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• W3096996838 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 advantageous in enhancing antigen avidity, thus allowing for the selection of cells that express low affinity antibodies (11Breous-Nystrom E. Schultze K. Meier M. Flueck L. Holzer C. Boll M. Seibert V. Schuster A. Blanusa M. Schaefer V. Grawunder U. Martin-Parras L. van Dijk M.A. Retrocyte Display® technology: generation and screening of a high diversity cellular antibody library.Methods. 2014; 65 (24036249): 57-6710.1016/j.ymeth.2013.09.003Crossref PubMed Scopus (18) Google Scholar). Because purified antigen must be applied to cells in solution or coupled to particles, the target is typically restricted to proteins or protein domains that are soluble and relatively stable; thus, identifying antibodies to membrane proteins remains challenging. Although whole-cell panning methods can be used with phage display to enrich for phage that bind to complex membrane proteins (12Nikfarjam S. Tohidkia M.R. Mehdipour T. Soleimani R. Rahimi A.A.R. Nouri M. Successful application of whole cell panning for isolation of phage antibody fragments specific to differentiated gastric cancer cells.Adv. Pharm. Bull. 2019; 9 (31857967): 624-63110.15171/apb.2019.072Crossref PubMed Scopus (6) Google Scholar, 13Jones M.L. Alfaleh M.A. Kumble S. Zhang S. Osborne G.W. Yeh M. Arora N. Hou J.J. Howard C.B. Chin D.Y. Mahler S.M. Targeting membrane proteins for antibody discovery using phage display.Sci. Rep. 2016; 6 (27189586)2624010.1038/srep26240Crossref PubMed Scopus (50) Google Scholar), the phage require reformatting, and this process can frequently ablate binding activity, thereby reducing the number of positive binders for subsequent analysis. This process also nonspecifically enriches for phage against unrelated, nontarget, cell surface proteins. The technology to screen large naïve libraries within a mammalian setting would clearly confer a significant advantage by improving the compatibility and developability of identified antibodies with mammalian cell manufacturing systems, without the requirement to use two distinct discovery methods. Furthermore, because many important therapeutic targets are membrane proteins, the ability to screen against a membrane antigen in its native configuration within a cellular membrane environment would ensure that only physiologically relevant epitopes are presented, thus giving a greater likelihood of identifying functional antibodies. To provide a mammalian display technique with both these major advantages, we here describe a method by which we package large antibody libraries with diversities of ∼109 into lentiviral particles and use these to transduce CHO cells that have been engineered to express the target membrane protein. This allows much larger library sizes to be sampled than with existing methods (by at least 100-fold) and is only limited by the number of cells that can be cultured in the laboratory. Our strategy results in individual CHO cells expressing the target antigen on their cell surface while co-expressing and secreting a variant from the high diversity scFv library. Thereby, CHO cells that express an scFv variant capable of binding the target undergo self-labeling, thus allowing them to be isolated, reselected, and eventually sequenced. Reported strategies have identified co-expression of scFvs and antigen in a single cell culture as a method to screen for antibodies, for example in a bacterial display system (14Guo X. Cao H. Wang Y. Liu Y. Chen Y. Wang N. Jiang S. Zhang S. Wu Q. Li T. Zhang Y. Zhou B. Yin J. Li D. Ren G. Screening scFv antibodies against infectious bursal disease virus by co-expression of antigen and antibody in the bacteria display system.Vet. Immunol. Immunopathol. 2016; 180 (27692095): 45-5210.1016/j.vetimm.2016.09.004Crossref PubMed Scopus (6) Google Scholar) and for affinity maturation in mammalian cells (15Eguchi A. Nakakido M. Nagatoishi S. Kuroda D. Tsumoto K. Nagamune T. Kawahara M. An epitope-directed antibody affinity maturation system utilizing mammalian cell survival as readout.Biotechnol. Bioengineer. 