Developing a workflow for the isolation of hybridoma cells producing fully human antigen-specific antibodies using a surface IgG detection method

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However, the reduction in sIgG expression on B cells during plasma cell differentiation presents challenges as it enables Ab production from only a small subset of B cells (e.g., memory B cells). The present study aimed to addressed this problem by developing a workflow to isolate human-IgG-secreting hybridoma cells produced by cell fusion, the majority of which express sIgG. We showed that our sIgG-based antigen-coated bead separation method efficiently enriched hybridoma cells expressing antigen-specific Abs with a yield of 83.5% (from the cell fusion pool) and a positive rate of 73.2%. Furthermore, because the separation could be performed after only a short (1−2-day) culture period following cell fusion, diverse hybridoma clones could be obtained, minimizing clonal selection and the incidence of duplicates. Given that the expression of membrane-bound IgG and sIgG are regulated by different splicing mechanisms, we speculate that the cell fusion step potentially attenuated the suppression of human sIgG expression. Overall, our proposed method is expected to markedly improve the efficiency of therapeutic Ab candidate production, which will have important clinical implications. Biological sciences/Biotechnology Biological sciences/Biotechnology/Biologics Biological sciences/Biotechnology/Biologics/Antibody therapy Hybridoma technology surface immunoglobulin G fully human monoclonal antibodies antigen-coated magnetic bead separation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The first step in the generation of monoclonal antibodies (mAbs) is the generation of rodent hybridoma cells via the fusion of B cells and myeloma cells 1 . The resulting rodent mAbs are then refined using chimerization or humanization techniques, which modify the rodent genetic sequences to generate therapeutic human mAbs 2–6 . We have previously reported the generation of trans-chromosomic mice producing fully human mAbs (TC-mAb mice), which have an artificial chromosome harboring full-length human immunoglobulin (Ig) loci (human heavy and light kappa chain). Moreover, we demonstrated the ability of these mice to efficiently produce therapeutic human mAb candidates via hybridoma technology 7,8 . Vast numbers of Ab-producing cells can be obtained from an immunized TC-mAb mouse by cell fusion. For example, one immunized TC-mAb mouse can generate over 30,000 hybridoma cells 8 . As a result, the process of screening to obtain the mAbs of interest is costly and time-consuming. To overcome these challenges, B cell-derived surface IgG (sIgG, also known as membrane-anchored IgG 9 ) molecules have recently been developed for the rapid production of therapeutics against COVID-19 and other infectious viral diseases 10–14 . This strategy uses fluorescently labeled antigens as baits to separate antigen-specific mAb-producing B cell fractions from the pool of peripheral blood mononuclear cells containing memory B cells via cell sorting 10–12,15,16 . This approach has markedly facilitated the development of therapeutic Ab candidates. However, since sIgG expression is downregulated during the differentiation of terminal B cells into plasma cells 9,17 , only a fraction of antigen-specific Abs produced in the immunized animals are available for harvesting; this presents a considerable barrier to obtaining therapeutic mAbs. The expression of sIgGs and secreted IgGs is controlled by alternative splicing machinery and the B cell maturation status 9 . Memory B cells are the major source of sIgGs in the spleen 18 . However, low-affinity B cells (i.e., those with low somatic hypermutation [SHM] rates) can readily enter the memory B cell pool from the germinal center before affinity maturation has been completed 19 . Therefore, strategies for obtaining high-affinity mAbs by enriching antigen-specific B cells expressing sIgG are urgently needed. The production of antigen-specific mAbs using sIgG from hybridoma cells derived from wild-type mice has previously been reported 20 . However, the number of hybridoma cells that can be processed at any given time is limited by the capacity of the cell-sorting instrument. Furthermore, because this method includes a 7-day cell culture period and a dead cell removal step after cell fusion, the same clones are repeatedly amplified. In this study, we developed a method of rapidly and accurately selecting hybridoma cells expressing antigen-specific mAbs on the basis of their sIgG expression. The extra selection step markedly enriched the number of hybridoma cells capable of producing antigen-specific mAbs, thereby improving screening efficiency. This approach could be successfully implicated because TC-mAb-mouse-derived hybridoma cells expressing anti-specific human IgG also express high levels of sIgG; moreover, their antigen-reactivity was readily detectable by flow cytometry. We showed that all the analyzed sIgG-producing hybridoma cell clones obtained from the TC-mAb mice expressed adequate levels of human IgG in response to labeled antigen. Thus, we demonstrated that antigen-specific human-mAb-producing hybridoma cells were highly enriched from a cell fusion pool using sIgG and antigen as bait. Specifically, we showed that over 80% of cells expressing antigen-specific sIgG were recovered from the cultured hybridoma cell pool. Furthermore, we were able to establish single-clone hybridoma cells approximately 2 weeks after cell fusion by optimizing the seeding density; this bypassed the need for laborious mass cell cloning. Collectively, our findings suggest that the workflow for hybridoma generation presented in this study (Fig. 1) has the potential to considerably advance the production of highly sought-after therapeutic mAb candidates. Results Detection of human sIgGs on hybridoma cells derived from TC-mAb mice We began by evaluating the expression and antigen-reactivity of sIgGs expressed by the already established hybridoma cell lines. Analysis of 16 anti-EpCAM mAb clones from TC-mAb mice 7,8 , which included the IgG1, IgG3, and IgG4 subclasses, showed a sIgG-associated signal shift in the presence of the His-tagged EpEX antigen relative to the negative control samples (Supplementary Fig. 1). Since the intensity of the signal shift depends on the sIgG expression levels and the affinity of the mAb, a range of weak to strong reactions to the EpEX antigen were detected. This result suggested that all the TC-mAb-mouse-derived hybridoma clones expressing human IgG displayed an amount of sIgG for selection. The antigen-specific hybridoma cells could therefore be enriched on the basis of their sIgG-mediated antigen-reactivity. Effective separation of hybridoma cell populations expressing antigen-specific IgG We next attempted to enrich the antigen-specific-mAb-expressing hybridoma cells from the hybridoma cell pool after cell fusion. The human angiotensin-converting enzyme 2 (hACE2) (EC 3.4.17.23., IUBMB) was used as a model protein. The full-length gene encoding the hACE2 extracellular domain was fused to the gene encoding the human Fc region to generate a construct expressing the recombinant hACE2-Fc protein. hACE2-Fc was expressed and purified as previously described 21 (Supplementary Fig. 2 and 3). Two TC-mAb mice were immunized with the purified hACE2-Fc; analysis of their anti-sera titers showed the robust induction of anti-hACE2 Abs (Supplementary Fig. 4). After harvesting the splenocytes and lymphocytes from the mouse spleen and lymph nodes, respectively, 5.6 × 10 8 cells were fused with myeloma cells and cultured in 1/3 hypoxanthine-aminopterin-thymidine (HAT) medium for 2 days, ensuring that cell viability was maintained with little or no cell division. Some (2.5 × 10 6 ) of the fused cells were also seeded into a 96-well plate to compare the efficiency of antigen-specific hybridoma cell selection with or without the use of magnetic beads. Cells (3.4 × 10 8 ) collected after 2 days of culture were incubated with the biotinylated hACE2-Fc protein. Then, the antigen-bound cell fraction, which included hybridoma cells and memory B cells, was separated using the streptavidin (StAv)-magnetic bead method, yielding 4.7 × 10 6 cells (1.4%) from a total of 3.4 × 10 8 cells (Table 1). The separated cells were seeded into eleven 96-well plates based on the predicted growth rate of hybridoma cells, with the aim of seeding one cell per well. After 12 days of culture, colony formation was observed in about 500 wells, with most of the wells containing single colonies. Table 1. Summary of the hybridoma isolation workflow Antigen (individual TC-mAb rats) hACE2-Fc (ndividual A and B) Bead separation No bead separation Total number of splenocytes 5.9 × 10 8 2.0 × 10 6 Number of cells after 2 days of culture 3.4 × 10 8 No treatment Number of cells obtained following bead separation 4.7 × 10 6 - Yield from **precleared cells 1.4% - Hypoxanthine-aminopterin-thymidine medium selection (number of plates) 11 1 Colonies/plate 45 156 Total number of colonies *500 156 Number of ELISA wells with a positive signal 366 1 Ratio of number of positive wells/number of colonies 73.9% 0.6% *Total colony count was calculated by multiplying the average number of colonies obtained from six 96-well plates by the number of plates. **Preclered cells are a pre-washed cell fraction of cells that bind nonspecifically to the beads by treatment with antigen-free beads. The supernatants removed from the wells containing hybridoma colonies were analyzed for their reactivity against hACE2-Fc by enzyme-linked immunosorbent assay (ELISA). Of the ~500 wells that contained hybridoma cell colonies, 366 wells (73.2%) generated a positive signal, which was defined as an absorbance value > 3-fold greater than that of the negative control (Fig. 2). Immunocytochemistry was then used to show that supernatants from 11 hybridoma cell colonies reacted with intact hACE2 expressed on unfixed HEK293-hACE2 cells (an hACE2-overexpressing cell line) . Six of the 11 clones were further evaluated using a combination of immunocytochemistry and flow cytometry (Fig. 3 and Supplementary Fig. 5). The