Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis.

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.

Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment.

The mechanism by which angiogenic factors recruit bone marrow (BM)-derived quiescent endothelial and hematopoietic stem cells (HSCs) is not known. Here, we report that functional vascular endothelial growth factor receptor-1 (VEGFR1) is expressed on human CD34(+) and mouse Lin(-)Sca-1(+)c-Kit(+) BM-repopulating stem cells, conveying signals for recruitment of HSCs and reconstitution of hematopoiesis. Inhibition of VEGFR1, but not VEGFR2, blocked HSC cell cycling, differentiation and hematopoietic recovery after BM suppression, resulting in the demise of the treated mice. Placental growth factor (PlGF), which signals through VEGFR1, restored early and late phases of hematopoiesis following BM suppression. PlGF enhanced early phases of BM recovery directly through rapid chemotaxis of VEGFR1(+) BM-repopulating and progenitor cells. The late phase of hematopoietic recovery was driven by PlGF-induced upregulation of matrix metalloproteinase-9, mediating the release of soluble Kit ligand. Thus, PlGF promotes recruitment of VEGFR1(+) HSCs from a quiescent to a proliferative BM microenvironment, favoring differentiation, mobilization and reconstitution of hematopoiesis.

Dose-dependent increases in circulating TGF-alpha and other EGFR ligands act as pharmacodynamic markers for optimal biological dosing of cetuximab and are tumor independent.

PURPOSE: The objective of this study was to characterize treatment-induced circulating ligand changes during therapy with epidermal growth factor receptor (EGFR) inhibitors and evaluate their potential as surrogate indicators of the optimal biological dose. EXPERIMENTAL DESIGN: Conditioned medium from human tumor cell lines, ascites fluid from tumor xenografts, and plasma samples from normal mice, as well as colorectal cancer patients, were assessed for ligand elevations using ELISA, following treatment with cetuximab (Erbitux), an anti-mouse EGFR neutralizing antibody, or a small-molecule EGFR tyrosine kinase inhibitor. RESULTS: A rapid elevation in human transforming growth factor alpha (TGF-alpha) was observed in all cell lines after treatment with cetuximab, but not with small-molecule inhibitors. The elevation showed a dose-response effect and plateau that corresponded to the maximal decrease in A431 proliferation in vitro and HT29 tumor growth in vivo. The TGF-alpha increase was exacerbated by ongoing ligand production and cleavage from the plasma membrane but did not involve transcriptional up-regulation of TGF-alpha or the matrix metalloproteinase tumor necrosis factor-alpha-converting enzyme/ADAM17. Elevations in plasma TGF-alpha, amphiregulin, and epiregulin were also detected in normal mice treated with an anti-mouse EGFR monoclonal antibody, illustrating a host tissue-dependent component of this effect in vivo. Finally, circulating TGF-alpha increased in the plasma of six patients with EGFR-negative colorectal tumors during cetuximab treatment. CONCLUSIONS: Treatment-induced increases in circulating ligands, particularly TGF-alpha, should be serially assessed in clinical trials of anti-EGFR therapeutic antibodies as potential biomarkers to aid in determination of the optimal biological dose.

Prioritization of EGFR/IGF-IR/VEGFR2 combination targeted therapies utilizing cancer models.

BACKGROUND: Rational strategies utilizing anticancer efficacy and biological principles are needed for the prioritization of specific combination targeted therapy approaches for clinical development, from among the many with experimental support. MATERIALS AND METHODS: Antibodies targeting epidermal growth factor receptor (EGFR) (cetuximab), insulin-like growth factor-1 receptor (IGF-IR) (IMC-A12) or vascular endothelial growth factor receptor 2 (VEGFR2) (DC101), were dosed alone or in combination, in 11 human tumor xenograft models established in mice. Efficacy readouts included the tumor burden and incidence of metastasis, as well as tumor active hypoxia inducible factor-1 (HIF-1), human VEGF and blood vessel density. RESULTS: Cetuximab and DC101 contributed potent and non-overlapping benefits to the combination approach. Moreover, DC101 prevented escape from IMC-A12 + cetuximab in a colorectal cancer model and cetuximab prevented escape from DC101 therapy in a pancreatic cancer model. CONCLUSION: Targeting VEGFR2 + EGFR was prioritized over other treatment strategies utilizing EGFR, IGF-IR and VEGFR2 antibodies. The criteria that proved to be valuable were a non-overlapping spectrum of anticancer activity and the prevention of resistance to another therapy in the combination.

