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Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells

Purpose: More than half of the diagnostic and therapeutic recombinant protein production depends on mammalian-based expression system. However, the generation of recombinant antibodies remains a challenge in mammalian cells due to the disulfide bond formation and reducing cytoplasm. Therefore, the production of functional recombinant antibodies in target cell line is necessary to be evaluated before used in therapeutic application such intrabodies against HIV-1. Methods: The work was to test expression of a single-chain variable fragment (scFv) antibody against HIV-1 Capsid p24 protein in a human mammalian-based expression system using HEK293T and Jurkat T cells as a model. Three expression plasmid vectors expressing scFv 183-H12-5C were generated and introduced into HEK293T. Expression of the scFv was analyzed, while ELISA and immunoblotting analysis verified its binding. The evaluation of the recombinant antibody was confirmed by HIV-1 replication and MAGI infectivity assay in Jurkat T cells. Results: Three plasmid vectors expressing scFv 183-H12-5C was successfully engineered in this study. Recombinant antibodies scFv (~29 kDa) and scFv-Fc (~52 kDa) in the cytoplasm of HEK293T were effectively obtained by transfected the cells with engineered pCDNA3.3-mu-IgGk-scFv 183-H12-5C and pCMX2.5-scFv 183-H12-5C-hIgG1-Fc plasmid vectors respectively. scFv and scFv-Fc are specifically bound recombinant p24, and HIV-1 derived p24 (gag) evaluated by ELISA and Western blot. Jurkat T cells transfected by pCDNA3.3-scFv 183-H12-5C inhibit the replication-competent NL4-3 viral infectivity up to 60%. Conclusion: Anti-p24 scFv 183-H12-5C antibody generated is suitable to be acted as intrabodies and may serve as a valuable tool for the development of antibody-based biotherapeutics against HIV-1.


Introduction
The development of smaller antibody fragments, which maintain the antigen affinity of the parental antibody, is preferable for better antibody production. Furthermore, the advance in smaller fragments production is the primary source for the in vitro antibody generation systems. [1][2][3] The Fv fragment that comprises of variable (V) regions is the smallest antigen-binding fragment that maintains the parental antibodies antigenicity properties. To connect the V regions, a soluble and flexible amino acid peptide linker is applied to a scFv (single chain fragment variable) fragment. [4][5][6] Nowadays, two standard formats of recombinant antibodies that are produced in eukaryotes expression system are scFv and Fab that show relevant for the clinical evaluation purposes. 7 Moreover, a fusion of the Fc domain towards antibody fragments result in regaining effector functions, stability, and avidity. 8,9 Recombinant antibodies targeting HIV-1 virus proteins have shown a potential in inhibiting the virus replication and infectivity. The recombinant antibodies are acting either as an intracellular antibody (intrabody) or transbody to achieve the requirement effects. Since early 1990 until 2016, four leading groups and few small laboratories had developed eight intrabodies against HIV-1 virus proteins. Marasco group focused on the generation of intrabodies like anti-Tat, anti-gp120, and anti-MA p17. [10][11][12][13][14] Meanwhile, Pomerantz and colleague worked on the production of anti-Rev, integrase, and reverse transcriptase intrabodies. [15][16][17] Designing of anti-CCR5, anti-Vif, and integrase intrabodies is the main focusing by Barbas III and Goncavales group. [18][19][20] In our knowledge, there is no works about the establishment of anti-CA p24 from anti-p24 hybridoma cell line and used as intrabodies in inhibiting the HIV-1 virus replication and infectivity. Here, we are the first group reporting the intrabody. Mammalian cells are predominantly used as expression hosts for heterologous protein production despite being harder to maintain compared to bacteria or yeast. Industrial pharmaceutical showed almost up to 50-60% of approved recombinant proteins and antibodies were produced in mammalian-based cell system. 21 HEK293T is among several cell lines derived from a human mammalian cell that used for the production of recombinant antibody. Frank Graham established human embryonic kidney 293 (HEK293) cells in 1973 at experiment number 293 by using DNA of Adenovirus 5. 22 DuBridge and the group then developed a cell line that stably transfected with the simian virus 40 (SV40) large T antigen for use in transient production process. 23 The cell line allows the episomal propagation thus enhancing the total yield of production process. 24 The cell line also resists against geneticin (G418) as result of the expression of neomycin phosphotransferase (Neo) selection marker. Therefore, stable transfection with any plasmids containing Neo is not possible with this cell line. 25 For last ten years, approximately 15% of scientific publications have used HEK cells, and the percentage will expectedly increase in the next decade. 26 The used of relevant cells in biology assessment is important to mimic the real situation of HIV-1/AIDS progression. Jurkat T cells are human acute lymphoblastic leukemic T cell line that is stably producing human interleukin 2. 27,28 The cell line derived from Fred Hutchinson Cancer Research Center and had designated as Jurkat-FHCRC. The cells were then subjected for limiting dilution to obtain Jurkat Clone E6-1 that now distributes by ATCC and NIH AIDS Reagent Program companies. 29 The cells have been popularly used for biology evaluations with nearly 17,000 Pubmed references for Jurkat from late 1980 until 2016. There are about 1,320 Pubmed literatures related with Jurkat and HIV-1 suggesting the model is a gold standard for assessment of HIV-1 replication and infectivity. HIV-1 Tat increased the CD4 receptor expression of Jurkat cells that led susceptibility to HIV-1 virus infection. 30 In fact, HIV-1 infected CD4 + T can produce up to 10x10 9 virions per day in an infected person and bear a half-life of 2.2 days. 31 In this study, anti-p24 scFv was expressed in the human mammalian-based system, and its functional activity was evaluated with the hope that antibody-based biotherapeutics can be developed as an alternative treatment against HIV-1/AIDS.

