OBJECTIVETo obtain 1-4 IgG-like domains of mouse vascular endothelial growth factor receptor 2 (VEGFR2) fusion protein (mVEGFR2D1-4/GST) and identify its antiginicity and biological activity.

METHODSThe gene of mVEGFR2D1-4 was amplified by RT-PCR from 14-days embryos of Balb/c mice. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mVEGFR2D1-4, which was transformed into E. coli BL21 (DE3) strain for mVEGFR2D1-4/GST expression. The fusion protein was identified by SDS-PAGE and Western blotting, and the antigenicity of the protein purified by affinity chromatography was characterized by ELISA. The VEGF blocking effect of the purified protein in human umbilical vein endothelial cells (HUVECs) were evaluated in in vitro cell cultures.

RESULTSThe mVEGFR2D1-4 gene was obtained, which had an identical sequence to that retrieved in GenBank. The prokaryotic expression vector for mVEGFR2D1-4 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the antigenicity of the purified mVEGFR2D1-4 fusion protein, which obviously blocked the effect of VEGF in promoting HUVEC proliferation in vitro.

CONCLUSIONThe mVEGFR2D1-4/GST fusion protein obtained shows a strong antigenicity and biological activity to facilitate further study of active anti-tumor immunotherapy targeting VEGFR2.

Animals ; Cell Proliferation ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Genetic Vectors ; Human Umbilical Vein Endothelial Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Plasmids ; Recombinant Fusion Proteins ; genetics ; immunology ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology ; isolation & purification

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

Anti-angiogenesis mechanism plays a vital role in tumor targeting immunotherapy. Based on the amino acid sequence of an anti-VEGFR-2 scFv-Fc fusion antibody (AK404R-Fc), this article is aimed to generate an anti-VEGFR-2 human IgG1-like full length antibody (Mab-04). Firstly, the light chain (L-chain) and heavy chain (H-chain) were obtained by overlap PCR and then linked to eukaryotic expression vector pcDNA3.1, separately. The recombinant plasmids (pcDNA3.1-L-chain and pcDNA3.1-H-chain) were then co-transfected into CHO-k cells using liposome transient transfection. Subsequently, Mab-04 antibody was expressed and purified by Protein A affinity chromatography. Western blotting was applied to identify the expression of Mab-04 and its affinity was detected by ELISA assay. DNA sequencing revealed the successful construction of recombinant plasmids and Western blotting assay proved the successful expression of full-length antibody (1 microg x mL(-1)). Finally, ELISA assay illustrated that the binding of the antibody to its antigen was in a concentration-dependent manner (IC50: 50 nmol x L(-1)). These outcomes above indicated that Mab-04 was successfully expressed and assembled, which laid the foundation for further preparation and antineoplastic activity study.
Animals ; Antibodies, Monoclonal, Humanized ; analysis ; CHO Cells ; Cricetulus ; Genetic Vectors ; Immunoglobulin Fc Fragments ; genetics ; Immunoglobulin G ; genetics ; immunology ; Plasmids ; Recombinant Fusion Proteins ; analysis ; genetics ; Single-Chain Antibodies ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology

Abstract: This study is to improve the affinity of scFv-AK404R against VEGFR2. The secondary mutational library was constructed by hydrophilic shuffling in CDR3 region of the heavy chain. VEGFR2-specific screening was performed by phage display technology and the protein of mutants was expressed in periplasm of E.coli HB2151 and purified by affinity chromatography. The affinity constant of scFvs was measured by competitive ELISA, and the structure of scFvs was analyzed by bioinformatics. The result showed that a library with 6.4x10(5) scFv members was established by electro-transformation. Two mutated clones with high absorbance value were isolated after screening. After purification by affinity chromatography, electrophoretically pure scFv proteins were obtained. The competitive ELISA showed that the affinities of WZ01 and WZ02 were three times higher than that of the parental AK404R, and bioinformatics analysis showed that the enlarged contact surface and fitted closely with KDR3 surface may be the reasons for improved affinity. These results suggest that introducing hydrophilic amino acids to the heavy chain CDR3 region is an effective approach to improve the affinity of scFv.
Amino Acid Sequence ; Antibody Affinity ; Chromatography, Affinity ; Computational Biology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Hydrophobic and Hydrophilic Interactions ; Peptide Library ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology ; metabolism

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

BACKGROUNDOverwhelming evidences on chronic myeloid leukemia (CML) indicate that patients harbor quiescent CML stem cells that are responsible for blast crisis. While the hematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated.

METHODSWe have previously isolated fetal liver kinase-1-positive (Flk1(+)) cells carrying the BCR/ABL fusion gene from the bone marrow of Ph(+) patients with hemangioblast property. In this study, we isolated CML patient-derived Flk1(+)CD31(-)CD34(-) mesenchymal stem cells (MSCs) and detected their biological characteristics and immunological regulation using fluorescence in situ hybridization (FISH) analysis, fluorescence activated cell sorting (FACS), enzyme-linked immunoadsorbent assay, mixed lymphocyte reaction assays; then we compared these characters with those of the healthy donors.

RESULTSCML patient-derived Flk1(+)CD31(-)CD34(-) MSCs had normal morphology, phenotype and karyotype while appeared impaired in immuno-modulatory function. The capacity of patient Flk1(+)CD31(-)CD34(-) MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro.

CONCLUSIONSCML patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than hematopoietic stem cells (HSCs). MSCs might be a potential target for developing efficacious treatment for CML.

Adolescent ; Adult ; Antigens, CD34 ; genetics ; metabolism ; Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; Immunomodulation ; In Situ Hybridization, Fluorescence ; Karyotype ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; enzymology ; immunology ; metabolism ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mesenchymal Stromal Cells ; cytology ; immunology ; Middle Aged ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; T-Lymphocytes ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism ; Young Adult

In carcinoma ex pleomorphic adenoma (CXPA), pleomorphic adenoma (PA) and diverse carcinoma components showing luminal (ductal) or non-luminal (myoepithelial) differentiation coexist. To elucidate the clinicopathological implications of cellular differentiation in CXPA and the potential role of p53, vascular endothelial growth factor (VEGF), c-erbB-2, c-kit, and glucose transporter 1 (Glut-1) in carcinogenesis, we analyzed 11 CXPAs with luminal differentiation (CXPAs-LD) and 6 CXPAs with non-luminal differentiation (CXPAs-NLD) and compared protein expressions in residual PAs and carcinomas by immunohistochemistry. Among the CXPAs-LD, 5 were invasive and 8 were histologically high-grade tumors. The 5-year survival rate was 72.7%. P53, c-erbB-2, VEGF, and Glut-1 were more immunoreactive in carcinoma components than in PAs (P = 0.008, 0.004, 0.002, and 0.024, respectively); c-erbB-2 overexpression was associated with high histological grade (P = 0.024). Carcinoma components frequently lacked c-kit expression (P = 0.009). CXPAs-NLD were all low-grade and invasive with a larger mean tumor size (5.2 cm) than CXPAs-LD (3.3 cm) (P = 0.040). The patients remained disease-free without significant immunohistochemical expression. The immunoprofiles and clinical course of CXPA differed according to cellular differentiation. Therefore, it is important to report the histological subtype and to assess potential biomarkers in diagnostic and therapeutic trials.
Adenoma, Pleomorphic/*immunology/metabolism/*pathology ; Adult ; Aged ; Carcinoma/*immunology/metabolism/*pathology ; Cell Differentiation ; Female ; Glucose Transport Proteins, Facilitative/metabolism ; Humans ; Male ; Middle Aged ; Proto-Oncogene Proteins c-kit/metabolism ; Receptor, erbB-2/metabolism ; Salivary Gland Neoplasms/*immunology/metabolism/*pathology ; Tumor Markers, Biological/*analysis ; Tumor Suppressor Protein p53/metabolism ; Vascular Endothelial Growth Factor A/metabolism

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

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