Inhibition of glioblastoma growth in a highly invasive nude mouse model can be achieved by targeting epidermal growth factor receptor but not vascular endothelial growth factor receptor-2.

PURPOSE: Major shortcomings of traditional mouse models based on xenografted human glioblastoma cell lines are that tumor cells do not invade and that genetic alterations, such as amplification of the epidermal growth factor receptor (EGFR) gene, are not maintained. Such models are thus of limited value for preclinical studies. We established a highly invasive model to evaluate the effect of antibodies against EGFR (cetuximab) and vascular endothelial growth factor receptor-2 (antibody DC101). EXPERIMENTAL DESIGN: After short-term culture, glioblastoma spheroids were implanted into the brains of nude mice. Animals were treated either i.c. with cetuximab or i.p. with DC101. Tumor burden was determined histologically using image analysis of 36 different landmark points on serial brain sections. RESULTS: Invasive xenografts were obtained from nine different glioblastomas. Three of seven cases treated with cetuximab responded with significant tumor growth inhibition, whereas four did not. All responsive tumors were derived from glioblastomas exhibiting EGFR amplification and expression of the truncated EGFRvIII variant, which were maintained in the xenografts. All nonresponsive tumors lacked EGFR amplification and EGFRvIII expression. The proportion of apoptotic cells was increased, whereas proliferation and invasion were decreased in responsive tumors. None of four xenograft cases treated with DC101 responded to treatment, and the diffusely invading tumors grew independent of angiogenesis. CONCLUSIONS: Inhibition of glioblastoma growth and invasion can be achieved using i.c. delivery of an anti-EGFR antibody, but tumor response depends on the presence of amplified and/or mutated EGFR. Antiangiogenic treatment with DC101 is not effective against diffusely invading tumors.

Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth.

Vascular endothelial growth factor receptor 3 (VEGFR-3) binds VEGF-C and VEGF-D and is essential for the development of the lymphatic vasculature. Experimental tumors that overexpress VEGFR-3 ligands induce lymphatic vessel sprouting and enlargement and show enhanced metastasis to regional lymph nodes and beyond, whereas a soluble form of VEGFR-3 that blocks receptor signaling inhibits these changes and metastasis. Because VEGFR-3 is also essential for the early blood vessel development in embryos and is up-regulated in tumor angiogenesis, we wanted to determine if an antibody targeting the receptor that interferes with VEGFR-3 ligand binding can inhibit primary tumor growth. Our results show that antibody interference with VEGFR-3 function can inhibit the growth of several human tumor xenografts in immunocompromised mice. Immunohistochemical analysis showed that the blood vessel density of anti-VEGFR-3-treated tumors was significantly decreased and hypoxic and necrotic tumor tissue was increased when compared with tumors treated with control antibody, indicating that blocking of the VEGFR-3 pathway inhibits angiogenesis in these tumors. As expected, the anti-VEGFR-3-treated tumors also lacked lymphatic vessels. These results suggest that the VEGFR-3 pathway contributes to tumor angiogenesis and that effective inhibition of tumor progression may require the inhibition of multiple angiogenic targets.

Long-term progression and therapeutic response of visceral metastatic disease non-invasively monitored in mouse urine using beta-human choriogonadotropin secreting tumor cell lines.

