
==== Front
Protein Sci
Protein Sci
10.1002/(ISSN)1469-896X
PRO
Protein Science : A Publication of the Protein Society
0961-8368
1469-896X
John Wiley & Sons, Inc. Hoboken, USA

10.1002/pro.5121
PRO5121
Research Article
Research Article
Engineering and physicochemical characterization of a novel, stable, symmetric bispecific antibody with dual target‐binding using a common light chain
Saito et al.
Saito Seiji https://orcid.org/0000-0002-7433-9141
1 seiji.saitou.yd@kyowakirin.com

Nakayama Makoto 2
Yamazaki Kaori 1
Miyamoto Yuya 1
Hiraishi Keiko 1
Tomioka Daisuke 1
Takagi‐Maeda Sayaka 3
Usami Katsuaki 3
Takahashi Nobuaki 4
Nara Shinji 1
Imai Eiichiro 1
1 Molecular Analysis Center, R&D Division Kyowa Kirin Co., Ltd. Tokyo Japan
2 Research Core Function Laboratories, R&D Division Kyowa Kirin Co., Ltd. Tokyo Japan
3 Modality Research Laboratories, R&D Division Kyowa Kirin Co., Ltd. Tokyo Japan
4 R&D Division Kyowa Kirin Co., Ltd. Tokyo Japan
* Correspondence
Seiji Saito, Tokyo Research Park, Kyowa Kirin Co., Ltd., Machida‐shi, Tokyo, Japan.
Email: seiji.saitou.yd@kyowakirin.com

14 9 2024
10 2024
14 9 2024
33 10 10.1002/pro.v33.10 e512107 7 2024
28 11 2023
09 7 2024
© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Abstract

Bispecific antibodies (BsAbs) have emerged as a major class of antibody therapeutics owing to their substantial potential in disease treatment. While several BsAbs have been successfully approved in recent years, ongoing development efforts continue to focus on optimizing various BsAbs tailored to particular antigens and action mechanisms, aiming to achieve favorable physicochemical properties. BsAbs generally encounter challenges due to their unfavorable physicochemical characteristics and poor manufacturing efficiencies, highlighting the need for optimization to achieve reliable productivity and developability. Herein, we describe the development of a novel symmetric BsAb, REGULGENT™ (N‐term/C‐term), comprising two Fab domains, using a common light chain. The heavy chain fragment encoded two antigen‐binding determinants in one chain. The design and production of REGULGENT™ (N‐term/C‐term) are simple owing to the use of the same light chain, which does not induce heavy and light chain mispairing, frequently observed with the asymmetric BsAb format. REGULGENT™ (N‐term/C‐term) exhibited high expression and low aggregation characteristics during cell culture and stress treatment under low pH conditions. Differential scanning calorimetric data indicated that REGULGENT™ molecules had high conformational stability, similar to that of stabilized monoclonal antibodies. Surface plasmon resonance data showed that REGULGENT™ (N‐term/C‐term) could bind to two antigens simultaneously and exhibited a high affinity for two antigens. In summary, the symmetric BsAb format of REGULGENT™ confers its desirable IgG‐like physicochemical properties, thus making it an excellent candidate for commercial development. The findings demonstrate a novel BsAb with substantial development potential for clinical applications.

acid‐induced aggregate
aggregation
antigen affinity
bispecific antibody
DSC
NS‐EM
productivity
SPR
stability
source-schema-version-number2.0
cover-dateOctober 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:14.09.2024
Saito S , Nakayama M , Yamazaki K , Miyamoto Y , Hiraishi K , Tomioka D , et al. Engineering and physicochemical characterization of a novel, stable, symmetric bispecific antibody with dual target‐binding using a common light chain. Protein Science. 2024;33 (10 ):e5121. 10.1002/pro.5121

Seiji Saito and Makoto Nakayama share first authorship.

Review Editor: Aitziber L. Cortajarena
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pmc1 INTRODUCTION

The idea of bispecific antibodies (BsAbs) was first proposed by Nisonoff and his colleagues more than 60 years ago (Nisonoff et al., 1960). BsAbs can simultaneously bind two different antigens or two different epitopes of the same antigen. BsAbs are superior in selectivity or functionality compared to a single mAb or a mixture of two different monoclonal antibodies (mAbs), which subsequently enhances the safety and antitumor efficacy of the therapy (Godar et al., 2018). More than 100 types of BsAbs have been designed, and more than 20 types have reached the clinical development stage (Huang et al., 2020; Lim, 2020). Additionally, BsAb technology platforms have been established for development. Currently, seven molecules have successfully been launched into the market (Kang et al., 2022).

However, BsAb structures are more complex and diverse than mAbs. BsAbs are typically difficult to express and are prone to aggregation, fragmentation, and various charge variants during cell culture (Qin et al., 2022; Sinharoy et al., 2020; Spiess et al., 2015). Despite the diverse range of design strategies, only a few formats of BsAbs have advanced beyond the clinical phase of Phase 1 (Gong et al., 2017). Therefore, for novel commercial BsAb format development, elements of the biomanufacturing process must be considered for clinically sustainable implementation.

With respect to heterogeneity, BsAbs are hybrid molecules that are artificially engineered. Heterogeneity problems, such as incorrect random pairing between the heavy and light chains, have been reported (Brinkmann & Kontermann, 2017; Wang et al., 2019). Aggregation also represents an important factor in BsAb development that affects immunogenicity and the manufacturing process, including production, purification, formulation, and storage which also represents risk steps regarding aggregation formation (Moussa et al., 2016). Some BsAb formats produce many aggregates during production (Brinkmann & Kontermann, 2017; Cao et al., 2018; Swope et al., 2020). Therefore, reducing BsAbs heterogeneity and aggregation is vital for therapeutic BsAbs development.

Varied and easy BsAb format selection for target binding is also critical in BsAbs therapeutics. BsAbs are used to bind two different antigens or two different epitopes of one antigen (Huang et al., 2020; Labrijn et al., 2019). Therefore, varied and simple BsAb format selection appropriate to optimal target binding is also important for therapeutic application.

In this study, we describe a new, symmetric BsAb design termed REGULGENT™ (N‐term/C‐term), which uses a common light chain and heavy chain‐like mAbs. Through the engineering and development of REGULGENT™ molecules, we demonstrated that both the N‐ and C‐term of REGULGENT™ (N‐term/C‐term) exhibited favorable drug‐like properties and manufacturing efficiency for commercial development. Depending on the mechanism of action (MOA) and antigen target, the REGULGENT™ format can select the N‐term, C‐term, and reversed Fab domain order format with reliable manufacturability and stability.

