Hualiang Huang

Targeting TNF-α with a tetravalent mini-antibody TNF-TeAb

Anti-TNF-α [anti-(tumour necrosis factor-α)] therapy is widely considered to be among the most efficient treatments available for rheumatoid arthritis, psoriatic arthritis and inflammatory bowel disease. In the present study a tetravalent mini-antibody, named ‘TNF-TeAb’, was constructed by fusing the tetramerization domain of human p53 to the C-terminus of an anti-TNF-scFv [anti-(TNF-α–single-chain variable fragment)] via a long and flexible linking peptide derived from human serum albumin. TNF-TeAb was overexpressed as inclusion bodies in the cytoplasm of Escherichia coli, purified to homogeneity by immobilized- metal affinity chromtaography under denaturing conditions and produced in functional form by using an in vitro refolding system. In vitro bioactivity assays suggested that tetramerization of TNF-scFv resulted in an enormous gain in avidity, which endowed TNF-TeAb with a stronger ability to inhibit both receptor binding and cytolytic activity of TNF-α. TNF-α targeting therapy in rats with collagen-induced arthritis demonstrated that TNF-TeAb provided a much more significant therapeutic effect than did TNF-scFv in suppressing arthritis progression, attenuating inflammation and destruction in joints, and down-regulating pro-inflammatory cytokines and anti-(type II collagen) antibody. The conclusions are therefore (i) that multimerization of the antibody fragment by a self-association peptide is an efficient way to increase its avidity and (ii) that TNF-TeAb has potential applicability for anti-TNF-α therapy.

One‐step on‐column purification and refolding of a single‐chain variable fragment (scFv) antibody against tumour necrosis factor α

Single‐chain variable fragment (scFv) is a low‐molecular‐mass recombinant antibody and is usually expressed as inclusion bodies in Escherichia coli. Highly efficient purification and refolding methods are required to provide enough active proteins for therapeutic or diagnostic use. In the present study, an anti‐TNFα (tumour necrosis factor α) scFv (TNF‐scFv) was constructed and expressed in E. coli BL21(DE3) star as inclusion bodies, and a convenient procedure of one‐step on‐column purification and refolding was provided for it. Briefly, denatured TNF‐scFv was firstly captured by immobilized metal (Ni) affinity chromatography, and then non‐denaturing detergent (Triton X‐100)‐containing and β‐cyclodextrin‐containing solutions were loaded in turn on to the column to perform ‘artificial chaperone‐assisted refolding’ after removing impurities. More than 77% of denatured TNF‐scFv protein was refolded successfully with a purity of more than 95%. Activity assays showed that refolded TNF‐scFv could bind to rhTNFα (recombinant human TNFα) specifically with high affinity. It could inhibit rhTNFα from binding to TNF receptors and neutralize the cytolytic activity of rhTNFα against L929 cells effectively. A conclusion was obtained that this refolding method is time‐saving and suitable for industrial production. It may also be applicable to other scFvs or other recombinant proteins.

A novel bivalent single‐chain variable fragment (scFV) inhibits the action of tumour necrosis factor α

Suppression of TNFα (tumour necrosis factor α) activity is widely considered to be among the most efficient treatments available for chronic inflammatory diseases. Here, a bivalent scFv (single‐chain variable fragment) fragment, named TNF‐BAb, was engineered by fusing two anti‐TNFα scFV fragments in tandem via a long and flexible linking peptide derived from human serum albumin and produced in functional form from Escherichia coli inclusion bodies. The bioactivity assays demonstrated that TNF‐BAb gained enormously in avidity and showed a much stronger ability to inhibit the biological action of TNFα, indicating that TNF‐BAb may become a good candidate for anti‐TNFα therapy.

Fusion of chemotactic peptide to a single-chain bi-specific antibody (scBsAb) potentiates its cytotoxicity to target tumour cells

Anti‐tumour BsAb (bi‐specific antibody) has been proved very effective in killing tumour cells both in vitro and in vivo. In order to enhance its ability to recruit and activate T‐lymphocytes and then improve tumour‐specific cytolysis, an anti‐ovarian carcinoma/CD3 BsAb, BHL‐I, was fused to N‐terminal 18 peptide of CCL21 (CC chemokine ligand 21) to produce a new chemotactic BsAb, named 18TBHL. It was expressed in soluble form in the cytoplasm of Escherichia coli and purified with DEAE anion‐exchange chromatography and immobilized‐metal‐ion affinity chromatography. The chemotactic ability of 18TBHL to PBLs (peripheral‐blood lymphocytes) was detected by Boyden chamber chemotaxis assay. The specific ability to bind to ovarian carcinoma cells, SKOV3, and PBLs was tested by ELISA and flow cytometry. Just as expected, the enhanced tumour‐specific cytolysis of 18TBHL was validated by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] method and flow cytometry. The results indicated that fusion of chemotactic peptide to BsAb potentiated its cytotoxicity to tumour cells in vitro. It suggests that 18TBHL may be a promising candidate agent in cancer immunotherapy.

ISOLATION OF A VERNALIZATION-RELATED CDNA CLONE (VRC) USING MRNA DIFFERENTIAL DISPLAY IN WINTER WHEAT

Vernalization is an essential factor which affects the flowering development in cold-requiring plants. There is a key stage of nucleic acid and protein metabolism in the process of vernalization in winter wheat. To probe into the molecular determinants of vernalization, we examined mRNA populations in differently-treated plumules of winter wheat (Triticum aestivum L. cv Yanda 1817) using mRNA differential display. One vernalizationrelated cDNA clone (VRC), VRC54, was identified and was only expressed at the key stage of 20 d vernalization, rather than at other stages of nonvernalization, 4 d vernalization and devernalization. Northern blot and sequence analysis indicated that VRC54 was a novel vernalization-related clone found in higher plant which not only might play an important role in the floral induction in vernalization-requiring plants but also was different from the cold-acclimatized genes.

RESEARCH ADVANCES IN GENETIC ENGINEERING OF PLANTIBODY

The combination of engineering antibody and plant biotechnology creates the plantibody. It has been reported that the engineering antibodies expressed in plant, no matter whether they are intact or small-molecular antibodies, keep their antigen-binding specificity, which means they are functional. This trait makes plantibody notable. Now the researches are focused mainly on the following three aspects: (i) Therapeutic antibody in clinic. It is expected to offer the opportunity of large-scale antibody procluction in agriculture systems, for the purpose of decreasing the cost greatly. Moreover, the property of multiple sexual hybridization between plants can be used to produce bior multi-functional antibodies and create new antibody medicines. (ii) Plant physiology. Engineering antibodies against plant hormones or some active materials can block their activities in plant, so the trait of plantibody could be used to study the growth and development of plant. (iii) Plant disease-resistance. The gene of antibody against plant virus can be transferred into plant to defend the intrusion of virus in order to cure the disease.