{"paper_id":"42d76624-56c8-4d3a-a9ed-adefb18178ea","body_text":"Plant-Derived Recombinant Macromolecular PAP-IgG Fc as A Novel Prostate Cancer Vaccine Candidate Eliciting Robust Immune Responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plant-Derived Recombinant Macromolecular PAP-IgG Fc as A Novel Prostate Cancer Vaccine Candidate Eliciting Robust Immune Responses Yangjoo Kang, Deuk-Su Kim, Hyunjoo Hwang, Young-Jin Seo, Peter Hinterdorfer, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5286242/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Mar, 2025 Read the published version in Transgenic Research → Version 1 posted 9 You are reading this latest preprint version Abstract Prostatic acid phosphatase (PAP) is a specific protein that is highly expressed in prostate cancer. In this study, we constructed two recombinant PAP fusion genes: PAP fused to the immunoglobulin G (IgG) Fc fragment (designated PAP-Fc) and PAP-Fc fused to the endoplasmic reticulum retention sequence KDEL (designated PAP-FcK). Transgenic Nicotiana tabacum plants expressing these recombinant macromolecular proteins (MPs) were generated using Agrobacterium-mediated transformation, and the presence of both genes was confirmed through genomic PCR. Western blot analysis validated the expression of PAP-Fc and PAP-FcK MPs, which were successfully purified via protein A affinity chromatography. Size-exclusion high-performance liquid chromatography revealed dimeric peaks for PAP-Fc (PAP-Fc P ) and PAP-FcK (PAP-FcK P ). Bio-transmission electron microscopy demonstrated 'Y'-shaped protein particles resembling antibody structures. Moreover, PAP-Fc P and PAP-FcK P exhibited a high association rate with human FcγR and FcRn. Vaccination of mice with both PAP-Fc P and PAP-FcK P resulted in increased total IgG against PAP and enhanced activation of CD4 + T cells, comparable to mice immunized with PAP, which served as a positive control. These findings indicate that both plant-derived MPs can effectively induce adaptive immunity, positioning them as promising candidates for prostate cancer vaccines. Overall, plants expressing PAP-Fc and PAP-FcK represent a viable production system for antigenic macromolecule-based prostate cancer vaccines. Prostate acid phosphatase Prostate cancer Prostate specific antigen transgenic plant vaccine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Plants have been considered an efficient expression platform for the production of recombinant pharmaceutical macromolecular proteins (MPs) (Lee et al. 2023 ; Jin et al. 2023 ; Kang et al. 2023b ; Park et al. 2024 ; Lee et al. 2024 ). They make a convenient system that offers advantages such as no human pathogen contamination, cost-effective large-scale production with correct folding, and glycosylation patterns similar to eukaryotes. Improvements in expression levels using stable and transient plant expression methods make the plant-based production system promising for producing a variety of recombinant biological proteins (Kang et al. 2023a ; Oh et al. 2023 ; Twyman et al. 2003 ). Vaccination with antigens provides a possible advantage in monitoring specific vaccine-induced immune responses (Jäger et al. 2003 ). Immunization with cancer-associated antigens is a potential approach to cancer prevention and treatment (Jäger et al. 2003 ; Tagliamonte et al. 2014 ). Prostate cancer is commonly diagnosed in males in developed countries, and its incidence is rising among men under 50 years of age (Saif et al. 2014 ; Jemal et al. 2008 ). It has been reported that prostatic acid phosphatase (PAP), prostate-specific antigen, and prostate-specific membrane antigen express in both normal and cancerous prostatic tissues (Fujio et al. 2015 ; Roos et al. 2005 ; Fong et al. 2001 ; Fong et al. 1997 ). A number of targeted antigens have been clinically tested for safety and immunotherapeutic effectiveness (Tagliamonte et al. 2014 ; Geary et al. 2013 ; Tarassoff et al. 2006 ). PAP is a potential target antigen for immunotherapy; it is a prostate-specific protein overexpressed in 95% of prostate tumors. Prostate cancer therapeutic approaches based on PAP protein include DNA vaccines (McNeel et al. 2009 ), cell-based medicine (Di Lorenzo et al. 2012 ; Kawalec et al. 2012 ), and peptide antigen vaccines (Matsueda et al. 2005 ; Machlenkin et al. 2005 ). In previous studies, the fusion of immunoglobulin G (IgG) Fc to a GA733 vaccine candidate against colorectal cancer was successfully expressed in Nicotiana tabacum transgenic tobacco plants (Park et al. 2015 ; Lim et al. 2015 ; Lu et al. 2012 ). The plant-derived