Volume 27, Issue 3 (Summer 2021)                   Intern Med Today 2021, 27(3): 400-417 | Back to browse issues page

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Moazzami R, Mirzahosaini H, Naddafi F, Davami F. A Comparative Investigation of the Bispecific Antibody: Expression in Expi293F Cells and E.coli. Intern Med Today 2021; 27 (3) :400-417
URL: http://imtj.gmu.ac.ir/article-1-3596-en.html
1- Iran Medical Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
2- Pharmaceutical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
3- Iran Medical Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. , f.davami@gmail.com
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1. Introduction
pplying monoclonal Antibodies (mAb) to diagnose and treat various diseases, including cancer, has become a standard and effective method. There exist >70 monoclonal antibodies on the world pharmaceutical market, and numerous are in various stages of testing [1]. However, in some cases, monoclonal antibodies have limitations, e.g., the size and complexity of the molecule reduce its potential to penetrate tumor tissues; the flat structure of the interacting segments complicates catalytic cavities to penetrate deep into enzymes [2 ,3]. To overcome such limitations and increase the antitumor activity of antibodies, there are various approaches, like antibody engineering to increase affinity; the invention of antibody conjugate antibodies or the use of antibody fragments; finally, the creation of bispecific antibodies. Binding to two different targets by a single antibody molecule is an intriguing idea in cancer treatment research. Such molecules can perform several functions, as follows: the inhibition of the cell surface receptor; simultaneous blockade of two ligands; the cross-linking of two surface receptors, and the placement of a T cell adjacent to the tumor [4]. Three dual-character antibodies have been licensed by legal authorities; catumaxomab [5], blinatumomab, and emicizumab [6]. The benefits of dual-character compounds include their bilateral effect on different tumor targets and their cumulative or synergistic drug effects due to the overlap of cancer signaling pathways. These antibodies come in a wide variety of formats, including those with and without Fc [4]. The BiTE Bispecific T cell engager format consists of two single antibody chains linked together by a polypeptide linker. In BiTEs, one arm is targeted against CD3 and one arm against the tumor antigen. The features of this format include the ability of Tcell-cell polyclonal antigen-dependent activation and the induction of T cell proliferation. Thus, BiTE has a great ability to lysis the target cell. Blinatumomab, or CD3 × CD19bsAb anti, is the first drug in the BiTE family to be introduced by Amgen in 2014 to treat Acute Lymphoblastic Leukemia (ALL) and Non-Hodgkin’s Lymphoma (NHL) [7].
Blinatombe is a protein fusion with a molecular weight of 55 KD that contains two scFv (Single Chain Fragment Variable) antibody chains. This protein consists of 4 variable domain cDNAs plus 3 linker peptides, 2 long linkers to bind to make the scFv fragment, and a short linker to bind the two fragments. This structure provides the flexibility and freedom of action required to connect each arm to the target epitopes located on the surface of the two cells. In the N-terminal part of this protein is scFv binding to murine CD19 antibody derived from mAb HD37. Moreover, in the C-terminal part of the scFv is terminal binding to 3 CD antibody derived from mAb L2K murine antibody [8]. The presence of a hexahistidine sequence at the C-terminus of this protein allows it to be purified by affinity resins Immobilized Metal Ion Affinity Chromatography (IMAC) [9].
 The persistent and high expression of CD19 molecule on malignant B cells and its role in the survival and proliferation of these cells made it a target marker on B cells. Unlike CD20 and CD22, the CD19 receptor is expressed in almost all developmental stages of B cell lineage. It is a reliable marker for treating this category. Furthermore, in patients, almost 100% of malignant cells express this antigen [10]. CD19 is a PI3 kinase activator. This enzyme is a key element in signaling in malignant cells [11]. The in vitro performance of CD3 × CD19 anti in T cell and CD19 expression cells was strongly significant in simultaneous culture experiments. The minimum concentration required for lysis of CD19 target cells was approximately pg/mL [12].