Historically, the use of mouse models of metastatic disease to evaluate anticancer therapies has been hampered because of difficulties in detection and quantification of such lesions without sacrificing the mice, which in turn may also be dictated by institutional or ethical guidelines. Advancements in imaging technologies have begun to change this situation. A new method to non-invasively measure tumor burden, as yet untested to monitor spontaneous metastases, is the use of transplanted tumors expressing secretable human beta-chorionic gonadotropin (beta-hCG) that can be measured in urine. We describe examples of beta-hCG-transfected tumor cell lines for evaluating the effect of different therapies on metastatic disease, which in some cases involved monitoring tumor growth for >100 days. We used beta-hCG-tagged mouse B16 melanoma and erbB-2/Her-2-expressing human breast cancer MDA-MB-231 models, and drug treatments included metronomic low-dose cyclophosphamide chemotherapy with or without a vascular endothelial growth factor receptor 2-targeting antibody (DC101) or trastuzumab, the erbB-2/Her-2-targeting antibody. Both experimental and spontaneous metastasis models were studied; in the latter case, an increase in urine beta-hCG always foreshadowed the development of lung, liver, brain, and kidney metastases. Metastatic disease was unresponsive to DC101 or trastuzumab monotherapy treatment, as assessed by beta-hCG levels. Our results also suggest that beta-hCG levels may be set as an end point for metastasis studies, circumventing guidelines, which have often hampered the use of advanced disease models. Collectively, our data indicates that beta-hCG is an effective noninvasive preclinical marker for the long term monitoring of untreated or treated metastatic disease.

IMC-EB10, an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.

The class III receptor tyrosine kinase FLT3 is expressed on the blasts of >90% of patients with B-lineage acute lymphoblastic leukemias (ALL). In addition, it is expressed at extremely high levels in ALL patients with mixed lineage leukemia rearrangements or hyperdiploidy and is sometimes mutated in these same patients. In this report, we investigate the effects of treating ALL cell lines and primary samples with human anti-FLT3 monoclonal antibodies (mAb) capable of preventing binding of FLT3 ligand. In vitro studies, examining the ability of two anti-FLT3 mAbs (IMC-EB10 and IMC-NC7) to affect FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. FLT3 phosphorylation was consistently inhibited by IMC-NC7 treatment, but in some cell lines, IMC-EB10 actually stimulated FLT3 activation, possibly as a result of antibody-mediated receptor dimerization. Through antibody-dependent, cell-mediated cytotoxicity, such an antibody could still prove efficacious against leukemia cells in vivo. In fact, IMC-EB10 treatment significantly prolonged survival and/or reduced engraftment of several ALL cell lines and primary ALL samples in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This occurred even when IMC-EB10 treatment resulted in FLT3 activation in vitro. Moreover, fluorescence-activated cell sorting and PCR analysis of IMC-EB10-treated NOD/SCID mice surviving 150 days post-leukemic cell injection revealed that FLT3 immunotherapy reduced leukemic engraftment below the level of detection in these assays (<0.001%). Furthermore, in vivo IMC-EB10 treatment did not select for resistant cells, because cells surviving IMC-EB10 treatment remain sensitive to IMC-EB10 cytotoxicity upon retransplantation. In vivo studies involving either partial depletion or activation of natural killer (NK) cells show that most of the cytotoxic effect of IMC-EB10 is mediated through NK cells. Therefore, such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

Inhibitory anti-FLT3 antibodies are capable of mediating antibody-dependent cell-mediated cytotoxicity and reducing engraftment of acute myelogenous leukemia blasts in nonobese diabetic/severe combined immunodeficient mice.

Aberrant FLT3 expression and/or mutation plays a significant role in leukemogenesis. This has prompted the development of selective small molecule tyrosine kinase inhibitors against FLT3. However, like most tyrosine kinase inhibitors, those against FLT3 are not completely specific and at the doses required to completely inhibit target, significant toxicities may occur. In addition, tyrosine kinase inhibitors for other kinases have been shown to select for cells that become resistant. To overcome some of these limitations we developed two fully human phage display monoclonal antibodies against FLT3 (IMC-EB10 and IMC-NC7). These antibodies inhibited ligand-mediated activation of wild-type FLT3 and constitutively activated mutant FLT3 and in most cell types affected downstream STAT5, AKT, and mitogen-activated protein kinase activation. In addition to interfering with FLT3 signaling, IMC-EB10 and, to a significantly lesser extent, IMC-NC7 initiated antibody-dependent cell-mediated cytotoxicity on FLT3-expressing cells. When IMC-EB10 was used in vivo to treat nonobese diabetic/severe combined immunodeficient mice given injections of primary FLT3/ITD acute myelogenous leukemia samples or myeloid cell lines with FLT3 expression, it significantly decreased engraftment of leukemic cells and increased survival, respectively. In contrast, IMC-EB10 treatment did not reduce engraftment of normal human CD34+ cord blood cells nor did it show any significant inhibition of normal murine hematopoiesis. Thus, these types of antibodies have the potential to be safe and effective new therapeutic agents for acute myelogenous leukemia and possibly other FLT3-expressing malignancies.

