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Biomedical subjects

Zhenping Zhu

Publications and source records attributed to Zhenping Zhu.

At least 55 records · Page 3Linked to original sources
PubMed2004

Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C.

In this report we utilize a novel antagonist antibody to the human VEGFR-3 to elucidate the role of this receptor in in vitro tubular morphogenesis of bovine and human endothelial cells (EC cells) induced by VEGF-C. The antibody hF4-3C5 was obtained by panning a human phage display library on soluble human VEGFR-3. The binding affinity constant of hF4-3C5 significantly exceeds that of the interaction of VEGFR-3 with VEGF-C. hF4-3C5 strongly inhibits the binding of soluble VEGFR-3 to immobilized VEGF-C and abolishes the VEGF-C-mediated mitogenic response of cells that expresses a chimeric human VEGFR-3-cFMS receptor. In fluorescence experiments, hF4-3C5 reactivity is observed with human lymphatic endothelial cells (LECs) and human umbilical vein endothelial cells (HUVECs). Binding of hF4-3C5 shows that about half of bovine aortic endothelial (BAE) cells express VEGFR-3 and cells in this subpopulation are primarily responsible for the chemotactic response to the mature form of VEGF-C (VEGF-C(DeltaNDeltaC)). This response was strongly inhibited by the addition of hF4-3C5. In vitro tube formation by BAE cells induced by VEGF-C(DeltaNDeltaC) was reduced by greater than 60% by hF4-3C5 whereas the response to VEGF(165) was unaffected. Addition of hF4-3C5 together with an antagonist antibody to VEGFR-2 completely abolished the response to VEGF-C(DeltaNDeltaC). Similar results were obtained with HUVECs. Together, these findings point to a role for VEGFR-3 in vascular tubular morphogenesis and highlight the utility of hF4-3C5 as a tool for the investigation of the biology of VEGFR-3.

Animals↗
PubMed2004

Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody.

FMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, is expressed at high levels in the blasts of approximately 90% of patients with acute myelogenous leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and point mutations in the kinase domain of FLT3 are found in approximately 37% of AML patients and are associated with a poor prognosis. We report here the development and characterization of a fully human anti-FLT3 neutralizing antibody (IMC-EB10) isolated from a human Fab phage display library. IMCEB10 (immunoglobulin G1 [IgG1], kappa) binds with high affinity (KD=158 pM) to soluble FLT3 in enzyme-linked immunosorbent assay (ELISA) and to FLT3 receptor expressed on the surfaces of human leukemia cell lines. IMC-EB10 blocks the binding of FLT3 ligand (FL) to soluble FLT3 in ELISA and competes with FL for binding to cell-surface FLT3 receptor. IMC-EB10 treatment inhibits FL-induced phosphorylation of FLT3 in EOL-1 and EM3 leukemia cells and FL-independent constitutive activation of ITD-mutant FLT3 in BaF3-ITD and MV4;11 cells. Activation of the downstream signaling proteins mitogen-activated protein kinase (MAPK) and AKT is also inhibited in these cell lines by antibody treatment. The antibody inhibits FL-stimulated proliferation of EOL-1 cells and ligand-independent proliferation of BaF3-ITD cells. In both EOL-1 xenograft and BaF3-ITD leukemia models, treatment with IMC-EB10 significantly prolongs the survival of leukemia-bearing mice. No overt toxicity is observed with IMC-EB10 treatment. Taken together, these data demonstrate that IMC-EB10 is a specific and potent inhibitor of wild-type and ITD-mutant FLT3 and that it deserves further study for targeted therapy of human AML.

Animals↗
PubMed2004

Inhibition of human B-cell lymphoma by an anti-CD20 antibody and its chimeric F(ab')2 fragment via induction of apoptosis.

Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully used in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. Despite all clinical success the exact mechanisms of action of various anti-CD20 antibodies remains mostly unclear. Several mechanisms have been proposed to be responsible for the therapeutic activity of anti-CD20 antibodies, including antibody-dependent cell-mediated cytotoxicity, complement-mediated cytotoxicity, and direct inhibition of tumor growth via induction of apoptosis. We previously produced an anti-CD20 mAb, HI47, and showed that the antibody effectively blocked human B-cell proliferation in vitro and inhibited xenografted B-cell lymphoma in nude mice. In this study, we engineered the chimeric versions of both the Fab and F(ab)'2 fragments of HI47 and produced the fragments in E. coli. Both fragments competed efficiently with HI47 for binding to CD20+ B cells, and inhibited proliferation of B-lymphoma cells in a dose-dependent manner. Mechanistic studies revealed that both antibody fragments induced significant degree of B-cell apoptosis that is independent of any cross-linking agents. Further, both the F(ab)'2 and Fab fragments when administered in vivo significantly inhibited the growth of human B-cell lymphoma xenografts in nude mice. The bivalent F(ab)'2 fragment showed consistently better efficacy compared to its monovalent Fab counterpart in inducing apoptosis and inhibiting B-cell lymphoma growth both in vitro and in vivo. Taken together, these observations suggest that HI47 and its fragments most likely exert their antitumor activity through induction of cell apoptosis, and cross-linking/dimerization of CD20 molecules on B- cell surface is an important, but not essential, process for therapeutic efficacy of HI47 and its fragments.

