Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently ...available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.
EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness ...of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies.
To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with ...the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5–600
nM to obtain
k
a
information. Second, to define the
k
d
of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1
h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average
k
a
and
k
d
for the PSA/mAb interaction as calculated from the 22 analyses were (4.1
±
0.6)
×
10
4
M
−1
s
−1 and (4.5
±
0.6)
×
10
−5
s
−1, respectively. Overall, the experimental standard errors in the rate constants were only ∼14%. Based on the kinetic rate constants, the affinity (
K
D
) of the PSA/mAb interaction was 1.1
±
0.2
nM.
The purpose of this study was to engineer a bivalent single-chain anticarcinoembryonic antigen (CEA) antibody and an interleukin 2 (IL-2) fusion protein derivative for selective tumor targeting of ...cytokines. The variable domains of a high affinity anti-CEA antibody, T84.66, were used to form a single-gene-encoded antibody single-chain variable fragment joined to the crystallizable fragment, Fc (scFvFc). The fusion protein (scFvFc.IL-2) consisted of mouse IL-2-fused to the COOH-terminal end of the scFvFc. The engineered proteins were assembled as complete molecules and were similar to the intact anti-CEA monoclonal antibody (Mab) in antigen-binding properties. Based on IL-2 content of the fusion protein, its ability to support proliferation of CTLL-2 cells was identical with that of IL-2. Despite a molecular size similar to that of the intact Mab, the blood clearance of the fusion protein was markedly faster than that of the intact Mab or scFvFc. Incubation of radiolabeled scFvFc.IL-2 but not the intact or scFvFc antibodies in mouse serum was accompanied by the appearance of complexes, suggesting that the latter may contribute to the accelerated clearance of the fusion protein. Biodistribution and tumor targeting studies were carried out in CEA-transgenic mice bearing CEA-positive murine tumors as well as the antigen-negative parental tumor. The bivalent anti-CEA scFvFc had tumor localization properties similar to those of the intact Mab. Although fusion of IL-2 to the COOH-terminal end of the bivalent scFvFc altered its pharmacokinetic properties, the fusion antibody was able to target tumors specifically. Maximum uptake of the intact Mab, scFvFc, and scFvFc.IL-2 in CEA-positive tumors was 29.3 +/- 5.0, 19.5 +/- 2.1, and 6.6 +/- 0.9% injected dose/g, respectively. Maximum tumor localization ratios (CEA-positive/CEA-negative tumor) were similar for all three antibody types (4.6-6.0), demonstrating the antigen specificity of the tumor targeting. Significant antigen-specific targeting to CEA-positive normal tissues of transgenic mice was not observed. Although the tumor-targeting properties of the fusion protein were low, the growth of CEA-expressing (P = 0.01) but not antigen-irrelevant (P = 0.22) syngeneic tumor cells was inhibited after treatment of transgenic mice with the anti-CEA-IL-2 antibody. Therapy of CEA-expressing tumors was improved after i.v. administration of the fusion protein (P = 0.0001). These studies indicate that anti-CEA antibody-directed cytokine targeting may offer an effective treatment for CEA-expressing carcinomas. The availability of an immunocompetent CEA transgenic mouse model will also help to determine the immunotherapeutic properties of these fusion proteins.
Currently, classifying a population of specific antigen-reactive monoclonal antibodies (mAbs) according to their epitope-binding properties has been limited to competition assays. Such assays are ...time consuming, labor intensive and restricted to the number of mAbs in the experiment. To overcome this problem, a differential antigen disruption-based antibody profiling procedure was developed. This procedure rapidly classifies specific antigen-reactive mAbs into epitope-related groups by measuring the binding signal of the antibodies to a set of structurally disrupted antigens and then clustering the antibodies according to the similarity of their binding profiles. The clustering results generated by differential antigen disruption showed a significant concordance with those generated by competition experiments. Therefore, differential antigen disruption method opens an opportunity to assess the entire population of antigen-reactive mAbs according to their epitope-binding properties. In doing so, a set of representative antibodies can be drawn to describe the epitope complexity for systematically exploring their functions.
