Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy. To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains. The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host. After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting. The yield of scDb protein was 570 microg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR. In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.
Antibodies, Bispecific ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Humans ; Plasmids ; Protein Binding ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective alternatives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-based regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics.
Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Antigens, CD20 ; immunology ; Humans ; Immunoconjugates ; therapeutic use ; Neoplasms ; drug therapy ; immunology ; Protein Engineering ; RANK Ligand ; immunology ; Receptor, Epidermal Growth Factor ; immunology ; Receptor, ErbB-2 ; immunology ; Vascular Endothelial Growth Factor A ; immunology

OBJECTIVETo investigate whether fetal bone marrow stromal cells have hemangioblastic characteristics.

METHODSHuman fetal bone marrow stromal cells (hfMSCs) were isolated and cultured. Immunophenotypes of hfMSCs were tested by FACS. hfMSCs seeded in the matrigel were induced with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in vitro. Vascularization and hematopoiesis were detected with immunohistochemistry and electron microscope.

RESULTSThe typical properties of this CD34- stromal cell population were that 99% cells expressed Flk1 (vascular endothelial cell growth factor receptor 2) and tube structure was formed. In the process of induction, hfMSCs could give rise to CD34+ round cells.

CONCLUSIONSWe have demonstrated that fetal bone marrow stroma-derived Flk1+ CD34- cells could differentiate into vascular endothelial cells and hematopoietic cells, indicating that fetal bone marrow stroma-derived Flk1+ CD34- cells have hemangioblastic characteristics.

Antigens, CD34 ; immunology ; Bone Marrow Cells ; cytology ; immunology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Fetus ; Fibroblast Growth Factor 2 ; metabolism ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; immunology ; metabolism ; Humans ; Immunophenotyping ; Stromal Cells ; cytology ; immunology ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; physiology

OBJECTIVETo prepare a neutralizing monoclonal antibody (McAb) against vascular endothelial growth factor receptor KDR and study its biological activity.

METHODSExtracellular immunoglobulin (Ig)-like domain III of KDR (KDR III) was expressed in E. coli and purified by affinity chromatograph. Monoclonal antibody against KDR III was prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [(3)H]-TdR incorporation assay were also used to detect the activity of anti-KDR McAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on VEGF-induced mitogenesis of human endothelial cells.

RESULTSMcAb Ycom1D3 against KDR III was prepared which bound specifically to both the soluble KDR III and the cell-surface expressed KDR. It effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated activation of KDR expression on human endothelial cells. Furthermore, Ycom1D3 efficiently neutralized VEGF-induced mitogenesis of human umbilical vascular endothelial cells.

CONCLUSIONMcAb Ycom1D3 against KDR III may suppress the action of VEGF by blocking native vascular endothelial growth factor receptor KDR. It has potential clinical applications in the treatment of cancers and other diseases where pathological angiogenesis is involved.

Antibodies, Monoclonal ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; Humans ; Neovascularization, Physiologic ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

The suppressive effect of anti-KDR antibody against VEGF on proliferation of hemangioma-derived vascular endothelial cells (HVECs) was investigated. HVECs from one case of hemangioma in proliferative phase were cultured. Both primary culture and sub-culture were conducted in M199 medium. The HVECs of passage 3 were divided into 4 groups based on the concentrations of anti-KDR antibody. Cell count was performed and inhibitory rate of HVECs was measured before and 9 days after interference. The results showed that the number of HVECs in the anti-KDR antibody-treated groups was significantly decreased and the inhibitory rate of HVECs by anti-KDR antibody (50, 10 and 2 microg/mL) was 84%, 63% and 39% respectively at 9th day after interference, with the difference being significant. In the control group, the number of HVECs was increased significantly. In was concluded that the anti-KDR antibody could suppress the activity of VEGF through blocking the KDR, indicating the potential clinical applications of anti-KDR antibody in the treatment of hemangioma.
Antibodies/*pharmacology ; Cell Proliferation/drug effects ; Endothelium, Vascular/*pathology ; Hemangioma/*pathology ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor Receptor-2/*immunology

OBJECTIVETo investigate the effect of vasoactive intestinal peptide (VIP) on angiogenesis after focal cerebral ischemia.