2 RESULTS

2.1 Design of new, symmetric, stabilized BsAbs

We used antitumor necrosis factor‐related apoptosis‐inducing ligand receptor 2 (TRAIL‐R2) and prostate‐specific membrane antigen (PSMA) BsAbs as a model. The REGULGENT™ protein has binding sites for two membrane proteins, the human antigen TRAIL‐R2(T) and the human antigen PSMA(P). TRAIL‐R2, also known as death receptor 5, has a high affinity to TRAIL and can trigger apoptosis through TRAIL‐R2‐induced cross‐linking in tumor cells (Snajdauf et al., 2021). PSMA is overexpressed on the membrane of aggressive forms of prostate cancer (Boinapally et al., 2021). To construct the anti‐T/P REGULGENT™ (N‐term) molecule of the heavy chain, the variable heavy chain 1 (VH1) sequences against antigen T were connected using an IgG4 constant heavy chain 1 (CH1) linker (98 amino acids in length) and were fused in tandem with the variable heavy chain 2 (VH2) sequences against antigen P (Figure 1a,b, Table 1). The VH1‐Linker‐VH2 sequences were connected to CH1‐Hinge‐CH2‐CH3 sequences (Figure 1b). For the light chain, we used common sequences for antigen T and P (Figure 1b,c). The variable light chain (VL) sequences were fused with a constant light chain. The anti‐T/P REGULGENT™ (N‐term) Fab domain was tandemly connected with the N‐terminal of the Fc region. The mAb with its Fab domain located at the N‐terminus of the heavy chain, distal from the Fc region, targeted the anti‐T/P of T, whereas the mAb with its Fab domain situated proximal to the Fc region targeted the anti‐T/P of P. The anti‐T/P REGULGENT™ (C‐term) of the variable domain 1 (VD1) Fab domains (anti‐T) was connected with N‐term and variable domain 1 (VD2) Fab domains (anti‐P) were connected with the C‐term of the heavy chain (Figure 1c). The Fc region is a stable IgG4 subclass (S228P/S235E/R409K), which was described by Namisaki et al. (2020). The IgG4 mAb was stable under low pH conditions with no ADCC/CDC activity. The cotransfection of mammalian cells using expression vectors encoding two chains (heavy and light) of each REGULGENT™ protein led to the secretion of a single species of an IgG‐like molecule. The purity after the purification of protein A and the corresponding molecular weight were confirmed via SDS‐PAGE (Figure S1A). The high‐molecular‐weight species (HMWS) and low‐molecular‐weight species (LMWS) ratios of the REGULGENT™ protein were determined with ultrahigh pressure size exclusion chromatography (UHP‐SEC) (Figure S1B). The precise molecular weight of REGULGENT™ BsAbs was determined using LC–MS. The nonreduced and reduced deglycosylated REGULEGENT™ masses matched the expected mass within the method error (Figure S1C). Ion exchange chromatography analysis was also performed to identify the heterogeneity of the REGULGENT™ proteins (Figure S2). No major impurities resulting from the mispairing of the heavy chain/light chain in either the N‐terminal or C‐terminal format were observed, suggesting that the REGULGENT™ protein format exhibited low heterogeneity, predominantly manifesting as homogenous BsAb.

FIGURE 1 Design of the symmetric stabilized bispecific antibody. (a) Detailed schematic diagram of a REGULGENT™ (N‐term). (b) Heavy and light chain DNA domain construct designs of a REGULGENT™ (N‐term/C‐term). (c) Construct design of the antibodies used in this experiment. All antibodies shared the variable regions of an anti‐T or P antibody and the light chain constant region of the Ck isotype. mAb, monoclonal antibody.

TABLE 1 REGULGENT™ protein identities.

REGULGENT™ protein name	Heavy chain linker sequence	CH1‐hinge‐CH2‐CH3	
P/T REGULGENT™ (N‐term)	IgG4 CH1 (98a.a.) *ASTKGP···DKRV	IgG4 (S228P/S235E/R409K)	
T/P REGULGENT™ (N‐term)	IgG4 CH1 (98a.a.) *ASTKGP···DKRV	IgG4 (S228P/S235E/R409K)	
P/T REGULGENT™ (C‐term)	–	IgG4 (S228P/S235E/R409K)	
T/P REGULGENT™ (C‐term)	–	IgG4 (S228P/S235E/R409K)	
*ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTKTYTCNVDHKPSNTKVDKRV	
	VH1–VH2 linker sequence	VL1–VL2 linker sequence	
DVD‐Ig	ASTKGPSVFPLAP (13a.a)	TVAAPSVFIFPP (12a.a.)	

2.2 Productivity of the REGULGENT™ BsAbs

The expression titers of anti‐T/P and anti‐P/T REGULGENT™ (N‐term/C‐term) antibodies, which were transiently transfected into the Chinese hamster ovary (CHO) cell line FreeStyle™ CHO‐S Cells, were analyzed using the amount of protein eluted from the protein A column (Figure 2a). Anti‐T and anti‐P mAbs, whose Fc region is a stable IgG4 subclass (S228P/S235E/R409K), were produced as parental mAbs control. The expression titer of anti‐T/P and anti‐P/T REGULGENT™ (N‐term/C‐term) on day 8 was between 250 and 400 mg/L (Figure 2a). Although transient expression productivity was lower than that of the parental mAb (60%–90%), the productivity of each of the four constructs was similar to the high levels reported previously for various IgG1 mAbs (Zhong et al., 2019).

FIGURE 2 Productivity of the monoclonal and bispecific antibody. (a) Transient expression productivity (mg/L) on day 8 of the monoclonal antibody, REGULGENT™ (N‐term/C‐term) with Expi CHO‐S. (b) Productivity of anti‐P monoclonal antibody and anti‐T/P REGULGENT™ (N‐term) with a stable Chinese hamster ovary (CHO) cell line productivity (mg/L). Productivity was determined by Protein A chromatography. Data are presented as mean ± standard deviation (SD) and all experiments were performed in triplicate.

To determine the manufacturing feasibility of REGULGENT™ for commercial development, we established stably transfected CHO cells, the most commonly used mammalian cell for commercial antibody production. For anti‐T/P REGULGENT™ (N‐term) antibodies and anti‐P mAb, both Fc regions were in the same stable IgG4 subclass (S228P/S235E/R409K) (Namisaki et al., 2020). The cell culture was analyzed every 2–3 days, and the productivity of the stable cell lines was evaluated using the amount of protein eluted from the protein A affinity column (Figure 2b). The anti‐T/P REGULGENT™ (N‐term) antibody titer reached 1.5 g/L on day 14 and the same level of the anti‐P parental mAb, which indicated a high expression level, similar to mAb.

