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R&D(Research and Development)

Innovations in Monoclonal Antibody Development

Medical & Biological Laboratories Co., Ltd. (MBL) was established in 1969 as the first Japanese company to manufacture antibodies. Since then, MBL has been involved in the research, development, manufacture, and sales of a broad range of diagnostics and research reagents. MBL has been engaged not only in the worldwide sales of over 8,000 antibodies developed in-house or marketed by foreign and domestic alliance partners but also in the contract manufacturing of custom-made polyclonal and monoclonal antibodies. In addition, MBL has extended its business to sales of in vitro diagnostic reagents for autoimmune diseases, cancers, and aberrant metabolic disorders. In Japan, MBL is the leading manufacturer of products involved in the diagnosis of autoimmune diseases. The core technology of MBL includes monoclonal antibody (mAb) development. In order to further reinforce our core competence, we constantly work towards the advancement and challenge of antibody development technology by improving the quality of antibodies. Moreover, we actively adopt novel techniques that have originally been developed by Japanese immunologists. By focusing on innovative and unique technologies of antibody production, MBL's business opportunities will expand beyond the continuous improvement of the quality of our own products, towards the development of novel antibodies for diagnostics, therapeutic antibodies, and revenue generating of license agreements.

Mouse mAb Development

The hybridoma technique for the establishment of mouse mAbs was first reported by Drs. Georges Köhler and César Milstein in 1975. In 1984, they were awarded the Nobel Prize in Physiology or Medicine for the discovery of this revolutionary technology. However, there has been almost no significant progress in this technology since its discovery. MBL commenced development of mouse mAbs in 1983, however, it it struggled with technological innovation for over 20 years, similar to the case of researchers worldwide. In 2004, Dr. Takeshi Watanabe reported the artificial lymph node technology in Nature Biotechnology1. At the time, Dr. Watanabe was a unit leader at the Research Center for Allergy and Immunology, RIKEN. Currently, he is a research professor at Kyoto University Graduate School of Medicine. This technology is a substantial landmark development in mAb generation. We adopted this technology as the cornerstone of mAb research, and currently, MBL is developing the know-how for the establishment of high-performance antibodies.

Artificial Lymph Node Technology

Artificial lymph node technology enables the creation of secondary lymphoid organs in mouse kidneys by transplanting stromal cells embedded in biocompatible scaffolds. Artificial lymph nodes are unique in that it contains only the targeted antigen-specific immune cell populations (cell groups producing specific antibodies), while normal lymph nodes contain various types of immunocompetent cells. Therefore, if we transplant artificial lymph nodes constructed with stromal cells from mice immunized with specific antigens into other mice, it will be possible to selectively transplant the entire immune system aimed at the targeted antigen. Further, severe combined immunodeficient (SCID) mice can be used as recipients in order to generate mice with an immune system derived exclusively from the artificial lymph nodes. When antigenic stimuli are applied to these mice, the serum antibody titer increased by 10- to 100-fold the level in normal mice1). Under the guidance of Prof. Watanabe, we applied this technology to mAb development, and we were able to obtain target mAb clones 10-fold greater than those obtained using conventional methods. Remarkably, the efficiency of isolation of these ultra-high-affinity antibodies was high, without any bias regarding the usage of VH genes. Therefore, this innovative technology facilitates to generate a wide variety of high-quality mAbs in large quantities. Currently, we are improving the technology in order to produce antibodies of even higher quality. Simultaneously, we are also challenging the development of antibodies directed at molecules, which is of a higher degree of difficulty.

Immunogenicity-enhancing Antigen Technology

The IEA (Immunogenicity-Enhancing Antigen) is our innovation in which genetic engineering of the target proteins (antigens) used for immunization is employed to enhance immunogenicity in mice. We used such modified antigens to successfully produce a dramatic increase in the mAb titer and the establishment of mAbs for proteins, which is difficult to accomplish by conventional methods. When combined with artificial lymph node technology, synergistic effects facilitate the isolation of a variety of mAbs. This technology has many applications and is particularly useful for the establishment of high-quality antibodies for any proteins.

Human mAb Development

In recent years, chimeric, humanized, and fully human antibodies have been used as therapeutic antibodies. Of these, fully human antibodies are regarded as highly safe antibodies for therapeutic use because they are not antigenic in human. The efficient methods of preparation of fully human antibodies include: phage display of cloned human antibody genes, antibody generation by immunization of transgenic mice with human immunoglobulin genes, immortalization of human antibody-producing cells by Epstein-Barr virus, and fusion of human peripheral blood mononuclear cells with fusion partners. The MBL group is well equipped to develop fully human antibodies by using methods such as the fusion partner method and the phage display method.

