ABSTRACT
Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Subject(s)
Animals , Humans , Mice , Antibody Affinity , Cells, Cultured , Cytidine Deaminase , Genetics , Metabolism , HEK293 Cells , Immunoglobulin Variable Region , Genetics , Allergy and Immunology , Mutation , Single-Chain Antibodies , Chemistry , Genetics , Allergy and Immunology , Somatic Hypermutation, Immunoglobulin , Genetics , Allergy and Immunology , Tumor Necrosis Factor-alpha , Allergy and ImmunologyABSTRACT
As an intracellular non-histone chromosomal binding protein, High mobility group Box-1 ( HMGB1 ) plays an important role in maintaining the stability of the nuclear body and DNA recombination, duplication, repair and genetic transcription. Besides, extracelluar HMGB1 may serves as a "late inflammatory mediator", which start inflammatory response in the late stage of inflammation. Recent studies have found that,HMGB1 is closely related with the growth, infiltration and migration of pancreatic carcinoma. Also, HMGB1 can promote the degradation of extracelluar matrix, the infiltration and the migration of pancreatic carcinoma.
ABSTRACT
Tumor necrosis factor alpha(TNF-?) is a multi-functional cytokine that plays a significant role in many autoimmune diseases. The key biological functional domain of mouse TNF-? was determined by identifying the binding site of mouse TNF-? neutralizing antibody 9C6. Using yeast surface display technology, it was determined that 9C6 can recognize the linear amino acid fragment 29 ～40 of mature mouse TNF-? protein. Through mutagenesis experiments of this TNF-? region, the critical amino acids necessary for 9C6 binding were identified. Finally, wild-type mouse TNF-? and mutant variants that loses binding ability to 9C6 were expressed in Escherichia coli, purified, and used in a L929 cell killing assay. The assay results proved that the key amino acids for 9C6 binding were consistent with mouse TNF-? functional domain.
ABSTRACT
As an effective method of studying soluble protein-protein interactions, yeast display system is now widely used for affinity maturation of single-chain antibodies.Due to the strong homology recombination machinery of yeast and the high-throughput nature of FACS detection, a rapid scan for interaction between antigen-antibody pairs could be easily achieved.Based on this system, a novel and reliable method for determining conformational epitopes was developed.Different fragments of macrophage migration inhibitory factor(MIF) and several point mutations of MIF were displayed on yeast cell surface using homologous recombination technology.Three MIF monoclonal antibodies, 10C3, 2A12 and 4E10, were screened for their binding affinity to each displayed peptide.Utilizing this technology, the key amino acids of MIF that bind to the MIF monoclonal antibodies were easily identified.
ABSTRACT
Previous studies have suggested that common breast cancers are associated with EBV. We used a highly sensitive quantitative real-time PCR method to screen whole tumor sections of breast cancers for the presence of the EBV genome. EBV DNA was detected in 19 of 92 (21%) tumors, but viral load was very low in positive samples (mean = 1.1 copy EBV/1000 cells, maximum = 7.1 copies EBV/1000 cells). Importantly, quantitative real-time PCR failed to detect the EBV genome in microdissected tumor cells from any case. Using a monoclonal antibody (2B4-1) reactive against the EBV nuclear antigen-1, we noted strong staining of tumor nuclei in a proportion of those breast cancers that had tested negative for the presence of the EBV genome. Because nuclear staining with the 2B4-1 antibody was previously observed more frequently in poor prognosis breast cancers, we examined a larger series of breast cancers with complete clinical follow-up. Strong punctate staining of tumor cell nuclei was observed in 47 of 153 (31%) breast cancers; 2B4-1-positive tumors were significantly more likely to be ER-negative (P < 0.0001), to be of higher grade (P = 0.001) and larger (P = 0.03), to involve more regional lymph nodes (P = 0.01), and to have higher Nottingham Prognostic Index scores (P = 0.0003). Conclusions are: (a) EBV can be regularly detected in whole sections of breast cancers but viral copy number is very low; (b) in these cases, tumor cells do not harbor virus; and (c) reactivity with the monoclonal antibody 2B4-1 is detectable in the absence of the EBV genome and is strongly associated with ER-negative breast tumors and with prognostically unfavorable disease. Additional studies should be directed to the identification of this protein and to elucidation of its role in breast cancer.
Subject(s)
Antibodies, Monoclonal/chemistry , Breast Neoplasms/virology , Epstein-Barr Virus Nuclear Antigens/immunology , Breast Neoplasms/pathology , Female , Genome, Viral , Herpesvirus 4, Human/genetics , Humans , Immunohistochemistry , In Situ Hybridization , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Viral LoadABSTRACT
The Epstein-Barr virus (EBV) has recently been associated with hepatocellular carcinoma (HCC) arising in Japanese patients. We analyzed 82 cases of HCC from Germany and the U.K. for the presence of EBV DNA and viral gene products within tumor cells. Initial screening of whole sections using quantitative (Q)-PCR detected EBV DNA in 9/58 U.K. cases and in 9/24 German cases; in positive cases viral load was very low, ranging between 1.4 and 49.1 copies of the EBV genome/1000 cell equivalents, compared to much higher values for EBV-positive Hodgkin's disease and nasopharyngeal carcinoma controls (range, 714-3259/1000 cells). EBV DNA was not detected in the tumor cells of any of the Q-PCR-positive cases either by Q-PCR of pure tumor cell populations isolated by laser capture microdissection or by isotopic in situ hybridization. Furthermore, none of the German or U.K. HCC tumors tested positive for EBER or EBNAI expression in tumor cells. Our results provide strong evidence that HCCs from the U.K. or Germany are not associated with EBV.