Bacterial strains and culture conditions
The E. coli strain DH5α (NEB, #C2987) was used for cloning and plasmid propagation purposes. Bacterial strains were grown aerobically in Luria-Bertani (LB) broth, or on LB agar at 30 o C, and in the presence of Ampicillin (100 µg mL -1 ). Bacterial strains were stored in glycerol (50%) -supplemented LB broth at -80°C.

General molecular biology techniques
PCR amplifications of DNA fragments intended for cloning purposes was carried out using Q5® High-Fidelity DNA Polymerase (NEB, #M0491), whereas Taq DNA Polymerase (NEB, #M0273) was used for routine colony PCR. PCR amplified or restricted DNA fragments were purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co, #740609). Plasmid DNA was purified using Wizard® Plus SV Minipreps DNA Purification System (Promega, #A1465). Vector and insert were mixed in 1:3 molar ratios (unless otherwise specified) and ligated in the presence of T4 DNA ligase (NEB, #M0202) at 4 o C for 18 h. 100 μl of E.coli DH5α as competent cells was transformed with 5 μl of vector-insert ligation mix and selected on LB agar plates containing 100 μg/mL ampicillin at 30°C overnight incubation. For colony PCR, at least ten randomly selected bacterial colonies were PCR amplified using a vector -specific and an insert -specific primer. Plasmid DNA was isolated from positive transformants that contained amplicon of expected size by PCR and then verified using DNA restriction.