2019; 116: 1742-175110.1002/bit.26965Crossref PubMed Scopus (4) Google Scholar). However, we describe for the first time the screening of a large naïve scFv library fully in a mammalian system and identify binders to a membrane protein antigen presented on the cell surface. We present here data showing the isolation of CHO cells that secrete influenza hemagglutinin epitope (HA)–tagged scFvs that specifically bind to the membrane protein EpCAM. EpCAM represents a type I transmembrane glycoprotein and has been previously identified as a tumor-associated antigen, most notably because of its overexpression in rapidly expanding epithelial tumors (16Raffel A. Eisenberger C.F. Cupisti K. Schott M. Baldus S.E. Hoffmann I. Aydin F. Knoefel W.T. Stoecklein N.H. Increased EpCAM expression in malignant insulinoma: potential clinical implications.Eur. J. Endocrinol. 2010; 162 (20097833): 391-39810.1530/EJE-08-0916Crossref PubMed Scopus (26) Google Scholar). In addition, this oncogene has been shown to contribute to several other biological processes including cell migration, cell adhesion, and proliferation (17Trzpis M. McLaughlin P.M. de Leij L.M. Harmsen M.C. Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule.Am. J. Pathol. 2007; 171 (17600130): 386-39510.2353/ajpath.2007.070152Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 18Huang L. Yang Y. Yang F. Liu S. Zhu Z. Lei Z. Guo J. Functions of EpCAM in physiological processes and diseases.Int. J. Mol. Med. 2018; 42 (30015855): 1771-178510.3892/ijmm.2018.3764PubMed Google Scholar). In brief, self-labeling cells were enriched by magnetic activated cell sorting (MACS), a common method used to isolate and purify specific cell populations (19Sheng X. Li Z. Wang D.-L. Li W.-B. Luo Z. Chen K.-H. Cao J.-J. Yu C. Liu W.-J. Isolation and enrichment of PC-3 prostate cancer stem-like cells using MACS and serum-free medium.Oncol. Lett. 2013; 5 (23426586): 787-79210.3892/ol.2012.1090Crossref PubMed Scopus (22) Google Scholar). This was followed by FACS to isolate a cell population that exhibited FRET in the presence of fluorophore-labeled anti-EpCAM and anti-HA antibodies. FRET has previously been used to monitor protein–protein interactions within the context of flow cytometry and thereby facilitates the assessment of interactions in large cell numbers (20Banning C. Votteler J. Hoffmann D. Koppensteiner H. Warmer M. Reimer R. Kirchhoff F. Schubert U. Hauber J. Schindler M. A flow cytometry-based FRET assay to identify and analyse protein–protein interactions in living cells.PLoS One. 2010; 5 (20179761)e934410.1371/journal.pone.0009344Crossref PubMed Scopus (115) Google Scholar). Because of the proximity requirements of the donor and acceptor fluorophores, a FACS-based FRET assay enabled CHO cells expressing specific antigen binding scFvs to be distinguished from those that bound endogenous cell surface proteins. In this way, single cell clones were isolated, screened, and sequenced leading to the identification of novel scFvs that specifically bound to the EpCAM protein on the cell surface. We further demonstrated the utility of the identified scFvs for the targeting of CAR-T cells to EpCAM-positive cells. CHO cells expressing human EpCAM under a doxycycline inducible promoter were generated by transduction of a parental suspension CHO-X cell line with a lentivirus encoding the EpCAM gene and a puromycin resistance marker. The pool of CHO cells then underwent puromycin selection, and cells that showed the highest levels of doxycycline-induced EpCAM expression, determined by surface EpCAM staining, were sorted into single cells by FACS and subsequently expanded (Fig. S1). These clonal cell lines were then screened for EpCAM expression and a high-expressing CHO–EpCAM clone was identified for use in further experiments (data not shown). Doxycycline-induced and noninduced CHO–EpCAM cells were analyzed for EpCAM expression by flow cytometry using an anti-EpCAM FITC-conjugated antibody (Fig. 1a). The results showed 92% of cells positive for EpCAM upon induction relative to control cells and uninduced cells showed 39% of cells positive for EpCAM as compared with control cells, suggesting that some baseline expression of EpCAM was present in the uninduced state. CHO–EpCAM cells were then transduced with a second lentiviral construct containing a known anti-EpCAM scFv with a C-terminal HA tag. After culturing for 3 days either in the presence or absence of doxycycline, the cells were stained with an anti-HA–phycoerythrin (PE) antibody, which showed that, although the CHO–EpCAM cells exhibited leaky EpCAM expression in the absence of doxycycline (Fig. 1a), a >100-fold increase in self-labeling signal was observed in the presence of doxycycline (Fig. 1b). A large culture (1.5 liters) of EpCAM expressing CHO cells (1.35 × 109 cells) was transduced with a lentivirus preparation packaging an untrained scFv library at a multiplicity of infection (MOI) of 1. The scFv library used was generated by shuffling of synthesized antibody fragments comprising the human germline repertoire of V, D, and J domains of the heavy chain and of V and J domains of the light chain. This synthetic library, named CHESS (CDRs from Human Efficiently Shuffled to form scFvs), is therefore a synthetic re-creation of the human naïve repertoire and comprises a natural-like repertoire of framework regions and diversity within the CDRs in terms of both sequence and length (Fig. 2). The theoretical maximum diversity of the library was 8.6 × 108 and was experimentally determined to contain ∼6.0 × 108 variants by counting colony-forming units by limited dilution of transformants containing the lentiviral packaging plasmid used for lentiviral preparation. To select for the cells expressing potential EpCAM binders, the scFv library transduced cells were cultured for 4 days with doxycycline to induce EpCAM expression and subsequently subjected to three rounds of enrichment by MACS using an automated system (AutoMACS, Miltenyi Biotec). In each round, the cells were incubated with PE-labeled anti-HA tag antibodies prior to decoration with anti-PE–coated MACS MicroBeads and capture on magnetic columns. The positive cell fraction was eluted from the magnetic columns and allowed to recover for 3–4 days before repeating EpCAM induction and MACS selection (Fig. 3a). In the first round, the self-labeled CHO–EpCAM cells were captured from a total cell population of 4 × 109 cells with an output of 4.2 × 107 cells. After allowing the cells to recover, 7 × 108 cells were labeled and selected as before with an output of 2.0 × 107 cells. For the third round of MACS selection, 1 × 108 cells were labeled and selected with an output of 7.6 × 106 cells. After three rounds of selection, the HA-staining positive cell fraction was 86% (Fig. 3b). At this stage, the enriched cell fraction was likely to contain three populations: the intended CHO–EpCAM population that had undergone self-labeling by EpCAM-specific scFv (self-labeling), cells labeled by EpCAM-specific scFvs secreted by neighboring cells (cross-labeling), and cells labeled by scFvs specific for off-target endogenous CHO cell surface proteins (off-target labeling). Therefore, FACS-based strategies were designed to specifically enrich for cells self-labeled with EpCAM-specific scFvs as follows. To minimize cross-labeling, the cells enriched by three rounds of MACS were co-cultured with an excess (1:9) of “decoy” CHO–EpCAM cells not expressing scFv, i.e. the same cell line used for lentiviral library transduction. Thus, when the cell mix was cultured in the presence of doxycycline to induce target expression, the decoy cells would capture the specific and nonspecific scFvs released by the enriched cells. Decoy cells were prelabeled with the live-cell dye CellTrackerTM Blue CMAC so that they could be sorted away from the MACS-enriched population. To minimize off-target labeling, a FRET assay was implemented to isolate the cell population that exhibited FRET in the presence of a donor fluorophore, anti-HA–PE, and an acceptor fluorophore, anti-EpCAM–Alexa Fluor 647 (Fig. 4a). Because the donor and acceptor fluorophores have to be in close proximity to generate a FRET signal, specific antigen interactions are more likely to generate a signal than off-target interactions; therefore, this method enables distinction of CHO cells exhibiting specific antigen binding scFvs from those that bind endogenous cell surface proteins. The output from the magnetic enrichments was subjected to two rounds of FACS on an SH800 cell sorter (Sony) by selecting the cells exhibiting a FRET signal in the far-red wavelength (665/30 nm) by proximity of the Alexa Fluor 647 fluorophore to the PE donor fluorophore upon co-binding to the target EpCAM. The cell sample was simultaneously co-cultured with CMAC-labeled CHO–EpCAM decoy cells to reduce the extent of cross-labeling as outlined above (Fig. 4b). 