Human Lymphocyte Fusion Partner SPYMEG Cell Line

In collaboration with Assoc. Prof. Naomasa Yamamoto of Ohu University School of Pharmaceutical Sciences, we have successfully generated a novel human lymphocyte fusion partner cell line, designated SPYMEG (PCT/JP2007/058129). SPYMEG was generated fusing SP2/O myeloma cells of murine original with MEG-01 human megakaryoblastic leukemia cells. It can overcome the problems of human chromosome loss upon preparation of hybridomas. We fused human B cells with SPYMEG cells to obtain hybridomas exhibiting efficient and stable IgG production. Further, the efficiency of these hybridomas has been shown to be considerably greater than that generated from any of the human fusion partners reported so far. Through cooperative studies, we have isolated human antibodies effective against viral infections such as HIV and influenza. We fused SPYMEG cells with the peripheral blood mononuclear cells obtained from volunteers vaccinated against influenza. We were able to obtain hybridomas producing neutralizing antibodies against influenza A H3N2 vaccine strains that were isolated during 1968–2005 and during 1989–2005 or influenza B strains that were isolated during 1987–20052). These results demonstrated the potential application of SPYMEG technology in isolating unique antibodies. Thus, this technology could be extended to the development of therapeutic antibodies against viral infections. SPYMEG cells can be used to establish hybridomas with neutralizing antibody-producing B cells isolated from patients recovering from infectious diseases or those who have not yet developed any symptoms. It is expected that human neutralizing antibodies produced by the hybridomas will be useful in medical treatments and the information obtained from these antibodies may lead to the elucidation of the mechanisms of infectious diseases or to the development of new prophylactic and therapeutic methods.

Phage Display Technology

Development of fully human antibodies using phage display involves isolating high-affinity antibodies in vitro. MBL possesses multiple libraries 1010 to 1011 in size, comprising naïve libraries and human immune libraries against antigens that are difficult to prepare in vivo, lethal toxin molecules that cannot be used as immunogenic agents, and low-molecular-weight compounds. We used phage display technology to develop fully human antibodies. In addition, we devised an original screening method termed ICOS (Isolation of antigen-antibody Complexes through Organic Solvents)3), which has been designed to select antibodies reactive to cell surface molecules. Moreover, we have identified more than 30 cancer-related molecules and successfully isolated more than 600 clones of antibodies against these molecules. We have also successfully isolated antibodies capable of neutralizing infectious viruses and anti-carbohydrate antibodies. Major pharmaceutical manufacturers from other countries have expressed an interest in our projects.

mAb Development Using Rabbits and Chickens

Since antibodies developed in animals are based on the immune systems of those animals, antibody titers against the proteins of the immunized animals (i.e., "autoantigens") may not increase due to immunological tolerance. Since mice and humans are conventionally subjects of research, the need for antibodies against their proteins accounts for the majority of the antibody requirement in research. Mice are representative immunized animals in which mAb development technologies have been established. Consequently, it is difficult to obtain antibodies against proteins that are highly homologous to mouse counterparts. In other words, if laboratory animals that are phylogenetically distant from human and mouse are used, the problem of immune tolerance can be avoided and a broader range of antibodies can be obtained. To this end, we are developing techniques for the acquisition of rabbit and chicken mAbs. In particular, chickens are very useful to generate antibodies against target proteins in human, mouse, and by extension, all mammals in general. Since we have a chicken fusion partner, MBL is the only company that can develop chicken mAbs based on the hybridoma method. mAbs are currently used as therapeutic antibodies, diagnostic agents, and research reagents, and their roles in the post-genome era will further expand in the future. Meanwhile, "depletion of targets" is a frequently used phrase in the field of therapeutic mAb development, wherein certain researchers hold the extreme viewpoint that since mAbs have been developed actively worldwide, almost all possible antibodies that can be obtained using ordinary methods have already been obtained. However, the development of novel and original methods such as the ones stated above, which include antibody development technologies using artificial lymph nodes, have enabled the generation of high-quality antibodies with higher sensitivity than those obtained with conventional techniques, and antibodies with unique properties and functions that have not yet been reported elsewhere. We believe that the numerous proprietary technologies owned by MBL can help overcome this situation, contribute to healthcare and related research, and lead to further technological developments.

References

1) Suematsu S. and Watanabe T. Nature Biotechnology. 22:1539-1545 (2004) [PMID:55680191]
2) Kubota-Koketsu, R. et al. Biochem Biophys Res Commun. 387(1):180-5(2009) [PMID:19580789]
3) Akahori, Y. et al. Biochem Biophys Res Commun. 378(4):832-5(2009) [PMID:19071089 ]