Generation of novel respiratory syncytial virus vaccine candidate antigens that can induce high levels of prefusion-specific antibodies.

Respiratory syncytial virus (RSV) infection is an acute respiratory infection caused by RSV. It occurs worldwide, and for over 50 years, several attempts have been made to research and develop vaccines to prevent RSV infection; effective preventive vaccines are eagerly awaited. The RSV fusion (F) protein, which has gained attention as a vaccine antigen, causes a dynamic structural change from the preF to postF state. Therefore, the structural changes in proteins must be regulated to produce a vaccine antigen that can efficiently induce antibodies with high virus-neutralizing activity. We successfully discovered several mutations that stabilized the antigen site Ø in the preF state, trimerized it, and improved the level of protein expression through observation and computational analysis of the RSV-F protein structure and amino acid mutation analysis of RSV strains. The four RSV-F protein mutants that resulted from the combination of these effective mutations stably conserved a wide range of preF- and trimeric preF-specific epitopes with high virus-neutralizing activity. Absorption assay using human serum revealed that mutants constructed bound to antibodies with virus-neutralizing activity that were induced by natural RSV infection, whereas they hardly bound to anti-postF antibodies without virus-neutralizing activity. Furthermore, mouse immunization demonstrated that our constructed mutants induced a high percentage of antibodies that bind to the preF-specific antigen site. These characteristics suggest that the mutants constructed can be superior vaccine antigens from the viewpoint of RSV infection prevention effect and safety.

Sar1A overexpression in Chinese hamster ovary cells and its effects on antibody productivity and secretion.

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.

Establishment of a novel cell line, CHO-MK, derived from Chinese hamster ovary tissues for biologics manufacturing.

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG1)-producing cells were generated, and the IgG1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing.

Construction of a novel kinetic model for the production process of a CVA6 VLP vaccine in CHO cells.

Bioprocess development benefits from kinetic models in many aspects, including scale-up, optimization, and process understanding. However, current models are unable to simulate the production process of a coxsackievirus A6 (CVA6) virus-like particle (VLP) vaccine using Chinese hamster ovary cell culture. In this study, a novel kinetic model was constructed, correlating (1) cell growth, death, and lysis kinetics, (2) metabolism of major metabolites, and (3) CVA6 VLP production. To construct the model, two batches of a laboratory-scale 2 L bioreactor cell culture were prepared and various pH shift strategies were applied to examine the effect of pH shift. The proposed model described the experimental data under various conditions with high accuracy and quantified the effect of pH shift. Next, cell culture performance with various pH shift timings was predicted by the calibrated model. A trade-off relationship was found between product yield and quality. Consequently, multiple objective optimization was performed by integrating desirability methodology with model simulation. Finally, the optimal operating conditions that balanced product yield and quality were predicted. In general, the proposed model improved the process understanding and enabled in silico process development of a CVA6 VLP vaccine. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00598-8.

Model-based control system design to manage process parameters in mammalian cell culture for biopharmaceutical manufacturing.

To enhance the robustness and flexibility of biopharmaceutical manufacturing, a paradigm shift toward methods of continuous processing, such as perfusion, and fundamental technologies for high-throughput process development are being actively investigated. The continuous upstream process must establish an advanced control strategy to ensure a "State of Control" before operation. Specifically, feedforward and feedback control must address the complex fluctuations that occur during the culture process and maintain critical process parameters in appropriate states. However, control system design for industry-standard mammalian cell culture processes is still often performed in a laborious trial-and-error manner. This paper provides a novel control approach in which controller specifications to obtain desired control characteristics can be determined systematically by combining a culture model with control theory. In the proposed scheme, control conditions, such as PID parameters, can be specified mechanistically based on process understanding and control requirements without qualitative decision making or specific preliminary experiments. The effectiveness of the model-based control algorithm was verified by control simulations assuming perfusion Chinese hamster ovary culture. As a tool to assist in the development of control strategies, this study will reduce the high operational workload that is a serious problem in continuous culture and facilitate the digitalization of bioprocesses.

Effects of genome instability of parental CHO cell clones on chromosome number distribution and recombinant protein production in parent-derived subclones.

Chinese hamster ovary (CHO) cells are the de facto standard host cells for biopharmaceuticals, and there is great interest in developing methods for constructing stable production cell lines. In this study, clones with a wide chromosome number distribution were selected from isolated antibody-producing strains, and subclones obtained from these clones were evaluated. The transgene copy number varied between the subclones. Even among subclones with similar copy numbers of antibody genes and maintained insertion sites, clones with different productivity were generated. Although the chromosome number distribution differed between these subclones, there was no correlation between the variability in chromosome number after cloning (genome instability) and productivity. Most of the subclones obtained from a parental strain with a wide chromosome number had the same wide chromosome number distribution as the parental strain. Less frequently, cells with less variation (remaining in one distribution) in chromosome number were isolated from cells with a wide chromosome number distribution, from which subclones with less variation in chromosome number were obtained when subcloning was performed again. These results imply that the characteristics of clones with chromosomal instability are inherited by subclones, and thus provide a better understanding of cell line stability/instability.

Enhancement of recombinant adeno-associated virus activity by improved stoichiometry and homogeneity of capsid protein assembly.

Studies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles. Capillary gel electrophoresis showed high-density particles possessing a designed DNA encapsidated in the capsid composed of (VP1 + VP2)/VP3 = 0.27, whereas low-density particles have the same DNA but with a different capsid composition of (VP1 + VP2)/VP3 = 0.31, supported by sedimentation velocity-analytical ultracentrifugation and charge detection-mass spectrometry. In vitro analysis demonstrated that the low-density particles had 8.9% higher transduction efficacy than that of the particles before fractionation. Further, based on our recent findings of VP3 clip, we created rAAV2 single amino acid variants of the transcription start methionine of VP3 (M203V) and VP3 clip (M211V). The rAAV2-M203V variant had homogeneous particles with higher (VP1+VP2)/VP3 values (0.35) and demonstrated 24.7% higher transduction efficacy compared with the wild type. This study successfully provided highly functional rAAV by the extensive fractionation from the mixture of rAAV2 full particles or by the single amino acid replacement.

Artificial induction of chromosome aneuploidy in CHO cells alters their function as host cells.

Chinese hamster ovary (CHO) cells are major host cells for biopharmaceuticals. During culture, the chromosome number of CHO cells alters spontaneously. Here, we investigated the effects of artificial changes in the chromosome number on productivity. When cell fusion between antibody-producing CHO-K1-derived cells was induced, we observed a wide range of aneuploidy that was not detected in controls. In particular, antibody productivities were high in clone-derived cell populations that retained a diverse chromosome number distribution. We also induced aneuploid cells using 3-aminobenzamide that causes chromosome non-disjunction. After induction of aneuploidy by 3-aminobenzamide, cells with an increased chromosome number were isolated, but cells with a decreased chromosome number could not be isolated. When antibody expression vectors were introduced into these isolated clones, productivity tended to increase in cells with an increased chromosome number. Further analysis was carried out by focusing on clone 5E8 with an average chromosome number of 37. When 5E8 cells were used as host, the productivity of multiple antibodies, including difficult-to-express antibodies, was improved compared with CHO-K1 cells. The copies of exogenous genes integrated into the genome were significantly increased in 5E8 cells. These findings expand the possibilities for host cell selection and contribute to the efficient construction of cell lines for recombinant protein production.

Effect of co-overexpression of the cargo receptor ERGIC-53/MCFD2 on antibody production and intracellular IgG secretion in recombinant Chinese hamster ovary cells.

Therapeutic antibodies are attractive biopharmaceuticals because of their high therapeutic effects, fewer side effects, and prolonged half-life in the blood. Chinese hamster ovary (CHO) cells are the most widely used host cell lines to produce therapeutic antibodies in industries. High-producing recombinant CHO cells can be established via overexpression of endogenous proteins. In this study, we focused on the intracellular traffic of an antibody-producing CHO cell line, CHO-HcD6. Assembled antibodies were accumulated in the endoplasmic reticulum (ER) in the cell. We hypothesized that the accumulation was due to the insufficient number of cargo receptors in the cell and focused on a cargo receptor, the ERGIC-53-MCFD2 complex, which transports expressed proteins from the ER to the Golgi apparatus. Overexpression of the cargo receptor transport was expected to improve antibody production. Exogenous ERGIC-53 and MCFD2 were transfected into CHO-HcD6 cells, and overexpressing CHO-HcD6 cells were constructed. As a result of overexpression, antibody productivity increased in batch cultivation. However, the chase assay results and immunofluorescence microscopic observations revealed intracellular IgG accumulation in the overexpressing cells. These results suggest that overexpression of cargo receptors not only promoted extracellular secretion but also enhanced the retention of intracellular antibodies.

[Research and Development of Manufacturing of Biologics-the Activities of Manufacturing Association of Biologics].

The industrial application of mammalian cells can be divided into two categories, where (1) the cells are mediators (i.e., the cell produces a desired product), or (2) the cells themselves are the product. The main application of cell-produced products is in biopharmaceuticals (biologics), and these include therapeutic enzymes, cytokines, antibodies, vaccines, and vectors for gene therapy. Among the 291 biopharmaceuticals launched in Europe and the United States by 2018, the number of products produced using mammalian cells exceeded 60%, with Chinese hamster ovary (CHO) cells being used for 131 products. The production of mammalian cells requires a comprehensive approach, including cell line development, cell culture, culture media, bioreactors, scale-up, separation and purification, process development, quality analysis and control, and research and development related to safety. In this manuscript, the activities of the "Manufacturing Technology Association of Biologics (MAB)" are introduced. MAB is a research organization composed of several companies, organizations, and academics that focuses on advanced manufacturing research for the production of biologics.

Secretion of a low-molecular-weight species of endogenous GRP94 devoid of the KDEL motif during endoplasmic reticulum stress in Chinese hamster ovary cells.

GRP94 (glucose-regulated protein 94) is a well-studied chaperone with a lysine, aspartic acid, glutamic acid and leucine (KDEL) motif at its C-terminal, which is responsible for GRP94 localization in the endoplasmic reticulum (ER). GRP94 is upregulated during ER stress to help fold unfolded proteins or direct proteins to ER-associated degradation. In a previous study, engineered GRP94 without the KDEL motif stimulated a powerful immune response in vaccine cells. In this report, we show that endogenous GRP94 is naturally secreted into the medium in a truncated form that lacks the KDEL motif in Chinese hamster ovary cells. The secretion of the truncated form of GRP94 was stimulated by the induction of ER stress. These truncations prevent GRP94 recognition by KDEL receptors and retention inside the cell. This study sheds light on a potential trafficking phenomenon during the unfolded protein response that may help understand the functional role of GRP94 as a trafficking molecule.

Production of monoclonal shark-derived immunoglobulin new antigen receptor antibodies using Chinese hamster ovary cell expression system.

Cartilaginous fishes such as sharks have adaptive immune systems based on immunoglobulins similar to those in mammals. During their evolution, cartilaginous fishes individually have acquired their adaptive immune system called immunoglobulin new antigen receptor (IgNARs). IgNARs maintain their functions in the harsh environment of shark serum, which contains a high concentration of urea to prevent water loss in seawater. Therefore, IgNARs have high structural stability, and are expected to be used as next-generation antibodies in applications different from those of conventional IgG antibodies. However, no recombinant expression system for IgNAR, which has a molecular weight of approximately 147 kDa as a dimer and multiple N-glycosylation sites, has yet been constructed. This has stalled research into IgNAR development. Here, we constructed a recombinant expression system for IgNAR using Chinese hamster ovary (CHO) cells, widely used as hosts for IgG antibody production. Using this system, IgNAR was successfully expressed and purified as a human IgG Fc fusion protein and showed antigen-binding ability. After Protein A affinity purification, followed by specific cleavage and removal of the human Fc-region, the final yield of IgNAR was 1.07 mg/L-medium. Moreover, this CHO cell expression system modified IgNAR with various N-glycans, including high-mannose and complex types. This expression system will allow us to analyze the structure, physicochemical properties, and biological functions of IgNAR. This fundamental information will advance the development of IgNARs for industrial and biotechnological applications.

Violacein improves recombinant IgG production by controlling the cell cycle of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are used as host cells for industrial monoclonal antibody (mAb) production. Cell cycle control is an effective approach to increase mAb production in the cell culture. Violacein, a purple-colored pigment produced by microorganisms, has diverse bioactive properties and has been proposed for various industrial applications. In this study, we evaluated the potency of violacein for cell cycle control and improvement of recombinant immunoglobulin G (IgG) production in CHO cells. Compared with the control, 0.9 µM violacein in a 14-day fed-batch culture increased the maximum IgG concentration by 37.6% via increasing the specific production rate and cell longevity. Cell cycle analysis showed that violacein induced G1 and G2/M phase arrest. However, the G1 arrest was observed only on day 1, while G2/M arrest lasted more than 3 days, suggesting that G2/M arrest mediated the violacein-induced enhanced IgG production. Moreover, in line with the increased protein expression, the expression levels of IgG mRNA and nutrient metabolic rates were also increased. N-Linked glycosylation and charge variant profiles were barely affected by violacein treatment. Our results indicate that violacein affects the cell cycle of CHO cells and increases IgG production without changing product quality, showing promise as a mAb production enhancer in CHO cells. The study provides insight into violacein utilization in industrial mAb manufacturing and can help develop advanced, effective mAb production technologies using CHO cell cultures.

Single-cell transcriptome analyses reveal heterogeneity in suspension cultures and clonal markers of CHO-K1 cells.

Cell-to-cell variability in cell populations arises from a combination of intrinsic factors and extrinsic factors related to the milieu. However, the heterogeneity of high cell density suspension cultures for therapeutic protein production remains unknown. Here, we illustrate the increasing heterogeneity in the cellular transcriptome of serum-free adapted CHO K1 cells during high cell density suspension culture over time without concomitant changes in the genomic sequence. Cell cycle-dependent subpopulations and cell clusters, which typically appear in other single-cell transcriptome analyses, were not found in these suspension cultures. Our results indicate that cell division changes the intracellular microenvironment and leads to cell cycle-dependent heterogeneity. Whole mitochondrial single-cell genome sequencing showed cell-to-cell mitochondrial genome variation and heteroplasmy within cells. The mitochondrial genome sequencing method developed here is potentially useful for the validation of cell clonality. The culture time-dependent increase in cellular heterogeneity observed in this study did not show any attenuation in this increasing heterogeneity. Future advances in bioengineering such as culture upscaling, prolonged culturing, and complex culture systems will be confronted with the need to assess and control cellular heterogeneity, and the method described here may prove useful for this purpose.

Establishment of fast-growing serum-free immortalised cells from Chinese hamster lung tissues for biopharmaceutical production.

Chinese hamster (Cricetulus griseus) ovary-derived Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the industrial production of recombinant therapeutics because of their ability to fold, assemble, and perform post-translational modifications, such as glycosylation, on proteins. They are also valuable for their ability to grow in serum-free suspension cultures. In this study, we established a cell line derived from lung tissue of Chinese hamsters, named Chinese hamster lung (CHL)-YN cells. The biosafety of CHL-YN cells was confirmed by in vitro sterility testing, mycoplasma detection, and reverse transcriptase assays. One of the key characteristics of CHL-YN cells was their doubling time of 8.1 h in chemically defined culture medium; thus, they proliferate much faster than conventional CHO cells and general mammalian cells. Transgenes could be introduced into CHL-YN cells with high efficiency. Finally, between 50% to > 100% of the amount of glycosylated immunoglobulin G (IgG)1 produced by CHO-K1 cells was produced by CHL-YN cells over a shorter period of time. In summary, fast-growing CHL-YN cells are a unique cell line for producing recombinant proteins.

Effect of the disulfide isomerase PDIa4 on the antibody production of Chinese hamster ovary cells.

Therapeutic monoclonal antibodies recognize and bind specific molecules on the surface of target cells, stimulating the immune system, which can attack these targeted cells. These antibodies are produced by mammalian cells, including Chinese hamster ovary (CHO) cells, because the formation of antibodies requires complicated posttranslational modifications, including peptidyl-prolyl cis/trans isomerization, disulfide bond formation, and glycosylation. Currently, it is thought that the efficient production of secretory proteins is limited by posttranslational processes. The ER is the biosynthesis site of all secreted and membrane proteins. The accumulation of unfolded proteins in the ER causes the ER stress response. During the ER stress state, various molecular chaperones are expressed to prevent proteins from the aggregate formation. The molecular chaperone involved in ER stress likely plays an essential role in the production of secretory proteins. The purpose of this study was to improve the production of monoclonal antibodies by cells. We elucidated the function of ER chaperones in the production of a monoclonal antibody. First, we quantitatively measured the mRNA expression levels of protein disulfide-isomerase family members. In CHO HcD6 cells treated with tunicamycin, the expression level of pdia4 was significantly increased. Second, we investigated the relationship between PDIa4 and antibody productivity in pdia4-knockdown cells. Both a decrease in the amount of secreted antibody and the accumulation of immature antibodies inside the cells were observed. Recombinant PDIa4 was able to refold the antibodies and Fabs. These results indicate that PDIa4 affects the production of monoclonal antibodies by catalyzing disulfide bond formation in these antibodies in CHO cells.

Low-concentration staurosporine improves recombinant antibody productivity in Chinese hamster ovary cells without inducing cell death.

Chinese hamster ovary (CHO) cells are used as host cells for biopharmaceutical production, including monoclonal antibodies (mAbs). Arresting the cell cycle with chemical compounds is an effective approach to improve biopharmaceutical productivity. In a previous study, potential new cell cycle-arresting compounds were screened from marine-derived microorganism culture extracts, and it was suggested that staurosporine might improve mAb productivity in CHO cells via cell cycle arrest. The purpose of this study was to demonstrate the effectiveness of staurosporine as a cell-cycle arresting compound to improve mAb productivity. The optimal staurosporine concentration range was initially investigated using batch cultures. Thereafter, the effects on the culture profile and mAb productivity were evaluated using fed-batch cultures. Staurosporine at concentrations ≥10 nM induced cell death, but at concentrations ≤5 nM did not. In the range of 2-4 nM, cell growth was inhibited, whereas the specific production rate (Qp) and cell longevity were improved in a dose-dependent manner. The Qp and maximum mAb concentration with 4 nM staurosporine improved by 36.3 and 5.2%, respectively, compared to those with control conditions. Cell viability post-culture without staurosporine was 40.0 ± 0.3%, whereas with 4 nM staurosporine, it was 90.1 ± 1.0%. Flow cytometric analysis indicated cell-cycle arrest at the G1/G0 phase with 4 nM staurosporine addition. The present study highlighted the efficacy of staurosporine in improving mAb production by causing cell-cycle arrest. Further research into staurosporine analogs and how to use them will lead to development of more effective industrial production technologies of biopharmaceuticals.

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kL a ratio as a direct indicator of gas stripping conditions.

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO2 ) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO2 was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the kL a values of O2 (kL a[O2 ]) and CO2 [kL a(CO2 )] in the SS bioreactors as a scale-up factor for dCO2 reduction. The kL a ratios [kL a(CO2 )/kL a(O2 )] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the kL a ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO2 trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This kL a ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The kL a ratios for the SUBs were evaluated and conditions that provided kL a ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the kL a ratio is a powerful scale-up factor useful to control dCO2 during fed-batch cultures.

Screening of new cell cycle suppressive compounds from marine-derived microorganisms in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are active pharmaceutical ingredients in antibody drugs, produced mainly using recombinant Chinese hamster ovary (CHO) cells. The regulation of recombinant CHO cell proliferation can improve the productivity of heterologous proteins. Chemical compound approaches for cell cycle regulation have the advantages of simplicity and ease of use in industrial processes. However, CHO cells have genetic and phenotypic diversity, and the effects of such compounds might depend on cell line and culture conditions. Increasing the variety of cell cycle inhibitors is a promising strategy to overcome the dependency. Marine microorganisms are a vast and largely undeveloped source of secondary metabolites with physiological activity. In this study, we focused on secondary metabolites of marine microorganisms and evaluated their effectiveness as cell cycle inhibitory compounds. Of 720 extracts from microorganisms (400 actinomycetes and 320 filamentous fungi) collected from the Okinawan Sea, we identified nine extracts that decreased the specific growth rate and increased the specific production rate without reducing cell viability. After fractionating the extracts, the components of active fractions were estimated using time-of-flight mass spectrometry analysis. Then, four compounds, including staurosporine and undecylprodigiosin were deduced to be active compounds. These compounds have been reported to exert a cell cycle inhibitory effect on mammalian cells. These compounds might serve as additives to improve mAb production in CHO cells. This study indicates that secondary metabolites of marine microorganisms are a useful source for new cell cycle inhibitory compounds that can increase mAb production in CHO cells.

Build-up functionalization of anti-EGFR × anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats.

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential.