Isolation of antigen/antibody complexes through organic solvent (ICOS) method Yasushi Akahori a , Gene Kurosawa a , Mariko Sumitomo b , Miwa Morita a , Chiho Muramatsu b , Keiko Eguchi b , Miho Tanaka c , Kazuhiro Suzuki d , Mototaka Sugiura c , Yoshitaka Iba a Atsushi Sugioka e , Yoshikazu Kurosawa a, * a Division of Antibody Project, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan b 21st Century COE Research Center, Fujita Health University, Toyoake, Aichi 470-1192, Japan c Department of Internal Medicine, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan d Institute for Antibody Ltd., Toyoake, Aichi 470-1192, Japan e Department of Surgery, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan article info Article history: Received 6 November 2008 Available online 9 December 2008 Keywords: Phage-display library of antibodies Antigen/antibody complex Tumor-associated antigen Hydrogen bond Organic solution abstract We developed a method termed ICOS (isolation of antigen–antibody complexes through organic solvent) for comprehensive isolation of monoclonal antibodies (mAbs) bound to molecules on the cell surface. By mixing a large number of phage particles of an antibody (Ab) library with living cells, antigen (Ag)–Ab complexes were formed on the cell surface. The mixture was overlaid on organic solution in a tube and subjected to centrifugation. Phages bound to cells were recovered from the precipitate. The phage fraction isolated turned out to contain mAbs that bind to very heterogeneous epitopes and show strong binding activity to Ags. The ICOS method was applied to isolation of human mAbs that may be therapeu- tic against cancers. Sixty percent of clones isolated by the screening of a phage Ab library against cancer cells turned out to bind to various kinds of tumor-associated Ags. The precise protocol of ICOS method and the rationale of efficient screening were described. Ó 2008 Elsevier Inc. All rights reserved. Since antibody (Ab) libraries were constructed by using phage-display technology [1], it has been expected that many useful monoclonal Abs (mAbs) against various antigens (Ags) will be isolated from them. In order to isolate mAbs against membrane proteins the procedure consisting of the following steps has conventionally been adopted [2,3]. (1) Phage particles of an Ab library expressing a single-chain Fv (scFv) are mixed with cells in an aqueous solution and stored at room tempera- ture until completion of Ag/Ab complex formation. (2) Cells are collected by centrifugation. (3) The cells are suspended in buffer. (4) Steps 2 and 3 are repeated several times to wash out free phages. (5) Phages bound to the cells are eluted by an acid solu- tion. (6) The phages are infected into Escherichia coli, grown and prepared. The whole process from step 1 to 6 is repeated three to four times. Finally, E. coli infected with the phages are spread on a nutrient agar plate and colonies are picked up. If the size of Ab repertoire in the library is large enough for covering many epitopes, it seems to be reasonable that we are able to comprehensively isolate mAbs specifically bound to mol- ecules present on the cell surface. However, many researchers including us experienced the following problems in actual screenings performed according to the conventional procedure. The number of clones with different sequences in the pool of mAbs finally picked up after several rounds of screening cycle described above tended to be limited. Moreover, the affinity of isolated clones to the Ags was not so high as expected. The majority showed K d values ranged between 0.1 and 1 lM. One may argue that since mAbs in phage libraries should be gener- ally naive to Ags we could not expect presence of Abs with high affinity to the Ags in the libraries. In this paper, we described a method for isolation of mAbs termed ICOS method (isolation of Ag–Ab complexes through or- ganic solvent). Although this method was similar to the BRASIL (biopanning and rapid analysis of selective interactive ligands) method developed by Giordano et al. [4], we improved it in various ways. Development of the ICOS method enabled us to comprehen- sively isolate mAbs against many membrane proteins. In fact, we succeeded in identification of 21 distinct tumor-associated Ags (TAAs) and in isolation of more than 400 human mAbs that specif- ically bound to one of the 21 TAAs [5]. Some of the mAbs showed strong antitumor activity in vitro and in vivo. Since we described the details of this method in this paper, it will be applicable to iso- lation of mAbs against various membrane proteins. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.129 Abbreviations: Ab, antibody; ADCC, antibody-dependent cell-mediated cytotoxicity; Ag, antigen; BRASIL, biopanning and rapid analysis of selective interactive ligands; ELISA, enzyme-linked immunosorbent assay; ICOS, isolation of Ag/Ab complexes through organic solvent; mAb, monoclonal Ab; scFv, single-chain Fv; TAA, tumor- associated Ag. * Corresponding author. Fax: +81 562 93 8835. E-mail address: kurosawa@fujita-hu.ac.jp (Y. Kurosawa). Biochemical and Biophysical Research Communications 378 (2009) 832–835 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc