Development of a novel mammalian display system for selection of antibodies against membrane proteins

Abstract

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies. Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies. Mammalian display was originally conceived for affinity maturation of single-chain variable fragments (scFv) expressed on the surface of human cells (1Ho M. Nagata S. Pastan I. Isolation of anti-CD22 Fv with high affinity by Fv display on human cells.Proc. Natl. Acad. Sci. U.S.A. 2006; 103 (16763048): 9637-964210.1073/pnas.0603653103Crossref PubMed Scopus (133) Google Scholar) and has been further developed for screening full-length antibody cell surface–expressed libraries (2Akamatsu Y. Pakabunto K. Xu Z. Zhang Y. Tsurushita N. Whole IgG surface display on mammalian cells: application to isolation of neutralizing chicken monoclonal anti-IL-12 antibodies.J. Immunol. Methods. 2007; 327 (17719061): 40-5210.1016/j.jim.2007.07.007Crossref PubMed Scopus (54) Google Scholar, 3Zhou C. Jacobsen F.W. Cai L. Chen Q. Shen W.D. Development of a novel mammalian cell surface antibody display platform.mAbs. 2010; 2 (20716968): 508-51810.4161/mabs.2.5.12970Crossref PubMed Scopus (68) Google Scholar). The use of mammalian cell display (4Zhou Y. Chen Z.-R. Li C.-Z. He W. Liu S. Jiang S. Ma W.-L. Tan W. Zhou C. A novel strategy for rapid construction of libraries of full-length antibodies highly expressed on mammalian cell surfaces.Acta Biochim. Biophys. Sinica. 2010; 42: 575-58410.1093/abbs/gmq055Crossref PubMed Scopus (11) Google Scholar) has some important advantages over other display systems (e.g. phage/yeast), in particular in relation to the manufacturability of the identified antibodies; in mammalian display, antibodies are produced using the endogenous eukaryotic secretion machinery enabling correct folding and biophysical properties and are therefore more likely to be compatible with mammalian cell production systems. However, a disadvantage of using mammalian display is that only a relatively small library size (usually up to 107) can be interrogated. The library sizes that are available for mammalian systems are typically limited by low transfection efficiency, although recent advances have improved this, for example by using CRISPR–Cas9 integration methods (5Parola C. Neumeier D. Friedensohn S. Csepregi L. Di Tacchio M. Mason D.M. Reddy S.T. Antibody discovery and engineering by enhanced CRISPR–Cas9 integration of variable gene cassette libraries in mammalian cells.mAbs. 2019; 11 (31478465): 1367-138010.1080/19420862.2019.1662691Crossref PubMed Scopus (10) Google Scholar, 6Parthiban K. Perera R.L. Sattar M. Huang Y. Mayle S. Masters E. Griffiths D. Surade S. Leah R. Dyson M.R. McCafferty J. A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing.mAbs. 2019; 11 (31107136): 884-89810.1080/19420862.2019.1618673Crossref PubMed Scopus (22) Google Scholar). Alternatively, library size can be effectively expanded by first utilizing phage display (7Frenzel A. Schirrmann T. Hust M. Phage display-derived human antibodies in clinical development and therapy.mAbs. 2016; 8 (27416017): 1177-119410.1080/19420862.2016.1212149Crossref PubMed Scopus (198) Google Scholar, 8Frenzel A. Kügler J. Helmsing S. Meier D. Schirrmann T. Hust M. Dübel S. Designing human antibodies by phage display.Transfus. Med. Hemother. 2017; 44 (29070976): 312-31810.1159/000479633Crossref PubMed Scopus (58) Google Scholar) to screen much larger naïve libraries before converting to mammalian cell display after one or two rounds of selection or by using libraries derived from immunized animals, in which initial antibody selection and maturation has occurred in vivo (6Parthiban K. Perera R.L. Sattar M. Huang Y. Mayle S. Masters E. Griffiths D. Surade S. Leah R. Dyson M.R. McCafferty J. A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing.mAbs. 2019; 11 (31107136): 884-89810.1080/19420862.2019.1618673Crossref PubMed Scopus (22) Google Scholar). With current mammalian display methods, the cells displaying the antibodies are incubated with the target antigen, which must be available in a soluble format and used either free in solution or bound to paramagnetic beads (1Ho M. Nagata S. Pastan I. Isolation of anti-CD22 Fv with high affinity by Fv display on human cells.Proc. Natl. Acad. Sci. U.S.A. 2006; 103 (16763048): 9637-964210.1073/pnas.0603653103Crossref PubMed Scopus (133) Google Scholar, 9Beerli R.R. Bauer M. Buser R.B. Gwerder M. Muntwiler S. Maurer P. Saudan P. Bachmann M.F. Isolation of human monoclonal antibodies by mammalian cell display.