hDM-C6 MH3B1 is selleck chemicals llc relatively stable in the presence of serum at 37°C. Comparison of the structures of hDM and the wild type enzyme as well as analysis of potential MHCII binding peptides generated as a result of fusion of two proteins and the
Glu201Gln:Asn243Asp mutations suggest that hDM-αH-C6.5 MH3B1 should have minimal immunogenicity in humans. Therefore, the hDM-C6 MH3B1-F-dAdo combination addresses many of the current limitations of ADEPT and provides an excellent candidate for treatment of HER2/neu expressing tumors with minimum systemic toxicity or immunogenicity. Methods AZD6244 solubility dmso Materials Guanosine and F-Ade were purchased from Sigma-Aldrich (St. Louis, MO), and F-dAdo was purchased from Berry & Associates (Dexter, MI). CT26 was purchased from ATCC ( Manassas, VA). Construction and characterization of CT26HER2/neu is described previously . MCF7-HER2  was a gift from Dr. Dennis Slamon (University of California, Los-Angeles). Cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; GIBCO, Carlsbad, CA) containing 5% calf-serum (GIBCO) for CT26 and CT26HER2/neu and IMDM containing 10% fetal bovine serum (GIBCO), 1% non-essential amino acids (GIBCO) and 1% sodium pyruvate Tucidinostat (GIBCO) for MCF-7HER2 cells. The expression vector for production of ECDHER2 was a gift from Dr. James Marks (University of California, San-Francisco). Plasmid construction and protein purification
Cloning of hPNP and hDM with αH linker at its C-terminus was Thymidylate synthase described previously . To construct hPNP-αH-C6.5 MH3B1 or hDM-αH-C6.5 MH3B1 genes,
first PNP-αH was amplified using 5′ gcggccgc gataccaccgatatccaccatggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggagacgagaatggatac acctatgaagattataagaac3′ and 5′taaagaggccgcagccaaagcgcaggtgcagctggtgcagtctgg3′ as forward and reverse primers respectively. The forward primer contains a NotI site, Kozak sequence and signal peptide, and the beginning of the PNP gene. The reverse primer encodes the αH linker and the beginning of C6.5 MH3B1. The sequence for the signal peptide is gatatccaccatggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggagac. The amino acid sequence for the αH linker is AEAAAKEAAAKA. The C6 MH3-B1 gene was PCR amplified with the forward primer complementary to the reverse primer used for PNP amplification encoding for αH linker, and the beginning of the C6.5 MH3B1 sequence. 5′ggagggaccaaggtcaccgtcctaggtcgttaataa tctaga 3′, which encodes the C-terminus of scFv and an XbaI site, was used for the reverse primer. The PCR product of each gene was purified, annealed and used as template for the final PCR amplification using PNP forward primer containing a NotI site and C6.5 MH3B1 reverse primer containing a XbaI site. The PCR product was cloned into the TOPO-Blunt vector (Invitrogen, Carlsbad, CA) and the sequence confirmed.