Hing C. Wong

In vitro and in vivo characterization of a novel antibody-like single-chain TCR human IgG1 fusion protein.

We have constructed a protein composed of a soluble single-chain TCR genetically linked to the constant domain of an IgG1 H chain. The Ag recognition portion of the protein binds to an unmutated peptide derived from human p53 (aa 264–272) presented in the context of HLA-A2.1, whereas the IgG1 H chain provides effector functions. The protein is capable of forming dimers, specifically staining tumor cells and promoting target and effector cell conjugation. The protein also has potent antitumor effects in an in vivo tumor model and can mediate cell killing by Ab-dependent cellular cytotoxicity. Therefore, single-chain TCRs linked to IgG1 H chains behave like Abs but possess the ability to recognize Ags derived from intracellular targets. These fusion proteins represent a novel group of immunotherapeutics that have the potential to expand the range of tumors available for targeted therapies beyond those currently addressed by the conventional Ab-based approach.

Design and analysis of PCR primers for the amplification and cloning of human immunoglobulin Fab fragments

We have developed PCR primers for the amplification and cloning of the genes encoding human antibody fragments. Two sets of primers were designed to amplify the coding sequence of the complete variable region and the first constant domain of immunoglobulin heavy chains. One set of primers was designed to amplify the coding sequence of complete kappa light chains. These three sets of primers were used in PCR amplifications with cDNA prepared from EBV transformed B cells as the template. The amplified fragments were cloned and their nucleotide sequences were determined. Analysis of the DNA sequences of such PCR generated antibody fragments suggested that our primer sets were able to amplify the major subsets of the heavy and light chain variable region genes. Comparison between the estimated frequency of the different subsets of heavy chain variable region genes and the distribution of our subclones further indicated that one set of our primers was able to proportionally amplify the subsets of the heavy chain family.