5 × 106 MACS-enriched cells were mixed with 4.5 × 107 decoy cells and cultured for 2–3 days in doxycycline-containing culture media. The sort gate was created in a bidimensional plot representing anti-HA–PE (600/60 nm) and anti-EpCAM–AF647 (665/30 nm) fluorescence of double stained cells upon blue laser excitation at 488 nm (Fig. 4b). In the first FACS round, 36,000 FRET-positive cells (7.23%) were sorted into a pool and recovered until outgrowth of 6 × 106 cells was achieved. These cells were then sorted a second time, using FRET in the presence of labeled decoy cells, into single cells in 96-well plates (600 cells in total), using a more restrictive gate (1.35%) (Fig. 4c), and expanded over a 3-week period. Outgrowth of clones was monitored on a Cell Metric® imaging system (Solentim) coupled to a Microlab STAR robot (Hamilton). The surviving clones were picked on day 18 postsorting and cultured in standard shaking conditions in 96–deep well plates. CHO–EpCAM clones that survived sorting and outgrowth (n = 107) were assessed for their ability to self-label with scFvs in the presence and absence of antigen induction by doxycycline. Induced and noninduced cells were stained with an anti-HA tag PE antibody, followed by flow cytometric analysis (Fig. S2). Of 93 evaluable clones, 26 were selected on the basis of showing a higher signal on doxycycline-induced cells compared with noninduced cells, suggesting EpCAM specificity. These clones were further analyzed using a cross-labeling study, whereby culture supernatants containing secreted scFvs from each clone were tested against target antigen expressing CHO–EpCAM cells and nonexpressing control CHO-X cells (Fig. S3). As a result (21Morgan R.A. Dudley M.E. Wunderlich J.R. Hughes M.S. Yang J.C. Sherry R.M. Royal R.E. Topalian S.L. Kammula U.S. Restifo N.P. et al.Cancer regression in patients after transfer of gentically engineered lymphocytes.Science. 2006; 314 (16946036): 126-12910.1126/science.1129003Crossref PubMed Scopus (2107) Google Scholar), candidate CHO clones were selected because of their apparent secretion of scFvs specific to EpCAM; 11 clones showed strong binding to CHO–EpCAM cells with no binding to control CHO-X cells and were therefore EpCAM-positive; one clone showed strong binding to both CHO–EpCAM cells and CHO-X cells and was therefore specific for a CHO cell surface protein; the remaining 14 clones showed more variable or weak binding, but an additional 10 clones were selected on the basis of displaying some degree of differential binding between antigen-positive and -negative CHO cells (Fig. S3b). From the output of the second round of FACS enrichment, the variable domain genes from the 21 selected EpCAM-positive binders were PCR-amplified and sequenced. As a result, we identified six sequence distinct clones (represented by SP12-E10, SP12-F2, SP14-C8, SP16-E7, SP17-F7, and SP18-C11), four of which were observed in more than one clone, with the sequence of SP12-F2 being represented in eight independently selected clones (SP12-F2, SP14-G8, SP15-B10, SP15-D3, SP15-D9, SP15-E11, SP17-E7, and SP18-F6). Three clones (SP12-E10, SP14-C8, and SP17-F7) shared a common heavy chain sequence but had different light chain sequences. All six sequences were recloned as both scFv and full-length IgG1 into expression plasmids and transfected into suspension HEK293 cells modified to express EBNA1 (293OX-EBNA). The supernatants from the transfected cells were once again challenged against CHO–EpCAM cells or CHO-X control cells to confirm their specificity. Only SP12-E10, SP14-C8, and SP17-F7 (Fig. 5a) were found to bind specifically to CHO–EpCAM cells when formatted as both scFv and whole IgG1; the SP16-E7 clone exhibited no binding in either format, whereas SP12-F2 or SP18-C11 were found to bind weakly when formatted as scFv but did not bind in the IgG1 format (data not shown). This result with SP12-F2 was particularly surprising because the initial cross-labeling assay results were very clear and positive (Fig. S3), and this sequence had been heavily selected for during panning because it was present eight times in the sequenced clones. The three positive clones displayed different binding characteristics either as scFv or whole IgG1 to the CHO–EpCAM cells with clone SP14-C8 showing the strongest binding in each case. The IgG1 converted clones were then purified via a protein A affinity column and assessed by SDS-PAGE and SEC-HPLC (Figs. S4 and S6). The antibodies were