recombinant protein GA733-Fc fused to the endoplasmic reticulum (ER) retention signal (KDEL motif) GA733-Fc P showed immune response efficacy in animals (Lim et al. 2015 ; Lu et al. 2012 ). Recombinant IgG Fc fusion proteins may have considerable potential, including enhanced antigen uptake and vaccination processing. This occurs by targeting Fc receptors on antigen-presenting cells and enhancing their plasma half-life. Furthermore, to avoid β(1,2)-xylose (Xyl) and α(1,3)-fucose (Fuc) plant specific glycan residues, an oligomannose glycan structure of the recombinant proteins was generated by retaining the protein in the ER. Fusion of IgG Fc to KDEL (Munro and Pelham 1987 ; Lu et al. 2012 )could retain the protein inside the ER and reduce the degradation of recombinant fusion protein in plant cells, eventually enhancing the production level. This study investigated the functional expression of a PAP-IgG Fc fusion macromolecular protein as a recombinant vaccine for prostate cancer in N. tabacum transgenic tobacco plants. Materials & Methods Construction of plant expression vectors for recombinant PAP fusion proteins The PAP cDNA (GenBank accession no. M34840.1) was synthesized to fuse the Fc region of human IgG 1 (GenBank accession No. AY172957.1) to construct the PAP-Fc fusion protein, which was further tagged with the ER retention signal KDEL to generate PAP-FcK. These fusion proteins were cloned under the control of an enhanced cauliflower mosaic virus (CaMV) 35S promoter (Fig. 1 ). The PAP-Fc and PAP-FcK expression cassettes were cloned into the plant binary vector pBI121 using Hind III and Eco RI restriction enzyme sites to generate pBI PAP-Fc and pBI PAP-FcK, respectively, and transformed in competent Escherichia coli DH5α cells for amplification (Fig. 1 ). The synthetic PAP complementary DNA (cDNA) fragment sequence comprised a 30 amino acid plant ER signal peptide from N. plumbaginifolia (Lu et al. 2012 ; So et al. 2013 ; Ko et al. 2003 ). Transformation of tobacco plant The pBI PAP-Fc and pBI PAP-FcK plant expression vectors were transferred into Agrobacterium tumefaciens strain LBA4404 using electroporation. Transgenic tobacco ( N. tabacum L. cv. Xanthi) plants were generated using Agrobacterium -mediated transformation [30, 31]. The A. tumefaciens strain LBA4404 carrying pBI PAP-Fc and pBI PAP-FcK vectors and the tobacco leaf explants were cultivated in co-cultivation media [Murashige and Skoog (MS) including B5 vitamin (4.8 g·L − 1 ), sucrose (30 g·L − 1 ), plant agar (8 g·L − 1 ) (Duchefa Biochemie, Haarlem, Netherlands), 6-benzylaminopurine solution (6-BAP) (1 mg·L − 1 ), 1-naphthylacetic acid (NAA) (100 µg·L − 1 ), and acetosyringone (100 µM)] at 25°C in dark for 3 days. The tobacco leaf explants were then transferred to a regeneration medium [MS medium (4.8 g·L − 1 ), 6-BAP (1 mg·L − 1 ), NAA (100 µg·L − 1 ), acetosyringone (100 µM), cefotaxime (250 mg·L − 1 ), and kanamycin (100 mg·L − 1 )] to induce shoot and callus formation. N. tabacum transgenic tobacco plant lines were selected on MS medium containing 100 mg·L − 1 kanamycin. Confirmation of PAP-Fc and PAP-FcK transgenic lines by polymerase chain reaction (PCR) Genomic DNA was isolated from the leaves (100 mg) of transgenic and non-transgenic plants using HiYield™ Genomic DNA Mini Kit (Plant) (RBC, Taipei, Taiwan). Genomic DNA was amplified using PCR to confirm the presence of recombinant PAP-Fc (1,905 bp) and PAP-FcK (1,917 bp) genes using forward and reverse primers: forward (5′-GGGGTACCATGGCTA CTCAACGAAGGGC-3′) and reverse (5′- GGACTAGTATCTGTACTGTCCTCAG-3′) primer sets were used for PAP fragment of PAP-Fc and PAP-FcK; forward (5′-CCTACTCTGGCAGCCCATC-3′) and reverse (5′-CCATTGCTCTCCCACTCCAC-3′) primer sets were used for IgG Fc fragment of PAP-Fc and PAP-FcK; forward (5′-CCTACTCTGGCAGCCCATC-3′) and reverse (5′-TCAGAGTTCATCTTTACCCGG-3′) primer sets were used for IgG Fc fragment tagged to KDEL of PAP-FcK. The PCR was conducted as follows: 94°C for 120s, 28 cycles 94°C for 20s, 62°C for 20s, 72°C for 70s, and 72°C for 5 min. A non-transgenic (NT) plant was used as a negative control, and pBI 121 vectors containing the PAP-Fc and PAP-FcK genes were used as positive controls. Reverse transcription (RT)-PCR amplification RT-PCR was conducted to confirm the presence of PAP-Fc and PAP-FcK mRNA transcripts in transgenic plants. Total RNA was extracted from leaf samples of transgenic plants containing PAP-Fc and PAP-FcK according to an RNeasy Plant Mini Kit protocol (Qiagen, Valencia, CA, USA). Genomic DNA was removed from the isolated total RNA, and cDNA was synthesized according