2.3 REGULGENT™ BsAbs aggregate formation in the cell culture

Next, we evaluated the aggregation level of the REGULGENT™‐transient‐producing CHO cell line. An aggregation analysis was performed using UHP‐SEC. The transient expression cells exhibited that the anti‐T/P and anti‐P/T REGULGENT™ (N‐term/C‐term) aggregation level on day 8 was <5% of HMWS, which is comparable to a stable IgG4 mAb (Namisaki et al., 2020). We also produced anti‐T/P dual variable domain immunoglobulin (DVD‐Ig), which is representative of the symmetric BsAbs for comparison. The CH1 amino acid length of DVD‐Ig of the VH1‐VH2 linker was 13 and that of the VL1‐VL2 linker was 12, the same sequence reported in Table 1 (Jakob et al., 2013). The DVD‐Ig aggregation levels were notably higher at 25% (Figure 3a). The LMWS of the parental mAb and REGULGENT™ (N‐term/C‐term) were <1% (Figure 3b). Additionally, we evaluated the stable CHO cell line expression product on day 14 of anti‐T/P REGULGENT™ (N‐term), which showed a low level of aggregation (<10% HMWS), comparable to that of the anti‐P parental mAb (Figure 3c). In summary, these data indicate that REGULGENT™ exhibits favorable manufacturability properties for potential drug development.

FIGURE 3 High‐molecular‐weight species (HMWS) and low‐molecular‐weight species (LMWS) formation of REGULGENT™ Bispecific antibodies (BsAbs) in cell culture. (a) HMWS formation of monoclonal antibody, REGULGENT™ (N‐term/C‐term) and dual variable domain immunoglobulin (DVD‐Ig) with transient expression Expi Chinese hamster ovary (CHO)‐S cell on day 8 in the cell culture. (b) LMWS formation of the transient expression cell on day 8 in the cell culture. (c) HMWS and LMWS formation of anti‐P monoclonal antibody and anti‐T/P REGULGENT™ (N‐term) with a stable CHO cell line on day 14 in the cell culture. Data are presented as mean ± SD and all experiments were performed in triplicate.

2.4 REGULGENT™ BsAbs aggregate formation under low pH stress

The stability of the REGULGENT™ and parental mAbs at low pH stress was investigated using UHP‐SEC. The proportion of HMWS increase of both anti‐T and anti‐P mAbs under low pH stress was <2% compared with the initial levels (Figure 4). This was consistent with previously reported stabilized IgG4 data (Namisaki et al., 2020). The anti‐T/P and anti‐P/T REGULGENT™ (N‐term/C‐term) showed <5% of HMWS formation, following incubation at the most severe conditions (pH 3.5, 37°C for 60 min). These data indicate that the REGULGENT™ (N‐term/C‐term) form is stable under low pH stress, and the aggregation formation level is the same as that of the stabilized IgG4 mAb format, which has the same stability as stable IgG1 subclass mAb (Namisaki et al., 2020).

FIGURE 4 High‐molecular‐weight species (HMWS) percentage increase in the monoclonal antibody and REGULGENT™ (N‐term/C‐term) antibodies under conditions of low pH stress, as determined by ultrahigh pressure size exclusion chromatography. Antibodies were incubated at pH 3.5 for 60 min at 25°C and 10 or 60 min at 37°C. Data are presented as mean ± SD, and all experiments were performed in triplicate.

2.5 Long‐term stability of REGULGENT™ antibodies

Next, we evaluated the long‐term stability of REGULGENT™ and the parental mAbs, at various temperatures at a concentration of 1 mg/mL, after up to 1 month of incubation at various temperatures (4, 25, and 40°C) (Figure 5). Apart from the standard storage conditions at 4°C, the product was stored for 1 month at 25°C (accelerated conditions) and 40°C (stressed conditions). Relative comparisons with stable mAbs were made at 1‐, 2‐, and 4‐week intervals during this period. All REGULGENT™ (N‐term and C‐term) formats showed a <5% increase in HMWS under every condition (Figure 5a). LMWS of the parental mAbs and REGULGENT™ format showed a <2% increase in LMWS (Figure 5b) during the 1‐, 2‐, and 4‐week intervals for relative comparison with stable mAbs. These data indicate that REGULGENT™ is stable in long‐term storage and exhibits favorable drug‐like properties for commercial development.

FIGURE 5 Long‐term (1 month) stability analysis of REGULGENT™ (N‐term/C‐term) antibodies at various temperatures: 4, 25, and 37°C in phosphate‐buffered saline (PBS) buffer with a 1 mg/mL concentration. (a) High‐molecular‐weight species (HMWS) increase with 1‐month storage as determined by ultrahigh pressure size exclusion chromatography (UHP‐SEC). (b) Low‐molecular‐weight species (LMWS) increase with 1‐month storage as determined by UHP‐SEC.

2.6 Thermodynamic stability of REGULGENT™ antibodies

The mechanistic basis for stability against low pH stress was investigated by performing differential scanning calorimetry (DSC) analysis for REGULGENT™ in neutral (pH 7.4) as well as low (pH 3.5) pH buffer (Figure 6). We discovered that the unfolding onset temperatures (T onset) of REGULGENT™ (N‐term/C‐term) were comparable for stable IgG4 mAb at pH 7.4 and 3.5 (Figure 6b). The first unfolding transition temperatures (i.e., T m1) of all four REGULGENT™ (N‐term/C‐term) were similar to those of stable parental mAbs at both pHs (pH 7.4 and 3.5), which implies that the CH2 domain conformational stabilities of all four formats were similar to those of stable mAbs. In relation to the second unfolding transition temperatures (i.e., T m2) of the REGULGENT™ (N‐term/C‐term), some REGULGENT™ showed two peaks (i.e., T m2 and T m2‐2), which were assumed to be two different Fab regions (VD1 and VD2). The T m2 of the REGULGENT™ (C‐term) was higher than that of the REGULGENT™ (N‐term) at both pH 7.4 and 3.5. In particular, anti‐P/T REGULGENT™ (N‐term), whose anti‐P Fab domain exists in the outer domain, showed a decreased T m2 of the Fab region peaks at pH 3.5. These data indicate that REGULGENT™ (N‐term/C‐term) exhibited different T m values compared with the Fab region, and it depends on the format (N‐term/C‐term) and order of the VD region. The T m3 values of the CH3 domain with all four REGULGENT™ (N‐term/C‐term) were similar at pH 7.4 and 3.5. Additionally, the T m3 values of the CH3 domain of all REGULGENT™ (N‐term/C‐term) and stable mAb were similar at both pH 7.4 and 3.5 (except for anti‐P mAb of pH 3.5).

