Purpose: Transforming growth factor β (TGFβ) is a pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. We investigated the therapeutic efficacy of anti-TGFβ receptor II (TGFβ RII) antibody in controlling metastasis and tumor growth as well as enhancing antitumor immunity in preclinical tumor models.Experimental Design: We generated neutralizing antibodies to TGFβ RII and assessed the antibody effects on cancer, stroma, and immune cells in vitro. The efficacy and mechanism of action of the antibody as monotherapy and in combination with chemotherapy in suppression of primary tumor growth and metastasis were evaluated in several tumor models.Results: Anti-TGFβ RII antibody blocked TGFβ RII binding to TGFβ 1, 2, and 3, and attenuated the TGFβ-mediated activation of downstream Smad2 kinase, invasion of cancer cells, motility of endothelial and fibroblast cells, and induction of immunosuppressive cells. Treatment with the antibody significantly suppressed primary tumor growth and metastasis and enhanced natural killer and CTL activity in tumorbearing mice. Immunohistochemistry analysis showed cancer cell apoptosis and massive necrosis, and increased tumor-infiltrating T effector cells and decreased tumor-infiltrating Gr-1+ myeloid cells in the antibody-treated tumors. Fluorescence-activated cell sorting analysis indicated the significant reduction of peripheral Gr-1+/CD11b+ myeloid cells in treated animals. Concomitant treatment with the cytotoxic agent cyclophosphamide resulted in a significantly increased antitumor efficacy against primary tumor growth and metastasis.Conclusions: These preclinical data provide a foundation to support using anti-TGFβ RII antibody as a therapeutic agent for TGFβ RII-dependent cancer with metastatic capacity.
Membrane transporter proteins play critical physiological roles in the cell and constitute 5-10% of prokaryotic and eukaryotic genomes. High-resolution structural information is essential for understanding the functional mechanism of these proteins. A prerequisite for structural study is to overexpress such proteins in large quantities. In the last few years, over 20 bacterial membrane transporters were overexpressed at a level of 1 mg/l of culture or higher, most often in Escherichia coli. In this review, we analyzed those factors that affect the quantity and quality of the protein produced, and summarized recent progress in overexpression of membrane transporters from bacterial inner membrane. Rapid progress in genome sequencing provides opportunities for expressing several homologues and orthologues of the target protein simultaneously, while the availability of various expression vectors allows flexible experimental design. Careful optimization of cell culture conditions can drastically improve the expression level and homogeneity of the target protein. New sample preparation techniques for mass spectrometry of membrane proteins have enabled one to identity the rigid protein core, which can be subsequently overexpressed. Size-exclusion chromatography on HPLC has proven to be an efficient method in screening detergent, pH an other conditions required for maintaining the stability and monodispersity of the protein. Such high-quality preparations of membrane transporter proteins will probably lead to successful crystallization and structure determination of these proteins in the next few years.
Objective. To investigate whether agonist antiplatelet-derived growth factor receptor ␣ (anti-PDGFR␣) antibodies are present in the serum of patients with systemic sclerosis (SSc; scleroderma).Methods. Sera were obtained from healthy subjects and scleroderma patients. An electrochemiluminescence binding assay was performed for detection of serum autoantibodies to PDGFR␣, PDGFR, epidermal growth factor receptor (EGFR), and colonystimulating factor receptor 1 (CSFR1). Serum immunoglobulin was purified by protein A/G chromatography. To assess Ig agonist activity, PDGFR␣-expressing cells were incubated with pure Ig and the level of receptor phosphorylation determined in an enzyme-linked immunoassay, as well as by Western blotting. Ig agonist activity was also assessed in a mitogenic assay and by MAP kinase activation in a PDGFR␣-expressing cell line.Results. Sera from 34.3% of the healthy subjects and 32.7% of the SSc patients contained detectable autoantibodies to PDGFR␣ and PDGFR, but not EGFR or CSFR1. Purified Ig from these sera was shown to retain PDGFR binding activity and, at 200-1,000 g/ml, exhibited no agonist activity in a cell-based PDGFR␣ phosphorylation assay and did not stimulate a mitogenic response or MAP kinase activation in a PDGFR␣-expressing cell line. Two purified Ig samples that were unable to bind PDGFR␣ did exhibit binding activity to a nonglycosylated form of PDGFR␣.Conclusion. Although approximately one-third of sera from scleroderma patients contained detectable autoantibodies to PDGFR, these antibodies were not specific to scleroderma, since they were also detected in a similar percentage of samples from normal subjects. PDGFR␣ agonist activity was not demonstrated when purified Ig from these sera was tested in cell-based assays.
The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.
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