFG-induced migration of both HL60 and HEL cells. As expected, the anti-EGFR antibody, IMC-C225, showed no effect on VEGF-induced migration of human leukemia cells.

Figure 4
figure4

Inhibition of VEGF-stimulated migration of human leukemia cells by anti-KDR antibodies. The leukemia cells were first incubated in the inserts with various concentrations of the anti-KDR antibodies for 30 min at 37°C, after which the inserts were placed into the wells of 24-well plates containing 0.5 ml of serum-free RPMI 1640 medium in the presence of VEGF at 200 ng/ml. The migration was carried out at 37°C, 5% CO2 for 4 h. Migrated cells were collected from the lower compartments and counted with a Coulter counter. Each bar represents the mean±s.d. of triplicate determinations.

Inhibition of leukemia growth in vivo with anti-KDR antibodies

We first examined the antileukemia effect of IMC-1C11 in vivo in NOD-SCID mice inoculated with HL60 cells. In this experiment, each mouse received an intravenous injection of 5 × 106 HL60 cells, and was treated 3 day later with IMC-1C11 via intraperitoneal injections. As shown in Figure 5a, IMC-1C11 prolonged the survival of NOD-SCID mice in a dose-dependent fashion, with 50% of the mice treated with the highest dose of the antibody (800 μg) survived over 100 days. All mice eventually died, however, of leukemia as confirmed by postmortem histopathological examinations (not shown, also see refs 18 and 25).

Figure 5
figure5

Inhibition of leukemia growth in vivo in NOD-SCID mice by anti-KDR antibodies. (a) Sublethally irradiated NOD-SCID mice were inoculated intravenously with 5 × 106 HL60 cells. At 3 days after tumor inoculation, groups of six mice were treated twice weekly with various doses of IMC-1C11 via intraperitoneal injection. Mice were observed daily for signs of toxicity and recorded for time of survival. (b) Sublethally irradiated NOD-SCID mice were inoculated intravenously with 2 × 107 HL60 cells. At 3 days after tumor inoculation, groups of 7–9 mice were treated twice weekly with various doses of IMC-1C11, IMC-2C6 or IMC-1121 via intraperitoneal injection. Mice were observed daily for signs of toxicity and recorded for time of survival.

In the second experiment, each mouse was inoculated with 20 × 106 HL60 cells, followed by treatment with IMC-1C11, IMC-2C6 or IMC-KDR-1121 at different doses. All untreated mice died within 17 days (Figure 5b, mean time of survival, 14±3 days), much earlier than those mice inoculated with 5 × 106 HL60 cells (Figure 5a). At this high tumor load, treatment with IMC-1C11 at 200 μg/mouse/injection moderately increased the survival, but all mice died within 35 days (mean±s.d. and the median time of survival, 21±7 and 19 days, respectively. P=0.03 compared to the control group). IMC-2C6, given at the same dose of 200 μg/mouse/injection, significantly prolonged the mouse survival to 34±12 days (median=29 days. P<0.01 compared to the control and P=0.01 compared to the IMC-1C11-treated group). The antibody with the highest affinity, IMC-1121, demonstrated a much stronger antileukemia effect than did IMC-1C11 and IMC-2C6; the mice treated with IMC-1121 survived 63±12 days (median=60 days. P<0.01 compared to both IMC-1C11and IMC-2C6-treated group). At a lower antibody dose tested, 100 μg/mouse/injection, IMC-1121 was also more efficacious than IMC-2C6; mice treated with IMC-1121 survived 46±16 days (median=41 days) compared to that of 29±11 days (median=26 days, P=0.03) in mice treated with IMC-2C6. No overt toxicities were observed in any of the antibody-treated animals throughout the course of the experiment. As seen previously, none of the antibody treatments, however, resulted in long-term disease-free survival of the leukemia-inoculated NOD-SCID mice.