Transient transfection assay X-tremeGENE HP DNA Transfection
The day before transfection, 293T cells was cultured at 37°C in 5% CO 2 to a density of about 5×10 5 cells/ml in 6-well plates in DMEM medium that contained 10% heat-inactivated FBS. The cultures were 80% confluent on the day of transfection. The scFv vector was transfected into 293T cells using the X-tremeGene or calcium phosphate. Briefly, X-tremeGENE HP DNA Transfection Reagent was used to deliver pCDNA3.3mu-IgGk-scFv 183-H12-5C plasmid into HEK293T cells. HEK293T cells were seeded at a cell density of 5 x 10 5 cells per plate onto a 60-mm tissue culture dishes in 5 mL DMEM medium containing 10% FBS the day before transfection. 5 μg of pCDNA3.3-mu-IgGk-scFv 183-H12-5C (stock 1mg/mL) plasmid was gently mixed with 500 μL of DMEM-serum free medium in 1.5 mL tube. Ten μL of X-tremeGENE HP DNA Transfection Reagent (2:1) was added directly into diluent without any contact with the wall of the tube. The complex was gently mixed by pipetting up and down. The transfection reagent: DNA complex was incubated for 15 minutes at room temperature. The transfection complex was added to the cells in a dropwise manner, and the cells were incubated for 24 and 48 hours before measuring protein expression in the culture supernatant.

Calcium Phosphate Transfection
Calcium Phosphate transfection was used to deliver pCMX2.5-scFv 183-H12-5C-hIgG1-Fc plasmid into HEK293T cells. HEK293T cells were seeded at a cell density of 5 x 10 5 cells per plate onto a 60-mm tissue culture dishes in 5 mL DMEM medium containing 10% FBS the day before transfection. Next day, medium on cells was changed three hours before beginning transfection. 2 μg of plasmid was gently mixed with 296 μL of filtered deionized water in 4 mL clear round-bottom tube by vortexing on a low setting (1000 rpm for 10 seconds). 37 μL of 2 M calcium chloride was added to the DNA/water mixture and gently mixed by vortexing on a low setting. 300 μL of 2X HBS was added into new clear round bottom tube and the DNA/water/CaCl2 mixture we dropwise added into it and vortexing on a low setting. The tube was incubated at room temperature for 30 minutes. The mixture was mixed and dropwise added to the prewarmed medium on the cells with gently swirling. The cells were incubated for 16 hours before changed with fresh medium. The culture supernatant was collected after 48, 72 and 96 hours before continuing with purification.

Neon Transfection System
For difficult transfected cells such human Jurkat T cell, introducing plasmid into this cell was done by electroporation using Neon Transfection system. The cells were washed with 1X PBS once and diluted to 3x10 6 cells/30uL in R-Buffer. 0.3x10 6 cells (10 μL) were gently mixed with pCDNA3.3-scFv 183-H12-5C (3 μg) in a sterile 0.2 mL tube. 10 μL of the mixture that represents 0.2x10 6 cells and 2 μg plasmids were used for electroporation with parameters; pulse voltage=1300, pulse width=10, and pulse number=3. The transfected cells were maintained at 0.2x10 6 cells/mL in pre-warmed RPMI 1640 growth medium and incubated at 37°C for 48 hours to allow cells to recover. The cells were used for antiviral assessment.

Purification of scFv 183-H12-5C and scFv-Fc 183-H12-5C
The Finally, the purified samples (scFv and scFv-Fc 183-H12-5C) were dialyzed against PBS. Dialysis was performed three times with an exchange of fresh PBS in each round. The dialyzed products were lyophilized and reconstituted in deionized H 2 O followed by quantification using Qubit Protein fluorometer. The purity of the eluted antibody fraction was analyzed by SDS-PAGE on 10% gels under reducing conditions. Protein bands were visualized by Coomassie blue staining. After electrophoresis, the proteins in the gel were transferred to a nitrocellulose membrane (GE), which was then blocked with 5% skim milk in TBST (TBS with 0.05% Tween 20). The membrane was incubated with a 1：10,000 dilution of HRP-conjugated goat anti-human IgG (KPL) for 1 h at room temperature and then washed three times with TBST. After that, the immunoreactive bands were visualized using an enhanced chemiluminescence detection system (ECL; Pierce Thermo).

