ARTICLE Coupling Recombinase-Mediated Cassette Exchange With Somatic Hypermutation for Antibody Affinity Maturation in CHO Cells Chuan Chen,1,2 Nan Li,1,2 Yun Zhao,1 Haiying Hang1 1 Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; telephone: þ86-10-64888473; fax: þ86-10-64888473; e-mail: [email protected] 2 University of Chinese Academy of Sciences, Beijing, China Introduction ABSTRACT: Heterologous expression of activation-induced cytidine deaminase (AID) can induce somatic hypermutation Technologies such as phage (de Bruin et al., 1999; Huse et al., 1992; (SHM) for genes of interest in various cells, and several research Smith, 1985; Winter et al., 1994), yeast (Boder and Wittrup, 1997; groups (including ours) have successfully improved antibody affinity in mammalian or chicken cells using AID-induced SHM. These Feldhaus et al., 2003), and bacterial displays (Francisco and affinity maturation systems are time-consuming and inefficient. In Georgiou, 1994; Mazor et al., 2009; Qiu et al., 2010) have been used this study, we developed an antibody affinity maturation platform in for antibody affinity maturation in vitro. In recent years, Chinese hamster ovary (CHO) cells by coupling recombinase- mammalian cell surface display has also been developed (Akamatsu mediated cassette exchange (RMCE) with SHM. Stable CHO cell et al., 2007; Higuchi et al., 1997; Ho et al., 2006; Wolkowicz et al., clones containing a single copy puromycin resistance gene (PuroR) expression cassette flanked by recombination target sequences (FRT 2005); it has advantages in protein folding, post-translational and loxP) being able to highly express a gene of interest placed in the modification, and code usage (Ho et al., 2006). Several groups have cassette were developed. The PuroR gene was replaced with an successfully coupled cell surface display with SHM to improve antibody gene by RMCE, and the antibody was displayed on the cell antibody affinity in either B cell lines (Akamatsu et al., 2007; surface. Cells displaying antibodies on their membrane were Arakawa et al., 2008; Cumbers et al., 2002; Ho et al., 2006; Seo et al., transfected with the AID gene, and mutations of the antibody gene were accumulated by AID-mediated hypermutation during cell 2005) or non-B cell lines (Bowers et al., 2011; Chen et al., 2012; proliferation followed by flow cytometric cell sorting for cells bearing McConnell et al., 2012). SHM is induced by activation-induced antibody mutantswith improved affinity.Affinity improvementswere cytidine deaminase (AID), which converts cytosine to uracil to detected after only one round of cell sorting and proliferation, mutant introduce mutations and amino acid substitutions during antibody fi fi clones with15-fold af nity improvement were isolated within ve affinity maturation (Martin and Scharff, 2002; Maul and Gearhart, rounds of maturation (within 2 months). CHO cells are fast growing, stress-resistant and produce antibody with glycosylations suitable for 2010; Shivarov et al., 2008). B cell lines with endogenous AID therapy. Our antibody-evolution platform based on CHO cells makes expression can generate SHM themselves (Seo et al., 2006; Todo antibody-affinity maturation more efficient and is especially et al., 2006), while other cell lines without endogenous AID convenient for therapeutic antibody affinity improvement. expression were transfected with the AID gene to induce SHM Biotechnol. Bioeng. 2016;113: 39–51. (Bowers et al., 2011; Chen et al., 2012; Yoshikawa et al., 2002). ß 2015 Wiley Periodicals, Inc. Episomal and non-episomal vectors were used to display KEYWORDS: activation-induced cytidine deaminase (AID); antibodies (Bowers et al., 2011; Ho et al., 2006). Episomal vectors fi somatic hypermutation (SHM); af nity maturation; recombinase- cannot be integrated in chromosomes, and it takes a longtime and mediated cassette exchange (RMCE); flow cytometric cell sorting multiple flow cytometric sortings to enrich a cell population that stably displays antibodies at high levels. High-level antibody displaying cell populations or cell clones can be generated by random integration of an antibody-expression vector, but it also takes a longtime and multiple flow cytometric sortings to reach this goal. Even if cells with an antibody highly displayed on the surface were generated, multiple copies of antibody genes often coexist in a Correspondence to: Dr. Hai-Ying Hang and Dr. Yun Zhao cell (Baer et al., 2000; Horlick et al., 2000). The mixed antibody Received 3 July 2014; Revision received 7 October 2014; Accepted 17 November 2014 display makes antibody affinity maturation inefficient, and more Accepted manuscript online 15 January 2015; time is needed to identify the best antibody gene. Article first published 31 July 2015 online in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25541/abstract). CHO cell lines are widely used in antibody