Animals↗
PubMed2004

Inhibition of both the autocrine and the paracrine growth of human leukemia with a fully human antibody directed against vascular endothelial growth factor receptor 2.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Here we show that certain "liquid" tumors such as acute myeloid leukemia not only produce VEGF but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. In addition, the leukemia-derived VEGF can also stimulate the production of growth factors, including interleukin 6 (IL6) and granulocyte-macrophage colony stimulating factor (GM-CSF), by human endothelial cells, which in turn further promotes the growth of leukemia cells (the paracrine loop). A fully human anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-2C6, strongly blocks KDR/VEGF interaction and neutralizes VEGF-stimulated activation of KDR in endothelial cells. In a system where leukemia cells are co-cultured with endothelial cells, IMC-2C6 inhibits both the production of IL6 and GM-CSF by endothelial cells and the growth of leukemia cells. Finally, IMC-2C6 effectively blocks VEGF-induced migration of KDR+ human leukemia cells, and when administered in vivo, significantly prolonged survival of mice inoculated with KDR+ human leukemia cells. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and certain types of leukemia.

Animals↗
PubMed2004

Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed.

Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.

Animals↗
PubMed2003

A fully human monoclonal antibody to the insulin-like growth factor I receptor blocks ligand-dependent signaling and inhibits human tumor growth in vivo.

The insulin-like growth factor I receptor (IGF-IR) is overexpressed in many diverse tumor types and is a critical signaling molecule for tumor cell proliferation and survival. Therapeutic strategies targeting the IGF-IR may therefore be effective broad-spectrum anticancer agents. Through screening of a Fab phage display library, we have generated a fully human antibody (A12) that binds to the IGF-IR with high affinity (4.11 x 10(-11) M) and inhibits ligand binding with an IC(50) of 0.6-1 nM. Antibody-mediated blockade of ligand binding to the IGF-IR inhibited downstream signaling of the two major insulin-like growth factor (IGF) pathways, mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/Akt, in MCF7 human breast cancer cells. As a result, the mitogenic and proliferative potential of IGF-I and IGF-II were significantly reduced. A12 did not block insulin binding to the insulin receptor but could block binding to atypical IGF-IR in MCF7 cells. In addition, A12 was shown to induce IGF-IR internalization and degradation on specific binding to tumor cells, resulting in a significant reduction in cell surface receptor density. In xenograft tumor models in vivo, IGF-IR blockade by A12 was shown to occur rapidly, resulting in significant growth inhibition of breast, renal, and pancreatic tumors. Histological analysis of tumor sections demonstrated a marked increase in apoptotic tumor cells in antibody-treated animals. These results demonstrate that A12 possesses strong antitumor activity in vitro and in vivo and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on IGF-IR signaling for growth and survival.

Animals↗
PubMed2003

Monoclonal antibody therapeutics and apoptosis.

The potential for disease-specific targeting and low toxicity profiles have made monoclonal antibodies attractive therapeutic drug candidates. Antibody-mediated target cell killing is frequently associated with immune effector mechanisms such as antibody-directed cellular cytotoxicity, but they can also be induced by apoptotic processes. Antibody-directed mechanisms, including antigen crosslinking, activation of death receptors, and blockade of ligand-receptor growth or survival pathways, can elicit the induction of apoptosis in targeted cells. Depending on their mechanism of action, monoclonal antibodies can induce targeted cell-specific killing alone or can enhance target cell susceptibility to chemo- or radiotherapeutics by effecting the modulation of antiapoptotic pathways. This review will focus on the mechanisms by which antibodies are capable of eliciting programmed cell death either directly or indirectly within tumor cells.

Animals↗
PubMed2003

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.