Discussion

VEGF is a member of an enlarging growth factor family that also includes PIGF, VEGF-B, VEGF-C and VEGF-D. Of the three structurally related kinase receptors, VEGF binds and exerts its biological activity via both Flt-1 and KDR, whereas the activities of PIGF/VEGF-B and VEGF-C/VEGF-D are mainly mediated through Flt-1 and VEGFR3 (also known as Flt-4), respectively.1,2,3 Accumulating evidence suggest that of the two VEGF receptors, KDR is the major receptor that mediates the biological activities of VEGF including induction of cell proliferation, migration, differentiation, promotion of tube formation, increase of vascular permeability and maintenance of vascular integrity.1,2,3 On the other hand, although Flt-1 has been implicated in VEGF and PIGF-induced cell migration, its functional role in angiogenesis in adult remained most unclear. Several recent reports have demonstrated that, however, Flt-1 may play an important role in promoting hematopoietic stem cell (HSC) mobilization35 and angiogenesis in both ischemic tissues36 and tumors.37 Taken together, these results suggest that both KDR and Flt-1 may represent good targets for developing pro- and angiogenic therapeutics.

In our previous studies, we have demonstrated that a neutralizing rat anti-mouse VEGFR2 antibody, DC101, effectively blocked VEGF-induced receptor signaling and endothelial cell mitogenesis, and inhibited growth of a variety of solid tumors, both syngeneic and human xenografts, in vivo via an antiangiogenic mechanism.38,39 Other studies have shown that inhibition of solid tumor growth in animal models could also be achieved with anti-VEGF antibodies,40,41 small molecular inhibitors to KDR tyrosine kinase,42,43 and ribozymes to KDR and Flt-1.44 In addition to their role in solid tumors, VEGF and its receptors are being increasingly implicated in the tumorigenesis and propagation of certain ‘liquid’ tumors including leukemia and lymphomas.7,8,9,10,11,12,13,14 It is well established that leukemia originate from HSC at different stages of their maturation and differentiation. A recent report has shown that VEGF may regulate HSC survival by an autocrine mechanism via the surface-expressed VEGFR.45 Since both Flt-1 and KDR have been shown expressed on HSC,35,46 it remains a subject of further investigation whether a single receptor or both are responsible for the autocrine signaling. Similarly, several studies have shown that a number of leukemia cell lines as well as primary leukemia cells from certain percentage of AML patients not only secrete VEGF, but also express on their cell surface functional VEGFR.18,19,20,21 VEGF stimulation of these VEGFR+, particularly, KDR+ leukemia cells resulted in receptor phosphorylation, cell migration and proliferation, 18 indicating the existence of an autocrine loop. In this report, we demonstrated that treatment with anti-KDR antibodies strongly inhibited VEGF-stimulated migration of KDR+ leukemia cells in vitro (Figure 4) and significantly prolonged the survial of leukemia-inoculated mice (Figure 5). It is noteworthy that the Flt-1-specific antibody, MAB 612, also inhibited VEGF-induced migration of HL60 and HEL cells (both are KDR+/Flt-1+) (Figure 4). We have previously shown that anti-KDR antibodies were efficacious inhibitors of PIGF (a Flt-1 specific ligand)-induced migration of HL60 and HEL cells.47 A recent report suggested that Flt-1 regulates cell migration through modulating actin reorganization, whereas KDR exerts 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.48 A dual functional antibody, obtained by combining both anti-KDR and anti-Flt-1 antibodies through genetic engineering, was more potent in inhibiting both VEGF-and PlGF-induced cell migration than either individual antibody.47 Taken together, these observations suggest that, in KDR/Flt-1 coexpressing leukemia cells, the two receptors mediate the chemotatic effects of 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 crosstalk between other components of the intracellular signal transduction pathways, or at the extracellular level, the formation of KDR/Flt-1 heterodimers. Since all the anti-KDR antibodies are human receptor (KDR)-specific, that is, they do not crossreact with murine VEGFR2 on mouse vasculature and do not directly inhibit tumor-induced angiogenesis in mice, the in vivo antileukemia activity of these antibodies is likely because of a direct inhibition of leukemia growth via blockade of the VEGF/KDR autocrine loop in human leukemia cells.

The anti-KDR antibody therapy did not, however, cure any of the leukemia-inoculated mice, suggesting, in addition to the autocrine loop, the existence of other pathways that support leukemia growth and propagation in vivo. One of these potential pathways may involve the paracrine mechanism between human leukemia cells and the host endothelial cells: the elevated blood VEGF level caused by the proliferating leukemia cells may stimulate host angiogenesis via mouse VEGFR2, and promote endothelial cells to secrete more hematopoietic growth factors, such as GM-CSF and IL6, which in turn support further leukemia growth.18,24,25 Anti-KDR antibodies may not affect this paracrine loop because they do not block VEGFR2 expressed on mouse endothelial cells. In support of this notion, combinational therapy using both anti-KDR (human leukemia-specific) and anti-murine VEGFR2 (host endothelium-specific) antibodies, aiming to simultaneously block both the autocrine and the paracrine mechanisms, resulted in long-term survival (>200 days) of 40% of human leukemia-inoculated NOD-SCID mice.25 Apart from leukemia, accumulating evidence suggest that the dual autocrine/paracrine mechanism may also play an important role in the growth and metastasis of certain solid tumors. For example, the VEGF/VEGFR autocrine loop was proposed in mediating growth and metastasis of several types of tumors, including carcinomas of prostate,49 ovaries,50 pancreas,51 breast52 and melanoma.53 The VEGFR-expressing tumor cells bind radiolabeled VEGF and respond to VEGF stimulation by undergoing receptor phosphorylation, cell proliferation and cell migration/invasion.49,51 Taken together, these observations suggest that effective therapies of VEGF-producing/VEGFR-expressing tumors may require efficient blockade of both autocrine (antitumor) and paracrine (antiangiogenesis) mechanisms. Anti-KDR antibodies, owing to their capability in blocking receptor expressed on both human endothelial and tumor cells, may have great clinical potential in the treatment of both solid tumors and leukemia via dual antiangiogenic and antitumor mechanisms.

One of the major obstacles in developing antibody-based therapeutics has been the immunogenicity of the nonhuman-derived MAb.28,29 Technological development in molecular engineering of antibodies, plus the availability of human antibody phage display libraries and human antibody transgenic mice, has enabled us to tailor-make human or human-like antibodies along with other desired characteristics, such as size and valency, to suit the intended applications.54,55 Here we produced two fully human anti-KDR antibodies from Fab fragments originally isolated from a large antibody phage display library. The human antibodies bind to KDR with high affinity and efficiently neutralized the biological activities of VEGF at nanomolar concentrations. These human antibodies should be much less immunogenic than those of nonhuman origin, and therefore be well tolerated in human therapy. It is further noteworthy that, although all three antibodies showed similar potency in blocking KDR binding to VEGF in vitro, the antibody with the highest affinity, IMC-1121, demonstrated the most potent biological activity in inhibiting VEGF-stimulated HUVEC mitogenesis and in prolonging survial of the leukemia-inoculated mice. Antibodies with higher affinity are likely able to bind more tightly to their targets (receptors) for a prolonged period of time, thus efficiently preventing the growth factors, such as VEGF, from interacting with their receptors. In this regard, IMC-1121 represents the preferable choice for further development as antiangiogenic therapeutics.

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Acknowledgements

This work was supported in part by grants from Leukemia and Lymphoma Society and American Cancer Society to S. R.

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Correspondence to Z Zhu.

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Keywords

  • angiogenesis
  • leukemia
  • VEGF/KDR
  • autocrine tumor growth
  • human antibody
  • antibody affinity
  • antibody engineering

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