Engineering of pCDNA3.3 -scFv 183-H12-5C
The inverse PCR of pCDNA3.3 vector resulted in the generation of vector plasmid with 5.5 kb ( Figure 1B). Amplification of insert-specific scFv 183-H12-5C from pHEN2-scFv 183-H12-5C (46) plasmid resulted in production of insert with 783 bp ( Figure 1B). Twelve colonies were randomly selected using a combination primer set and nine out of twelve were amplified around 850 bp scFv 183-H12-5C-6His ( Figure 1C). Three positive colonies were then selected and verified with SfiI and NotI restriction enzyme respectively. All clones were successfully restricted by detection of linearized plasmids as showed in Figure 1D.

Engineering of pCDNA3.3-mu-IgGk-scFv 183-H12-5C
The inverse PCR of pCDNA3.3-scFv 183-H12-5C vector resulted in the generation of vector plasmid with 6.19 kb. Oligo annealing of mouse IgG kappa chain signal peptide resulted in ligation with the purified vector that was verified by amplification of scFv 183-H12-5C-6His with 780 bp from twelve randomly transformed colonies ( Figure 2B). Five positive colonies were then selected and further restricted with BsiWI and NotI restriction enzyme respectively. Two clones were successfully restricted by detection of ~5.4 kb (pCDNA3.3) and 855 bp amplicons as showed in Figure 2C.

Expression of secreted soluble scFv 183-H12-5C and scFv-Fc 183-H12-5C
Since the MBP-scFv 183-H12-5C produced from bacteria efficiently bound the recombinant and wild-type HIV-  Figure 4A). Western blot using anti-6His was performed to confirm the expression of soluble scFv 183-12H5C. Using standard protocol, the HEK293T cells transfected with pCDNA3.3-muIgGk-scFv 183-H12-5C vectors produced soluble scFv by detecting the ~29 kDa bands. In contrast, no detection of red fluorescence protein bands was detected since the protein was not tagged with 6His ( Figure 4B). The optimizing experiment showed 0.2 μg of pCDNA3.3-muIgGk-scFv 183-H12-5C plasmid is the suitable concentration to transfect HEK293T for expression of secreted-scFv 183-H12-5C. There was no difference between the scFv collected after 24 or 48 hours of post-transfection ( Figure 4C and 4D). The structure of the scFv-Fc 183-H12-5C antibody produced in the reducing environment of HEK293T was analyzed by SDS-PAGE and immunoblot. The scFv-Fc is expected to be demonstrated as a 52 kDa monomer under the reducing conditions as the interaction of CH 3 domains breaks in the presence of a potent surfactant such as SDS. As shown in Figure 5B, detection of 52 kDa bands confirmed that the scFv-Fc 183-H12-5C was produced in HEK293T cells.

Purification of scFv 183-H12-5C and scFv-Fc 183-H12-5C
Since scFvs 183-H12-5C has a hexahistidine tag at its amino terminal. Hence, the recombinant antibodies were purified using HisPur cobalt resin. As shown in Figure  5A, a simple spin-column method led to the recovery of highly purified scFv 183-H12-5C-6His from the supernatant of HEK293T cells. The incubation of the soluble fraction at 4°C for overnight resulted in efficient binding of scFv 183-H12-5C to the resin bed. Washing buffer containing 30 mM of imidazole with six times washing steps was able to remove unbound histidine protein. The bound histidine protein then eluted for four times under acidic condition. Detection of a prominent protein band of approximately 28 kDa under reducing conditions confirmed that the scFv 183-H12-5C-6His was produced in HEK293T cells.
Since scFv-FC 183-H12-5C has a Fc portion at its amino terminal. Hence, the recombinant antibody was purified using Protein G based on Streptococcal protein G bind to human Fc fragments at β-strand 3 and the α-helix. As shown in Figure 5B, purified scFv-Fc 183-H12-5C were obtained from the supernatant of HEK293T cells using a fast protein liquid chromatography method. Almost all of the scFv-Fc 183-12H-5 protein was bound to the Protein G when the supernatant was flowed in protein G cartridge column with flow rate at 1min/mL. 12 mL wash buffer was able to remove the unrelated protein and scFv-Fc was successfully eluted under acidic condition. The purity of the purified scFv-Fc 183-H12-5C was more than 85%, and scFv-Fc was used for all subsequent immunoblot analysis.