FIGURE 6 Differential scanning calorimetry (DSC) and nano differential scanning fluorimetry (nanoDSF) analysis of the thermodynamic stability of antibody domains in various pH buffers of phosphate‐buffered saline (PBS). (a) DSC charts of monoclonal antibodies (mAbs) and REGULGENT™ bispecific antibodies (BsAbs) at two different pH conditions (pH 3.5/7.4). (b) The melting temperature (T m, T m1, T m2, and T m3) and onset temperature (T onset) of REGULGENT™ BsAbs were determined from the DSC analysis. (c) Temperature of aggregation onset (T agg) value of mAbs and REGULGENT™ BsAbs using nanoDSF.

We also analyzed the temperature of aggregation onset (T agg) of REGULGENT™ using nano differential scanning fluorimetry (nanoDSF) (Figure 6c). NanoDSF can recognize protein aggregation by detecting the back‐reflection intensity of a light beam that passes the sample twice. Compared with anti‐T/P mAbs, the REGULGENT™ (N‐term/C‐term) T agg value was lower by 2–4°C; however, all four anti‐T/P, P/T REGULGENT™ (N‐term/C‐term) of T agg values were similar at ca. 73°C.

2.7 Antigen‐ and FcRn‐binding and cellular activity of the REGULGENT™ antibody variants

First, we examined the antigen‐binding affinity of anti‐T/P and anti‐P/T REGULGENT™ (N‐term/C‐term) to each antigen (T, P) using surface plasmon resonance (SPR) (Figure 7a). The SPR experiments showed that the VD1 (outer domain against the antigen) of anti‐T/P and anti‐P/T REGULGENT™ (N‐term) had a comparable affinity to each mAb. The VD2 (inner domain) affinity of anti‐T/P and anti‐P/T REGULGENT™ (N‐term) against both antigens was lower compared to each mAb (5–10 times). Next, we examined the antigen‐binding affinity of anti‐T/P and anti‐P/T REGULGENT™ (C‐term) and found that the affinity of the N‐term domain against the antigens was the same as that of each mAb. The C‐term domain affinity was lower compared to that of each parental mAb (2–3 times).

FIGURE 7 Binding activity of bispecific antibodies (BsAbs) (a) Binding kinetics parameters of BsAbs to two antigens, prostate‐specific membrane antigen (PSMA) and tumor necrosis factor‐related apoptosis‐inducing ligand receptor 2 (TRAIL‐R2). Each experiment was repeated twice and consisted of three replicates, and the results shown are geometric means ± SD. (b) REGULGENT™ BsAbs tetravalent dual‐specific binding ability was assessed using the Biacore™ system. REGULGENT™ BsAbs had the ability to simultaneously bind two antigens (TRAIL‐R2 and PSMA). (c) Binding affinity of REGULGENT™ BsAbs with human FcRn at pH 6.0 and 7.4 using the Biacore™ system. Each experiment was repeated three times, and the results shown are geometric means ± SD. None of the antibodies bound to the human FcRn at pH 7.4 (N.B., no binding). (d) Effects of REGULGENT™ (N‐term) on the viability of Expi293F cells and PSMA‐expressing Expi293F cells. Expi293F cells and PSMA‐expressing Expi293F cells were incubated with the indicated concentrations of anti‐T/P REGULGENT™ (N‐term) and anti‐DNP/DNP REGULGENT™ (N‐term). After a 24‐h incubation, the viability of cells was determined with the WST‐8 assay‐based Cell Counting Kit.

To confirm if the REGULGENT™ (N‐term/C‐term) can bind two antigens simultaneously, we conducted simultaneous binding experiments using SPR. The REGULGENT™ (N‐term) and REGULGENT™ (C‐term) were captured onto the sensor, binding to the antigen TRAIL‐R2. When the antigen PSMA was injected into the sensor with the REGULGENT/TRAIL‐R2 complex, the binding response was observed (Figure 7b). These results indicated that the REGULGENT can bind both antigen TRAIL‐R2 and PSMA simultaneously.

Furthermore, the SPR system was used to analyze the binding profiles of REGULGENT™ to a neonatal receptor (FcRn), which is a crucial receptor for in vivo pharmacokinetics (Figure 7c). The REGULGENT™ (N‐term) and REGULGENT™ (C‐term) showed the same order and similar affinity to FcRn at pH 6.0 as that of mAbs. Neither the REGULGENT™ (N‐term/C‐term) nor parental mAbs bound to FcRn at pH 7.4. These results indicate that REGULGENT™ exhibits consistent pharmacokinetics, likely comparable to those of mAbs in terms of their potential efficiency.

FIGURE 8 Structure of REGULGENT™ (N‐term/C‐term) bispecific antibodies (BsAbs) using negative stain electron microscopy (NS‐EM). mAb, monoclonal antibody.

Additionally, we analyzed the functionality of anti‐T/P REGULGENT™ (N‐term) and control REGULGENT™ (N‐term) (anti‐2,4‐dinitrophenol [DNP]/DNP REGULGENT™ [N‐term]) by performing a cellular viability assay. We used Expi293F cells and HEK293 derivative (Sun et al., 2023), which endogenously expressed TRAILR‐2 (Trebing et al., 2014) and transiently expressed PSMA (Expi293F + PSMA), for the assay. The anti‐T/P REGULGENT™ (N‐term) formats and control REGULGENT™ (N‐term) did not show any cytotoxic effects against Expi293F cells. However, anti‐T/P REGULGENT™ (N‐term) reduced the viability of Expi293F cells that transiently expressed PSMA, whereas the control REGULGENT™ (N‐term) did not (Figure 7d).

2.8 Structural visualization of REGULGENT™ antibodies via negative stain electron microscopy

We conducted the structural visualization of anti‐T parental mAb and anti‐T/P REGULGENT™ (N‐term/C‐term) using negative stain electron microscopy (NS‐EM). The anti‐T/P REGULGENT™ (N‐term) of the two Fab regions within each arm were bent at an angle of at least 90° relative to each other. The Fc and Fab arms have been labeled, thereby indicating the distinction between Arm 1 (obtuse Angle 2) and Arm 2 (acute Angle 2). Regarding the REGULGENT™ (C‐term), only a few class averages revealed a well‐defined structure. Most averages consisted of domains that were blurry or of low density, suggesting that high flexibility resulted in averaging out these domains. Some of the classes appeared to consist of five substructures approximately the size and shape of the Fc or Fab region. There could be a central Fc region, with two Fab regions extending at various angles from one end and two Fab regions extending at various angles from the opposite end. These data reveal the structure characterization of REGULGENT™ (N‐term) and REGULGENT™ (C‐term) and the difference to the stable IgG4 subclass mAb. The visualization structure helps the understanding of the Fab arm domain angle, flexibility, and Fab‐Fab distance of REGULGENT™ (N‐term/C‐term) BsAb format that makes it possible to consider the advantage of this structure and also selection of N‐term or C‐term of REGULGENT™ and VD order fit for target MOA (Figure 8).