results indicated that the mAbs from all six clones reacted with the hACE2 expressed on the surface HEK293-hACE2 cells. The steps involved in the separation of antigen-specific hybridoma cells are summarized in Table 1. Epitope distribution analysis showed that at least three different hACE2 epitopes were recognized by the mAbs obtained (Fig. 4). Four clones, namely hACE2-A008, hACE2-A038, hACE2-A077, and hACE2-A134, were subsequently selected for the next phase of analyses. We found that clones hACE2-A008, hACE2-A038, and hACE2-A134 differed in their heavy and light chain sequences, while clones hACE2-A038 and hACE2-A077 had identical sequences. In accordance, the ELISA and flow cytometry results indicated that hACE2-A038 and hACE2-A077 but not hACE2-A008, hACE2-A028, hACE2-A048, and hACE2-A134 had similar levels of reactivity to the hACE antigen, suggesting that our workflow reduced the likelihood of clonal duplication (Fig. 3). These results indicate that the effective separation of hybridoma cells expressing antigen-specific sIgG could be achieved using hACE2-Fc as a bait protein. Evaluation of mAb recovery rates achieved using the antigen-coated bead separation method We next evaluated the yield of hybridoma cells generated by cell fusion to determine the effectiveness of the magnetic bead separation method. To this end, after cell fusion, a portion (2.0 × 10 7 ) of the fused cells was cultured in HAT medium for 7 days to obtain a bulk hybridoma cell sample containing a mixture of hybridoma cells. The fraction of the cells that did not bind to the antigen-coated beads was also cultured in HAT medium for 7 days to obtain an unbound hybridoma cell sample. To detect the hybridoma cells expressing antigen-specific mAbs, the bulk and unbound hybridoma samples were stained with biotinylated hACE2-Fc. After incubating the cells with fluorescence-labeled StAv and anti-human IgG-Fc, flow cytometric analysis revealed that the frequencies of antigen-specific hybridoma cells in the bulk and unbound cell fractions were 21.4% and 7.1%, respectively (Fig. 5 and Supplementary Fig. 6). After adjusting for background binding using the hACE2-Fc-biotin-untreated negative control, the yield of antigen-specific mAb expressing hybridoma cells from a cell fusion pool was determined to be 83.5%. Thus, the majority of hybridoma cells expressing antigen-specific sIgG was recovered using the antigen-coated magnetic beads. In addition, analysis of all the hybridoma clones obtained showed that the presence of sIgG with anti-hACE2-Fc reactivity was readily detectable on the hybridoma cell surface (Fig. 6 and Supplementary Fig. 7). Discussion In this study, we established a workflow for the efficient production of antigen-specific mAb-producing hybridoma cells by exploiting the reactivity of sIgG to antigen on the cell surface (Fig. 1). The combination of biotinylated antigen and StAv-coated magnetic beads achieved a recovery rate of over 80% (Fig. 5). Furthermore, the laborious cloning step could be omitted by seeding the cells at a density of one cell per well of a 96-well plate. These measures reduced the time required to establish human-mAb-producing hybridoma cells and increased the efficiency of antigen-specific fully human mAb generation from TC-mAb mice (Table 1). Cell fusion is a conventional method for obtaining secretory-Ab-expressing cells. It uses the plasmablasts, plasma cells, and memory B cells with high SHM rates to generate hybridoma cells that express high-affinity mAbs 22 . The fact that all of the hybridoma clones analyzed expressed sIgG on their surface suggests that the class switching of membrane-bound and secretory forms of IgG 23,24 was potentially attenuated by cell fusion with myeloma cells. To verify this notion, we also isolated hybridoma cells from a pool of fused cells originating from immunized TC-mAb mice using anti-human IgG beads. This led to the recovery of 47.4%–55.6% of the cells (Supplementary Table 1), suggesting that the cell fusion process enabled the production of a large number of sIgG-expressing cells. The switching machinery of the membrane and secretory forms of IgG is regulated by alternative splicing during B cell differentiation. Moreover, the heterogenous nuclear RNA-binding protein LL (hnRNPLL) is specifically induced in terminally differentiated lymphocytes, including plasma cells 25 . Therefore, the mechanism of alternative splicing regulation by cell fusion can be investigated in plasma cells by tracing changes in the expression of proteins such as hnRNPLL when the plasma cells (CD138 + TACI + lymphocytes 26 ) are fused with myeloma cells. Such an analysis allowed us to determine whether all B cells that fused with myeloma cells expressed sIgG on their surface. As a result, the real recovery rate of antigen-specific B cells produced in each immunized TC-mAb mouse could be calculated. Whether the re-expression of sIgG in hybridoma cells occurs in the Ig gene of wild-type mice is currently unknown. If mouse sIgG re-expression is also induced by cell fusion, the utility of the present results will increase dramatically. The workflow presented here led to the successful recovery of more than 80% of the antigen-specific-mAb-expressing hybridoma cells (Fig. 5 and Table 1). In addition, the culture period after cell fusion was limited to 2 days to increase the recovery rate while minimizing the possibility of obtaining clonal duplicates. The fact that 366 different clones were obtained from two TC-mAb mice (Fig. 2 and Table 1) indicates that this workflow yielded a sufficient number of antigen-specific hybridoma cells for the selection of therapeutic mAb candidates. The analysis of epitope distribution and mAb-coding gene sequences (Fig. 3 and 4) confirmed that the established clones were highly diverse. Furthermore, since the bead separation method has a capacity to process up to 10 9 cells simultaneously, all splenocytes (~ 4 × 10 8 cells/mouse) from the two immunized TC-mAb mice could be screened in one procedure for Abs of interest. In the present study, we were able to process 5.9 × 10 8 cells from two immunized TC-mAb mice simultaneously (Table 1). We estimated that without the magnetic bead selection method ~35,000 hybridoma cells would have been obtained (Table 1), all of which would require screening. The fact that we were able to reduce this number to only 500 colonies indicates that our method considerably streamlined the mAb screening process. In this study, all hybridoma cells derived from the two TC-mAb mice expressed sIgG, indicating that they could be effectively screened on this basis and their antigen-reactivity. Thus, our method is applicable to the screening of cross-reactive Abs, which recognize mutation-prone antigens such as the coronavirus spike proteins and homologous antigens from different animal species. Thus, the workflow developed in this study markedly facilitated the generation of fully human antigen-specific Abs from TC-mAb mice via hybridoma technology. This method is expected to markedly speed up the development of therapeutic Ab candidates, which are in high demand for various therapeutic applications worldwide. Methods Ethics declarations This study was approved by the Animal Care and Use Committee of Tottori University (Permit Number: 20-Y-13, 20-Y-31, 21-Y-26, 22-Y-36, and 22-Y-63). All experiments were carried out in compliance with the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations. Mice were sacrificed by cervical dislocation prior to tissue collection, and all efforts were made to minimize their suffering. Antigen preparation, immunization, and cell fusion The expression of the hACE2-Fc fusion protein and the associated vector construction methods have been previously published 21 . In brief, a full-length ACE2 extracellular domain coding sequence (amino acids 18–740) was fused to the human IgG1-Fc via a linker with a GGGGS sequence. Mutations were introduced to prevent the activation of the human Fc from affecting the immune response when the TC-mAb mice were immunized (Supplementary Fig. 2). Next, Expi293F TM cells (Thermo Fisher Scientific, Waltham, MA, USA) were transfected with the hACE2-Fc expression vector and cultured for 5 days. The culture supernatant was then harvested and applied to a Protein G Sepharose FF column (Cytiva, Tokyo, Japan) to purify the recombinant protein. The denatured protein was analyzed by SDS-PAGE (Supplementary Fig. 3). Two TC-mAb mice were then immunized with the hACE2-Fc recombinant protein (60 μg per mouse) as previously described 7 . The protocol used for the fusion of lymphocytes to myeloma cells (P3X63Ag8.653, ATCC CRL-1580, Manassas, VA, USA) to generate hybridomas has also been previously published 7,8 . The mice used in this study were kept in a designated animal facility, under specific pathogen-free conditions, in a controlled ambient temperature environment with a 12 h light/dark cycle. Mice underwent isoflurane-induced anesthesia for all blood draws and other sampling. All animal experiments were approved by the Animal Care and Use Committee of Tottori University and conducted in strict accordance with ARRIVE guidelines. Preparation of biotinylated hACE2-Fc The EZ-Link Sulfo-NHS-LC-Biotin kit (Thermo Fisher Scientific) was used to biotinylate hACE2-Fc, according to the manufacturer’s protocol. Briefly, 4 μl of 10 mM Sulfo-NHS-LC-LC-biotin (Thermo Fisher Scientific) solubilized in distilled water were added to 3 ml of 1 mg/ml hACE2-Fc in phosphate-buffered saline (PBS) (Nacalai Tesque, Kyoto, Japan). After incubation at room temperature for 30 min, 300 μl of 1M Tris-HCl (pH 8.0) (Merck, Billerica, MA, USA) was added to stop the reaction. The biotinylated sample was applied to an ultracentrifuge column (Amicon Ultra, 30 kDa, Merck Millipore) and concentrated by replacing the buffer with PBS. The molar ratio of biotin to hACE2-Fc was estimated as 4.6 using the biotin quantification kit containing 4'-hydroxyazobenzene-2-carboxylic acid (Thermo Fisher Scientific). Separation of antigen-specific-mAb-expressing hybridoma cells After 2 days of cell culture in 1/3 HAT medium (1:2 HAT to hypoxanthine-thymidine (HT) medium), the cells were harvested and incubated with StAv-magnetic beads (BD IMag™ Streptavidin