Functional Fv fragment of an antibody specific for CD28: Fv‐mediated co‐stimulation of T cells

Abstract

The most predominant co-stimulation pathway, which is critical for T cell activation and proliferation, is the CD28-B7 pathway. The anti-CD28 monoclonal antibody (mAb) also provides a co-stimulatory signal to T cells. In order to construct a functional Fv fragment (complex of VH and VL domains) of anti-CD28 antibody using a bacterial expression system, cDNA encoding the variable regions of immunoglobulin from 15E8 hybridoma cells was cloned and expressed in Escherichia coli. The Fv fragment was obtained as a soluble protein from the periplasmic fraction and showed a binding pattern similar to parental IgG. The Fv fragment induced proliferation of peripheral blood mononuclear cells in the presence of anti-CD3 or anti-CD2 mAb and enhanced anti-tumor activity of anti-MUC1U Uanti-CD3 bispecific antibody when tested with lymphokine-activated killer cells with T cell phenotype. Thus, the anti-CD28 Fv fragment will be promising not only for the study of costimulation, but also for cancer immunotherapy. ß

Multimerization of anti‐(epidermal growth factor receptor) IgG fragments induces an antitumor effect: the case for humanized 528 scFv multimers

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Anti‐EGFR scFv tetramer (tetrabody) with a stable monodisperse structure, strong anticancer effect, and a long in vivo half‐life

“…Bacterial expression vectors for h528 scFv in the V H – V L order with a 16‐amino acid linker (HLG3), with a 6‐amino‐acid linker (HLG1), and without a linker (HLG0) were constructed previously . The scFvs were expressed individually in E. coli strain BL21(DE3) (Life Technologies, Carlsbad, CA, USA) and purified from bacterial supernatant plus periplasmic fraction using immobilized metal ion affinity chromatography . The scFvs were also prepared from ICS fraction using BugBuster reagent (Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions.…”

Antitumor activity of interleukin‐21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli

“…Recent advances in genetic engineering have made it easier to prepare recombinant proteins such as cytokines, chemokines, co‐stimulatory molecules, and fragments of variable regions of antibodies (Fv) [3–6]. The successful construction of several proteins by using bacterial expression systems has been reported, and some proteins are now used for therapeutic purposes [7–9]. Overproduction of proteins, however, often leads to the formation of insoluble aggregates, referred to as inclusion bodies, in the cytoplasmic or periplasmic space.…”