Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons

Abstract

In the present study, we describe the production of transgenic silkworms expressing a recombinant mouse mAb in their cocoons. Two transgenic lines, L‐ and H‐, were generated that carried cDNAs encoding the L‐ and H‐chains of a mouse IgG mAb, respectively, under the control of the enhancer‐linked sericin‐1 promoter. Cocoon protein analysis indicated that the IgG L‐ or H‐chain was secreted into the cocoons of each line. We also produced a transgenic line designated L/H, which carried both cDNAs, by crossing the L‐ and H‐lines. This line efficiently produced the recombinant mAb as a fully assembled H2L2 tetramer in its cocoons, with negligible L‐ or H‐chain monomer and H‐chain dimer production. Thus, the H2L2 tetramer was synthesized in, and secreted from, the middle silk gland cells. Crossing of the L/H‐line with a transgenic line expressing a baculovirus‐derived trans‐activator produced a 2.4‐fold increase in mAb expression. The recombinant mAb was extracted from the cocoons with a buffer containing 3 m urea and purified by protein G affinity column chromatography. The antigen‐binding affinity of the purified recombinant mAb was identical to that of the native mAb produced by a hybridoma. Analysis of the structure of the N‐glycans attached to the recombinant mAb revealed that the mAb contained high mannose‐, hybrid‐ and complex‐type N‐glycans. By contrast, insect‐specific paucimannose‐type glycans were not detected. Fucose residues α‐1,3‐ and α‐1,6‐linked to the core N‐acetylglucosamine residue, both of which are found in insect N‐glycans, were not observed in the N‐glycans of the mAb.

Transgenic silkworms that weave recombinant proteins into silk cocoons

“…Each L-chain is linked to a H-chain by a disulfide bond, and the H-chains are linked to each other by other disulfide bonds. To investigate the synthesis of this relatively complex molecule in the silkworm, three transgenic lines, L-, H-, and L/H-, were generated, which synthesized the mouse IgG L-chain, H-chain, or both the L-and H-chains, respectively (Iizuka et al 2009). The L-line silkworm secreted the L-chain as a monomer into the cocoon, whereas the H-line silkworm secreted the H-chain as a dimer and higher molecular aggregates.…”

Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands

“…However, using iv ERT with recombinant human enzyme preparations produced by mammalian cell lines to treat LSDs has several disadvantages, i.e. i) large-scale production of recombinant human lysosomal enzymes by mammalian cells is limited and costly in comparison to the large-scale production of therapeutic antibodies (5)(6)(7) and ii) the risk of pathogen infection and the risk of an immune response and adverse effects including production of neutralizing antibodies against enzyme preparations in patients with an LSD and an enzyme deficiency as a result of continuous administration (18). Alternative human glycoprotein expression systems need to be developed as more effective, safer, and cheaper lysosomal enzyme sources to overcome these problems.…”

Melanoma antigen family A4 protein produced by transgenic silkworms induces antitumor immune responses

“…Many host systems, including bacteria, yeast and insect cells, are now used in the production of recombinant proteins, and the preferred method of production varies according to the purpose of use (7). The TG silkworm system has been used for the expression of recombinant proteins since 2000, and the expression of a number of recombinant proteins in TG silkworms, including green fluorescent protein, human serum albumin, membrane receptors and monoclonal antibodies has been documented (23)(24)(25). The TG silkworm system has a number of advantages, including its lower cost, mass breeding capacity, availability of multiple tissues for expression, suitability for large scale production and reduced endotoxin contamination.…”