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.

A recombinant, fully human, bispecific antibody neutralizes the biological activities mediated by both vascular endothelial growth factor receptors 2 and 3.

Vascular endothelial growth factors (VEGF) and their receptors (VEGFR) have been implicated to play important roles in tumor-associated angiogenesis and lymphangiogenesis, and hence in tumor growth and metastasis. We previously produced a number of fully human antibodies directed against VEGF receptor 2 (VEGFR2) and VEGF receptor 3 (VEGFR3) and showed that these antibodies are capable of inhibiting growth factor (VEGF and VEGF-C)-induced receptor activation, migration, and proliferation of human endothelial cells. In this report, we constructed and produced a bispecific antibody, a diabody, using the variable domain genes of two neutralizing antibodies, IMC-1121 to VEGFR2 and hF4-3C5 to VEGFR3. The diabody binds to both VEGFR2 and VEGFR3 in a dose-dependent manner, and blocks interaction between VEGF/VEGFR2, VEGF-C/VEGFR2, and VEGF-C/VEGFR3. In cell-based assays, the diabody neutralized both VEGF and VEGF-C-stimulated activation of VEGFR2, VEGFR3, and p44/p42 mitogen-activated protein kinase in endothelial cells. Furthermore, the diabody was able to inhibit both VEGF and VEGF-C-induced migration of endothelial cells. Taken together, our results suggest that a dual blockade of both VEGFR2 and VEGFR3 simultaneously may represent a more potent approach to effective cancer therapy.

Targeting the platelet-derived growth factor receptor alpha with a neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: implications as a potential therapeutic target.

Platelet-derived growth factor receptor alpha (PDGFRalpha) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRalpha, which did not cross-react with the beta form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRalpha. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRalpha. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P=0.0004) and SKLMS-1 (P <0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRalpha.

DEGA/AMIGO-2, a leucine-rich repeat family member, differentially expressed in human gastric adenocarcinoma: effects on ploidy, chromosomal stability, cell adhesion/migration and tumorigenicity.

We have discovered DEGA, a novel cDNA differentially expressed in human gastric adenocarcinomas. The DEGA gene product contains a signal peptide, five leucine-rich repeat motifs and a single IgG, and transmembrane domain, suggesting its residence on the plasma membrane. Transfection of 293 cells with a DEGA-GFP fusion construct confirmed its cell surface localization. Although the cytosolic portion of the DEGA gene product does not contain known protein domains, approximately one-fifth of these residues are either a serine or a threonine, suggesting that DEGA may play a role in signal transduction. BLAST searches revealed DEGA to be an exact match to AMIGO-2, a recently identified, but functionally uncharacterized protein related to AMIGO, a leucine-rich repeat containing cell adhesion molecule implicated in axon tract development. In this report, we show that DEGA/AMIGO-2 mRNA is differentially expressed in approximately 45% of tumor versus normal tissue from gastric adenocarcinoma patients. Stable expression of a DEGA/AMIGO-2 antisense construct in the gastric adenocarcinoma cell line, AGS, led to altered morphology, increased ploidy, chromosomal instability, decreased cell adhesion/migration, and a nearly complete abrogation of tumorigenicity in nude mice. These findings suggest a potential etiologic role for DEGA/AMIGO-2 in gastric adenocarcinoma.

Clinical development of angiogenesis inhibitors to vascular endothelial growth factor and its receptors as cancer therapeutics.

