Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology

Abstract

The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

Targeting the 4-1BB costimulatory molecule through single chain antibodies promotes the human T-cell response

“…For cell treatment, 500 × 10 3 purified T-cells were seeded into 24-and 48-well plates and treated with 10 μg/ml different scFvs for 72 h (Fig. 1c) [28].…”

“…A minimum of 100,000 events were acquired. All data analysis was performed using the FlowJo software (v.7.2.5; Tree Star) [28].…”

“…We previously used phage display technology to isolate four anti-4-1BB single-chain fragment variable antibodies (scFvs), called PI.12, PI.42, PII. 16 and PII.29 [28]. These antibody fragments specifically bind to 4-1BB, activate T cells and increase IL-2 production.…”

Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA1) from a M13 Phage Display Library Using Purified LPA1 Stabilized in Nanodiscs

“…Sequence analysis of these two clones showed distinct sequences at the CDR regions (Figure (c)), indicating that they might bind to different sites of LPA 1 . The number of scFv clones specific to LPA 1 was relatively low compared to the numbers recorded in other reports of biopanning experiments . However, there was a significant number of scFv clones specific to both P9 and P9‐LPA 1 (data not shown), although the library for biopanning had been pretreated with an immobilized nanodisc containing P9 protein.…”

Construction of immune human library and screening of neutralizing antibodies specific to glycoprotein of severe fever with thrombocytopenia syndrome virus

“…After four rounds of panning, the single clone of eluted phages was picked up to produce phage antibody, and tested for their binding activity to Gn or Gc by phage-ELISA [14]. For the positive clones, the genes of V H and V L chains were sequenced, and their corresponding amino acid sequences were aligned.…”