3 DISCUSSION

BsAbs have unique characteristics, such as binding two different targets or the same target with different epitopes, facilitating their use by various MOAs such as T‐cell activation, immuno‐oncology, agonistic, and antigen clearance (Huang et al., 2020; Wei et al., 2022). Generally, the BsAb format is selected considering the MOA and antigen target for therapy (Yu et al., 2020). A wide variety of BsAb format selections with good manufacturing is important and required for therapeutic antibody development. Challenges still exist, especially in manufacturing, because of the more complex format than mAbs (Wang et al., 2019).

The major challenge with using the IgG‐like bispecific format is the mispairing of heavy chains and light chains (Wang et al., 2019) and the formation of aggregates (>40% in some cases) (Sinharoy et al., 2020). Regarding the heavy chain/light chain mispairing, the REGULGENT™ antibodies format uses the same light chain for VDs 1 and 2; therefore, only two expression cassettes are required for antibody generation, as for mAbs. All four REGULGENT™ formats, including N‐term and C‐term and the reversed VDs, require only two DNA constructs for the heavy and light chain transfections in the manufacturing process. As for the point in terms of manufacturing, the levels of aggregation and expression are the critical points for the successful production of the therapeutic BsAbs (Xu et al., 2022). All four REGULGENT™ formats, including the N‐term and C‐term and the reversed VDs of the aggregation, exhibited low levels of aggregation during cell culture and low pH treatment where the antibody is exposed during protein A elution and viral inactivation in the manufacturing process (Figures 3 and 4) (Mazzer et al., 2015). We also assessed the protein concentration of both the protein‐A‐purified and low‐pH‐treated samples before and after centrifugation. Our results indicated no changes in the total protein content before and after centrifugation (Figure S3A). To further evaluate samples larger than 100 nm, we performed HIAC (Figure S3B). Results demonstrated that the REGULGENT™ format exhibited particle counts and size distribution comparable to those of the stabilized mAb.

The expression level of the desired BsAbs is often noted with standard mAbs (Rios & Lundberg, 2020). Our REGULGENT™ format showed the desired expression level with stable cell line expression at ca. 1 g/L, similar to that of mAbs. Like mAbs, the optimization of the gene sequence, heavy chain/light chain vector ratio, selection of high productivity clones, and optimization of the culture and purification method, could improve BsAb expression (Zhang et al., 2022).

To examine the high stability of the REGULGENT™ format, we analyzed the conformational stability of the REGULGENT™ format using DSC and nanoDSF. We observed that some T m and T agg values decreased with REGULGENT™ format compared with parental mAbs. For example, the T m2 (assumed to be the Fab domain) value of REGULGENT™ (N‐term) at pH 7.4 and 3.5 (Figure 6). Although the REGULGENT™ (N‐term/C‐term) formats have lower T m2 and T agg values compared with the parental mAb, the long‐term stability and low pH stress data (Figures 4 and 5) showed that the HMWS formation of the REGULGENT™ (N‐term/C‐term) was <5%, similar to that of stable mAbs. Furthermore, Bailly et al. (2020) reported that T onset and T m1 (assumed to be the CH2 domain) are important parameters for predicting the stability of the antibody. REGULGENT™ (N‐term/C‐term) had a similar T onset value and T m1 value compared to those of stable mAbs. This may be the reason that all four REGULGENT™ (N‐term/C‐term) formats have the same high stability as that of stable IgG4 mAbs under the stress conditions employed in this study. Additionally, linker length and composition affect the stability of the aggregation format (Brinkmann & Kontermann, 2017; Le Gall et al., 2004; Schanzer et al., 2011). Therefore, the adjustment of linker length and composition in the REGULGENT™ (N‐term/C‐term) format is an area for future study.

In relation to PK, we analyzed the FcRn binding of REGULGENT™ and found that REGULGENT™ had the same order and a similar FcRn binding affinity as that of monoclonal IgG4. Binding to FcRn in vitro is a prerequisite for PK measurements; therefore, further research is necessary to fully comprehend the PK profiles of REGULGENT™ molecules with reliable PK properties.

The REGULGENT™ (N‐term/C‐term) can bind two antigens simultaneously (Figure 7b). However, both the VD2 (inner domain) of REGULGENT™ (N‐term) and VD2 (C‐term domain) of REGULGENT™ (C‐term) had decreased binding affinity compared with the parent mAb (Figure 7a). In contrast, we also observed that the antigen‐binding affinity recovered to the level of each parent mAb by reversing the VD1 and VD2 domain order in both REGULGENT™ (N‐term) and REGULGENT™ (C‐term) (Figure 7a). Previous reports have emphasized the significance of reducing antibody affinity for achieving agonist activity with certain receptors, such as Fas receptor, CD40, 4‐1BB, and PD‐1 (Chodorge et al., 2012; Yu et al., 2023). For instance, in the case of Fas receptor, lower‐affinity antibodies have demonstrated a substantial increase in agonist activity. This is attributed to the crucial role of partial dissociation of a bivalent antibody from the Fas receptor, which is essential for recruiting additional Fas monomers to the receptor cluster (Chodorge et al., 2012). Consequently, the choice of VD order (VD order) can be tailored to the specific target antigen, aligning with the intended objective.

Regarding the linker sequence and length, we used a 98‐amino‐acids‐long sequence of IgG4 CH1 for the VH1 and VH2 linker for the REGULGENT™ (N‐term). A previous study reported that linker length and composition affect the affinity of BsAbs (Jakob et al., 2013). We evaluated the minimum required linker length and discovered that the 14th Cys of the linker (ASTKGPSVFPLAPC) is essential for proper antibody structure formation and production (Figure S4). This is because Cys, present at the 14th position within the heavy chain linker, is needed for disulfide bond formation with the C‐terminal Cys of the light chain. Therefore, a CH1 linker sequence containing more than 14 amino acids that include the Cys for disulfide bond can be used for the linker sequence. Therefore, the REGULGENT™ (N‐term) format can select the IgG4 CH1 linker sequence from 14 to 98 amino acid length, and also from IgG/IgA/IgM/IgE/IgD‐derived CH1 sequences that include the Cys residue capable of forming disulfide bond formation with the light chain, to select the affinity of the REGULGENT™ format fit for purpose.