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18812621): 14336-1434110.1073/pnas.0805942105Crossref PubMed Scopus (138) Google Scholar, 10Bowers P.M. Horlick R.A. Kehry M.R. Neben T.Y. Tomlinson G.L. Altobell L. Zhang X. Macomber J.L. Krapf I.P. Wu B.F. McConnell A.D. Chau B. Berkebile A.D. Hare E. Verdino P. et al.Mammalian cell display for the discovery and optimization of antibody therapeutics.Methods. 2014; 65 (23792919): 44-5610.1016/j.ymeth.2013.06.010Crossref PubMed Scopus (47) Google Scholar). The latter system can be in antigen thus allowing for the selection of cells that express low affinity antibodies E. K. Meier M. L. C. M. A. M. L. and screening of a high antibody 2014; 65 PubMed Scopus Google Scholar). antigen must be to cells in solution or to the target is typically to proteins or protein that are soluble and relatively identifying antibodies to membrane proteins methods can be used with phage display to for phage that to membrane proteins S. M.R. T. R. M. application of cell for isolation of phage antibody fragments specific to 2019; PubMed Scopus Google Scholar, S. Zhang S. M. N. membrane proteins for antibody discovery using phage 2016; PubMed Scopus Google the phage and this can thereby the of binders for for phage against cell surface The to screen large naïve libraries within a mammalian a by the and of identified antibodies with mammalian cell the to use two discovery important targets are membrane proteins, the to screen against a membrane antigen in native configuration within a membrane environment that only are thus a of identifying functional provide a mammalian display with we a method by which we large antibody libraries with of and use to cells that have been to express the target membrane much larger library sizes to be with methods and is only limited by the of cells that can be in the strategy in cells expressing the target antigen on their cell surface and a from the high scFv cells that express an scFv of the target thus allowing to be and have identified co-expression of and antigen in a cell as a method to screen for for example in a display system X. Y. Liu Y. Chen Y. N. Jiang S. Zhang S. Wu Q. Li T. Zhang Y. Zhou B. J. Li D. scFv antibodies against by co-expression of antigen and antibody in the display Immunol. 2016; PubMed Scopus Google Scholar) and for affinity maturation in mammalian cells A. M. S. D. K. T. M. antibody affinity maturation system utilizing mammalian cell as 2019; PubMed Scopus Google Scholar). However, we for the first the screening of a large naïve scFv library in a mammalian system and identify binders to a membrane protein antigen on the cell surface. We present the isolation of cells that secrete that to the membrane protein a and has been identified as a antigen, of in epithelial A. K. M. I. in clinical J. 2010; PubMed Scopus Google Scholar). this has been to to other cell cell and M. P.M. cell adhesion more a and adhesion J. 2007; PubMed Scopus Google Scholar, L. Y. Liu S. Z. Z. J. of in and J. Med. Google Scholar). cells by magnetic cell sorting a method used to and specific cell X. Li Z. Li Z. Chen C. Liu Isolation and enrichment of cells using and PubMed Scopus (22) Google Scholar). was by to a cell population that in the of and has been used to within the of flow and thereby the of in large cell C. J. D. M. R. J. M. A flow to identify and in 2010; PubMed Scopus Google Scholar). of the of the and a cells expressing specific antigen to be from that bound endogenous cell surface this cell screened, and to the of novel that bound to the protein on the cell surface. 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Neumeier D. Friedensohn S. Csepregi L. Di Tacchio M. Mason D.M. Reddy S.T. Antibody discovery and engineering by enhanced CRISPR–Cas9 integration of variable gene cassette libraries in mammalian cells.mAbs. 2019; 11 (31478465): 1367-138010.1080/19420862.2019.1662691Crossref PubMed Scopus (10) Google with in a the of was to and on the cells to a the cells expanded by a of C. Neumeier D. Friedensohn S. Csepregi L. Di Tacchio M. Mason D.M. Reddy S.T. Antibody discovery and engineering by enhanced CRISPR–Cas9 integration of variable gene cassette libraries in mammalian cells.mAbs. 2019; 11 (31478465): 1367-138010.1080/19420862.2019.1662691Crossref PubMed Scopus (10) Google and of a The cells expanded in cell and cells in and to a of K. Perera R.L. Sattar M. Huang Y. Mayle S. Masters E. Griffiths D. Surade S. Leah R. Dyson M.R. McCafferty J. A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing.mAbs. 2019; 11 (31107136): 884-89810.1080/19420862.2019.1618673Crossref PubMed Scopus (22) Google cells with target cell a of for The cells with and with for The cells three with by of on an flow with are within the We the for with the with single-chain fragment variable Chinese hamster ovary epithelial cell adhesion molecule chimeric antigen receptor T cell magnetic cell sorting of cell surface

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