scFv 183-H12-5C inhibits the replication-competent NL4-3 viral infectivity in Jurkat CD4+ T cell line.
To determine the effect of the recombinant antibody on HIV-1 viral replication in CD4 + T cell, multi-round infectivity assay was performed. T-cell adapted laboratory strain virus such NL4-3 can be propagated in CD4 + T cell such Jurkat. The introduction of scFv 183-H12-5C in this cell line by electroporation was hypothesized might affect the HIV viral replication. Jurkat T cells were transiently transfected with a plasmid expressing scFv 183-H12-5C by the electroporation method. Transfected cells then were infected with molecular clone of NL4-3 and the virus infectivity was monitored by MAGI infectivity assay. At MOI 0.1 with mimicking natural infection process, virus infectivity of Jurkat expressing scFv were reduced by more than 60% from day one until day 4 ( Figure 7A and 7B). Together this preliminary data showed that the scFv acted efficiently in inhibition of virus infectivity producing from CD4 + T cells.

Discussion
Many studies highlighting the use of antibody from commercial hybridoma cell (clone 183-H12-5C) in detecting the HIV-1 capsid protein p24, 32,33 however until now it has yet to be used for therapeutic purpose of HIV-1/AIDS. With the fact that monoclonal antibodies (mAbs) are highly specific and stable, it becomes an attractive candidate for in vivo treatment. Since 1992 once FDA approved the first therapeutic mAb muromonab-CD3, 61 therapeutic mAbs approved by FDA until mid-2016. 34 Although the number of approved mAbs is encouraging, most of them are limited to target surface antigens that make an intracellular protein such HIV-1 p24 is challenging. The initial part of developing antibody-based biotherapeutic is to test the antigenicity of anti-p24 mAb inside the target cells. However, antibodies do not readily permeate cell membranes, and thus the use of smaller fragment like scFv which easily expressing in the cytoplasm of mammalian is an option. Previously, the monovalent scFv antibody fragment against HIV-1 CA p24 had successfully isolated by phage display technology (data not shown). Expression of the scFv fusion in bacterial cells had shown high potential for production of functional scFv which then being used as detection reagent (data not shown).
Although there was one recombinant antibody scFv against HIV-1 CA p24 produced using bacterial-based system had been reported recently, however no antiviral assays were performed using the format in order to validate its function in HIV replication and infectivity. 35 Here the expression of scFv in the human mammalianbased system was reported and discussed for establishment of antibody-based biotherapeutic against HIV-1.
Recombinant antibodies against HIV-1 p24 were recovered under two different forms: (i) as secreted soluble scFv molecules from the supernatant of pCDNA3.1-mIgG kappa-scFv 183-H12-5C-transfected HEK293T, and (ii) as scFv-Fc in the culture medium of pCMX2.5-hIgG1-scFv 183-H12-5C-Fc-transfected HEK293T. Both forms could be used as biological tools for different purposes such as soluble scFv potentially can be used as an intracellular antibody (intrabody) and scFv-Fc as a detection reagent. Here the study showed that the format expressed in mammalian reducing environment cytoplasm of HEK293T was able to react with recombinant p24 and HIV-1 derived p24 (gag). These findings suggest that the transient expression of scFv 183-H12-5C in HEK293T cells is not toxic to the cells and functionally active. In the case of soluble scFv-Fc 183-H12-5C, the format could serve in conventional diagnostic assays for HIV-1 Gag detection, through specific recognition of the conserved CA p24 epitope. The format can replace the used of full-length IgG clone 183-H12-5C produced from hybridoma system that expensive and time consuming. Indeed, it has been reported that hybridoma cells tend to lose their mAb productivity during long-term cultivation. 