METHODSFocal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 120 min in adult SD rats with intracerebroventricular VIP administration at the beginning of reperfusion. Immunohistochemistry was performed to assay BrdU immunoreactive endothelial cells, expressions of VEGF, flt-1 and flk-1 in the ischemic zone, and the protein expressions of vascular endothelial growth factor (VEGF) in the brain was measured using Western blotting.

RESULTSImmunohistochemical staining revealed significantly increased BrdU immunoreactive endothelial cells on the margins of the ischemic lesion in rats treated with VIP as compared with that in the control rats (P<0.05). VIP significantly increased the number of VEGF immunoreactive cells and flt-1- and flk-1-positive endothelial cells in comparison with the control group (P<0.01). Western blotting showed that VIP treatment resulted in significantly increased VEGF protein level in the ipsilateral hemisphere (P<0.05).

CONCLUSIONSVIP enhances angiogenesis in the ischemic brain by increasing the expressions of VEGF in the brain tissue and its receptors flt-1 and flk-1 in the endothelial cells.

Animals ; Brain ; drug effects ; metabolism ; Brain Ischemia ; immunology ; metabolism ; pathology ; physiopathology ; Endothelial Cells ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Male ; Neovascularization, Physiologic ; drug effects ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-1 ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Vasoactive Intestinal Peptide ; pharmacology

OBJECTIVETo prepare a new targeted liposome ultrasonic contrast agent with anti-KDR antibody that binds specifically with KDR as the main receptor of VEGF and evaluate its physical characteristics, biological activity and specific binding capability in vitro.

METHODSA sonicator was used to prepare the biotinylated lipid micro-bubbles (MB-B), and biotin-avidin-mediated technique was used for attachment of anti-mouse KDR monoclonal antibody to the micro-bubble shell to generate MB-BAB-KDR. MB-BAB-KDR was incubated with fluorescent second antibody to assess the link condition, and the control groups were the MB-B and micro-bubbles with the antibody alone (MB-B-KDR). A parallel plate flow chamber system was used to characterize micro-bubbles attachment efficiency to KDR Fc.

RESULTSThe surface of the micro-bubbles could carry KDR antibody through the biotin-avidin bridge and MB-BAB-KDR were spherical and well-distributed. After incubation with the second antibody, MB-BAB-KDR could be observed to emit bright green fluorescence (Grade II) as compared with little or weak fluorescence in the control MB-B group (Grade 0) and MB-B-KDR group (Grade I). Targeted micro-bubbles bound to KDR Fc increased as the KDR Fc concentration increased (P<0.05).

CONCLUSIONThe targeted liposome contrast agent linked with KDR antibody by biotin-avidin bridge we prepared shows an increased binding number as the KDR Fc concentration increases, which provides a novel approach to molecular imaging study of endometrial receptivity.

Animals ; Antibodies, Monoclonal ; Contrast Media ; chemistry ; Endometrium ; physiology ; Female ; Liposomes ; chemistry ; Mice ; Microbubbles ; Molecular Imaging ; Sonication ; Ultrasonography ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

OBJECTIVETo investigate the specific cytotoxity of tumor infiltrating lymphocytes (TIL) transfected with chimeric T cell receptor (CTCR) on cells which express KDR.

METHODSA recombinant retroviral plasmid (pMSCVneo-Vhgamma) was constructed by cloning VEGF121-hinger-FcRgamma (Vhgamma) into retroviral vector pMSCVneo. After packaging by PT67, the virus with high titer was used to infect TIL isolated from liver cancer tissues, and then MSCVneo-Vhgamma-TIL was generated. TIL infected with MSCVneo was used as a control. The cytotoxicty of the transgenic TIL on NIH3T3 and HepG2 expressing no KDR and on ECV304 and A375 expressing KDR was detected with MTT colorimetric assay.

