Endothelial cell specific antibodies and uses thereof
A specific and antibody technology, applied in the direction of antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, specific peptide, etc.
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Embodiment 1
[0083] Endothelial Precursor Cells (EPC's)
[0084]Myeloid cells expressing the endothelial lineage markers AC133 and CD34 were stimulated with VEGF, bFGF and heparin in fibronectin or collagen monolayer cultures to differentiate into a phenotype described as endothelial precursor cells (EPCs). Briefly, in order to differentiate bone marrow cells into EPC's, the AC133+ / CD34+ bone marrow cell population was isolated using conventional methods. In suspension cultures, 50 ng / ml VEGF was used in IMDM medium supplemented with 15% FBS 165 , 10-50ng / ml bFGF, and 50ng / ml heparin stimulated AC133+ / CD34+ cells. Cells undergo rapid proliferation and differentiation. The resulting adherent cell populations were then used in in vitro assays examining TEM function. These EPCs can be expanded in culture for more than twelve passages and appear to be an intermediate population between bone marrow-derived stem cells and fully differentiated endothelial cells. Characterization of these EPCs...
Embodiment 2
[0086] TEM1 antibody
[0087] To generate mouse polyclonal antibodies to TEM1, C57B1 / 6 mice were inoculated intramuscularly with the pcDNA 3.1 human TEM1 (hTEM1) plasmid containing the full-length TEM1 human gene. The human TEM1 coding sequence was cloned from human fetal brain RNA by RT-PCR using 3' and 5' TEM1 specific primers. The 5' end primer was modified to include the consensus Kozak sequence from CCACC 5' to the ATG start codon. Sequencing confirmed the amplified product, a 2273 base pair (bp) fragment, corresponding to the nucleotide ( nt) 6-nt 2279, and subcloned into pCDNA3.1 vector (Invitrogen). This vector pcDNA3.1 hTEM1 encodes a full-length protein of 757 amino acids.
[0088] To generate polyclonal specificity for the TEM1 extracellular domain, nt102 to nt1963 of the full-length human gene of TEM1 was subcloned into the VV-1 vector (Genovac AG, Freiburg, Germany), with the 5' signal sequence located at the 5' end In the same reading frame as myc-TAG and the...
Embodiment 3
[0095] TEM17 antibody
[0096] To generate a polyclonal antibody specific for the extracellular structure of TEM17, nt 152 to nt 1318 of the full-length human gene of TEM17 was subcloned into the VV-1 vector (Genovac AGG, Freiburg, Germany), with the 5' signal sequence located at the 5' end and myc-TAGG and the GPI transmembrane anchor motif located at the 3' end are in the same reading frame, resulting in VV-1 TEM17. The extracellular portion included in the vector encodes TEM17 of amino acids 24 to 412 of SEQ ID NO. 230 of US Serial No. 09 / 918715 (Publication No. 20030017157). Rabbits were immunized with the VV-1 TEM17 vector.
[0097] To produce murine polyclonal antibodies to TEM17, C57B1 / 6 mice were immunized intramuscularly with the pcDNA 3.1 hTEM17 plasmid containing the full-length TEM17 human gene. The human TEM17 coding sequence was cloned from human fetal brain RNA by RT-PCR using 3' and 5' TEM17 specific primers. The 5' end primer was modified to include the con...
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