Additionally, CD3+, CD4+, or CD8+ T cells present a similar ability to lysis target cells and perform so with vigor. Of course, naïve T cells are an exception [13]. Moreover, anti-CD3 × CD19 is sufficient for T cell activation and requires no prior activation or stimulants. T cell activation leads to the expression of CD69 and C25 as well as the up-regulation of adhesion molecules, like CD2 on its surface. Besides, it releases inflammatory cytokines, such as IFNϒ, TNFα, IL2, IL6, IL1, finally the sequential proliferation of T cells [14]. The mechanism of T cell-cell killing in CD19 target cells involves the formation of a strong cytolytic synapse between the two cells, followed by the depletion of the toxic proteins perforin and granzymes from the secretory T vesicles on the target cell. The activation of caspase enzymes in the target cell indicates the significance of the apoptosis pathway parallel to the pathway of secretory vesicles to destroy the target cell [8].
 For producing therapeutic recombinant proteins, including monoclonal antibodies, various hosts are used, such as bacteria, baculovirus, yeast, plant cells, and mammalian cells [15]. Among these, the mammalian cell class due to its ability to produce protein by natural folding and creating proper post-translational modifications are especially important. Chinese Hamster Ovary (CHO) cell line is the most widely used cell line for generating antibodies; producing about two-thirds of recombinant therapeutic proteins in this class [16]. HEK293 human embryonic kidney is a human kidney embryonic cell. It is widely used for the transient expression of TGE recombinant proteins. Despite its epithelial origin, it adapts well to suspended culture conditions. Due to the human nature of this class, the recombinant proteins expressed in it are similar to human proteins concerning post-translational changes. Furthermore, the process of translation, folding, and maturation processes occur with considerable adequacy. The transient gene expression approach is commonly used to shortly produce large amounts of protein for the biochemical studies of the drug and to perform preclinical investigations [1718]. Expi 293 cell is a HEK293 cell-derived class for culture in high cell density suspension and serum-free culture medium. The chemically defined form of these properties makes this cell suitable for industrial production [19].
 The mammalian cell expression system is currently the most common method of producing this antibody; however, Escherichia coli has the potential to produce proteins without a glycosylation pattern, as well as the ease of ordering the production process and the cost-effectiveness of the bacterial culture raw materials has made this host a suitable host for the production of Fc-free antibodies and dual-specific antibodies [20]. Accordingly, a significant portion of therapeutic proteins without glycosylation pattern is produced in this host, and BL21 (DE3) is among the most common industrial and research strains. The long history of industrial use of E. coli, as well as the appropriate rules of the drug production supervisory departments to this host, are significant advantages of this production platform [21].
 Considering the single-chain and non-glycosylation of Blinatombe, the capacity of the bacterial expression system to produce this antibody was used; we compared it with the antibody produced in eukaryotic cells. This is because no such comparison has been made for the Blinatomb antibody. The great advantages of E. coli, as a suitable host include its industrial production parameters; the availability of inexpensive culture media for the production of biopharmaceuticals in this host; the expression of monoclonal antibodies of two BiTE family traits (due to no post-translational changes). The commercial manufacturer expressed Blinatomomb in the host of CHO. This study aimed to examine the expression and binding properties of antibodies expressed in both systems and compare them.
2. Materials and Methods
Cells and consumables