CM101, a polysaccharide isolated from the culture medium of Group B streptococcus, a neonatal pathogen, targets pathological
angiogenesis and inhibits tumor growth in mice and humans. CM101 also ...targets neonatal lung and adult sheep lung endothelial
cells. A gene encoding a transmembrane protein that interacts with CM101 was isolated from a sheep lung endothelial cell cDNA
library. The gene, termed sp55 , encodes a 495-amino acid polypeptide. COS-7 cells transfected with a vector containing sp55 express the SP55 protein-bound CM101 in a concentration-dependent manner. Stably transfected CHO cells also bound CM101.
The corresponding human gene, hp59 , was isolated from a human fetal lung cDNA library and had a predicted identity to SP55 of 86% over 495 amino acids. HP59
protein was shown by immunohistochemistry to be present in the pathological tumor vasculature of the lung, breast, colon,
and ovary, but not in the normal vasculature, suggesting that the protein may be critical to pathological angiogenesis. The
hp59 gene and/or the HP59 protein was not expressed in a variety of normal tissues, but was significantly expressed in human fetal
lung, consistent with the pathophysiology of Group B streptococcus infections in neonates. Mice immunized with HP59 and SP55
peptides showed significant attenuation of tumor growth. Immunization effectively inhibited both the tumor angiogenesis and
vasculogenesis processes, as evidenced by lack of both HP59- and CD34-positive vessels. These results and the immunohistochemistry
data suggest a therapeutic potential for the CM101 target protein HP59 both as a drug target and as a vaccine against pathoangiogenesis.
Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor
family complicate study of structure, metabolism, and function of single ...isoforms in mammalian cells. As an alternative, we
expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We
developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies
was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral
insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed
a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive
epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular
domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria.
The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop
I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively
sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms
are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like
loops in the extracellular domain of the two isoforms are presented.
Fibroblast growth factors (FGF), which have been implicated in tumor cell growth and angiogenesis, have biological activities that appear to be mediated by both heparinlike extracellular matrix sites ...and transmembrane tyrosine kinase receptor sites. In the present study, we demonstrated that inositolhexakisphosphate (InsP6) inhibits basic FGF (bFGF) binding to heparin. Our spectrofluorometric analyses demonstrated that InsP6not only bound to bFGF, presumably within the bFGF heparin-binding domain, but also protected bFGF from degradation by trypsin. Also, InsP6inhibited the cellular binding of bFGF and other fibroblast growth factor family members such as acidic FGF (aFGF) and K-FGF in a saturable and dose-dependent manner. Furthermore, concentrations as low as 100 μ M InsP6inhibited bFGF-induced DNA synthesis in AKR-2B fibroblasts, as well as the growth of bFGF-and K-FGF-transfected NIH/3T3 cells. Together, these results indicate that InsP6may serve as a useful antagonist of FGF activity.
Incubation of 16-kDa 125I-labeled heparin binding (acidic fibroblast) growth factor type one (HBGF-1) with human hepatoma cells and normal rat hepatocytes resulted in the appearance of a stable ...125I-labeled complex with an apparent molecular mass of 40 kDa. The complex could be isolated with specific antibodies against HBGF-1. In contrast to membrane receptor-bound 125I-HBGF-1, the complex was resistant to dissociation by detergents, acid, heat, and reducing or denaturing agents. Formation of a stable complex did not require treatment with cross-linking agents. Appearance of the 40-kDa complex was dependent on time, temperature, and enriched culture medium. Conditions that enhanced or reduced display of specific HBGF-1 membrane receptor sites enhanced or reduced the appearance of the 40-kDa complex. Dansylcadaverine, chloroquine, and staurosporine blocked the appearance of the 40-kDa complex concurrent with the blockage of internalization of the receptor-bound HBGF-1. Two-dimensional gel electrophoretic analysis, metabolic labeling with L-35Scysteine, and recovery of 16-kDa HBGF-1 from the 40-kDa complex after base treatment suggest involvement of a 24-kDa cellular protein in the complex formation. These results suggest a potentially novel receptor-dependent pathway for metabolism of HBGF-1.
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