RESULTSThe sequences of VEGF121 and hinger-FcRgamma were different from those reported, but the deduced amino sequences were identical to the reported ones. The cytotoxity of TIL infected with MSCVneo on target cell was similar to that of the control TIL; both only had mild cytotoxity on cancer cell line. No significant cytotoxity was found in TIL infected with MSCVneo-cTCR on NIH3T3, but its cytotoxity on ECV304 was significant. The cytotoxity on HepG2 was similar to that of MSCVneo-TIL and uninfected TIL, but cytotoxity on A375 was significantly higher.

CONCLUSIONChimeric T cell receptor permanently grafts TIL cell with predefined new specificity. TIL expressing Vhgamma can selectively recognize and kill vascular endothelial cell and tumor cells which express VEGF receptor KDR.

Animals ; Cytotoxicity, Immunologic ; Humans ; Lymphocytes, Tumor-Infiltrating ; immunology ; Mice ; NIH 3T3 Cells ; Plasmids ; Receptors, Antigen, T-Cell ; physiology ; Retroviridae ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; physiology

To date, more than 30 antibodies have been approved worldwide for therapeutic use. While the monoclonal antibody market is rapidly growing, the clinical use of therapeutic antibodies is mostly limited to treatment of cancers and immunological disorders. Moreover, antibodies against only five targets (TNF-alpha, HER2, CD20, EGFR, and VEGF) account for more than 80 percent of the worldwide market of therapeutic antibodies. The shortage of novel, clinically proven targets has resulted in the development of many distinct therapeutic antibodies against a small number of proven targets, based on the premise that different antibody molecules against the same target antigen have distinct biological and clinical effects from one another. For example, four antibodies against TNF-alpha have been approved by the FDA -- infliximab, adalimumab, golimumab, and certolizumab pegol -- with many more in clinical and preclinical development. The situation is similar for HER2, CD20, EGFR, and VEGF, each having one or more approved antibodies and many more under development. This review discusses the different binding characteristics, mechanisms of action, and biological and clinical activities of multiple monoclonal antibodies against TNF-alpha, HER-2, CD20, and EGFR and provides insights into the development of therapeutic antibodies.
Animals ; Antibodies, Monoclonal/*pharmacology/therapeutic use ; Antigens, CD20/immunology ; Drug Discovery ; Humans ; Immune System Diseases/*drug therapy/immunology ; *Immunotherapy/trends ; *Molecular Targeted Therapy ; Neoplasms/*drug therapy/immunology ; Receptor, Epidermal Growth Factor/immunology ; Receptor, erbB-2/immunology ; Tumor Necrosis Factor-alpha/immunology ; United States ; United States Food and Drug Administration ; Vascular Endothelial Growth Factor A/immunology

To date, more than 30 antibodies have been approved worldwide for therapeutic use. While the monoclonal antibody market is rapidly growing, the clinical use of therapeutic antibodies is mostly limited to treatment of cancers and immunological disorders. Moreover, antibodies against only five targets (TNF-alpha, HER2, CD20, EGFR, and VEGF) account for more than 80 percent of the worldwide market of therapeutic antibodies. The shortage of novel, clinically proven targets has resulted in the development of many distinct therapeutic antibodies against a small number of proven targets, based on the premise that different antibody molecules against the same target antigen have distinct biological and clinical effects from one another. For example, four antibodies against TNF-alpha have been approved by the FDA -- infliximab, adalimumab, golimumab, and certolizumab pegol -- with many more in clinical and preclinical development. The situation is similar for HER2, CD20, EGFR, and VEGF, each having one or more approved antibodies and many more under development. This review discusses the different binding characteristics, mechanisms of action, and biological and clinical activities of multiple monoclonal antibodies against TNF-alpha, HER-2, CD20, and EGFR and provides insights into the development of therapeutic antibodies.
Animals ; Antibodies, Monoclonal/*pharmacology/therapeutic use ; Antigens, CD20/immunology ; Drug Discovery ; Humans ; Immune System Diseases/*drug therapy/immunology ; *Immunotherapy/trends ; *Molecular Targeted Therapy ; Neoplasms/*drug therapy/immunology ; Receptor, Epidermal Growth Factor/immunology ; Receptor, erbB-2/immunology ; Tumor Necrosis Factor-alpha/immunology ; United States ; United States Food and Drug Administration ; Vascular Endothelial Growth Factor A/immunology