To address the theoretical considerations for the reason, REGULGENT™ (N‐term) technology has stable physicochemical properties, we further compared the REGULGENT™ (N‐term) linker, which has 13 amino acids (aa) and lacks the 14th Cys residue, with the 98‐aa CH1 linker. Data indicated that the T agg value (72.8°C) of the 98‐aa CH1 linker was better than that of the 13‐aa linker REGULGENT™ (N‐term) (70.2°C) (Figure S5). Furthermore, we compared the DVD‐Ig format, which connects VH1‐VH2 and VL1‐VL2 directly with the linker (ASTKGPSVFPLAP [13 aa] and TVAAPSVFIFPP [12 aa]). The results demonstrated ca. 25% HMWS formation with transient expression (Figure 3a). This indicated that the linker (CH1) sequence affected the stability of REGULGENT™ (N‐term). The T agg value of the DVD‐Ig format was 65.1°C. This indicated that structure differences between REGULGENT™ (N‐term) and DVD‐Ig affected the T agg value. The use of the full‐length CH1 linker (98 a.a) was considered to be the factor responsible for the high stability of this antibody similar to that of the mAb; however, the detailed mechanism of stability requires further elucidation.

Affinity and stability are important in antibody production with respect to the relationship between K d and T m. For anti‐T/P and anti‐P/T REGULGENT™ (C‐term), the change in the T m and K d values is small even if the position of the Fab domain is changed compared to that of the mAb. However, in anti‐T/P and anti‐P/T REGULGENT™ (N‐term), more pronounced changes in the K d and T m values were observed compared to those in mAbs, as the K d was reduced in the VD2 domains of both anti‐T/P and anti‐P/T in REGULGENT™ (N‐term). This suggests a change in the interaction with the solvent. The changes in both the K d and T m may be related to the accessibility of the antibody to the solvent. Our NS‐EM data revealed that the Fab arm of the REGULGENT™ (N‐term) was bent at an angle of ca. 90° (Figure 8). We calculated the molecular model using AlphaFold‐Multimer to obtain the structural insight into the antigen binding of the REGULGENT™ (N‐term) (Evans et al., 2021). The model structure showed that the Fab arm of the REGULGENT™ (N‐term) was bent at an angle of ca. 90° (Figure S6). This observation is in good agreement with our NS‐EM data. Therefore, our NS‐EM data may reflect the overall structure of the REGULGENT™ and suggest a unique structure.

We demonstrated that the anti‐TRAIL‐R2/PSMA REGULGENT™ (N‐term) BsAb exhibited in vitro activity specifically against Expi293F cells expressing both TRAIL‐R2 and PSMA (Figure 7d). The R2‐E11 clone of the TRAIL‐R2 antibody has been reported to display potent agonistic activity in the presence of anti‐human IgG, which promotes mAb oligomerization (Mori et al., 2004). Therefore, the observed in vitro activity of the anti‐TRAIL‐R2/PSMA REGULGENT™ (N‐term) BsAb may be attributed to the oligomerization of TRAIL‐R2 upon binding to PSMA. We have further evaluated the in vitro/in vivo activity of anti‐TRAIL‐R2/PSMA REGULGENT™ (N‐term) BsAb against prostate and non‐small cell lung cancer cells, and other REGULGENT™ (C‐term) against other antigens. This research is currently being prepared for submission.

4 CONCLUSION

We engineered new, stabilized, IgG‐like symmetric BsAbs, namely REGULGENT™, that have excellent manufacturing capabilities for commercial development as well as the desired IgG‐like physicochemical properties. REGULGENT™ also provides a new selection of BsAb repertoires for clinical applications.

5 MATERIALS AND METHODS

5.1 Antibody generation and purification

Kyowa Kirin Co., Ltd. (Tokyo, Japan) expressed and purified all the antibodies utilized in this study. The anti‐T (TRAIL‐R2) antibodies were R2‐E11 clones (Mori et al., 2004). To produce light‐chain common type BsAb, a single‐chain fragment variable library was prepared using the VL of R2‐E11, and the anti‐P (PSMA) antibody was obtained by phage display using an R2‐E11 VL fixed library (Krah et al., 2017). The VH gene of the library was extracted from the spleen of PSMA‐immunized human antibody transgenic mice (Ishida et al., 2002). For the anti‐T/P REGULGENT (N‐term) construction, the heavy chain of the anti‐T was fused through a linker of IgG4‐derived CH1 from 98 amino acids and serially fused to the N‐terminus of the anti‐P heavy chain (Table 1). For the anti‐T/P REGULGENT (C‐term) construction, the heavy chain of the anti‐P was fused to the C‐terminus of the anti‐T mAb heavy chain directly. The DNA encoding the above antibodies was generated via commercial gene synthesis (FASMAC Co., Ltd Japan). To construct expression vectors, the genes of full‐length heavy or light chains were separately cloned into a pCI expression vector (Promega, Madison, WI, USA). Thereafter, the two chains were cotransfected into CHO cells (ExpiCHO‐S™), followed by expression and protein A affinity purification. The buffer was replaced with Dulbecco's phosphate‐buffered saline (PBS; Gibco, Gaithersburg, MD, USA) in a NAP25 desalting column (Cytiva, Marlborough, MA, USA). The purity of the antibody was analyzed using SEC (Figure S1B).

5.2 Antigens and FcRn

Recombinant human TRAIL‐R2, human PSMA, and human FcRn were purchased from ACROBiosystems Inc. (Newark, DE, USA).

5.3 Generation of a stable cell line of CHOs

Cell lines stably expressing anti‐P mAb and anti‐T/P REGULGENT (N‐term) antibodies were generated. The stable cell lines were established via electroporation with the gene of interest followed by positive and negative selection pressure. The cells were cultured in Balan CD CHO Growth A supplemented with 7.5% BalanCD CHO Feed 1 (FUJIFILM Irvine Scientific, CA, USA). The expression titers of the antibodies were then assessed using Protein A affinity columns.

5.4 Expression titer

The expression titers of mAbs were determined using protein A chromatography (PA ID Sensor Cartridge, Applied Biosystems, MA, USA). The inner diameter and the length of the cartridge were 2.1 mm and 3.0 cm, respectively. The loading buffer comprised 100 mM of sodium citrate (pH 6.0) and flowed at 3 mL/min. We injected 50 μL of the sample, following which the bound protein was eluted using 100 mM of sodium citrate (pH 3.0). The UV absorbance was monitored at 280 nm. The 280‐nm antibody peak was integrated and compared with a calibration curve to calculate mAb concentration.