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. Here we constructed and produced two fully human recombinant bispecific antibodies (BsAb) that target both EGFR and IGFR, using two neutralizing human antibodies originally isolated from a phage display library. The BsAb not only retained the antigen binding capacity of each of the parent antibodies, but also were capable of binding to both targets simultaneously as demonstrated by a cross-linking enzyme-linked immunosorbent assay. Furthermore, the BsAb effectively blocked both ligands, EGF and IGF, from binding to their respective receptors, and inhibited tumor cell proliferation as potently as a combination of both the parent antibodies. More importantly, the BsAb were able to completely block activation of several major signal transduction molecules, including Akt and p44/p42 MAP kinases, by both EGF and IGF, whereas each individual parent antibody was only effective in inhibiting those signal molecules activated by the relevant single growth factor. The BsAb molecules retained good antigen binding activity after incubation with mouse serum at 37 degrees C for up to 6 days. Taken together, our results underscore the benefits of simultaneous targeting multiple growth factor receptor pathways for more efficacious cancer treatment. This report describes the first time use of a recombinant BsAb for targeting two tumor-associated molecules on either a single or adjacent tumor cells for enhanced antitumor activity.

Antibodies, Bispecific↗
PubMed2003

FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells.

The FLT3 receptor tyrosine kinase is highly expressed in most acute leukemias and frequently mutated in acute myeloid leukemia (AML). The mutated form of the receptor is constitutively activated and known to play an important role in AML, but the activation state of the overexpressed wild-type (wt) receptor is, at present, unknown. In this study, we examined the activation state of the wild-type receptor in AML. We found that the wild-type receptor was constitutively phosphorylated/activated in 8 of 12 primary AML samples and 4 of 13 leukemia cell lines. To explain why wtFLT3 is often activated, we investigated the expression of its ligand, FL, by these same cells. Coexpression of FL with FLT3 was a universal finding in both primary AML samples and leukemic-derived cell lines. To further prove that autocrine signaling was accounting for the activation, we showed that conditioned media but not fresh media was able to activate FLT3. In addition, an antibody that blocks binding of ligand to the receptor blocks FLT3 activation. Finally, depletion of FL from conditioned media is able to block the activation of FLT3. Taken together, these findings represent strong evidence that wtFLT3 is often constitutively activated in AML and thus, like its mutated form, might contribute to the altered signaling that characterizes leukemogenesis.

Base Sequence↗
PubMed2003

Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity.

Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We previously identified several fully human neutralizing anti-VEGF receptor 2 (or kinase inserting domain-containing receptor (KDR)) antibodies from a large antibody phage display library. These antibodies bind specifically to KDR, block VEGF/KDR interaction, and inhibit VEGF-induced proliferation of human endothelial cells and migration of KDR+ leukemia cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original naïve human library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for KDR. However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to KDR (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked VEGF/KDR interaction with an IC50 of approximately 1 nm, compared with that of 3-4 nm for the parent Fab fragments. Further, clone 1121 was more potent in inhibiting VEGF-stimulated KDR phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of KDR. Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are cysteine-constrained, whereas clone 1121 binds to both cysteine-constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides also cross-react with clone 1121, but none of the clone 1121-specific peptides binds to 2C6, indicating that clone 1121 retained part of the original binding epitope(s) of 2C6 while gaining new binding specificity. Taken together, our observation suggests that clone 1121 may have great clinical potential in anti-angiogenesis therapy. It further underscores the efforts to identify antibodies of high affinity for enhanced biological activities.

Amino Acid Sequence↗
PubMed2003

Vascular endothelial growth factor (VEGF) receptor-2 signaling mediates VEGF-C(deltaNdeltaC)- and VEGF-A-induced angiogenesis in vitro.

Angiogenesis and lymphangiogenesis are regulated by members of the vascular endothelial growth factor (VEGF) family of cytokines, which mediate their effects via tyrosine kinase VEGF receptors -1, -2, and -3. We have used wild-type and mutant forms of VEGFs -A, -B, and -C, a pan-VEGFR tyrosine kinase inhibitor (SU5416) as well as neutralizing anti-VEGFR-2 antibodies, to determine which VEGF receptor(s) are required for bovine endothelial cell invasion and tube formation in vitro. This was compared to the ability of these cytokines to induce expression of members of the plasminogen activator (PA)-plasmin system. We found that cytokines which bind VEGFR-2 (human VEGF-A, human VFM23A, human VEGF-C(deltaNdeltaC), and rat VEGF-C(152)) induced invasion, tube formation, urokinase-type-PA, tissue-type-PA, and PA inhibitor-1, invasion and tube formation as well as signaling via the MAP kinase pathway were efficiently blocked by SU5416 and anti-VEGFR-2 antibodies. In contrast, cytokines and mutants which exclusively bind VEGFR-1 (human VFM17 and human VEGF-B) had no effect on invasion and tube formation or on the regulation of gene expression. We were unable to identify cytokines which selectively stimulate bovine VEGFR-3 in our system. Taken together, these findings point to the central role of VEGFR-2 in the angiogenic signaling pathways induced by VEGF-C(deltaNdeltaC) and VEGF-A.

Animals↗
PubMed2003

Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats.

We tested the hypothesis that intravenous infusion of human bone marrow stromal cells (hMSCs) promotes vascular endothelial growth factor (VEGF) secretion, VEGF receptor 2 (VEGFR2) expression and angiogenesis in the ischemic boundary zone (IBZ) after stroke. hMSCs (1x10(6)) were intravenously injected into rats 24 hours after middle cerebral artery occlusion (MCAo). Laser scanning confocal microscopy (LSCM), immunohistochemistry and ELISA were performed to assay angiogenesis and levels of human and rat VEGF in the host brain, respectively. In addition, capillary-like tube formation was measured using mouse brain-derived endothelial cells (MBDECs). Morphological and three dimensional image analyses revealed significant (P<0.05) increases in numbers of enlarged and thin walled blood vessels and numbers of newly formed capillaries at the boundary of the ischemic lesion in rats (n=12) treated with hMSCs compared with numbers in rats (n=12) treated with PBS. ELISA measurements showed that treatment with hMSCs significantly (P<0.05) raised endogenous rat VEGF levels in the IBZ from 10.5+/-1.7 ng/mL in the control group to 17.5+/-1.6 ng/mL in the hMSC-treated group. In addition, treatment with hMSCs increased endogenous VEGFR2 immunoreactivity. In vitro, when MBDECs were incubated with the supernatant obtained from cultured hMSCs, capillary-like tube formation was significantly (P<0.01) induced. However, hMSC-induced capillary-like tube formation was significantly (P<0.01) inhibited when the endothelial cells were incubated with the supernatant from hMSCs in the presence of a neutralizing anti-VEGFR2. These data suggest that treatment of stroke with hMSCs enhances angiogenesis in the host brain and hMSC-enhanced angiogenesis is mediated by increases in levels of endogenous rat VEGF and VEGFR2.

Animals↗
PubMed2003

Nitric oxide enhances angiogenesis via the synthesis of vascular endothelial growth factor and cGMP after stroke in the rat.

We investigated the effects of NO on angiogenesis and the synthesis of vascular endothelial growth factor (VEGF) in a model of focal embolic cerebral ischemia in the rat. Compared with control rats, systemic administration of an NO donor, DETANONOate, to rats 24 hours after stroke significantly enlarged vascular perimeters and increased the number of proliferated cerebral endothelial cells and the numbers of newly generated vessels in the ischemic boundary regions, as evaluated by 3-dimensional laser scanning confocal microscopy. Treatment with DETANONOate significantly increased VEGF levels in the ischemic boundary regions as measured by ELISA. A capillary-like tube formation assay was used to investigate whether DETANONOate increases angiogenesis in ischemic brain via activation of soluble guanylate cyclase. DETANONOate-induced capillary-like tube formation was completely inhibited by a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). Blocking VEGF activity by a neutralized antibody against VEGF receptor 2 significantly attenuated DETANONOate-induced capillary-like tube formation. Moreover, systemic administration of a phosphodiesterase type 5 inhibitor (Sildenafil) to rats 24 hours after stroke significantly increased angiogenesis in the ischemic boundary regions. Sildenafil and an analog of cyclic guanosine monophosphate (cGMP) also induced capillary-like tube formation. These findings suggest that exogenous NO enhances angiogenesis in ischemic brain, which is mediated by the NO/cGMP pathway. Furthermore, our data suggest that NO, in part via VEGF, may enhance angiogenesis in ischemic brain.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2003

Di-diabody: a novel tetravalent bispecific antibody molecule by design.

The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods have been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. These recombinant antibody molecules possess dual antigen-binding capability with, in most cases, monovalency to each of their target antigens. Here, we describe an efficient approach for the production of a novel tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a bispecific/divalent diabody to, via the hinge region, the N-terminus of the CH(3) domain of an IgG. The novel BsAb, which we termed "di-diabody", represents a tetravalent diabody dimer resulting from dimerization between the hinge region and the CH(3) domains. A di-diabody was constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor, expressed both in a single Escherichia coli host and in mammalian cells, and purified to homogeneity by a one-step affinity chromatography. Compared to the bispecific/divalent diabody, the tetravalent di-diabody binds more efficiently to both of its target antigens and is more efficacious in blocking ligand binding to the receptors. The di-diabody retained good antigen-binding activity after incubation at 37 degrees C in mouse serum for 72 h, demonstrating good product stability. Finally, expression of the di-diabody in mammalian cells yielded higher level of production and better antibody activity. This design and expression for BsAb fragments should be applicable to any pair of antigen specificities.

Angiogenesis Inducing Agents↗