Generation and selection of immunized Fab phage display library against human B cell lymphoma

Abstract

npgThe approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94×10 7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (V H ) and variable light domains (V L ) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

Application of antibody phage display to identify potential antigenic neural precursor cell proteins

“…The immunized phage library that was created consists of 2.5 × 10 6 cfu and is considered to be moderate, since more but even less complex libraries have been reported. As a measure, an immunized phage display library contains 99% of all antibody molecules when 1 × 10 7 individual clones are represented [13,14]. Since the phagemids of the created library are stored, a freshly prepared library could be easily reamplified on demand.…”

Isolation of Osteosarcoma‐Associated Human Antibodies from a Combinatorial Fab Phage Display Library

“…This cannot be attributed to a bias in the Fab library, since all of the sequenced clones from rounds two or three had different VH and V κ sequences compared to the original library [10]. The emergence of clones with a restricted diversity of V gene fragments is a feature that accounts for the selection of specific ligands, as was reported when other antibody phage display libraries were used to isolate high affinity immunoglobulins [22, 24]. Clones sharing VH3-7 and A17 were the majority of the clones identified in our selection process.…”

Recombinant human arginase induced caspase-dependent apoptosis and autophagy in non-Hodgkin’s lymphoma cells

“…For western blot analysis, cells were harvested and washed with cold phosphate-buffered saline (PBS) and kept on ice for at least 20 min in Cell Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China). 41 The lysates were centrifuged and the supernatants were collected. Equal amounts of protein were run on SDS-PAGE gels and subsequently transferred to polyvinylidene fluoride membranes.…”