Aishun Jin

A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood.

Antigen-specific human monoclonal antibodies (mAbs) are key candidates for therapeutic agents. However, the availability of a suitable screening system for antigen-specific antibody–secreting cells (ASCs) is limited in humans. Here we present a unique method for detecting individual ASCs using microwell array chips, which enables the analysis of live cells on a single-cell basis and offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for identifying and recovering objective ASCs. We applied the system to detect and retrieve ASCs for hepatitis B virus and influenza viruses from human peripheral blood lymphocytes and produced human mAbs with virus-neutralizing activities within a week. Furthermore, we show that the system is useful for detecting ASCs for multiple antigens as well as for selection of ASCs secreting high-affinity antibodies on a chip. Our method can open the way for the generation of therapeutic antibodies for individual patients.

Cell-microarray analysis of antigen-specific B-cells: single cell analysis of antigen receptor expression and specificity.

The authors previously developed a cell‐microarray system that effectively detects antigen‐specific B‐cells by monitoring intracellular Ca2+ at single cell levels. Here they present a novel method to detect antigen‐specific B‐cells using cell‐microarray system. To detect antigen‐specific B‐cells, they arrayed live lymphocytes on a chip, stained cells with fluorescence‐labeled nonspecific proteins, and analyzed them with a fluorescence scanner to detect nonspecific protein binding to B‐cells. They then stained cells with fluorescence‐labeled antigen and analyzed them with the scanner. Cells stained with specific antigen, but not with nonspecific proteins, were determined as antigen‐specific B‐cells and harvested. Antibody cDNA was amplified from retrieved B‐cells by single‐cell RT‐PCR, inserted into expression vectors, and was examined for its specificity by ELISA. They could detect antigen‐specific B‐cells at a frequency of 0.01% in a model system using transgenic mice that express antibody to hen‐egg lysozyme on the surface of B‐cells. They applied this system to directly detect hepatitis B virus surface‐antigen (HBs‐Ag)‐specific B‐cells from peripheral blood in HBs‐Ag‐vaccinated volunteers and succeeded in producing HBs‐Ag‐specific monoclonal antibody. This novel system allows us to identify human antigen‐specific B‐cells of very low frequency and is a powerful tool to explore the candidates of antibody therapeutics.

A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days.

Antigen-specific T cell therapy, or T cell receptor (TCR) gene therapy, is a promising immunotherapy for infectious diseases and cancers. However, a suitable rapid and direct screening system for antigen-specific TCRs is not available. Here, we report an efficient cloning and functional evaluation system to determine the antigen specificity of TCR cDNAs derived from single antigen-specific human T cells within 10 d. Using this system, we cloned and analyzed 380 Epstein-Barr virus–specific TCRs from ten healthy donors with latent Epstein-Barr virus infection and assessed the activity of cytotoxic T lymphocytes (CTLs) carrying these TCRs against antigenic peptide–bearing target cells. We also used this system to clone tumor antigen–specific TCRs from peptide-vaccinated patients with cancer. We obtained 210 tumor-associated antigen–specific TCRs and demonstrated the cytotoxic activity of CTLs carrying these TCRs against peptide-bearing cells. This system may provide a fast and powerful approach for TCR gene therapy for infectious diseases and cancers.

Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array

Here we report a new method for isolating antigen-specific antibody-secreting cells (ASCs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ASCs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. This protocol can be completed in less than 7 h, including 3 h of cell culture. The method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (ELISPOT) but it also overcomes the limitations of ELISPOT in recovering ASCs that can be used to produce antigen-specific human monoclonal antibodies. This method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis.

Analysis of the epitope and neutralizing capacity of human monoclonal antibodies induced by hepatitis B vaccine.

Hepatitis B virus (HBV) is an infectious agent that is a significant worldwide public health issue. However, the mechanism by which vaccination-induced antibodies prevent HBV infection remains unclear. To investigate the mechanism by which antibodies induced by hepatitis B surface Ag (HBsAg)-vaccination prevent HBV infection in humans, we prepared human monoclonal antibodies (mAbs) against HBsAg using a novel cell-microarray system from peripheral blood B-lymphocytes from vaccinated individuals. We then characterized the IgG subclass, L-chain subtype, and V-gene repertoire of the H/L-chain, as well as affinities of each of these mAbs. We also determined the epitopes of the individual mAbs using synthesized peptides, and the HBV-neutralizing activities of mAbs using the hepatocyte cell line HepaRG. Consequently, IgG1 and kappa chain was mainly used as the mAbs for HBsAg. Seventy percent of the mAbs bound to the loop domain of the small-HBsAg and showed greater neutralizing activities. There were no relationships between their affinities and neutralization activities. A combination of mAbs recognizing the first loop domain showed a synergistic effect on HBV-neutralizing activity that surpassed conventional hepatitis B-Ig (HBIG) in the HepaRG cell line assay. These results may contribute to the development of effective mAb treatment against HBV infection replacing conventional HBIG administration.