36,37 Furthermore, the constant region (Fc) plays a crucial role in the structural stability of the binding site and antibody conformation. Usually, the human IgG1 hinge region comprises of CH2 and CH3 was molecularly conjugated to the C-terminus of the scFv to generate the bivalent recombinant antibody. The presence of conserved cysteine amino acids in the constant part leads to the formation of disulfide bond which eventually associates the Fc region into dimers. The resulting bivalent recombinant antibody is known as scFv-Fv closely be similar to the structure of intact IgG. 8 The modifications aimed to increase the affinity of the monovalent scFv-Fc 183-H12-5C against HIV-1 CA p24. The current results are consistent with the result obtained by Ray and group that showed a fusion of Fc region can enhance the activity of anti-GPRV scFv. 38 Moreover, a fusion of mouse origin scFv with human constant domains (CH2 and CH3) has proven to be beneficial in the clinical setting. Eliminating of the human anti-mouse antibody (HAMA) response can be simply achieved by generating the chimeric antibodies, humanized, and fully human antibodies. 39 The anti-CEA scFv-Fc (H310A) chimeric antibody exhibited a low incidence of the HAMA response. 40 These indicated that chimeric antibody is suitable for the assessment in the human clinical stage. Although the present study successfully obtained scFv-Fc 183-H12-5C, the reagent was used only for the detection analysis and not for biology assessment. The used of chimeric scFv-Fc 183-H12-5C in HIV-1 replication inhibition will provide a benefit to the research community. For a reason, the role of scFv-Fc against HIV-1 replication is under investigation. To act as an intrabody, the scFv must be expressed in the mammalian target cells. The plasmid pCDNA3.3-scFv 183-H12-5C-6His construct was transiently expressed in T cell line and the infectivity of HIV-1 was monitored. The finding revealed that intracellular expression of anti-p24 scFv resulted in the production of functionally active intracellular antibodies (intrabodies) that interacted with incoming HIV capsid and prevented cells from producing the infectious viruses. No genomic integration of the gene of scFv when this type of expression was applied. Since the scFv was expressed episomally, over time the expression of the scFv is lost. Increasing of the infectious virus after day 4 of propagation may reflect from the phenomenon. Despite that, the transient expression is very useful in this study since it directly proved that scFv construct could be expressed well in Jurkat cell and at the same time can react with NL4-3 virus which led to a decrease of infectious virus. The model system used was correlated with the report of effectiveness intrabodies against HIV-1 replication and infectivity. 14,19,20 Since the transient expression of scFv 183-H12-5C in Jurkat cell showed the potential clinical efficiency, stable expression was preferred for expressing the scFv. The correct integration that allows for scFv expression are small, and for that reason, several rounds of selective pressure in the form antibiotic supplementation needed to be applied for several weeks. However, stable production of scFv 183-H12-5C in Jurkat cells system under controlled of CMV promoter did not work. No expression of the recombinant antibody in developed cell line after selection process although the gene was still presented in the genome of the cells (data not shown). Integration of plasmid into chromosomes from cells grown under selective pressure analyzed by fluorescence in situ hybridization analysis (FISH) demonstrated that the integration constantly happened at a single site irrespective of the transfection method. 41 This finding suggests that electroporation method was not the cause of the failure in the establishment of stable expressing scFv anti-p24 Jurkat cell line. Currently, to overcome the limitation, engineering the plasmid having another promoter and reporter gene for stably expressing of scFv and transfection efficiency determination in Jurkat cells respectively is undergoing.

Conclusion
In this work, the mammalian plasmid vectors expressing anti-p24 scFv were successfully engineered. ScFv and scFv-Fc were specifically expressed in a human mammalian-based expression system. The purified scFv and scFv-Fc showed good binding activity and specificity towards recombinant p24 and HIV-1 derived p24 (gag). Intracellular expression in relevant cells also demonstrated the effectiveness of inhibiting HIV-1 replication and infectivity. The antibody could be used as candidates for the development of antibody-based biotherapeutics against HIV-1/AIDS.