Particles Plus–DM, BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Any cells that bound nonspecifically to the beads were removed. After incubating for 30 min in a rotator, the unbound fraction was collected using a magnetic stand. The cells were then incubated with 2.5 μg biotinylated hACE2-Fc or biotinylated anti-human IgG-Fc (Bethyl Laboratories, Montgomery, TX, USA) per 1 × 10 6 cells on ice for 1 h with rotation. After washing with PBS supplemented with 1% fetal bovine serum (FBS), 2 ml of StAv-magnetic beads were added to the cells, followed by another 30 min incubation at 4 °C with rotation. After four washes to remove nonspecifically bound cells, the remaining 4.65 × 10 6 cells were seeded into eleven 96-well plates (TPP Techno Plastic Products AG, Zollstrasse, Switzerland). ELISA The antibody titers were measured by ELISA as previously described 7 . In brief, 96-well immunoassay plates (Nunc Maxisorp, Thermo Fisher Scientific) were coated with 100 µl/well of antigen overnight and then blocked with PBS containing 5% skim milk (DB Biosciences, Franklin Lakes, NJ, USA) for 30 min at room temperature. After washing, 100 µl of each antibody sample were added to each well of an ELISA plate and incubated for 1 h at room temperature. The plates were washed again and incubated with 100 µl of goat anti-human IgG (H+L) cross-adsorbed secondary Ab (Abcam, Cambridge, UK) at a 10,000-fold dilution in TBS-T (Tris-buffered saline and 0.05% Tween 20) for 30 min at room temperature. The plates were washed once again and developed using 100 µl of o -phenylenediamine dihydrochloride (Fujifilm Wako, Osaka, Japan). The reaction was stopped with 25 µl of 1 M H 2 SO 4 (Nacalai Tesque). After the plates were allowed to develop for 10–30 min, the absorbance was read at 492 nm. Immunocytochemistry HEK293-hACE2 cells (Takara Bio Inc., Shiga, Japan) were purchased and maintained according to the manufacturer’s instructions. Cells cultured in 96-well plates were treated with 100 μl of hybridoma supernatant or purified mAb and incubated for 1 h on ice. After washing with ice-cold medium, 100 μl of goat anti-human IgG (H+L) cross-adsorbed secondary antibody, conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Abcam) and diluted 600-fold in medium, was added. After additional washing, 100 μl of PBS supplemented with 1% FBS was added. The samples were then analyzed on fluorescence microscope (BZ-X700; Keyence, Itasca, IL, USA) or IncuCyte S3 instrument (Sartorius, Göttingen, Germany). Flow cytometry Cultured HEK293-hACE2 cells were dissociated from the culture plate using Accutase (Innovative Cell Technologies, Inc., San Diego, CA, USA) and stained. All staining was performed on ice. The stained cells were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA). To prevent interactions between Fcg receptors and Abs, the cells were pretreated with Human Seroblock (Bio-Rad Laboratories Inc., Hercules, CA, USA) for 10 min before staining, according to the manufacturer’s instructions. To determine the antigen-reactivity of sIgGs expressed on the surface of the hybridoma cells, 3 μg of protein antigen (i.e., EpEX) were added to 1 × 10 6 cells in staining buffer (PBS with 1% FBS). At the same time, 0.5 μl of the biotinylated anti-human Ig light chain κ antibody (Biolegend, San Diego, CA, USA) or 4 μl of the PE/Cyanine7-conjugated anti-human IgG-Fc antibody (Biolegend) was added to detect sIgG on the hybridoma cell surface. After a 60 min incubation, the cells were washed three times with staining buffer. To detect biotinylated molecules, 0.1 μl (0.5 μg) of SAv-Brilliant Violet 650 (Biolegend) was added to the cells. To detect His-tagged protein binding, 0.25 μl of anti-His-tag mAb conjugated to Alexa Fluor 594 was added to the cells. After a 30 min incubation, the cells were washed three times with staining buffer and analyzed on a CytoFLEX S flow cytometer (Beckman Coulter). The splenocytes were stained as previously described 7 . Determination of human mAb subclasses The supernatants obtained from the wells of cultured hybridoma cells were analyzed using the rapid human antibody isotyping kit (Antagen Pharmaceuticals, Boston, MA, USA) according to the manufacturer’s instructions. Epitope distribution assay The distribution of epitopes to which the hybridoma cells bound was analyzed using a competitive assay. In brief, unlabeled purified mAbs were prepared using a protein G Sepharose 4 Fast Flow column. The purified mAbs were used as the capture Abs and the biotinylated mAbs were used as the detector Abs. 96-well immunoassay plates (Nunc Maxisorp) were coated with each capture Ab at 100 ng/well, and 1 h later blocked with PBS containing 5% skim milk for 30 min at room temperature. Ab biotinylation was performed using the EZ-Link Sulfo-NHS-LC-Biotin kit according to the manufacturer’s instructions. After washing, 100 µl of the biotinylated antibody samples was added to the wells and incubated for 1 h at room temperature. The plates were washed again and incubated with 100 µl of Streptavidin-HRP conjugate (Proteintech, Tokyo, Japan), diluted 3,000-fold in TBS-T, for 30 min at room temperature. The plates were washed once again and developed using o-phenylenediamine dihydrochloride. The reaction was terminated using 1 M H 2 SO 4 , and after a developing step, the absorbance was read at 492 nm. Declarations Data availability Sequence data that support the findings of this study have been deposited in the DNA Data Bank of Japan (DDBJ) with the accession numbers: LC804973 (ACE2A008_H), LC804974 (ACE2A008_L), LC804975 (ACE2A038_H), LC804976 (ACE2A038_L), LC804977 (ACE2A077_H), LC804978 (ACE2A077_L), LC804979 (ACE2A134_H), LC804980 (ACE2A134_L). Acknowledgments We thank Maki Takami for assisting with human Ab production and the generation of antigen-specific hybridoma cell lines. We also thank Dr. Hiroyuki Kugoh and Dr. Satoshi Abe at Tottori University, Kazuma Tomizuka at Tokyo University of Pharmacy and Life Sciences, and Dr. Yasuyuki Kurihara at Yokohama National University for critical discussions. This study was supported in part by the Japan Agency for Medical Research and Development (AMED) (Grant Numbers JP21am0101124, JP23ama121046, JP23gm1610006, JP23gm0010010, JP23am0401002, JP23gm1810008 all awarded to Y.K), the Joint Research of the Exploratory Research Center on Life and Living Systems (ExCELLS) (ExCELLS program No. 21-101 to Y.K.), and JST CREST (Grant Number JPMJCR18S4 to Y.K.). This research was partly performed at the Tottori Bio Frontier facility, managed by the Tottori prefecture, Japan. We thank Edanz (https://jp.edanz.com/ac) for editing the English text of a draft of this manuscript. Author contributions H.S. and W.Y. planned the study and optimized the hybridoma cell separation method; K.M. and H.Takayama maintained the TC-mAb mice and collected splenocytes and lymphocytes for cell fusion; H.Tanaka. and K.H. performed cell fusion; Y.W., H.S., H. Tu, and Y.Q. performed cell culture and mAb production experiments; H.S. performed mAb purification and epitope distribution analysis; H.S. performed cell staining experiments; H.S. characterized the antigen-specific B cells from TC-mAb mice; Y.W. and H.S. contributed to the analysis and discussion of the data and wrote the manuscript, S.I., X.G., M.O., and Y.K. supervised the study. Competing interests M.O. is a CEO, employee, and shareholder of Trans Chromosomics, Inc., and H.Tanaka, K.H., K.M., and H.Takayama are employees of Trans Chromosomics, Inc. S.I. is a CEO, employee, and shareholder of Purotech Bio Inc. The remaining authors declare no competing interests. References Köhler, G., Milstein, C. Cultivos continuos de células fusionadas que secretan anticuerpos de especificidad predefinida. Nature 256 , 495–497 (1975). Tian, Y., Hu, D., Li, Y. & Yang, L. Development of therapeutic vaccines for the treatment of diseases. Molecular Biomedicine 3 , 1–30 (2022). Brüggemann, M. et al. Human Antibody Production in Transgenic Animals. Arch Immunol Ther Exp (Warsz) 63 , 101–108 (2015). Lonberg, N. Human antibodies from transgenic animals. Nat Biotechnol 23 , 1117–1125 (2005). Lee, E. C. et al. Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery. Nat Biotechnol 32 , 356–363 (2014). Murphy, A. J. et al. Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice. Proceedings of the National Academy of Sciences 111 , 5153–5158 (2014). Satofuka, H. et al. Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci. Nat Commun 13 , 1–15 (2022). Satofuka, H. et al. Characterization of human anti ‑ EpCAM antibodies for developing an antibody – drug conjugate. Sci Rep 1–11 (2023) doi:10.1038/s41598-023-31263-x. Fusil, F. et al. A lentiviral vector allowing physiologically regulated membrane-anchored and secreted antibody expression depending on B-cell maturation status. Molecular Therapy 23 , 1734–1747 (2015). Brouwer, P. J. M. et al. Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability. Science (1979) 369 , 643–650 (2020). Liu, L. et al. Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. Nature 584 , 450–456 (2020). Hansen, J. et al. Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail. Science (1979) 369 , 1010–1014 (2020). Cao, Y. et al. Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells. Cell 182 , 73-84.e16 (2020). Keating, S. M. et al. Generation of recombinant hyperimmune globulins from diverse B-cell repertoires. Nat Biotechnol 39 , 989–999 (2021). Pedrioli, A. & Oxenius, A. Single B cell technologies for monoclonal antibody discovery. Trends Immunol 42 , 1143–1158 (2021). Onodera, T. et al. A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site. Immunity 54 , 2385-2398.e10 (2021). Akkaya, M., Kwak, K. & Pierce, S. K. B cell memory: building two walls of protection against pathogens. Nat Rev Immunol 20 , 229–238 (2020). Nojima, T. et al. In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo. Nat Commun 2 , (2011). Shinnakasu, R. et al. Regulated selection of germinal-center cells into the memory B cell compartment. Nat Immunol 17 , 861–869 (2016). Akagi, S., Nakajima, C., Tanaka, Y. & Kurihara, Y. Flow cytometry-based method for rapid and high-throughput screening of hybridoma cells secreting monoclonal antibody. J Biosci Bioeng 125 , 464–469 (2018). Wines, B. D. et al. Fc engineered ACE2-Fc is a potent multifunctional agent targeting SARS-CoV2. Front Immunol 13 , 1–15 (2022). Shinnakasu, R. et al. Regulated selection of germinal-center cells into the memory B cell compartment. Nat Immunol 17 , 861–869 (2016). Brown, S. L. & Morrison, S. L. Regulation of the production of secretory and membrane immunoglobulin during lymphocyte development. Clin Immunol Immunopathol 50 , 155–170 (1989). Liew, P. The Longevity of the Humoral Immune Response – Survival of Long-lived Plasma Cells. Akademeia 2 , ea0116 (2012). Chang, X., Li, B. & Rao, A. RNA-binding protein hnRNPLL regulates mRNA splicing and stability during B-cell to plasma-cell differentiation. Proc Natl Acad Sci U S A 112 , E1888–E1897 (2015). Pracht, K. et al. A new staining protocol for detection of murine antibody-secreting plasma cell subsets by flow cytometry. Eur J Immunol 47 , 1389–1392 (2017). Additional Declarations No competing interests reported. Supplementary Files 4.SupplementaryFiguresandTables.pptx Cite Share Download PDF Status: Published Journal Publication published 04 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 27 Jun, 2024 Reviews received at journal 23 Jun, 2024 Reviewers agreed at journal 14 Jun, 2024 Reviewers agreed at journal 24 Apr, 2024 Reviews received at journal 09 Apr, 2024 Reviewers agreed at journal 08 Apr, 2024 Reviewers invited by journal 08 Apr, 2024 Editor assigned by journal 08 Apr, 2024 Editor invited by journal 05 Apr, 2024 Submission checks completed at journal 05 Apr, 2024 First submitted to journal 27 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3993293","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":289179103,"identity":"6eab30c2-5657-4036-8f71-4074dd6d4149","order_by":0,"name":"Hiroyuki Satofuka","email":"","orcid":"","institution":"Tottori University","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Satofuka","suffix":""},{"id":289179105,"identity":"858d2d10-3fa2-4ef7-ba93-af31d87cffa4","order_by":1,"name":"Yayan Wang","email":"","orcid":"","institution":"Tottori University","correspondingAuthor":false,"prefix":"","firstName":"Yayan","middleName":"","lastName":"Wang","suffix":""},{"id":289179109,"identity":"beb338b8-f353-4cb1-987f-95bb835d5e6c","order_by":2,"name":"Hiroshi Tanaka","email":"","orcid":"","institution":"Trans Chromosomics Inc","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Tanaka","suffix":""},{"id":289179113,"identity":"3d7fe0a3-bae2-4952-a60a-01832838def2","order_by":3,"name":"Kei Hiramatsu","email":"","orcid":"","institution":"Trans Chromosomics Inc","correspondingAuthor":false,"prefix":"","firstName":"Kei","middleName":"","lastName":"Hiramatsu","suffix":""},{"id":289179114,"identity":"5c08ab16-df2d-49a0-8a02-0bf7dfb7a955","order_by":4,"name":"Kayoko Morimoto","email":"","orcid":"","institution":"Trans Chromosomics 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08:14:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3993293/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3993293/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-73770-5","type":"published","date":"2024-10-04T15:58:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54452780,"identity":"948011eb-b3cc-4553-b07a-af624d8005ac","added_by":"auto","created_at":"2024-04-10 18:28:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorkflow for the separation of antigen-specific-IgG-producing hybridoma cells on the basis of their surface IgG expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TC-mAb mice were immunized with antigen (Ag) to generate hybridoma cells, which were subsequently treated with antigen Ag-biotin-StAv-magnetic bead complexed to select the cells expressing antigen-specific monoclonal antibodies (mAbs). The hybridoma cells were then seeded into 96-well plates at approximately one cell per well to generate clonal colonies.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-3993293/v1/2b15d144e7321ad5de3a34b2.png"},{"id":54452782,"identity":"ba25791e-d574-4c65-93b3-1b044b7f12d3","added_by":"auto","created_at":"2024-04-10 18:28:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":256828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eELISA analysis of the magnetic-bead-enriched hybridoma cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The binding of monoclonal antibodies (mAbs) within the hybridoma cell culture supernatant to hACE2 was analyzed by ELISA. The threshold for a positive signal (i.e., an absorbance value \u0026gt; 0.6, indicated by the green line) was selected on the basis of being 3-fold higher than the negative control value. Representative images of ELISA plates \u003cstrong\u003e(b)\u003c/strong\u003e and the corresponding absorbance values \u003cstrong\u003e(c) \u003c/strong\u003eare shown. The plate numbers are indicated in the upper left corner of each plate. The wells that did not produce colonies are blank. Signals with absorbance values at 492 nm higher than 4 are simply shown as \"4\" because this is the detection limit of the instrument.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-3993293/v1/19faf06019a5b6bd6e234d3d.png"},{"id":54452783,"identity":"efd12fd9-116a-4ae3-8331-307c3a0ffd5b","added_by":"auto","created_at":"2024-04-10 18:28:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":167316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometric analysis of the monoclonal antibodies obtained.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Unfixed HEK293-hACE2 cells were stained with the purified monoclonal antibodies (mAbs) harvested from the hybridoma cell culture supernatants. The negative control (gray-filled histogram) was not incubated with a purified mAb. The HEK293-hACE2 cells were stained using 2-fold serial dilutions of a 10 μg/ml mAb preparation. \u003cstrong\u003e(b)\u003c/strong\u003eEach histogram represents the median fluorescence intensity (MFI) of a given mAb.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-3993293/v1/ce766f0305f058d8bc7da738.png"},{"id":54453573,"identity":"caccb4eb-0904-4585-bda2-2a836e1b124f","added_by":"auto","created_at":"2024-04-10 18:36:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of epitope distribution among monoclonal antibodies obtained.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Schematic representation of the sandwich ELISA method used to determine the epitope distribution of each monoclonal antibody (mAb). Of note, the mAbs obtained were used as both the capture and detection mAbs; however, the capture mAb was purified while the detector mAb was biotinylated. If the capture and detection mAbs bound to the same epitope, the capture mAb would obscure the antigenic epitope, preventing the detection mAb from binding to the antigen; in such a case, no signal would be observed. However, if the capture and detector mAbs targeted different epitopes, the capture-mAb-antigen complex would react with the detector mAb, producing a signal that can be detected following a secondary reaction with a StAv-HRP conjugate. \u003cstrong\u003e(b)\u003c/strong\u003e Epitope distribution analysis heatmap. \u003cstrong\u003e(c)\u003c/strong\u003e Venn diagram showing the epitopes among the mAbs obtained. The mAb clones of hACE2A008, 038, 077, and 134 recognize the same epitope. The epitopes of hACE2A008 and 048 are not identical but overlap partially.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-3993293/v1/cace8c8a8a0d72370e63945d.png"},{"id":54452784,"identity":"cbfda7fd-163f-49ba-a267-c75ae3708334","added_by":"auto","created_at":"2024-04-10 18:28:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":192375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometric analysis of the cultured hybridoma cells before and after magnetic bead separation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eA cartoon representation of the molecular interactions involved in the isolation of antigen-specific-Ab-expressing hybridoma cells. \u003cstrong\u003e(b)\u003c/strong\u003e Flow cytometric analysis of bulk hybridoma cell samples in the presence (right)or absence (left) of hACE2-Fc-biotin, which was used to reduce nonspecific binding.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-3993293/v1/fdf534fb43bcdad26f26086f.png"},{"id":54452786,"identity":"e902f886-a754-40f7-bba3-029a26eccbe7","added_by":"auto","created_at":"2024-04-10 18:28:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":222420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometric analysis of hybridoma cells on the basis of antigen-specific-surface IgG expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReactivity of hybridoma cells toward anti-human IgG-Fc-PC/Cyanine 7 (PC7) (y-axis) and antigen (x-axis). The hybridoma cells were stained with biotinylated hACE2-Fc (left dot plots) or left unstained (right dot plots), before being stained with a streptavidin-Alexa Fluor 594 conjugate. The overlaid histograms correspond to the data presented in the dot plots, in which the presence or absence of hACE2-Fc is indicated in blue and gray, respectively. The hybridoma clone name is indicated above each set of graphs. The y-axis of the histograms was set to %MAX to facilitate the comparison of different cell population sizes.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-3993293/v1/109922186d5ac03fdfeb9a9f.png"},{"id":66097706,"identity":"5fdf9b28-2c08-4887-8bd9-32709f9b073c","added_by":"auto","created_at":"2024-10-07 16:15:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1618498,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3993293/v1/28560878-07a7-4147-a995-e1d6ddd6f726.pdf"},{"id":54453574,"identity":"1b344fe6-62ec-4d71-a8a0-4623febe260b","added_by":"auto","created_at":"2024-04-10 18:36:38","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3306255,"visible":true,"origin":"","legend":"","description":"","filename":"4.SupplementaryFiguresandTables.pptx","url":"https://assets-eu.researchsquare.com/files/rs-3993293/v1/2e9bf95b875c915fce850540.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Developing a workflow for the isolation of hybridoma cells producing fully human antigen-specific antibodies using a surface IgG detection method","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe first step in the generation of monoclonal antibodies (mAbs) is the generation of rodent hybridoma cells via the fusion of B cells and myeloma cells\u003csup\u003e1\u003c/sup\u003e. The resulting rodent mAbs are then refined using chimerization or humanization techniques, which modify the rodent genetic sequences to generate therapeutic human mAbs\u003csup\u003e2\u0026ndash;6\u003c/sup\u003e. We have previously reported the generation of trans-chromosomic mice producing fully human mAbs (TC-mAb mice), which have an artificial chromosome harboring full-length human immunoglobulin (Ig) loci (human heavy and light kappa chain). Moreover, we demonstrated the ability of these mice to efficiently produce therapeutic human mAb candidates via hybridoma technology\u003csup\u003e7,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eVast numbers of Ab-producing cells can be obtained from an immunized TC-mAb mouse by cell fusion. For example, one immunized TC-mAb mouse can generate over 30,000 hybridoma cells\u003csup\u003e8\u003c/sup\u003e. As a result, the process of screening to obtain the mAbs of interest is costly and time-consuming. To overcome these challenges, B cell-derived surface IgG (sIgG, also known as membrane-anchored IgG\u003csup\u003e9\u003c/sup\u003e) molecules have recently been developed for the rapid production of therapeutics against COVID-19 and other infectious viral diseases\u003csup\u003e10\u0026ndash;14\u003c/sup\u003e. This strategy uses fluorescently labeled antigens as baits to separate antigen-specific mAb-producing B cell fractions from the pool of peripheral blood mononuclear cells containing memory B cells via cell sorting\u003csup\u003e10\u0026ndash;12,15,16\u003c/sup\u003e. This approach has markedly facilitated the development of therapeutic Ab candidates. However, since sIgG expression is downregulated during the differentiation of terminal B cells into plasma cells\u003csup\u003e9,17\u003c/sup\u003e, only a fraction of antigen-specific Abs produced in the immunized animals are available for harvesting; this presents a considerable barrier to obtaining therapeutic mAbs.\u003c/p\u003e\n\u003cp\u003eThe expression of sIgGs and secreted IgGs is controlled by alternative splicing machinery and the B cell maturation status\u003csup\u003e9\u003c/sup\u003e. Memory B cells are the major source of sIgGs in the spleen\u003csup\u003e18\u003c/sup\u003e. However, low-affinity B cells (i.e., those with low somatic hypermutation [SHM] rates) can readily enter the memory B cell pool from the germinal center before affinity maturation has been completed\u003csup\u003e19\u003c/sup\u003e. Therefore, strategies for obtaining high-affinity mAbs by enriching antigen-specific B cells expressing sIgG are urgently needed.\u003c/p\u003e\n\u003cp\u003eThe production of antigen-specific mAbs using sIgG from hybridoma cells derived from wild-type mice has previously been reported\u003csup\u003e20\u003c/sup\u003e. However, the number of hybridoma cells that can be processed at any given time is limited by the capacity of the cell-sorting instrument. Furthermore, because this method includes a 7-day cell culture period and a dead cell removal step after cell fusion, the same clones are repeatedly amplified.\u003c/p\u003e\n\u003cp\u003eIn this study, we developed a method of rapidly and accurately selecting hybridoma cells expressing antigen-specific mAbs on the basis of their sIgG expression. The extra selection step markedly enriched the number of hybridoma cells capable of producing antigen-specific mAbs, thereby improving screening efficiency. This approach could be successfully implicated because TC-mAb-mouse-derived hybridoma cells expressing anti-specific human IgG also express high levels of sIgG; moreover, their antigen-reactivity was readily detectable by flow cytometry.\u003c/p\u003e\n\u003cp\u003eWe showed that all the analyzed sIgG-producing hybridoma cell clones obtained from the TC-mAb mice expressed adequate levels of human IgG in response to labeled antigen. Thus, we demonstrated that antigen-specific human-mAb-producing hybridoma cells were highly enriched from a cell fusion pool using sIgG and antigen as bait. Specifically, we showed that over 80% of cells expressing antigen-specific sIgG were recovered from the cultured hybridoma cell pool. Furthermore, we were able to establish single-clone hybridoma cells approximately 2 weeks after cell fusion by optimizing the seeding density; this bypassed the need for laborious mass cell cloning. Collectively, our findings suggest that the workflow for hybridoma generation presented in this study (Fig. 1) has the potential to considerably advance the production of highly sought-after therapeutic mAb candidates.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDetection of human sIgGs on hybridoma cells derived from TC-mAb mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe began by evaluating the expression and antigen-reactivity of sIgGs expressed by the already established hybridoma cell lines. Analysis of 16 anti-EpCAM mAb clones from TC-mAb mice\u003csup\u003e7,8\u003c/sup\u003e, which included the IgG1, IgG3, and IgG4 subclasses, showed a sIgG-associated signal shift in the presence of the His-tagged EpEX antigen relative to the negative control samples (Supplementary Fig. 1). Since the intensity of the signal shift depends on the sIgG expression levels and the affinity of the mAb, a range of weak to strong reactions to the EpEX antigen were detected. This result suggested that all the TC-mAb-mouse-derived hybridoma clones expressing human IgG displayed an amount of sIgG for selection. The antigen-specific hybridoma cells could therefore be enriched on the basis of their sIgG-mediated antigen-reactivity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffective separation of hybridoma cell populations expressing antigen-specific IgG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next attempted to enrich the antigen-specific-mAb-expressing hybridoma cells from the hybridoma cell pool after cell fusion. The human angiotensin-converting enzyme 2 (hACE2) (EC 3.4.17.23., IUBMB) was used as a model protein. The full-length gene encoding the hACE2 extracellular domain was fused to the gene encoding the human Fc region to generate a construct expressing the recombinant hACE2-Fc protein. hACE2-Fc was expressed and purified as previously described\u003csup\u003e21\u003c/sup\u003e (Supplementary Fig. 2 and 3). Two TC-mAb mice were immunized with the purified hACE2-Fc; analysis of their anti-sera titers showed the robust induction of anti-hACE2 Abs (Supplementary Fig. 4). After harvesting the splenocytes and lymphocytes from the mouse spleen and lymph nodes, respectively, 5.6 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells were fused with myeloma cells and cultured in 1/3 hypoxanthine-aminopterin-thymidine (HAT) medium for 2 days, ensuring that cell viability was maintained with little or no cell division. Some (2.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) of the fused cells were also seeded into a 96-well plate to compare the efficiency of antigen-specific hybridoma cell selection with or without the use of magnetic beads.\u003c/p\u003e\n\u003cp\u003eCells (3.4 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e) collected after 2 days of culture were incubated with the biotinylated hACE2-Fc protein. Then, the antigen-bound cell fraction, which included hybridoma cells and memory B cells, was separated using the streptavidin (StAv)-magnetic bead method, yielding 4.7 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells (1.4%) from a total of 3.4 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells (Table 1). The separated cells were seeded into eleven 96-well plates based on the predicted growth rate of hybridoma cells, with the aim of seeding one cell per well. After 12 days of culture, colony formation was observed in about 500 wells, with most of the wells containing single colonies.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"565\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1. Summary of the hybridoma isolation workflow\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eAntigen (individual TC-mAb rats)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.389380530973455%\" colspan=\"2\"\u003e\n \u003cp\u003ehACE2-Fc (ndividual A and B)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003eBead separation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003eNo bead separation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eTotal number of splenocytes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e5.9 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e2.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eNumber of cells after 2 days of culture\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e3.4\u0026nbsp;\u0026times;\u0026nbsp;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003eNo treatment\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eNumber of cells obtained following bead separation\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e4.7 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eYield from **precleared cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e1.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eHypoxanthine-aminopterin-thymidine medium selection\u0026nbsp;(number of plates)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eColonies/plate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eTotal number of colonies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e*500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eNumber of ELISA wells with a positive signal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e366\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.610619469026545%\"\u003e\n \u003cp\u003eRatio of number of positive wells/number of colonies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e73.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.194690265486727%\"\u003e\n \u003cp\u003e0.6%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\"\u003e\n \u003cp\u003e*Total colony count was calculated by multiplying the average number of colonies obtained from six 96-well plates by the number of plates.