Research and Development Projects Adopted in FY2015

Development of the methods for high-performance isolation of T-cell receptor (TCR) like antibodies against intracellularly expressing tumor antigens

Project Leader:Isobe Masaharu

Professor, Graduate School of Science and Engineering for Research, University of Toyama

Isobe Masaharu

In the development of the therapeutic antibody for cancer treatment, an antigen molecule showing selective expression on the surface of the cancer cells is usually chosen as a target. Among cancer cells expressing antigens, “cancer antigens” are only expressed in cancerous cells, while “cancer-related antigens” are expressed in both cancer and normal cells. But the levels of those expressions in cancer cells are much higher than those of normal cells. So far more than several hundreds of such antigens have been found. Since the majority of such cancer (-related) antigens are only expressed within the cells but not on the surface of cancerous cells, it has been believed that those antigens are not suitable targets for cancer therapeutic antibodies, which ultimately cause a shortage of target antigen. Recent findings indicate that a part of peptides derived even from the protein only expressed within the cells are presented on the surface of cancer cells by forming a complex with major histocompatibility antigen (MHC) class I molecules. This complex is normally recognized by a T-cell receptor (TCR) expressed on the surface of cytotoxic T-cells (CTLs) leading to elimination of cancer cells, presenting a complex composed of a peptide derived from a cancer antigen and MHC class I molecules (cancer pMHC). More recently the use of the TCR-like monoclonal antibody (mAb) recognizing this cancer pMHC attracts attention due to its potential on the recognition of intracellular cancer antigen. However in spite of many efforts, the use of the TCR-like mAb has been disturbed till now by difficulty of its acquisition. The big possibility opens out for the development of the new therapeutic drug if one obtains the method for isolating TCR-like mAb more certainly. Thus the purpose of this project is the development of the method for generation of TCR-like antibody more reliably by improving our mAb isolation system described in Fig 1 and 2.

Recently the method termed Bi-specific T-cell Engager (BiTE) is shown to be effective to kill cancer cells. BiTE antibodies are designed to utilize the binding properties of two mAbs, one binds to an antigen on the surface of a cancer cell while the other binds to CD3 molecule on the surface of a T-cell. By bridging between a cancer cell and an endogenous cytotoxic T-cell with this BiTE antibody, it can induce the destruction of tumor cells at low concentration of BiTE antibody as described in Fig 3. Therefore we also aim the construction of such BiTE antibody if we succeed in obtaining TCR-like mAb against a cancer pMHC to evaluate its efficacy of a TCR-like mAb and its potential to the seed for therapeutic antibody.

<Figure1> The outline of the high-performance isolation system of antibodies from antigen-specific single plasma cells.

We have developed the most advanced system capable of rapid and large-scale isolation of antigen specific mAbs. Three key steps compose our system. The first step is an isolation of antigen specific antibody producing cells from immunized animals. The second step is a single cell-based cDNA synthesis. We have developed the method named MAGnetic reaction through Arrayed Hanging Droplets (MAGrahd) to synthesize cDNA reliably from hundreds of single antibody producing cells. The third step is simple and efficient way to make expression constructs of immunoglobulin genes named target selective joint polymerase chain reaction (TS-jPCR) without any purification steps. The resulting immunoglobulin-expression constructs from amplified V genes were ready to transfect into cultured cells without any purification and the media were used for the analysis of binding specificities of mAbs. Our new system significantly reduced the cost and the time for generation of mAbs so that it allows us to obtain hundreds of mAbs within four to five days. With this world fastest antibody isolation system in hand we aim the development of the methods suitable for generation of TCR-like mAbs.

<Figure2> The MAGrahd system.

To automatize cDNA synthesis from large numbers of single cells, we constructed a noncontact magnetic power transmission instrument that we call the MAGrahder. It has MAGrahder reactor trays and a desktop robot. It also has 12-channel, parallel magnetic rods on a robotic arm that transports and mixes nucleic acid-bound magnetic beads in the trays. This is followed by mRNA extraction, reverse transcription, and the homopolymer-trailing reaction that can handle up to 144 samples and only takes an hour to complete.


The outline of the strategy for attacking cancer cells by the Bi-specific T-cell Engager (BiTE) method.