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy.

A Novel Rabbit Immunospot Array Assay on a Chip Allows for the Rapid Generation of Rabbit Monoclonal Antibodies with High Affinity

Antigen-specific rabbit monoclonal antibodies (RaMoAbs) are useful due to their high specificity and high affinity, and the establishment of a comprehensive and rapid RaMoAb generation system has been highly anticipated. Here, we present a novel system using immunospot array assay on a chip (ISAAC) technology in which we detect and retrieve antigen-specific antibody-secreting cells from the peripheral blood lymphocytes of antigen-immunized rabbits and produce antigen-specific RaMoAbs with 10–12 M affinity within a time period of only 7 days. We have used this system to efficiently generate RaMoAbs that are specific to a phosphorylated signal-transducing molecule. Our system provides a new method for the comprehensive and rapid production of RaMoAbs, which may contribute to laboratory research and clinical applications.

Characterization of a fully human monoclonal antibody against extracellular domain of matrix protein 2 of influenza A virus

The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved between influenza A virus strains compared to hemagglutinin and neuraminidase, and has long been viewed as a potential and universal vaccine target. M2e induces no or only weak and transient immune responses following infection, making it difficult to detect M2e-specific antibodies producing B-cells in human peripheral blood lymphocytes. Recently, using a single-cell manipulation method, immunospot array assay on a chip (ISAAC), we obtained an M2e-specific human antibody (Ab1-10) from the peripheral blood of a healthy volunteer. In this report, we have demonstrate that Ab1-10 reacted not only to seasonal influenza A viruses, but also to pandemic (H1N1) 2009 virus (2009 H1N1) and highly pathogenic avian influenza A virus, and that the antibody-bound M2e of 2009 H1N1 inactivated the virus with high affinity (∼10(-10)M). More importantly, it inhibited 2009 H1N1 viral propagation in vitro. These results suggest that Ab1-10 might be a potential candidate for antibody therapeutics for a wide range of influenza A viruses.

Post-translational modification of TRAIL receptor type 1 on various tumor cells and the susceptibility of tumors to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and TRAIL-R2) are promising targets for tumor therapy. However, their clinical use is limited because some tumors show resistance to TRAIL-treatment. Here, we analyzed epitopes of nine TRAIL-R1-specific human monoclonal antibodies and demonstrated at least five tentative epitopes on human TRAIL-R1. We found that some of the five were post-translationally modified on some tumor cell lines. Interestingly, one of them, an epitope of TR1-272 antibody (TR1-272-epitope) disappeared on the tumor cells that are more susceptible to TRAIL-induced apoptosis compared to TR1-272-epitope positive cells. Treatment of TR1-272-epitope negative cells with TRAIL induced large cluster formation of TRAIL-R1, while treatment of TR1-272-epiope positive cells with TRAIL did not. These results suggest that TR1-272-antibody might distinguish the TRAIL-R1 conformation that could deliver stronger death signals. Further analysis of epitope-appearance and sensitivity to TRAIL should clarify the mechanisms of TRAIL-induced apoptosis of tumor cells and would provide useful information about tumor therapy using TRAIL and TRAIL-R signaling.

Generation of TRAIL-receptor 1-specific human monoclonal Ab by a combination of immunospot array assay on a chip and human Ab-producing mice.

Monoclonal antibodies have attracted attention clinically as promising therapeutic agents for immunotherapy against various malignancies because of their high specificity and low toxicity. In the clinical application of mAb, immunogenicity of mAb is one of the major concerns. We have developed immunospot array assay on a chip (ISAAC) technology using chips with an array of microwells [1], which enables direct identification of Ag-specific Ab-secreting cells (ASC) from human peripheral blood lymphocytes and rapid cloning of Ab cDNA, leading to easy, efficient and rapid production of human mAb. However, the limitation of immunizing volunteers with Ag hampers its application for the production of human mAb for desired Ag. In this study, we used the ISAAC method in combination with TransChromo (TC) mice that contain human chromosome fragments encoding the entire human immunoglobulin H-chain and kL-chain loci [2] for isolating fully human mAb specific to TNF-related apoptosis-inducing ligand (TRAIL)-receptors type 1 with the object of cancer immunotherapy. TRAIL receptors are expressed in a wide variety of tumor cell types as well as normal cell types. TRAIL induces apoptosis in a wide variety of human cancer cell lines by activating two functional receptors, TRAILFigure 1. Detection of TRAIL-R1-specific ASC and production of TRAIL-R1-specific Ab with ISAAC system. (A) Schematic diagram of rapid generation of fully human mAb from TRAIL-R1-immunized TC mice using ISAAC method. (B) Detection of single human TRAIL-R1-specific ASC using ISAAC system. (Left) Signals for Ag-specific Ab secreted from individual cells; (middle) cells stained with Oregon Green; (right) merged