well-expressed and showed only minimal signs of aggregation. These purified IgG variants were then applied to CHO–EpCAM and CHO-X control cells in a titration experiment to determine the EC50 on whole cells (Fig. S5). The IgG derived from the clone SP17-F7 revealed only very poor binding at the highest concentrations (5 μm); therefore an accurate EC50 could not be determined. However, the IgGs derived from SP14-C8 and SP12-E10 gave EC50 values of 10.8 ± 1.4 nm and 1.75 ± 0.36 μm, respectively. Single-cycle kinetic SPR analysis on a Biacore T200 of SP12-E10, SP14-C8, and SP17-F7 immobilized on a protein A surface using recombinant human extracellular domain of EpCAM as analyte gave affinities of 5.92, 0.76, and 58.9 nm, respectively (Table 1). The association rate constant (Ka) of SP14-C8 (1.28 × 105 1/Ms) was significantly faster than for the other two antibodies, whereas the dissociation rate constants (Kd) between all three antibodies varied by less than 2-fold.Table 1Summary of the antibody affinities measured by SPR and EC50 determination Open table in a new tab We repeated the single-cycle kinetic SPR analysis by immobilization of the soluble EpCAM extracellular domain via its C-terminal His6 tag with the SP12-E10, SP14-C8, and SP17-F7 mAbs acting as the analytes (Fig. S7). In this case we only observed 17–18% of the expected activity for SP14-C8, only 4% of the expected activity for SP12-E10, and no binding activity observed for SP17-F7. Possibly indicating that only a fraction of the EpCAM extracellular domain or antibody was active on the anti-His tag capture surface, or it binds through a different stoichiometry, or this may be due to a preferred orientation of the EpCAM extracellular domain presented on the capture surface. However, affinities for both SP14-C8 and SP12-E10 could be determined, providing values of 0.26 and 380 nm, respectively. The large difference between the two SPR experiments for SP12-E10 appeared to be partly caused by a large shift in the observed Ka from 9.5 × 103 1/Ms to 9.5 × 102 1/Ms. Therefore, using our mammalian display technique, we have isolated an anti-EpCAM specific antibody with an apparent subnanomolar affinity. The observed difference between the measured EC50 and SPR values may be due to the diverse conditions of the two experiments, such as the high concentration of the purified soluble domain of EpCAM used in SPR experiment inducing an additional avidity effect. This contrasts with the lower recombinant levels of cell surface expression on the CHO cells used in the EC50 assay. Given the great interest in therapeutic immunomodulation and targeting of tumor cells with autologous lymphocytes genetically manipulated to express an scFv-based chimeric antigen T cell receptor (21Morgan R.A. Dudley M.E. Wunderlich J.R. Hughes M.S. Yang J.C. Sherry R.M. Royal R.E. Topalian S.L. Kammula U.S. Restifo N.P. et al.Cancer regression in patients after transfer of gentically engineered lymphocytes.Science. 2006; 314 (16946036): 126-12910.1126/science.1129003Crossref PubMed Scopus (2107) Google Scholar), CAR-T cells were tested with our panel of three EpCAM binders. Two EpCAM-positive cell lines, MCF-7 (a breast cancer cell line) and the CHO–EpCAM cell line used for antibody selections, were used as targets and both Jurkat cells and CD3+ T cells derived from human peripheral blood mononuclear cells (PBMCs) were used as effector cells. CAR-T cells were generated by lentiviral transduction of either Jurkat cells or CD3+ T cells derived from human PBMC. Lentivirus was engineered to encode a second-generation CAR scaffold with anti-EpCAM scFv fused to a CD8-derived transmembrane region followed by a 4-1BB co-stimulatory domain and a CD3ζ chain. Transduced CD3+ T cells derived from human PBMC were expanded for 10 days before testing. Control CAR-T cells with the same architecture and an scFv recognizing CD19 were also generated. The antigen specificity of the CAR-T cells was tested in a co-culture with CHO-X and CHO–EpCAM cell lines. EpCAM–CAR-T cells displayed a significant increase in the expression of activation marker CD25 (36.5 and 29% by SP14-C8 an" @default.
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• W3096996838 title "Development of a novel mammalian display system for selection of antibodies against membrane proteins" @default.
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