To identify the interchangeability of V(H) and V(L) framework region (FR) residues, we artificially introduced random mutations at all residue positions in a chicken monoclonal antibody, which has only one functional V(H) and Vλ gene. When we classified the amino acids into 5 groups by their physicochemical properties, all FR residues could be replaced by another group except L23 (C), H36 (W), H86 (D), H104 (G), and H106 (G). Eighty-two (50.9%), 48 (29.8%), 17 (10.6%), and 9 FR residues (5.6%) could be replaced by 4, 3, 2, and 1 group(s), individually, without significant loss of reactivity. We also confirmed a similar level of versatility with 2 different chicken antibodies. This high level of versatility on FR residues has not been predicted because it has not been observed in the 150 chicken antibodies that we previously generated or in the 1,269 naïve chicken V(H) sequences publically available. In conclusion, chicken antibody FR residues are highly interchangeable and this property can be applied for improving the physicochemical property of antibody including thermal stability, solubility and viscosity.
Amino Acids ; Antibodies ; Chickens* ; Immunoglobulin Variable Region ; Solubility ; Somatic Hypermutation, Immunoglobulin ; Viscosity

The humoral immune response of B cells is the key to the protection of specific immunity, and immune aging reshapes its production and function. The decreased B cell immune function is an indicator of immune senescence. The impaired humoral immune function mediated by antibody secreted by B cells leads to a decline in the response of elderly individuals to the vaccine. These people are therefore more susceptible to infection and deterioration, and have a higher incidence of tumors and metabolic diseases. Activation-induced cytidine deaminase (AID) is an enzyme that triggers immunoglobulin class conversion recombination (CSR) and somatic high frequency mutation (SHM). It decreases during immune senescence and is considered to be a biomarker of decreased B cell function in aging mice and humans. Understanding the inherent defects of B-cell immune senescence and the regulation mechanism of AID in the aging process can provide new research ideas for the susceptibility, prevention and treatment of diseases in the elderly.
Animals ; Humans ; Mice ; Aging/metabolism* ; B-Lymphocytes/metabolism* ; Cytidine Deaminase/metabolism* ; Somatic Hypermutation, Immunoglobulin

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.
Animals ; Antibody Affinity ; Cells, Cultured ; Cytidine Deaminase ; genetics ; metabolism ; HEK293 Cells ; Humans ; Immunoglobulin Variable Region ; genetics ; immunology ; Mice ; Mutation ; Single-Chain Antibodies ; chemistry ; genetics ; immunology ; Somatic Hypermutation, Immunoglobulin ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo study the clonal rearrangements and mutation status of IgVH genes in classic Richter's syndrome, the relationship between molecular findings of IgVH gene and clinical outcome, and to deciper the possible molecular mechanism of transformation.

METHODSThe clonal rearrangements and mutation status of IgVH genes were analyzed in cases of classic Richter's syndrome by Genescan and sequencing. Immunohistochemical study for zeta-chain associated protein kinase 70 kDa (ZAP70), p53 and interferon regulation factor 4 (IRF-4) was also performed.

RESULTSSamples of 18 cases of B-chronic lymphocytic leukemia (B-CLL)/ diffuse large B-cell lymphoma (DLBCL,78. 3%) had identical tumor cell clones, whereas DLBCL developed as a clonally independent neoplasm in 5 patients (21.7%). Among the clonally related group, 12 cases carried unmutated VH genes in both B-CLL and DLBCL components and VH3-23, VH3-74 and VH1-2 were accounted for the B-CLL transformation to DLBCL. Immunohistochemical study showed that the transformed DLBCL expressed CD5 in 32.1% of cases, CD23 in 14.3%, ZAP70 in 23.8%, p53 in 80.6% and IRF-4 in 82.6% of the cases respectively. Follow-up data were available in 17 patients with classic Richter's syndrome. The median survival period was 7 months. No significant difference in survival rate was obtained between the clonally related or unrelated groups, between IgVH gene mutated or unmutated groups, and between the groups with or without expression of ZAP70, p53 and IRF-4.

CONCLUSIONSThe ratio of clonally related transformed DLBCL from B-CLL to clonally unrelated DLBCL is 2:1. Clonal transformation to DLBCL predominantly occurs in B-CLL patients carrying unmutated IgVH genes. The biased IgVH gene usage suggests antigens are involved in classic Richter's syndrome. Molecular differences of IgVH genes and very poor clinical outcome of this group of transformed DLBCL indicate that there cases may be regarded as a distinct subset of DLBCL.

Aged ; Aged, 80 and over ; B-Lymphocytes ; pathology ; Genes, p53 ; genetics ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; Lymphoma, B-Cell ; genetics ; Lymphoma, Large B-Cell, Diffuse ; genetics ; Male ; Middle Aged ; Somatic Hypermutation, Immunoglobulin ; genetics ; ZAP-70 Protein-Tyrosine Kinase ; genetics