BsAb Generation by Hybrid-hybridoma Technology

1. BsAb Generation by Hybrid-hybridoma Technology With manufacture, Creative Biolabs has become an incontestable global leader in the bispecific antibodies (BsAbs) market. Based on our well- established hybrid-hybridoma technology platform, Creative Biolabs can provide first-class BsAbs production services to meet your BsAbs development goal in a timely and cost-effective manner. ample experience in antibodies development and Hybridoma Technology Hybridoma technology has long been an outstanding and indispensable platform for producing high-quality monoclonal antibodies (mAbs). Hybridoma-derived mAbs have becoming the most rapidly expanding class of therapeutic biologics. Based on the development of mAb humanization and the production of transgenic- humanized mice, this technology has opened novel avenues for effectively producing humanized or fully human mAbs as therapeutics. Besides, in the early stage of BsAb development, BsAbs are produced by cell fusion of two hybridoma cell lines, forming a quadroma cell which secretes among other immunoglobulin combinations as well as BsAb.

2. Figure 1. The left part of this figure shows the monoclonal antibody production by hybridoma technology. The right part of this figure shows the BsAbs production by hybrid-hybridoma technology. (Moldenhauer, G. 2011) BsAbs from Hybrid Hybridoma Hybrid-hybridomas also known as quadromas or tetradomas secrete BsAbs with two different specificities which can be able to crosslink two distinct molecules. Hybrid-hybridomas are produced by fusing two permanently growing hybridoma lines, the goal of which is to select resulting hybrids and exclude intra-hybridoma fused cells and non- fused parental cells. The fusion usually enables to be achieved by cell sorting techniques or chemical selection procedures. Introduction of selection markers: such as the hypoxanthine guanine phosphoribosyl transferase (HGPRT) or neoR gene Selection by flow cytometry: the two hybridoma cell lines are labeled with different fluorescent markers Cell fusion by polyethylene glycol or electrofusion Screening hybrid Hybridoma cultures for specific antibody: ELISA, flow cytometry, and cytotoxicity are the most commonly used methods

3. Single-cell cloning of hybrid hybridomas Purification of bispecific antibody One major drawback of the initial hybrid-hybridoma technology is that fusion cells from two mouse hybridomas or two rabbit hybridomas produce mainly nonfunctional variants with mismatched heavy/light chains pairing. To address the issue, rat/mouse hybrid-hybridomas are developed. The resulting BsAbs, the concept of Triomab developed by some group, have correct species-restricted H/L chain pairing which reduces the mismatch variants. Features of BsAb from Hybrid Hybridoma The major function of a BsAb for tumor therapy is absolutely the recruitment of immune effector cells to the tumor site. Based on the type of effector cell, additional functional requirements have to be satisfied. BsAbs from hybrid hybridoma, one type of the IgG-like BsAbs, have nearly the same serum half life as conventional IgG antibodies, because they have the same size (about 150 kDa) and high stability as conventional IgG antibodies. For example, there is a rat-mouse hybrid IgG-like BsAb that was approved in Europe on 2009. This BsAb is able to simultaneously bind antigens CD3 and EpCAM, and is used for the treatment of malignant ascites in patients with EpCAM-positive cancer if a standard therapy is not work. With the well-established cell lines, Creative Biolabs is dedicated to offering first-class BsAbs production services to meet your development goal in a time-saving manner. Creative Biolabs also provides other various services regarding BsAbs development. Please feel free to contact us for more information and a detailed quote. References 1. Moldenhauer, G. Bispecific antibodies from hybrid hybridoma. In Bispecific Antibodies.Springer Berlin Heidelberg. 2011: 29-46.

4. 2. Pandey, S. Hybridoma technology for production of monoclonal antibodies. Hybridoma. 2010, 1(2): 017.