 Expi293F cell and related culture medium (Expi293 recession Expression Medium) and its specific transfection agent (ExpiFectamine™ 293 transfection Reagent), as well as Pen/Strep and L-Glu antibiotics and pcDNA3.1 (+) vector (Invitrogen; CA, USA), were prepared. Ni-NTA chromatographic resin was obtained from QIAGEN (USA). Trypan blue and 3-d3 aminobenzidine (DAB) and antihistamine conjugated with HRP and TMB were prepared from Sigma-Aldrich (USA). E.coli BL21 strain (DE3) and vector pET-22b were obtained from Novagen (USA). Restriction enzymes were collected from Thermo Fisher Scientific (USA). NALM-6 and Jurkat cell lines were obtained from the cell bank of the Pasteur Institute of Iran.
 Expressive structures and expression in E.Coli
 The expression constructs expressing the two antibodies of blinatumomab from the PGH vector in the pET-22b vector were cloned by HindIII and NcoI enzymes. BL21 (DE3) strain was used for protein expression. The pET-22b vector was designed for the periplasmic expression of the target protein. The strain transformed by the expressive vector in the LB medium was incubated to reach OD=0.5 in a wavelength of 600 nm. Then, 0.5mM IPTG was used for induction. The cells were isolated by centrifugation after 4 hours of culture. Besides, a culture batch without induction was considered as a negative control.
 Expression structure and the expression of Expi293F cell line
 The coding sequence of the blinatombe protein was cloned from the PGH vector into the pcDNA3.1 vector. This measure helped to construct the expression structure in the cellular system by two enzymes, NheI and HindIII. These expression constructs were then transfected into Expi293F cells. Expi293F cells were cultured in specific and without serum culture medium Expi293 ™ expression medium with penicillin-streptomycin (2mM) antibiotics. The cells were cultured in a CO2 incubator shaker in a humid environment and in rotating glass bottles at 125 rpm. The cells were passaged every three days at a density of approximately 3 × 10⁵ cells/mL. The cell count method with trypanblo was employed to calculate the frequency of cells. In short, the transfection was performed according to the manufacturer’s method, as follows: The day before the transfection, the cells were cultured in an additive-free medium. The next day, the transfectamine ™ 293 reagents and plasmid were mixed with specific relativity. Incubations were added to the cells. After 16-18 hours, ExpiFectamine ™ Transfection Enhancer 1 and 2 were added to the cells according to the manufacturer’s instructions. On the seventh day, the supernatant was isolated from the cells and stored to assess expression.
Protein purification
To purify the antibodies expressed by the Expi293F cell line, Ni-NTA (Ni-Nitrilotriacetic acid) column was used. Initially, the cell soup was filtered by a 0.45 μm filter. The column was then washed with a binding buffer [NaH2PO4 (50 mM), NaCl (300 mM), imidazole (10 mM), pH=8.0)]. Accordingly, NaH2PO4 (50 mM), NaCl (300 mM), imidazole (20 mM), pH=8.0 were washed and finally buffered by NaH2PO4 (50 mM), NaCl (300 mM), imidazole (250 mM; pH=8.0). The purification of the protein expressed in bacteria was similar to that of the cell. The difference concerned the chemical composition of the triple buffers, i.e., changed as follows:
Binding buffer: (100mM NaH2PO4, 10mM Tris, 8M Urea pH=8);
Washing buffer: (100mM NaH2PO4, 10mM Tris, 8M Urea pH=6.3);
Elution buffer: (100mM NaH2PO4, 10mM Tris, 8M Urea pH=4.5).
SDS PAGE and Western blot analysis
 SDS PAGE and Western blot analysis were implemented to evaluate protein expression. Kumasi blue staining was used to detect proteins. The proteins were electrophoresed in a 12% gel at 100 volts for 80 minutes. SDS PAGE was followed by Western blotting. This process was performed by a semi-dry transfer cell Trans-Blot (Biorad) using nitrocellulose membrane (GE Healthcare) to transfer proteins. After transfer, the membrane was blocked by 4% Bovine Serum Albumin (BSA). Next, the antibody against polyhistidine conjugated with HRP in a dilution of 1: 1500 was used. For the appearance of protein spots, 3 and 3 Deminobenzidine (DAB) methods were used. 
ELISA Assessment
 An ELISA test was used to evaluate the expression level of antibodies secreted by two different expression systems. For this purpose, CD19-expressing cell lines, called NALM-6, and CD3-expressing cell lines, called Jurkat were cultured in 96 cells overnight. The cells were then fixed using 3.7% formaldehyde. After washing the cells 3 times, the unoccupied surfaces of the well were blocked by 2% bovine serum albumin for one hour. Next, the cells were incubated overnight with the serial dilutions of the antibody expressed at 4°C (12.5, 6.25, 3.12, 1.56, 0.58 pg/mL). After performing the re-washing steps, 100 μL of antibody against HRP-conjugated polyhistidine was added to each well at a dilution of 1: 250 in 1% BSA. After one hour and re-rinsing, 100 μL of TMB (3,30,5,50 Tetramethylbenzidine) was added to each well to perform a dyeing reaction. Finally, the light absorption of each well was read with an ELISA reader at 450 nm. Cell line without CD19 and CD3 markers (CHO cell) was also used as a negative control.
3. Results
 The sequence encoding the blinatombe gene was successfully cloned into the pcDNA3.1 vector using two restriction enzymes NheI and HindIII. To confirm the cloning accuracy of the structure made by the two mentioned enzymes, an enzyme was digested which led to the creation of two pieces with a size of approximately 1600 and 5400 bp (Figure 1-A).