Angiogenesis, the formation of new blood vessels, is essential for both tumor growth and metastasis. Recent advances in our understanding of the molecular mechanisms underlying the angiogenesis process and its regulation have led to the discovery of a variety of pharmaceutical agents with anti-angiogenic activity. The potential application of these angiogenesis inhibitors is currently under intense clinical investigation. Compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptors play critical roles in tumor-associated angiogenesis, and that they represent potential targets for therapeutic intervention. This has been demonstrated in a variety of animal tumor models in which disabling the function of VEGF and its receptors was shown to inhibit both tumor growth and metastasis. A number of agents designed specifically for targeting VEGF and/or its receptors are being evaluated in various clinical trials in cancer patients. This review will discuss the biology of the VEGF and its receptors, the mechanisms of action as well as the current status in clinical development of antagonistic agents to VEGF and its receptors. Included in this review are antagonistic antibodies, ribozymes, immunotoxins, and synthetic small molecular inhibitors.

Fab-scFv fusion protein: an efficient approach to production of bispecific antibody fragments.

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. Here, we describe an efficient approach for the production of a novel bispecific antibody fragment by genetically fusing a single-chain Fv (scFv) to the C-terminus of either the light chain or the heavy chain of a Fab fragment of different antigen-binding specificity. The bispecific Fab-scFv fragments were expressed in a single Escherichia coli host and purified to homogeneity by a one-step affinity chromatography. Two different versions of the bispecific Fab-scFv fragments were constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor. These bispecific antibody fragments not only retained the antigen-binding capacity of each of the parent antibodies, but also are capable of binding to both targets simultaneously as demonstrated by a cross-linking ELISA. Further, the bispecific antibodies were comparable to their parent antibodies in their potency in blocking ligand binding to the receptors and in inhibiting ligand-induced biological activities. This design for BsAb fragments should be applicable to any pair of antigen specificities.

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.

Localization of VEGF receptor-2 (KDR/Flk-1) and effects of blocking it in oxygen-induced retinopathy.

PURPOSE: Vascular endothelial cell growth factor (VEGF) has been implicated in vascular development and in proliferative retinopathies. The goal of this study was to examine the immunohistochemical localization and relative levels of VEGF receptor-2 (KDR) in canine retina during postnatal vasculogenesis and during angiogenesis in oxygen-induced retinopathy (OIR) and to investigate the effects of neutralizing KDR on these processes. METHODS: Eyes from normal dogs ranging from 1 to 22 days of age and age-matched oxygen-treated animals were snap frozen for immunohistochemical analysis with antibodies against human KDR. To examine the effects of blocking KDR, 6-day-old air-reared control and oxygen-treated animals were surgically implanted with slow release polymer pellets containing control IgG or anti-KDR. Material eluted from pellets was assessed using a binding assay (measures binding to soluble KDR) to determine the kinetics of anti-KDR release and endothelial cell proliferation to measure bioactivity. Animals were killed at 22 days of age and tissues examined with adenosine diphosphatase (ADPase) histochemical staining of blood vessels. RESULTS: KDR immunoreactivity was only weakly associated with developing retinal vessels and was not observed in angioblasts throughout normal postnatal development. Immunoreactivity was very strong in reforming retinal vessels and intravitreal neovascularization in oxygen-treated animals. Anti-KDR had no effect on vessel morphology or growth in air-reared control animals. In oxygen-treated animals, anti-KDR significantly inhibited revascularization of the retina (P = 0.005) and formation of intravitreal neovascularization compared with control IgG pellet eyes (P < 0.04). CONCLUSIONS: KDR/Flk-1 was only weakly associated with normal developing primary retinal vessels but was strongly expressed by proliferating endothelial cells in reforming retinal vessels and intravitreal neovascularization after hyperoxic insult. Anti-KDR antibody delivered by slow-release pellets had no effect on normal vasculogenesis, but it inhibited the formation of intravitreal neovascularization and retinal vessel development in OIR. The study suggests that blocking KDR may be beneficial for treating pathologic angiogenesis in adult tissue.

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.

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.