\u003c/p\u003e\n \u003cp\u003e**Preclered cells are a pre-washed cell fraction of cells that bind nonspecifically to the beads by treatment with antigen-free beads.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe supernatants removed from the wells containing hybridoma colonies were analyzed for their reactivity against hACE2-Fc by enzyme-linked immunosorbent assay (ELISA). Of the ~500 wells that contained hybridoma cell colonies, 366 wells (73.2%) generated a positive signal, which was defined as an absorbance value \u0026gt; 3-fold greater than that of the negative control (Fig. 2). Immunocytochemistry was then used to show that supernatants from 11 hybridoma cell colonies reacted with intact hACE2 expressed on unfixed HEK293-hACE2 cells (an hACE2-overexpressing cell line) . Six of the 11 clones were further evaluated using a combination of immunocytochemistry and flow cytometry (Fig. 3 and Supplementary Fig. 5). The results indicated that the mAbs from all six clones reacted with the hACE2 expressed on the surface HEK293-hACE2 cells. The steps involved in the separation of antigen-specific hybridoma cells are summarized in Table 1.\u003c/p\u003e\n\u003cp\u003eEpitope distribution analysis showed that at least three different hACE2 epitopes were recognized by the mAbs obtained (Fig. 4). Four clones, namely hACE2-A008, hACE2-A038, hACE2-A077, and hACE2-A134, were subsequently selected for the next phase of analyses. We found that clones hACE2-A008, hACE2-A038, and hACE2-A134 differed in their heavy and light chain sequences, while clones hACE2-A038 and hACE2-A077 had identical sequences. In accordance, the ELISA and flow cytometry results indicated that hACE2-A038 and hACE2-A077 but not hACE2-A008, hACE2-A028, hACE2-A048, and hACE2-A134 had similar levels of reactivity to the hACE antigen, suggesting that our workflow reduced the likelihood of clonal duplication (Fig. 3). These results indicate that the effective separation of hybridoma cells expressing antigen-specific sIgG could be achieved using hACE2-Fc as a bait protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of mAb recovery rates achieved using the antigen-coated bead separation method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next evaluated the yield of hybridoma cells generated by cell fusion to determine the effectiveness of the magnetic bead separation method. To this end, after cell fusion,\u0026nbsp;a portion (2.0 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e) of the fused cells was cultured in HAT medium for 7 days to obtain a bulk hybridoma cell sample containing a mixture of hybridoma cells. The fraction of the cells that did not bind to the antigen-coated beads was also cultured in HAT medium for 7 days to obtain an unbound hybridoma cell sample. To detect the hybridoma cells expressing antigen-specific mAbs, the bulk and unbound hybridoma samples were stained with biotinylated hACE2-Fc. After incubating the cells with fluorescence-labeled StAv and anti-human IgG-Fc, flow cytometric analysis revealed that the frequencies of antigen-specific hybridoma cells in the bulk and unbound cell fractions were 21.4% and 7.1%, respectively (Fig. 5 and Supplementary Fig. 6). After adjusting for background binding using the hACE2-Fc-biotin-untreated negative control, the yield of antigen-specific mAb expressing hybridoma cells from a cell fusion pool was determined to be 83.5%. Thus, the majority of hybridoma cells expressing antigen-specific sIgG was recovered using the antigen-coated magnetic beads. In addition, analysis of all the hybridoma clones obtained showed that the presence of sIgG with anti-hACE2-Fc reactivity was readily detectable on the hybridoma cell surface (Fig. 6 and Supplementary Fig. 7).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we established a workflow for the efficient production of antigen-specific mAb-producing hybridoma cells by exploiting the reactivity of sIgG to antigen on the cell surface (Fig. 1). The combination of biotinylated antigen and StAv-coated magnetic beads achieved a recovery rate of over 80% (Fig. 5). Furthermore, the laborious cloning step could be omitted by seeding the cells at a density of one cell per well of a 96-well plate. These measures reduced the time required to establish human-mAb-producing hybridoma cells and increased the efficiency of antigen-specific fully human mAb generation from TC-mAb mice (Table 1).\u003c/p\u003e\n\u003cp\u003eCell fusion is a conventional method for obtaining secretory-Ab-expressing cells. It uses the plasmablasts, plasma cells, and memory B cells with high SHM rates to generate hybridoma cells that express high-affinity mAbs\u003csup\u003e22\u003c/sup\u003e. The fact that all of the hybridoma clones analyzed expressed sIgG on their surface suggests that the class switching of membrane-bound and secretory forms of IgG\u003csup\u003e23,24\u003c/sup\u003e was potentially attenuated by cell fusion with myeloma cells. To verify this notion, we also isolated hybridoma cells from a pool of fused cells originating from immunized TC-mAb mice using anti-human IgG beads. This led to the recovery of 47.4%\u0026ndash;55.6% of the cells (Supplementary Table 1), suggesting that the cell fusion process enabled the production of a large number of sIgG-expressing cells.\u003c/p\u003e\n\u003cp\u003eThe switching machinery of the membrane and secretory forms of IgG is regulated by alternative splicing during B cell differentiation. Moreover, the heterogenous nuclear RNA-binding protein LL (hnRNPLL) is specifically induced in terminally differentiated lymphocytes, including plasma cells\u003csup\u003e25\u003c/sup\u003e. Therefore, the mechanism of alternative splicing regulation by cell fusion can be investigated in plasma cells by tracing changes in the expression of proteins such as hnRNPLL when the plasma cells (CD138\u003csup\u003e+\u003c/sup\u003eTACI\u003csup\u003e+\u003c/sup\u003e lymphocytes\u003csup\u003e26\u003c/sup\u003e) are fused with myeloma cells. Such an analysis allowed us to determine whether all B cells that fused with myeloma cells expressed sIgG on their surface. As a result, the real recovery rate of antigen-specific B cells produced in each immunized TC-mAb mouse could be calculated. Whether the re-expression of sIgG in hybridoma cells occurs in the Ig gene of wild-type mice is currently unknown. If mouse sIgG re-expression is also induced by cell fusion, the utility of the present results will increase dramatically.\u003c/p\u003e\n\u003cp\u003eThe workflow presented here led to the successful recovery of more than 80% of the antigen-specific-mAb-expressing hybridoma cells (Fig. 5 and Table 1). In addition, the culture period after cell fusion was limited to 2 days to increase the recovery rate while minimizing the possibility of obtaining clonal duplicates. The fact that 366 different clones were obtained from two TC-mAb mice (Fig. 2 and Table 1) indicates that this workflow yielded a sufficient number of antigen-specific hybridoma cells for the selection of therapeutic mAb candidates. The analysis of epitope distribution and mAb-coding gene sequences (Fig. 3 and 4) confirmed that the established clones were highly diverse. Furthermore, since the bead separation method has a capacity to process up to 10\u003csup\u003e9\u003c/sup\u003e cells simultaneously, all splenocytes (~ 4 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells/mouse) from the two immunized TC-mAb mice could be screened in one procedure for Abs of interest. In the present study, we were able to process 5.9 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells from two immunized TC-mAb mice simultaneously (Table 1). We estimated that without the magnetic bead selection method ~35,000 hybridoma cells would have been obtained (Table 1), all of which would require screening. The fact that we were able to reduce this number to only 500 colonies indicates that our method considerably streamlined the mAb screening process.\u003c/p\u003e\n\u003cp\u003eIn this study, all hybridoma cells derived from the two TC-mAb mice expressed sIgG, indicating that they could be effectively screened on this basis and their antigen-reactivity. Thus, our method is applicable to the screening of cross-reactive Abs, which recognize mutation-prone antigens such as the coronavirus spike proteins and homologous antigens from different animal species. Thus, the workflow developed in this study markedly facilitated the generation of fully human antigen-specific Abs from TC-mAb mice via hybridoma technology. This method is expected to markedly speed up the development of therapeutic Ab candidates, which are in high demand for various therapeutic applications worldwide.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Animal Care and Use Committee of Tottori University (Permit Number: 20-Y-13, 20-Y-31, 21-Y-26, 22-Y-36, and 22-Y-63). All experiments were carried out in compliance with the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations. Mice were sacrificed by cervical dislocation prior to tissue collection, and all efforts were made to minimize their suffering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntigen preparation, immunization, and cell fusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of the hACE2-Fc fusion protein and the associated vector construction methods have been previously published\u003csup\u003e21\u003c/sup\u003e. In brief, a full-length ACE2 extracellular domain coding sequence (amino acids 18\u0026ndash;740) was fused to the human IgG1-Fc via a linker with a GGGGS sequence. Mutations were introduced to prevent the activation of the human Fc from affecting the immune response when the TC-mAb mice were immunized (Supplementary Fig. 2). Next, Expi293F\u003csup\u003eTM\u003c/sup\u003e cells (Thermo Fisher Scientific, Waltham, MA, USA) were transfected with the hACE2-Fc expression vector and cultured for 5 days. The culture supernatant was then harvested and applied to a Protein G Sepharose FF column (Cytiva, Tokyo, Japan) to purify the recombinant protein. The denatured protein was analyzed by SDS-PAGE (Supplementary Fig. 3). Two TC-mAb mice were then immunized with the hACE2-Fc recombinant protein (60 \u0026mu;g per mouse) as previously described\u003csup\u003e7\u003c/sup\u003e. The protocol used for the fusion of lymphocytes to myeloma cells (P3X63Ag8.653, ATCC CRL-1580, Manassas, VA, USA) to generate hybridomas has also been previously published\u003csup\u003e7,8\u003c/sup\u003e. The mice used in this study were kept in a designated animal facility, under specific pathogen-free conditions, in a controlled ambient temperature environment with a 12 h light/dark cycle. Mice underwent isoflurane-induced anesthesia for all blood draws and other sampling. All animal experiments were approved by the Animal Care and Use Committee of Tottori University and conducted in strict accordance with ARRIVE guidelines.