In the case of the expression construct in bacteria, the coding sequence was successfully cloned into the pET-22b vector. Enzymatic digestion in this case also indicated the formation of 5000 and 1600 bp formats (Figure 1-B). Finally, by sequencing the created structures, the cloning accuracy was confirmed in both cases.
The expression level in both expression systems was determined after purifying the protein with a nickel column (Figure 2).
Moreover, the purified protein from each system was determined to be concentrated. This was 2.3 mg/L for Expi293F cells and 100 mg/L for the bacterial expression system. SDS PAGE analysis, followed by Western blotting was performed per expression system; this measure indicated the expression of 55 kDa target protein at the respective site (Figure 3).

To evaluate the frequency of expression per case, a densitometric system was used to determine the density of each band in SDS PAGE gel. The obtained results revealed that the band related to the expression system in bacteria contained approximately 19.3% of the total proteins. Additionally, the band related to the mammalian cell expression system contains about 7.1% of the total cultured proteins (Table 1). 

Antibody binding ability expressed by ELISA was assessed. Two NALM-6 cell lines were applied for CD19 and Jurkat cell line for CD3. Both classes were treated with serial dilutions of antibodies (Figure 4).

The relevant results reflected that the extent of antibody binding expressed from the Expi293F cellular system was approximately twice as high as the antibody binding expressed in BL21 (DE3).
4. Discussion
The expression system of mammalian cells is of considerable significance in the biopharmaceutical industry. The special features of this system include high sensitivity; expensive culture media; the inherent complexity of eukaryotic cells, as well as the time-consuming process of culturing these cells. Such characteristics assist researchers in the field of industrial protein production research; they could use alternative systems in cases where simpler and cheaper hosts can be replaced. This also applies to producing antibody fragments or single-chain antibodies without a glycosylation pattern. A relevant challenge is to study the possibility of producing each molecule of a drug or antibody candidate protein in simpler expression systems, like bacteria. It is necessary to explore the possibility of production in cheaper systems for each protein separately; thus, the possibility of producing antibodies against the CD19 marker, as a powerful and expensive drug in the treatment of leukemia, was investigated (this antibody was available at the time of the release of the most expensive therapeutic antibody). This study compared and evaluated the produced protein using the method of rapid antibody production in the mammalian expression system (temporary expression system) and simultaneous expression in the bacterial system.
 After successful cloning and expression of dual-character antibody in both systems, this antibody was purified and its production was confirmed. The expression levels in bacteria and Expi293F cells equaled 100 mg/L and 2.3 mg/L, respectively. Mack et al. obtained the expression of 15 mg/L by the antibody expression of two anti-EpCAM × anti-CD3 properties in BiTE format in the bacterial host [22]. McCall et al. produced a dual-character antibody in a similar format against anti-HER2 / neu × anti-CD16 in bacteria with an expression level of about 3.7 mg/L. Kuo et al. expressed the anti-CD123 × anti-CD3 antibody in the CHO-K1 host in a format similar to BiTE at a rate of about 5 mg/L [23]. The level of expression in E.coli expression systems is generally higher than that in mammalian expression systems; however, Root et al. achieved a significant expression of 1300 mg/L [24]. De Nardis et al. also produced dual-specific antibodies against HER2 and HER3 in the CHO-DG44 host in IgG-like formulation with an expression level of about 1200 mg/L [25]. 
The examined bacterial system reflected the ability to produce higher amounts of antibodies; however, the generated antibody had a lower binding power, compared to mammalian cells. Examining antibody binding characteristics produced per system by ELISA test signified that the mammalian expression system is more efficient in antibody production than the bacterial production system. In other words, the binding rate to each of the target indices (i.e., CD19 & CD3) was averagely about twice as high. This is probably due to differences in folding systems and protein processing in mammalian cells and bacteria. Previous studies provided similar results [22]. Flow cytometric analysis can be used to more accurately evaluate the antibody binding power produced. Besides, a mixed lymphocyte culture system in the presence of target cells can be used to evaluate the cytotoxic effect of antibodies on the target cell [26 ,12]. Additionally, further studies are suggested to evaluate the expression of this antibody in other common expression systems, such as other mammalian cells or yeast cells. Another approach is to implement different expression vectors in the mammalian expression system.
5. Conclusion
 This study indicated that in the case of antibodies to two traits of the BiTE family, like Blinatombe, mammalian cells present a more efficient and successful expression system; although the bacterium can produce much larger amounts of the antibody.
 Authors: Reza Moazami (laboratory and research methods, data analysis and text writing and editing) Hassan Mirza Hosseini (data analysis and text writing and editing) Fatemeh Nadafi (laboratory and research methods, text writing and editing) Fatemeh Davami (Research idea, conceptualization, data analysis, and text writing and editing)

Ethical Considerations
Compliance with ethical guidelines

This research has obtained the ethics code IR.PII.REC.1399.008 from Pasteur Institute of Iran.

This research has been done using the research grant of Pasteur Institute of Iran and in this institute.

Authors' contributions
Laboratory and research methodology: Reza Moazami and Fatemeh Nadafi; Data analysis, writing – original draft, and writing – review & editing: All Authors; Conceptualization: Fatemeh Davami.

Conflicts of interest
The authors declared no conflict of interest.

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Type of Study: Original | Subject: Basic Medical Science
Received: 2020/10/6 | Accepted: 2021/01/20 | Published: 2021/06/22

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