expression as they are DOI 10.1002/bit.25541 fast growing, stress-resistant and have identical or similar post- ß 2015 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 113, No. 1, January, 2016 39 translational modifications to humans (Schmidt, 2004; Walsh and plasmid pCEP-Neo-mAID was created in two steps: first, the Jefferis, 2006), and it is convenient to generate cell lines with only hygromycin resistance gene between NruI and SalI in pCEP4 one copy of retargetable high-level expression cassette by RMCE in (Invitrogen, USA) was replaced with a neomycin resistance gene CHO cells (Kim and Lee, 2008; Qiao et al., 2009). A DNA fragment of from pCDNA3.1(þ) to form the pCEP-Neo plasmid, and then the interest can be efficiently and unidirectionally inserted into a mAID (mouse activation-induced deaminase (Chen et al., 2012) preferred pre-determined genomic locus flanked with FRTand loxP gene was inserted into pCEP-Neo between HindIII and XhoI. In sequences by simultaneously expressing Flp and Cre recombinases order to compare the stabilities of different vectors to display anti- (dual RMCE) (Anderson et al., 2012)). In this study, we generated a TNF-a antibody, the SP-HA-Ab-TM cassette was PCR amplified and CHO cell clone bearing such a retargetable expression cassette in a inserted into pCDNA3.1-hygro(þ) and pCEP4 vectors between genome locus that maintained high expression of puromycin HindIII and XhoI to generate pCDNA3.1-Ab and pCEP4-Ab. resistance protein; the puromycin resistance gene (PuroR) was then The seven chosen antibody mutants (Table II) were generated as replaced with an antibody gene. Cells highly displaying the antibody hybrid scFv (mouse scFv-human Fc, scFv-hFc) for affinity analysis. were obtained by flow sorting only after 2 days of culture. The cells The scFv-hFc expression cassettes were inserted into the pCEP4 were transfected with AID and a mutant antibody with 15-fold of vectors between HindIII and XhoI to generate pCEP4-scFv-hFC affinity improvement was generated by five rounds of flow sorting- expression vectors. cell proliferation within 2 months. The primers used in constructing the above plasmids are listed in Table I. All the constructed plasmids were confirmed by sequencing. Materials and Methods Cell Culture Vectors CHO/dhFr- cells (12200036, Cell Bank of the Chinese Academy of To establish cell clones in which the FRT-PuroR-loxP cassette is Sciences, Shanghai, China) and cell lines derived from CHO/dhFr- integrated in a chromosome site, which can then be replaced by an were propagated in IMDM medium (HyClone) containing 10% fetal antibody gene of interest, a series of plasmids were created as bovine serum (FCS, HyClone), 0.1 mM hypoxanthin, and 0.016 mM described below. To generate the plasmid pCDNA3.1/hygro(þ)- thymidine (HT, Gibco, USA), at 37 C in a 5% CO2 incubator. DHFR (named as pCD), a SV40 promoter-DHFR-SV40 polyA Subcultures were carried out every 2–3 days. CHO-S cells expression cassette (Huang et al., 2007) was inserted into the (Invitrogen, USA) were maintained in FreeStyleTM CHO Expression pCDNA3.1/hygro(þ) plasmid at the BglII site. The plasmid Medium (Invitrogen, USA) in a round bottle in an ES-W orbital þ pCDNA3.1/hygro( )-FRT-GFP-loxP-DHFR (named as pCDFGL) shaker (BIOTOP, Shanghai, China) at 130 rpm, 37 C, and 8% CO2. was created by inserting the FRT-EGFP-loxP cassette into pCD Flp-In CHO cells (Invitrogen, USA) were maintained in F12 medium between HindIII and XhoI. The FRTand loxP sequences were added (HyClone) with 10% fetal bovine serum (FCS, HyClone), at 37Cina on the two sides of the EGFP gene by PCR using two primers 5% CO2 incubator. containing FRTand loxP sequences on their 50 ends, respectively. To generate the dual recombinase expression plasmid pCI-Flp-2A-Cre Transfection, Stable Cell Line Establishment, and (named as pF2AC), we used the framework of the F2AC containing Antibody Affinity Maturation Flp-2A-Cre cassette (Anderson et al., 2012), but replaced Flpe and Cre with Flpo and iCre by overlapping PCR. The resulting cassette The plasmid pCDFGL was linearized by BglII digestion prior to was then inserted into the pCI plasmid (Promega, Madison, WI) electroporation. CHO/dhFr- cells (1E6) in log phase were collected between NheI and XhoI. Flpo is a variant of Flp, and it is more active by trypsinization and resuspended in 0.1 mlLopti-MEM medium than the wild type Flp (Raymond and Soriano, 2007); iCre is a (Invitrogen, USA) together with 2.5 mg linearized pCDFGL DNA. variant of Cre, and it is more active than the wild type Cre The mixture was then pulsed at 160 V,7.5 ms in a 0.2 cm gap cuvette (Shimshek et al., 2002); the 2Aused here is the TaV variant of the 2A (BIO-RAD Gene Pulser XcellTM). The electroporated cells were peptides, and it is more self-cleaving efficient than the other 2A plated into a 10 cm dish containing 10 mL IMDM medium with 10% peptides (Donnelly et al., 2001).