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003ePreparation of biotinylated hACE2-Fc\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe EZ-Link Sulfo-NHS-LC-Biotin kit (Thermo Fisher Scientific) was used to biotinylate hACE2-Fc, according to the manufacturer\u0026rsquo;s protocol. Briefly, 4 \u0026mu;l of 10 mM Sulfo-NHS-LC-LC-biotin (Thermo Fisher Scientific) solubilized in distilled water were added to 3 ml of 1 mg/ml hACE2-Fc in phosphate-buffered saline (PBS) (Nacalai Tesque, Kyoto, Japan). After incubation at room temperature for 30 min, 300 \u0026mu;l of 1M Tris-HCl (pH 8.0) (Merck, Billerica, MA, USA) was added to stop the reaction. The biotinylated sample was applied to an ultracentrifuge column (Amicon Ultra, 30 kDa, Merck Millipore) and concentrated by replacing the buffer with PBS. The molar ratio of biotin to hACE2-Fc was estimated as 4.6 using the biotin quantification kit containing 4\u0026apos;-hydroxyazobenzene-2-carboxylic acid (Thermo Fisher Scientific).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eSeparation of antigen-specific-mAb-expressing hybridoma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 2 days of cell culture in 1/3 HAT medium (1:2 HAT to hypoxanthine-thymidine (HT) medium), the cells were harvested and incubated with StAv-magnetic beads (BD IMag\u0026trade; Streptavidin Particles Plus\u0026ndash;DM, BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer\u0026rsquo;s instructions. Any cells that bound nonspecifically to the beads were removed. After incubating for 30 min in a rotator, the unbound fraction was collected using a magnetic stand. The cells were then incubated with 2.5 \u0026mu;g biotinylated hACE2-Fc or biotinylated anti-human IgG-Fc (Bethyl Laboratories, Montgomery, TX, USA) per 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells on ice for 1 h with rotation. After washing with PBS supplemented with 1% fetal bovine serum (FBS), 2 ml of StAv-magnetic beads were added to the cells, followed by another 30 min incubation at 4 \u0026deg;C with rotation. After four washes to remove nonspecifically bound cells, the remaining 4.65 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells were seeded into eleven 96-well plates (TPP Techno Plastic Products AG, Zollstrasse, Switzerland).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antibody titers were measured by ELISA as previously described\u003csup\u003e7\u003c/sup\u003e. In brief, 96-well immunoassay plates (Nunc Maxisorp, Thermo Fisher Scientific) were coated with 100 \u0026micro;l/well of antigen overnight and then blocked with PBS containing 5% skim milk (DB Biosciences, Franklin Lakes, NJ, USA) for 30 min at room temperature. After washing, 100 \u0026micro;l of each antibody sample were added to each well of an ELISA plate and incubated for 1 h at room temperature. The plates were washed again and incubated with 100 \u0026micro;l of goat anti-human IgG (H+L) cross-adsorbed secondary Ab (Abcam, Cambridge, UK) at a 10,000-fold dilution in TBS-T (Tris-buffered saline and 0.05% Tween 20) for 30 min at room temperature. The plates were washed once again and developed using 100 \u0026micro;l of \u003cem\u003eo\u003c/em\u003e-phenylenediamine dihydrochloride (Fujifilm Wako, Osaka, Japan). The reaction was stopped with 25 \u0026micro;l of 1 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (Nacalai Tesque). After the plates were allowed to develop for 10\u0026ndash;30 min, the absorbance was read at 492 nm.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eImmunocytochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293-hACE2 cells (Takara Bio Inc., Shiga, Japan) were purchased and maintained according to the manufacturer\u0026rsquo;s instructions. Cells cultured in 96-well plates were treated with 100 \u0026mu;l of hybridoma supernatant or purified mAb and incubated for 1 h on ice. After washing with ice-cold medium, 100 \u0026mu;l of goat anti-human IgG (H+L) cross-adsorbed secondary antibody, conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Abcam) and diluted 600-fold in medium, was added. After additional washing, 100 \u0026mu;l of PBS supplemented with 1% FBS was added. The samples were then analyzed on fluorescence microscope (BZ-X700; Keyence, Itasca, IL, USA) or IncuCyte S3 instrument (Sartorius, G\u0026ouml;ttingen, Germany).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultured HEK293-hACE2 cells were dissociated from the culture plate using Accutase (Innovative Cell Technologies, Inc., San Diego, CA, USA) and stained. All staining was performed on ice. The stained cells were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA). To prevent interactions between Fcg receptors and Abs, the cells were pretreated with Human Seroblock (Bio-Rad Laboratories Inc., Hercules, CA, USA) for 10 min before staining, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003eTo determine the antigen-reactivity of sIgGs expressed on the surface of the hybridoma cells, 3 \u0026mu;g of protein antigen (i.e., EpEX) were added to 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in staining buffer (PBS with 1% FBS). At the same time, 0.5 \u0026mu;l of the biotinylated anti-human Ig light chain \u0026kappa; antibody (Biolegend, San Diego, CA, USA) or 4 \u0026mu;l of the PE/Cyanine7-conjugated anti-human IgG-Fc antibody (Biolegend) was added to detect sIgG on the hybridoma cell surface. After a 60 min incubation, the cells were washed three times with staining buffer. To detect biotinylated molecules, 0.1 \u0026mu;l (0.5 \u0026mu;g) of SAv-Brilliant Violet 650 (Biolegend) was added to the cells. To detect His-tagged protein binding, 0.25 \u0026mu;l of anti-His-tag mAb conjugated to Alexa Fluor 594 was added to the cells. After a 30 min incubation, the cells were washed three times with staining buffer and analyzed on a CytoFLEX S flow cytometer (Beckman Coulter). The splenocytes were stained as previously described\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDetermination of human mAb subclasses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supernatants obtained from the wells of cultured hybridoma cells were analyzed using the rapid human antibody isotyping kit (Antagen Pharmaceuticals, Boston, MA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEpitope distribution assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe distribution of epitopes to which the hybridoma cells bound was analyzed using a competitive assay. In brief, unlabeled purified mAbs were prepared using a protein G Sepharose 4 Fast Flow column. The purified mAbs were used as the capture Abs and the biotinylated mAbs were used as the detector Abs. 96-well immunoassay plates (Nunc Maxisorp) were coated with each capture Ab at 100 ng/well, and 1 h later blocked with PBS containing 5% skim milk for 30 min at room temperature. Ab biotinylation was performed using the EZ-Link Sulfo-NHS-LC-Biotin kit according to the manufacturer\u0026rsquo;s instructions. After washing, 100 \u0026micro;l of the biotinylated antibody samples was added to the wells and incubated for 1 h at room temperature. The plates were washed again and incubated with 100 \u0026micro;l of Streptavidin-HRP conjugate (Proteintech, Tokyo, Japan), diluted 3,000-fold in TBS-T, for 30 min at room temperature. The plates were washed once again and developed using o-phenylenediamine dihydrochloride. The reaction was terminated using 1 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and after a developing step, the absorbance was read at 492 nm.\u003cbr\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in the DNA Data Bank of Japan (DDBJ) with the accession numbers: LC804973 (ACE2A008_H), LC804974 (ACE2A008_L), LC804975 (ACE2A038_H), LC804976 (ACE2A038_L), LC804977 (ACE2A077_H), LC804978 (ACE2A077_L), LC804979 (ACE2A134_H), LC804980 (ACE2A134_L).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Maki Takami for assisting with human Ab production and the generation of antigen-specific hybridoma cell lines. We also thank Dr. Hiroyuki Kugoh and Dr. Satoshi Abe at Tottori University, Kazuma Tomizuka at Tokyo University of Pharmacy and Life Sciences, and Dr. Yasuyuki Kurihara at Yokohama National University for critical discussions.\u003c/p\u003e\n\u003cp\u003eThis study was supported in part by the Japan Agency for Medical Research and Development (AMED) (Grant Numbers JP21am0101124, JP23ama121046, JP23gm1610006, JP23gm0010010, JP23am0401002, JP23gm1810008 all awarded to Y.K), the Joint Research of the Exploratory Research Center on Life and Living Systems (ExCELLS) (ExCELLS program No. 21-101 to Y.K.), and JST CREST (Grant Number JPMJCR18S4 to Y.K.). This research was partly performed at the Tottori Bio Frontier facility, managed by the Tottori prefecture, Japan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe thank Edanz (https://jp.edanz.com/ac) for editing the English text of a draft of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.S. and W.Y. planned the study and optimized the hybridoma cell separation method; K.M. and H.Takayama maintained the TC-mAb mice and collected splenocytes and lymphocytes for cell fusion; H.Tanaka. and K.H. performed cell fusion; Y.W., H.S., H. Tu, and Y.Q. performed cell culture and mAb production experiments; H.S. performed mAb purification and epitope distribution analysis; H.S. performed cell staining experiments; H.S. characterized the antigen-specific B cells from TC-mAb mice; Y.W. and H.S. contributed to the analysis and discussion of the data and wrote the manuscript, S.I., X.G., M.O., and Y.K. supervised the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.O. is a CEO, employee, and shareholder of Trans Chromosomics, Inc., and H.Tanaka, K.H., K.M., and H.Takayama are employees of Trans Chromosomics, Inc. S.I. is a CEO, employee, and shareholder of Purotech Bio Inc. The remaining authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eK\u0026ouml;hler, G., Milstein, C. Cultivos continuos de c\u0026eacute;lulas fusionadas que secretan anticuerpos de especificidad predefinida. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e256\u003c/strong\u003e, 495\u0026ndash;497 (1975).\u003c/li\u003e\n\u003cli\u003eTian, Y., Hu, D., Li, Y. \u0026amp; Yang, L. Development of therapeutic vaccines for the treatment of diseases. \u003cem\u003eMolecular Biomedicine\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1\u0026ndash;30 (2022).\u003c/li\u003e\n\u003cli\u003eBr\u0026uuml;ggemann, M. \u003cem\u003eet al.\u003c/em\u003e Human Antibody Production in Transgenic Animals. \u003cem\u003eArch Immunol Ther Exp (Warsz)\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 101\u0026ndash;108 (2015).\u003c/li\u003e\n\u003cli\u003eLonberg, N. Human antibodies from transgenic animals. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1117\u0026ndash;1125 (2005).\u003c/li\u003e\n\u003cli\u003eLee, E. C. \u003cem\u003eet al.\u003c/em\u003e Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 356\u0026ndash;363 (2014).\u003c/li\u003e\n\u003cli\u003eMurphy, A. J. \u003cem\u003eet al.\u003c/em\u003e Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 5153\u0026ndash;5158 (2014).\u003c/li\u003e\n\u003cli\u003eSatofuka, H. \u003cem\u003eet al.\u003c/em\u003e Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1\u0026ndash;15 (2022).\u003c/li\u003e\n\u003cli\u003eSatofuka, H. \u003cem\u003eet al.\u003c/em\u003e Characterization of human anti ‑ EpCAM antibodies for developing an antibody \u0026ndash; drug conjugate. \u003cem\u003eSci Rep\u003c/em\u003e 1\u0026ndash;11 (2023) doi:10.1038/s41598-023-31263-x.\u003c/li\u003e\n\u003cli\u003eFusil, F. \u003cem\u003eet al.\u003c/em\u003e A lentiviral vector allowing physiologically regulated membrane-anchored and secreted antibody expression depending on B-cell maturation status. \u003cem\u003eMolecular Therapy\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1734\u0026ndash;1747 (2015).\u003c/li\u003e\n\u003cli\u003eBrouwer, P. J. M. \u003cem\u003eet al.\u003c/em\u003e Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability. \u003cem\u003eScience (1979)\u003c/em\u003e \u003cstrong\u003e369\u003c/strong\u003e, 643\u0026ndash;650 (2020).\u003c/li\u003e\n\u003cli\u003eLiu, L. \u003cem\u003eet al.\u003c/em\u003e Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e584\u003c/strong\u003e, 450\u0026ndash;456 (2020).\u003c/li\u003e\n\u003cli\u003eHansen, J. \u003cem\u003eet al.\u003c/em\u003e Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail. \u003cem\u003eScience (1979)\u003c/em\u003e \u003cstrong\u003e369\u003c/strong\u003e, 1010\u0026ndash;1014 (2020).\u003c/li\u003e\n\u003cli\u003eCao, Y. \u003cem\u003eet al.\u003c/em\u003e Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients\u0026rsquo; B Cells. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e182\u003c/strong\u003e, 73-84.e16 (2020).\u003c/li\u003e\n\u003cli\u003eKeating, S. M. \u003cem\u003eet al.\u003c/em\u003e Generation of recombinant hyperimmune globulins from diverse B-cell repertoires. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 989\u0026ndash;999 (2021).\u003c/li\u003e\n\u003cli\u003ePedrioli, A. \u0026amp; Oxenius, A. Single B cell technologies for monoclonal antibody discovery. \u003cem\u003eTrends Immunol\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 1143\u0026ndash;1158 (2021).\u003c/li\u003e\n\u003cli\u003eOnodera, T. \u003cem\u003eet al.\u003c/em\u003e A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site. \u003cem\u003eImmunity\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 2385-2398.e10 (2021).\u003c/li\u003e\n\u003cli\u003eAkkaya, M., Kwak, K. \u0026amp; Pierce, S. K. B cell memory: building two walls of protection against pathogens. \u003cem\u003eNat Rev Immunol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 229\u0026ndash;238 (2020).\u003c/li\u003e\n\u003cli\u003eNojima, T. \u003cem\u003eet al.\u003c/em\u003e In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, (2011).\u003c/li\u003e\n\u003cli\u003eShinnakasu, R. \u003cem\u003eet al.\u003c/em\u003e Regulated selection of germinal-center cells into the memory B cell compartment. \u003cem\u003eNat Immunol\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 861\u0026ndash;869 (2016).\u003c/li\u003e\n\u003cli\u003eAkagi, S., Nakajima, C., Tanaka, Y. \u0026amp; Kurihara, Y. Flow cytometry-based method for rapid and high-throughput screening of hybridoma cells secreting monoclonal antibody. \u003cem\u003eJ Biosci Bioeng\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 464\u0026ndash;469 (2018).\u003c/li\u003e\n\u003cli\u003eWines, B. D. \u003cem\u003eet al.\u003c/em\u003e Fc engineered ACE2-Fc is a potent multifunctional agent targeting SARS-CoV2. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1\u0026ndash;15 (2022).\u003c/li\u003e\n\u003cli\u003eShinnakasu, R. \u003cem\u003eet al.\u003c/em\u003e Regulated selection of germinal-center cells into the memory B cell compartment. \u003cem\u003eNat Immunol\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 861\u0026ndash;869 (2016).\u003c/li\u003e\n\u003cli\u003eBrown, S. L. \u0026amp; Morrison, S. L. Regulation of the production of secretory and membrane immunoglobulin during lymphocyte development. \u003cem\u003eClin Immunol Immunopathol\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 155\u0026ndash;170 (1989).\u003c/li\u003e\n\u003cli\u003eLiew, P. The Longevity of the Humoral Immune Response \u0026ndash; Survival of Long-lived Plasma Cells. \u003cem\u003eAkademeia\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, ea0116 (2012).\u003c/li\u003e\n\u003cli\u003eChang, X., Li, B. \u0026amp; Rao, A. RNA-binding protein hnRNPLL regulates mRNA splicing and stability during B-cell to plasma-cell differentiation. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, E1888\u0026ndash;E1897 (2015).\u003c/li\u003e\n\u003cli\u003ePracht, K. \u003cem\u003eet al.\u003c/em\u003e A new staining protocol for detection of murine antibody-secreting plasma cell subsets by flow cytometry. \u003cem\u003eEur J Immunol\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 1389\u0026ndash;1392 (2017).\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hybridoma technology, surface immunoglobulin G, fully human monoclonal antibodies, antigen-coated magnetic bead separation ","lastPublishedDoi":"10.21203/rs.3.rs-3993293/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3993293/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The antigen-mediated B cell isolation method, based on the detection of surface IgG (sIgG), has increased the efficiency of therapeutic antibody (Ab) discovery. However, the reduction in sIgG expression on B cells during plasma cell differentiation presents challenges as it enables Ab production from only a small subset of B cells (e.g., memory B cells). The present study aimed to addressed this problem by developing a workflow to isolate human-IgG-secreting hybridoma cells produced by cell fusion, the majority of which express sIgG. We showed that our sIgG-based antigen-coated bead separation method efficiently enriched hybridoma cells expressing antigen-specific Abs with a yield of 83.5% (from the cell fusion pool) and a positive rate of 73.2%. Furthermore, because the separation could be performed after only a short (1−2-day) culture period following cell fusion, diverse hybridoma clones could be obtained, minimizing clonal selection and the incidence of duplicates. Given that the expression of membrane-bound IgG and sIgG are regulated by different splicing mechanisms, we speculate that the cell fusion step potentially attenuated the suppression of human sIgG expression. Overall, our proposed method is expected to markedly improve the efficiency of therapeutic Ab candidate production, which will have important clinical implications.","manuscriptTitle":"Developing a workflow for the isolation of hybridoma cells producing fully human antigen-specific antibodies using a surface IgG detection method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-10 18:28:33","doi":"10.21203/rs.3.rs-3993293/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-27T10:02:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-23T21:55:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150648118477388500610930852590697786157","date":"2024-06-14T18:37:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2c474044-fafc-4e79-93a1-97b2b284c106","date":"2024-04-24T10:39:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-09T06:21:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22ea769c-2783-412f-9b15-b9ef042d70e2","date":"2024-04-09T02:10:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-08T09:59:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-08T09:34:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-05T14:05:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-05T13:06:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-27T08:01:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4b591011-780a-44de-92dc-c779cb2091cc","owner":[],"postedDate":"April 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30447636,"name":"Biological sciences/Biotechnology"},{"id":30447637,"name":"Biological sciences/Biotechnology/Biologics"},{"id":30447638,"name":"Biological sciences/Biotechnology/Biologics/Antibody therapy"}],"tags":[],"updatedAt":"2024-10-07T16:12:29+00:00","versionOfRecord":{"articleIdentity":"rs-3993293","link":"https://doi.org/10.1038/s41598-024-73770-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-10-04 15:58:18","publishedOnDateReadable":"October 4th, 2024"},"versionCreatedAt":"2024-04-10 18:28:33","video":"","vorDoi":"10.1038/s41598-024-73770-5","vorDoiUrl":"https://doi.org/10.1038/s41598-024-73770-5","workflowStages":[]},"version":"v1","identity":"rs-3993293","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3993293","identity":"rs-3993293","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}