5.5 UHP‐SEC

The percentages of HMWS and LMWS of all the antibodies were analyzed by UHP‐SEC using an ACQUITY UPLC BEH200 SEC column (200 Å, 1.7 μm, 4.6 × 150 mm; Waters, MA USA). The mobile phase comprised 50 mM of sodium phosphate (pH 6.8) and 500 mM of sodium chloride. The experimental conditions were as follows: flow rate, 0.4 mL/min; detection wavelength, 215 nm; and analysis time, 8.0 min. A total of 2.5 μg of each antibody was injected into the column. The peaks of the monomers, HMWS, and LMWS were analyzed using the Empower 2 chromatography data software (Waters, MA, USA).

5.6 Evaluation of low pH stress stability

The pH of the antibody buffer solution was adjusted to 3.5 using 0.2 M citric acid and 0.05 M sodium chloride (pH 2.7). The antibodies were incubated at a temperature of 37°C and a pH of 3.5 for 10 or 60 min in a PCR Thermocycler (Applied Systems, CA, USA) before neutralization with a 200 mM phosphate, 0.05 M sodium chloride (pH 8.0). The samples were maintained at 4°C until UHP‐SEC analysis. The degrees of HMWS and LMWS were analyzed using UHP‐SEC, as previously described.

5.7 DSC analysis

The thermal stability of individual antibody domains was evaluated using DSC. Measurements were performed with 0.5 mg/mL of antibodies in PBS buffer (pH 3.5/7.4) using a Micro Cal VP‐Capillary DSC system (Malvern Instruments, Worcestershire, UK). Temperature scans were performed from 25 to 100°C at a scan rate of 1°C/min. A buffer reference scan was subtracted from each sample scan before normalizing the concentration. Baselines were established using Origin7.0 (OriginLab, MA, USA) and cubic interpolation of the pre‐ and post‐transition baselines.

5.8 NanoDSF analysis

NanoDSF was performed using Prometheus NT.48 equipped with back reflection mode (Nano Temper Technologies, München, Germany). Samples were loaded in nanoDSF grade standard capillaries (Nano Temper Technologies, München, Germany) and exposed at thermal stress from 20 to 95°C by thermal ramping rate of 1°C/min. T agg were calculated by PR.Therm Control software (Nano Temper Technologies, München, Germany).

5.9 SPR experiments

The antigen‐binding affinities of the antibodies were measured using Biacore™ T200 at 25°C with HBS‐EP+ pH 7.4 (Cytiva) as the buffer. The Biotin Capture Kit (Cytiva) was used to immobilize biotin‐labeled antigen proteins onto the sensor chip. Biotinylated antigens were injected, followed by the antibody solution in a single cycle. Sensorgrams were analyzed using the Biacore Evaluation Software (Cytiva), fitting a 1:1 binding model to determine the binding kinetics (k a, K d, KD).

Simultaneous binding was evaluated using Biacore™ T200 at 25°C with HBS‐EP+ pH 7.4 (Cytiva) as the buffer. Biotinylated TRAIL‐R2 was injected, followed by the antibody solution and the PSMA solution. Sensorgrams were analyzed using the Biacore Evaluation Software.

FcRn‐binding affinities were measured using Biacore™ 8 K at 25°C with HBS‐EP+ pH 7.4 and 6.0 (Cytiva) as the buffers. Biotinylated FcRn was injected, followed by the antibody solution in multiple cycles. Sensorgrams were analyzed using the Biacore Evaluation Software (Cytiva) with a steady‐state model to determine the KD value (Additional information is available in the Supplemental Information section).

5.10 Cell proliferation assay

PSMA antigens were expressed via transient transfection of Expi293F cells (Thermo Fisher, MA, USA) at 37°C overnight. The Expi293F and PSMA‐expressing Expi293F cells were seeded in 96‐well plates (10,000 cells/well). Antibodies were added at different concentrations (10‐fold dilution from 1000 to 0.01 ng/mL). After 24 h of incubation, cell proliferation was assessed using the WST‐8 assay and Cell Counting Kit‐8 (Dojindo, Kumamoto, Japan).

5.11 NS‐EM analysis

Negative staining imaging analysis was performed by Nano Imaging Services, Inc. Antibodies at 1 mg/mL were diluted 166‐fold with PBS and 3 μL applied to the EM grid and staining with 3 μL of a 1% uranyl formate solution. Negative stain TEM nanoimaging was performed using a FEI Tecnai T12 electron microscope. 2D classification analysis was performed by XMIPP (Sorzano et al., 2004).

AUTHOR CONTRIBUTIONS

Seiji Saito: Conceptualization; data curation; investigation; formal analysis; project administration; writing – original draft; writing – review and editing; visualization. Makoto Nakayama: Conceptualization; writing – review and editing; investigation; data curation; formal analysis. Kaori Yamazaki: Data curation; investigation; formal analysis; visualization. Yuya Miyamoto: Writing – review and editing; data curation; investigation; formal analysis; visualization. Keiko Hiraishi: Data curation; investigation; formal analysis; visualization. Daisuke Tomioka: Data curation; investigation; formal analysis; visualization. Sayaka Takagi‐Maeda: Data curation; investigation; formal analysis; visualization. Katsuaki Usami: Writing – review and editing; supervision. Nobuaki Takahashi: Conceptualization. Shinji Nara: Writing – review and editing; project administration. Eiichiro Imai: Writing – review and editing; project administration.

CONFLICT OF INTEREST STATEMENT

All authors are employees of Kyowa Kirin Co., Ltd.

Supporting information

Data S1. Supporting information.

ACKNOWLEDGMENTS

We would like to thank Dr. Yuya Sasajima for valuable discussion and suggestions. We also thank Editage (www.editage.jp) for their English language editing service.
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REFERENCES

Bailly M , Mieczkowski C , Juan V , Metwally E , Tomazela D , Baker J , et al. Predicting antibody developability profiles through early stage discovery screening. mAbs. 2020;12 :1743053.32249670
Boinapally S , Ahn HH , Cheng B , Brummet M , Nam H , Gabrielson KL , et al. A prostate‐specific membrane antigen (PSMA)‐targeted prodrug with a favorable in vivo toxicity profile. Sci Rep. 2021;11 :7114.33782486
Brinkmann U , Kontermann RE . The making of bispecific antibodies. mAbs. 2017;9 :182–212.28071970
Cao M , Wang C , Chung WK , Motabar D , Wang J , Christian E , et al. Characterization and analysis of scFv‐IgG bispecific antibody size variants. MAbs. 2018;10 :1236–1247.30130449
Chodorge M , Züger S , Stirnimann C , Briand C , Jermutus L , Grütter MG , et al. A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency. Cell Death Differ. 2012;19 :1187–1195.22261618
Evans R , O'Neill M , Pritzel A , Antropova N , Senior A , Green T , et al. Protein complex prediction with AlphaFold‐Multimer. bioRxiv. 2021. 10.1101/2021.10.04.463034
Godar M , de Haard H , Blanchetot C , Rasser J . Therapeutic bispecific antibody formats: a patent applications review (1994‐2017). Expert Opin Ther Pat. 2018;28 :251–276.29366356
Gong S , Ren F , Wu D , Wu X , Wu C . Fabs‐in‐tandem immunoglobulin is a novel and versatile bispecific design for engaging multiple therapeutic targets. MAbs. 2017;9 :1118–1128.28692328
Huang S , van Duijnhoven SMJ , Sijts AJAM , van Elsas A . Bispecific antibodies targeting dual tumor‐associated antigens in cancer therapy. J Cancer Res Clin Oncol. 2020;146 :3111–3122.32989604
Ishida I , Tomizuka K , Yoshida H , Tahara T , Takahashi N , Ohguma A , et al. Production of human monoclonal and polyclonal antibodies in TransChromo animals. Cloning Stem Cells. 2002;4 :91–102.12006160
Jakob CG , Edalji R , Judge RA , DiGiammarino E , Li Y , Gu J , et al. Structure reveals function of the dual variable domain immunoglobulin (DVD‐Ig™) molecule. MAbs. 2013;5 :358–363.23549062
Kang J , Sun T , Zhang Y . Immunotherapeutic progress and application of bispecific antibody in cancer. Front Immunol. 2022;13 :1020003.36341333
Krah S , Schröter C , Eller C , Rhiel L , Rasche N , Beck J , et al. Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display. Protein Eng Des Sel. 2017;30 :291–301.28062646
Labrijn AF , Janmaat ML , Reichert JM , Parren P . Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019;18 :585–608.31175342
Le Gall F , Reusch U , Little M , Kipriyanov SM . Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody. Protein Eng des Sel. 2004;17 :357–366.15126676
Lim SI . Fine‐tuning bispecific therapeutics. Pharmacol Ther. 2020;212 :107582.32450189
Mazzer AR , Perraud X , Halley J , O'Hara J , Bracewell DG . Protein a chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold. J Chromatogr A. 2015;1415 :83–90.26346187
Mori E , Thomas M , Motoki K , Nakazawa K , Tahara T , Tomizuka K , et al. Human normal hepatocytes are susceptible to apoptosis signal mediated by both TRAIL‐R1 and TRAIL‐R2. Cell Death Differ. 2004;11 :203–207.14576771
Moussa EM , Panchal JP , Moorthy BS , Blum JS , Joubert MK , Narhi LO , et al. Immunogenicity of therapeutic protein aggregates. J Pharm Sci. 2016;105 :417–430.26869409
Namisaki H , Saito S , Hiraishi K , Haba T , Tanaka Y , Yoshida H , et al. R409K mutation prevents acid‐induced aggregation of human IgG4. PLoS One. 2020;15 :e0229027.32182240
Nisonoff A , Wissler FC , Lipman LN . Properties of the major component of a peptic digest of rabbit antibody. Science. 1960;132 :1770–1771.13729245
Qin Y , Ma R , Li Y , Li Y , Chen G , Zhou W . Productivity and quality improvement for a symmetric bispecific antibody through the application of intensified perfusion cell culture. Antib Ther. 2022;5 :111–120.35719210
Rios M , Lundberg B . A light‐chain platform for developing bispecific antibodies. BioProcess Int. 2020;18:5–9.
Schanzer J , Jekle A , Nezu J , Lochner A , Croasdale R , Dioszegi M , et al. Development of tetravalent, bispecific CCR5 antibodies with antiviral activity against CCR5 monoclonal antibody‐resistant HIV‐1 strains. Antimicrob Agents Chemother. 2011;55 :2369–2378.21300827
Sinharoy P , Aziz AH , Majewska NI , Ahuja S , Handlogten MW . Perfusion reduces bispecific antibody aggregation via mitigating mitochondrial dysfunction‐induced glutathione oxidation and ER stress in CHO cells. Sci Rep. 2020;10 :16620.33024175
Snajdauf M , Havlova K , Vachtenheim J Jr , Ozaniak A , Lischke R , Bartunkova J , et al. The TRAIL in the treatment of human cancer: an update on clinical trials. Front Mol Biosci. 2021;8 :628332.33791337
Sorzano CO , Marabini R , Velázquez‐Muriel J , Bilbao‐Castro JR , Scheres SH , Carazo JM , et al. XMIPP: a new generation of an open‐source image processing package for electron microscopy. J Struct Biol. 2004;148 :194–204.15477099
Spiess C , Zhai Q , Carter PJ . Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 2015;67 :95–106.25637431
Sun H , Wang S , Lu M , Tinberg CE , Alba BM . Protein production from HEK293 cell line‐derived stable pools with high protein quality and quantity to support discovery research. PLoS One. 2023;18 :e0285971.37267316
Swope N , Chung WK , Cao M , Motabar D , Liu D , Ahuja S , et al. Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation. Biotechnol Bioeng. 2020;117 :1063–1071.31930476
Trebing J , El‐Mesery M , Schäfer V , Weisenberger D , Siegmund D , Silence K , et al. CD70‐restricted specific activation of TRAILR1 or TRAILR2 using scFv‐targeted TRAIL mutants. Cell Death Dis. 2014;5 :e1035.24481449
Wang Q , Chen Y , Park J , Liu X , Hu Y , Wang T , et al. Design and production of bispecific antibodies. Antibodies (Basel). 2019;8 :43.31544849
Wei J , Yang Y , Wang G , Liu M . Current landscape and future directions of bispecific antibodies in cancer immunotherapy. Front Immunol. 2022;13 :1035276.36389699
Xu T , Zhang J , Wang T , Wang X . Recombinant antibodies aggregation and overcoming strategies in CHO cells. Appl Microbiol Biotechnol. 2022;106 :3913–3922.35608667
Yu J , Song Y , Tian W . How to select IgG subclasses in developing anti‐tumor therapeutic antibodies. J Hematol Oncol. 2020;13 :45.32370812
Yu X , Orr CM , Chan HTC , James S , Penfold CA , Kim J , et al. Reducing affinity as a strategy to boost immunomodulatory antibody agonism. Nature. 2023;614 :539–547.36725933
Zhang JH , Shan LL , Liang F , Du CY , Li JJ . Strategies and considerations for improving recombinant antibody production and quality in chinese hamster ovary cells. Front Bioeng Biotechnol. 2022;10 :856049.35316944
Zhong X , Ma W , Meade CL , Tam AS , Llewellyn E , Cornell R , et al. Transient CHO expression platform for robust antibody production and its enhanced N‐glycan sialylation on therapeutic glycoproteins. Biotechnol Prog. 2019;35 :e2724.30299005