to the QuantiTect RT kit protocol (Qiagen, Valencia, CA, USA). Each RNA sample was used as a template for RT-PCR analysis, which was performed using a Maxime PCR Premix Kit (Intron Biotechnology, Seoul, Korea). cDNA was PCR-amplified to confirm the presence of recombinant PAP-Fc and PAP-FcK genes, using the genomic DNA PCR primer sets as described above. The RT-PCR was conducted as follows: 94°C for 120s; 30 cycles at 94°C for 20s, 56.5 ℃ for 10s, 72°C for 22s, and 72°C for 120s. The elongation factor 1-α gene ( EF-1α ) for plant growth was used as a reference gene. A NT plant leaf was used as a negative control. Western blot analysis To confirm the expression of PAP-Fc and PAP-FcK by western blotting, plant leaf (100 mg) was homogenized to extract total soluble protein in 300 µL 1× phosphate-buffered saline (PBS). Twenty microliters of the plant leaf extract samples were mixed with protein loading buffer [bromophenol blue (0.1%), glycerol (50%), 2-mercaptoethanol (5%), SDS (10%), and Tris-HCl (1 M)], run using 10% SDS-PAGE. The separated total proteins on the gel were transferred to a nitrocellulose botting membrane. Skim milk (5%) was used to block the membrane in 1×TBS-T buffer (0.005% Tween 20 1×TBS; v/v) for 2 h at room temperature (RT). The blots were incubated with either rabbit anti-human PAP antibody (Abcam, Cambridge, MA, USA) (1:5,000) or murine anti-human Fc γ antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch)(1:5,000). The blots were further incubated for 2 h at RT with goat anti-rabbit IgG antibody conjugated to horseradish peroxidase as a secondary antibody (Bethyl Laboratories, Montgomery, TX, USA) diluted in blocking buffer at 1:5,000. The specific protein was detected on an X-ray film using Clarity™ Western enhanced chemiluminescence substrate (Bio-Rad, Hercules, CA, USA). Non-transgenic tobacco plants and human recombinant PAP (PAP H ) (Sino Biological, Beijing, China) were used as negative and positive controls, respectively. Western blotting was performed more than three times to confirm the results. Purification of recombinant fusion proteins from transgenic plant leaves Purification of PAP-Fc and PAP-FcK recombinant protein was carried out as previously described (Park et al. 2015 ; Song et al. 2019 ) The tobacco leaves were homogenized in extraction buffer [15 mM EDTA (pH 8.0), 50 mM NaCl, 75 mM sodium citrate, (pH 6.7), 0.2% sodium thiosulfate, and 37.5 mM Tris-HCl (pH 7.5)] using an HMF-3250S aluminum blender (Hanil, Seoul, Korea). The leaf extracts were centrifuged (8,800 ×g, 30 min, 4°C). The supernatant was applied to Miracloth (Calbiotech, Sandiego, CA). The filtered solution was adjusted to pH 5.1 using 17.4 M glacial acetic acid (Duksan, Seoul, Korea), then centrifuged (10,200 ×g, 30 min, 4°C). The supernatant was adjusted to pH 7.0 using 3 M Tris-HCl. Subsequently, the supernatant was mixed with ammonium sulfate (Duchefa Biochemie) (8%) and incubated for 2 h at 4°C. After centrifugation (8,800 ×g, 30 min, 4°C), the supernatant was mixed with ammonium sulfate again at 40% saturation, followed by overnight incubation at 4°C and centrifugation (8,800 ×g, 30 min, 4°C). The protein pellet was dissolved in extraction buffer (1/10 of the original solution volume), and then centrifuged (10,200 ×g, 30 min, 4°C) (Park et al. 2015 ; Lim et al. 2015 ). The supernatant was filtered using a hydrophilic polyvinylidene difluoride 0.45 µm filter (Millipore, Billerica, MA, USA). PAP-Fc and PAP-FcK proteins were purified using HiTrap Protein A HP (GE Healthcare, Sweden). Elutes of plant-derived PAP-Fc and PAP-FcK proteins were dialyzed with 1×PBS (pH 7.4). The protein concentration was determined by an Epoch microplate spectrophotometer (Biotech, Winooski, VT, USA), and the purified proteins were visualized by SDS-PAGE. Aliquots of the purified proteins were stored at -80 ℃. Size-exclusion chromatography (SEC)-high-performance liquid chromatography (HPLC) SEC-HPLC was conducted to measure the molecular weight of the recombinant PAP-Fc P and PAP-FcK P fusion proteins by Agilent 1260 Infinity Quaternary LC (Agilent Technologies Inc., Santa Clara, CA, USA). PAP P , PAPK P , PAP-Fc P , and PAP-FcK P fusion proteins were separated using an Agilent Bio SEC-5 300 Å column (7.8 × 300 mm) (Agilent Technologies Inc.). 150 mM sodium phosphate buffer (pH 7.0) was applied to equilibrate the HPLC system and SEC column at a flow rate of 1 mL·min − 1 at 25°C. Plant-derived proteins were detected by monitoring the absorbance at 280 nm. AdvanceBio SEC 300 Å protein (Agilent Technologies Inc.) was used as the standard. Bio-transmission electron microscopy (Bio-TEM) Bio-TEM analysis was performed to analyze the structures of the purified PAP-Fc P and PAP-FcK P fusion proteins (dialyzed with 1×PBS). The negative staining of proteins was conducted at the Korea Basic Science Institute (KBSI), Korea. The protein solution (5 µL) was applied on to glow-discharged carbon-coated copper grids. The proteins were absorbed for 2 min and blotted off on Whatman filter paper (Sigma Aldrich, St. Louis, MO, USA). Then, uranyl acetate [2% (w/v)] ware treated to stain the proteins on the grids. The plant-derived recombinant proteins were imaged by a Tecnai G2 Spirit Twin fitted with an anti-contaminator (FEI, Hillsboro, OR, USA) operating at 120 kV. Images were recorded a CCD camera (4K94K Ultrascan 895) (Gatan Inc., Pleasanton, CA, USA) was used to record images at a magnification of 52,000. Surface plasmon resonance (SPR) analysis to confirm Fc gamma (Fcγ) receptor binding activity of PAP-Fc P and PAP-FcK P The affinity of PAP-Fc P and PAP-FcK P toward the Fcγ receptor (FcγR) was confirmed by SPR analysis at 25 ℃ using a general layer compact (GLC) chip on a ProteOn XPR36 SPR biosensor (Bio-Rad Labs, Hercules, CA, USA). For measuring affinity, the GCL sensor chip was used to immobilize 1,000–1,500 relative units (RU) of FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), and FcγRIIIb (CD16b) (Sino Biological, Beijing, China) in sodium acetate buffer (pH 5.0–5.5). The purified PAP-Fc P and PAP-FcK P proteins (1 µg) were dissolved in 300 µL of 1×PBS and applied to the immobilized Fcγ receptors at a flow rate of 30 µL·min − 1 at 25°C and pH 6.0. After each measurement, phosphoric acid buffer (pH 6.0) and 1×PBST buffer (0.135 mM KCl, 0.075 mM KH 2 PO 4 , 7 mM NaCl, 0.325 mM Na 2 HPO 4 .2H 2 O, and 0.05% Tween 20) (pH 7.4) were applied to the surface of the GLC sensor chip for its regeneration. Immunization of mice with PAP H , PAP-Fc P , PAP-FcK P proteins Eight-week-old male BALB/c mice (five per group) (Japan SLC, Inc. Hamamatsu, Shizuoka, Japan) were intraperitoneally (i.p.) injected with PAP H (Sino Biological, Beijing, China), PAP-Fc P , and PAP-FcK P in the presence of an adjuvant (aluminum hydroxide, Sigma, St. Louis, MO, USA). PAP fusion proteins (10 µg) were thrice injected to mice at two-week intervals for six weeks. Retro-orbital sinus bleeding method was used to collect blood samples before injection and 10 days after the second immunization. Retro-orbital plexus bleeding method was used for blood collection at 10 days after the third immunization [26]. After bleeding, the spleens were harvested (Fig. 5 ). All animal experiments for this were conducted by the Animal Ethics Committee (ACE) (#2017-00087) of Chung-Ang University, Korea. Enzyme-linked immunosorbent assay to quantify PAP-specific IgG PAP-specific IgG antibodies induced by PAP-Fc P and PAP-FcK P proteins were evaluated using ELISA. ELISA plates were coated with 0.5 µg·mL − 1 of PAP H antigen (Sino Biological, Beijing, China) and treated with 4–5 fold serial dilutions of mouse sera for the endpoint of the experiment. Goat anti-murine antibodies conjugated to horseradish peroxidase was used to detect PAP-specific whole IgG and IgG 1 responses (Jackson ImmunoResearch). Isolation of splenocytes The immunized mice were sacrificed to collect spleens for splenocytes. The homogenized spleen was passed through 70 µm cell strainer (SPL Life Sciences Co. Ltd., Seoul, South Korea). The spleen was treated with ammonium-chloride-potassium lysis buffer (1 mL). The spleen was homogenized using a 1 mL syringe plunger. To eliminate red blood cells, the released cells were incubated with ACK lysis buffer for 5 min at RT and washed twice with 10 mL 1×PBS. The released cells were centrifuged to collect pellets (680 ×g, 5 min, 4°C). The pellets were dissolved using Hyclone RPMI 1640 media (GE Healthcare, Little Chalfont, UK). Fat was removed by filtration using a strainer. 100 µL cell suspensions (2×10 6 cells/well) were placed on 96-well round bottom plates. Surface staining and flow cytometry Cell suspensions isolated from the spleen were centrifuged at 680 ×g for 5 min at RT and then resuspended in 200 µL/well of fluorescence-activated cell sorting buffer [1×PBS with 1% bovine serum albumin (BSA), 2 mM EDTA, and 10% sodium azide]. Cell suspensions were stained using FACS buffer containing murine-specific antibodies (1:200) directly conjugated to CD4 (PerCP/Cyanine7, Tonbo Bioscience, San Diego, CA, USA) and CD8a (APC, Tonbo Bioscience, San Diego, CA, USA) for 20 min at 4°C. Cell suspensions were then washed twice with 200 µL/well FACS buffer, resuspended in the same buffer, and transferred to a 1.5 mL micro tube for flow cytometry analysis. Results Generation of transgenic tobacco plants expressing PAP-Fc and PAP-FcK Agrobacterium -mediated transformation was applied to obtain transgenic plants using the expression vectors pBI PAP-Fc and pBI PAP-FcK (Fig. 1 . Left and Right). The PAP-Fc and PAP-FcK fusion genes placed downstream of the untranslated leader sequence from the alfalfa mosaic virus were controlled by the enhanced CaMV 35S promoter (E/35S-P) (Fig. 1 ). Among the regenerated plants, genomic DNA was isolated from three randomly selected transgenic lines for each type of expression vector (Fig. 2 A). The existence of PAP, IgG Fc fragment, and KDEL DNA sequences in PAP-Fc and PAP-FcK in the transgenic tobacco plant leaf tissues was confirmed by PCR using specific primers (Fig. 2 A). The transgenic plant lines with PAP-Fc and PAP-FcK showed the expected amplified bands at 1,173 bp (PAP), 1,185 bp (IgG Fc), and 1,905 bp (KDEL), respectively (Fig. 2 A). Likewise, semi-quantitative RT-PCR confirmed the mRNA expression of PAP, IgG Fc fragment, and KDEL of PAP-Fc and PAP-FcK in three randomly selected transgenic tobacco plants, and RT-PCR products of the expected sizes were detected at 199 bp (PAP), 224 bp (IgG Fc), and 166 bp (KDEL), respectively (Fig. 2 B). None of the PAP-Fc or PAP-FcK transgenes were detected in non-transgenic plants (Fig. 2 ). The EF-1α band as a internal control was observed at 67 bp (Fig. 2 B). Expression and purification of PAP-Fc and PAP-FcK in transgenic tobacco plants The expression of PAP-Fc and PAP-FcK in transgenic tobacco plants was confirmed by a protein band of 75 kDa in western blotting, (Fig. 2 CD). PAP H (45 kDa) as a positive control was detected using the anti-PAP antibody (Fig. 2 C), and the heavy chain of human IgG antibody as a positive control for IgG Fc was detected as a 50 kDa band with the anti-human Fcγ antibody (Fig. 2 D). In PAP-Fc and PAP-FcK proteins, protein bands of 45, 75, and 240 kDa were detected by anti-human PAP antibody (Fig. 2 C). Similarly, after treatment with anti-human Fcγ antibody, protein bands at 75 kDa and > 240 kDa were detected; however, protein band of 45 kDa was not observed (Fig. 2 D). PAP-Fc and PAP-FcK were expressed in tobacco plants and purified by protein A chromatography. The yields of PAP-Fc and PAP-FcK proteins were 10 and 15 mg·kg − 1 of fresh weight of tobacco plant leaves, respectively. In addition, SDS-PAGE showed expected molecular weights of 70.7 kDa and 71.2 kDa for PAP-Fc and PAP-FcK, respectively (Fig. 2EF, respectively) Molecular weight profiling and Bio-TEM images of plant-derived recombinant fusion proteins The PAP-Fc P and PAP-FcK P fusion proteins were analyzed by SEC-HPLC (Fig. 3 ). PAP P and PAPK P showed one protein peak at ~ 5.4 min (Fig. 3 B and C, PAP), whereas PAP-Fc P and PAP-FcK P showed two protein peaks (~ 5.4 and 7.45 min) (Fig. 3 D and E). The retention time for PAP-Fc P and PAP-FcK P was compared to those of the standard molecular weight proteins (Fig. 3 A), which indicated an increased molecular weight of PAP-Fc P and PAP-FcK P than expected. Bio-TEM analysis of plant-derived PAP-Fc P and PAP-FcK P showed that both the proteins were observed as 'Y' shape particles, similar to the antibodies (Fig. 3 FG, respectively). Binding affinity of PAP fusion proteins to Fcγ receptors The binding affinity activities of plant-derived PAP-Fc and PAP-FcK fusion proteins to Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) were observed by SPR analysis. The assay was performed with the receptors (1000–1500 RU) immobilized on the chip, and the proteins were injected into the fluid phase at 50 nM (Fig. 4 ). PAP-Fc and PAP-FcK showed high affinity for FcγRI (CD64), and neither protein dissociated on the surface during injection (Fig. 4 A). FcγRIIIa (CD16) showed a relatively high affinity at 250s after injection, with little dissociation (Fig. 4 D). In contrast, the fusion proteins bound weakly to FcγRIIa (CD32a) and could not bind to both FcγRIIb and FcγRIIIb (Fig. 4 B, C, and E). IgG response in mice immunized with PAP-Fc and PAP-FcK fusion proteins The immunogenicity of the PAP-Fc P and PAP-FcK P proteins was investigated in BALB/c mice, with blood samples collected to analyze the serum IgG response following immunization (Fig. 5 A). To confirm the induction of anti-EpCAM IgGs in the mice immunized with PAP-Fc P and PAP-FcK P recombinant proteins, ELISA analysis was performed on sera collected 10 days after the second and third immunizations (Fig. 5 A, B, and C). In the sample from the first blood draw after the second injection of PAP H , PAP-Fc P , and PAP-FcK P , the signal was undetectable (Fig. 5 B ‘1st’). However, in the second blood draw after the third immunization, increased optical density (OD) values were observed in all groups of mice, except those immunized with 1×PBS (Fig. 5 B ‘2nd’). Notably, both PAP-Fc P and PAP-FcK P proteins induced IgG responses similar to those elicited by PAP H (Fig. 5 C). Overall, the plant-derived PAP-Fc fusion proteins demonstrated comparable induction of anti-PAP IgG responses, similar to PAP H . T-cell responses in mice immunized with PAP-Fc fusion proteins Flow cytometry analysis was conducted to confirm activation of the CD8 and CD4 + T To investigate CD4 + helper and CD8 + cytotoxic T cell responses to PAP-Fc P or PAP-FcK P fusion proteins, we examined the activation state of splenic CD4 + and CD8 + T lymphocytes in lymphocytes isolated from splenocytes of the mice immunized with PAP H , PAP-Fc P , and PAP-FcK P at 10 days after the third immunization (Figs. 5 D, E, F, and G). The expression level of CD69, a T-cell activation marker, on CD4 + and CD8 + T cells was analyzed using the gating strategy. Immunization with commercial PAP H slightly increased the expression of CD69 on CD4 + T cells [mean fluorescence intensity (MFI): 135] compared to that on the negative control (1×PBS-treated group; MFI: 131) (Fig. 5 D and E). However, immunization with PAP-Fc P (MFI: 181) or PAP-FcK P (MFI: 201) markedly increased the expression of CD69 in CD4 + T cells (Figs. 5 D and E). In CD8 + T cells, a slight increase in CD69 expression was observed in all mouse groups (immunized with PAP H , PAP-Fc P , and PAP-FcK P ) than in the PBS-treated group (Fig. 5 F and G). Collectively, these results suggest that immunization with PAP-Fc P or PAP-FcK P fusion proteins efficiently induces CD4 + and CD8 + T cell-mediated immune responses. Discussion We demonstrated the potential of a vaccination approach against prostate cancer using the PAP antigen-IgG Fc fusion protein expressed in transgenic plants. In previous studies, the cancer vaccine candidates fused to the Fc fragment have been successfully expressed and assembled to induce immunogenicity in mice (Lim et al. 2015 ; Lu et al. 2012 ; Shin et al. 2019 ; Kang et al. 2017 ; Kang et al. 2016 ). In the current study, PAP-Fc and PAP-FcK fusion proteins were expressed in transgenic N. tabaccum plants and their purified forms were demonstrated as immunogenic candidate cancer vaccines in mouse experiments. The feasible expression and immune function of the recombinant protein are essential for the development of a plant production system for vaccine candidates (Lee and Ko 2017 ; Kamo et al. 2012 ) In plant expression systems, recombinant protein expression in the extracellular space involves initial protein entry into the ER by a signal peptide and passing through the Golgi complex in plant cells. In the ER, the recombinant proteins are folded, assembled, and glycosylated. The glycosylated protein them moves to the Golgi apparatus and secreted outside (Pagny et al. 1999 ; Kermode 1996 ). To enhance the accumulation of recombinant proteins in plants, subcellular targeting to ER is effective in retaining their biological characteristics (So et al. 2013 ; Song et al. 2018 ; Pires et al. 2012 ). In this study, two different transgenic tobacco plant lines expressing PAP-Fc and PAP-FcK were generated to investigate the effect of the Fc fragment and KDEL ER retention signal on PAP expression and its biological activity. Each transgenic plant with PAP-Fc and PAP-FcK was grown in vivo to investigate the transgene insertion and protein expression. PCR and RT-PCR revealed that all randomly tested plants had PAP-Fc and PAP-FcK transgenes insertion and mRNA transcripts of the transgenes. Western blot analysis confirmed the protein expression of PAP-Fc and PAP-FcK in transgenic plants. Protein band of 75 kDa was detected, and the molecular weight was similar to that predicted for the fusion of PAP (50 kDa) and IgG Fc fragment (25 kDa). PAP-FcK transgenic plants showed higher fusion protein expression than PAP-Fc transgenic plants. In PAP-FcK protein fused to the ER retention signal peptide KDEL, the protein was retained in the ER resulting in increased accumulation. This result is consistent with previous studies where the expression of recombinant proteins tagged to KDEL could be enhanced in transgenic plants (Kang et al. 2016 ; Song et al. 2018 ; Gomord et al. 2005 ; Schouten et al. 1996 ). Both transgenic plant lines expressing PAP-Fc and PAP-FcK showed > 240 kDa and 45 kDa protein bands. The > 240 kDa protein band could be due to the dimerization of the PAP-Fc and PAP-FcK monomers. The IgG Fc fragment can become dimeric (Park et al. 2015 ; Lim et al. 2015 ; Shin et al. 2019 ) .In addition, the 45 kDa protein bands were detected with anti-PAP but not with anti-Fc, suggesting that the 45 kDa band corresponds to PAP protein. These results indicate that the PAP-Fc and PAP-FcK fusion proteins are cleaved into PAP and Fc fragments. However, it is currently difficult to explain why the anti-Fc antibody did not detect the Fc fragment in western blotting. It is speculated that the Fc fragment itself cannot be recognized by the anti-Fc antibody. Both PAP-Fc and PAP-FcK proteins were purified from plant leaves and analyzed using SEC-HPLC and EM. SEC-HPLC indicated the presence of a protein peak corresponding to dimers in PAP-Fc P and PAP-FcK P . In addition, EM analysis confirmed the dimerization of PAP-Fc P and PAP-FcK P . However, the polymeric molecular weight peaks of PAP-Fc P and PAP-FcK P at unexpected larger sizes may result from multiple glycan residues or protein aggregation in the HPLC system. In Bio-TEM, the aggregation formation was more identified in PAP-Fc P compared to PAP-FcK P . It is speculated that high mannose type glycans in PAP-FcK P could reduce the aggregation of glycoproteins compared to PAP-Fc P , which harbors more plant-specific type glycans and less high mannose type glycans (Schaefer and Plückthun 2012 ). The capacity of the PAP-Fc P and PAP-FcK P fusion proteins to bind complement component 1q (C1q) was analyzed using ELISA (Data not shown). The Fc fragment can bind to C1q to initiate assembly of the complement membrane attack complex (MAC) to kill target cells (complement-dependent cytotoxicity) (Yang et al. 2018 ; Walport 2001 ). PAP-Fc P and PAP-FcK P proteins could bind to C1q in a concentration-dependent manner, forming immune complexes. These in vitro assay results demonstrated the binding activity of the PAP-Fc P and PAP-FcK P recombinant fusion proteins to C1q, indicating their potential for immunogenicity. Immune leukocytes exhibiting Fcγ receptors (FcγRs) on their surface membrane are recruited and activated by binding the IgG Fc fragment to FcγRs. The binding triggers antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated phagocytosis to kill and remove target cells (Yang et al. 2018 ; Woof and Burton 2004 ; Ravetch and Bolland 2001 ). We investigated whether PAP-Fc and PAP-FcK proteins showed a stronger association level for FcγRs (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb). Both PAP-Fc and PAP-FcK bound with high association level to human FcγRI, with slight differences. In addition, the fusion proteins could bind to FcγIIIa, but showing a slightly lower association level for FcγRIIa; however, the binding was poor for both FcγRIIb and FcγRIIIb. PAP-FcK P showed a higher association than PAP-Fc P but similar to PAP-Fc P . Further, we investigated the ability of fusion proteins to interact with neonatal Fc receptor (FcRn), which plays a role in maintaining the long half-life of antibodies in circulation. The PAP-Fc P and PAP-FcK P bound with a higher association level to human FcRn, and binding occurred only at a pH of 6.0, similar to the previous studies (Mekhaiel et al. 2011 ; Andersen et al. 2010 ). Overall, these results indicate that PAP-Fc P and PAP-FcK P exhibit a high association level for human FcγR and FcRn. Moreover, the association levels of PAP-Fc P and PAP-FcK P for FcRn and FcγRs were not markedly different. The recombinant PAP-Fc P and PAP-FcK P proteins induced antibody responses in mice, as demonstrated by the total IgG levels. The proteins were found to have a much higher IgG 1 response, suggesting a Th2 bias. Therefore, plant-derived recombinant proteins were immunogenic in mice; however, IgG 1 responses were quantitatively different. Notably, PAP-Fc P and PAP-FcK P proteins induced relatively similar responses to PAP H in mice, which possibly explained by Fc receptors or cross-reactivity to immune complex-capturing mechanisms in mice. In addition, these proteins elicited CD4 + and CD8 + T cell responses in mice. Indeed, CD4 + helper T cells regulate B cells to produce antibodies, including anti-tumor antibodies (Kim and Cantor 2014 ; Lai et al. 2011 ). An effective therapeutic cancer vaccine should generate T-cell responses (Kartikasari et al. 2018 ; Westdorp et al. 2014 ). The activities of CD4 + and CD8 + cells were determined by expression of CD69, which is significantly expressed on T-cells upon (Ziegler et al. 1994 ). PAP-Fc P and PAP-FcK P proteins induced the activity of CD4 + T cells rather than CD8 + T cells. Specifically, the cellular immune responses were more efficiently induced in the PAP-Fc P and PAP-FcK P proteins than PAP H , most probably due to the efficient binding to the Fc receptors. Proliferation and activation of effector CD4 + T cells are more crucial for induction of immune-mediated tumor control by cancer vaccines than CD8 + T cells (Kim and Cantor 2014 ; Melssen and Slingluff 2017 ; Kumai et al. 2017 ). CD4 + T cells promote anti-cancer immunity through various mechanisms, including T-cell activation, T-cell homing enhanced, antigen-presentation co-stimulation, effector function (Melssen and Slingluff 2017 ). Taken together, the PAP-FcK P protein was immunogenically more active than the PAP-Fc P protein. These results suggest that the efficacy of the vaccine could be increased due to the oligo-mannose glycan structure of ER retained proteins in plants (Lu et al. 2012 ; Schouten et al. 1996 ; Wandelt et al. 1992 ). Overall, plant-derived PAP-Fc and PAP-FcK proteins are immunogenic in mice and induce cellular immunity, which is required to prevent prostate cancer. Therefore, these plant-derived proteins can be potential candidates to develop vaccines for prostate cancer prevention. Declarations Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgements This research was funded by the National Research Foundation of Korea grant [RS-2024-00409554]. Funding None Author information Department of Medicine, Medicine of College, Chung-Ang University, 06974, Seoul, South Korea Yangjoo Kang, Deuk-Su Kim, Hyunjoo Hwang & Kisung Ko Department of Life Science, Chung-Ang University, 06974, Seoul, South Korea Young-Jin Seo Department of Applied Experimental Biophysics, Johannes Kepler University Linz, 4040, Linz, Austria Peter Hinterdorfer C ontributions Conceptualization: Yangjoo Kang, Deuk-Su Kim, Young-Jin Seo, and Kisung Ko; methodology: Yangjoo Kang, Deuk-Su Kim, and Hyunjoo Hwang; validation: Yangjoo Kang, Young-Jin Seo, and Kisung Ko; formal analysis: Yangjoo Kang, Young-Jin Seo, and Kisung Ko; investigation: Yangjoo Kang, Deuk-Su Kim, and Hyunjoo Hwang; writing—original draft preparation Yangjoo Kang, Deuk-Su Kim, and Kisung Ko; writing—review and editing: Yangjoo Kang, Young-Jin Seo, and Kisung Ko; supervision, Kisung Ko; project administration, Kisung Ko; funding acquisition, Kisung Ko. All authors read and approved the manuscript. Corresponding author Correspondence to Kisung Ko Ethics declarations Conflict of interests The authors declare that they have no competing financial interest References Andersen JT, Daba MB, Berntzen G, Michaelsen TE, Sandlie I (2010) Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding. J Biol Chem 285 (7):4826-4836. doi:10.1074/jbc.M109.081828 Di Lorenzo G, Ferro M, Buonerba C (2012) Sipuleucel-T (Provenge®) for castration-resistant prostate cancer. Bju Int 110 (2b):E99-E104. doi:10.1111/j.1464-410X.2011.10790.x Fong L, Brockstedt D, Benike C, Breen JK, Strang G, Ruegg CL, Engleman EG (2001) Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy. 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Cite Share Download PDF Status: Published Journal Publication published 26 Mar, 2025 Read the published version in Transgenic Research → Version 1 posted Editorial decision: Revision requested 15 Nov, 2024 Reviews received at journal 15 Nov, 2024 Reviewers agreed at journal 15 Nov, 2024 Reviews received at journal 04 Nov, 2024 Reviewers agreed at journal 21 Oct, 2024 Reviewers invited by journal 21 Oct, 2024 Editor assigned by journal 21 Oct, 2024 Submission checks completed at journal 20 Oct, 2024 First submitted to journal 17 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5286242\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":368878491,\"identity\":\"07576a8a-a2a9-429b-9532-54bc684a5e63\",\"order_by\":0,\"name\":\"Yangjoo Kang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yangjoo\",\"middleName\":\"\",\"lastName\":\"Kang\",\"suffix\":\"\"},{\"id\":368878492,\"identity\":\"6e4f8459-3f10-48e0-ab45-2abf37c13409\",\"order_by\":1,\"name\":\"Deuk-Su Kim\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Deuk-Su\",\"middleName\":\"\",\"lastName\":\"Kim\",\"suffix\":\"\"},{\"id\":368878493,\"identity\":\"3f07d993-ca1d-448b-9600-6442c2f00f48\",\"order_by\":2,\"name\":\"Hyunjoo Hwang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hyunjoo\",\"middleName\":\"\",\"lastName\":\"Hwang\",\"suffix\":\"\"},{\"id\":368878494,\"identity\":\"2e0e089e-bef1-433a-bf4a-f424ba5d0c1f\",\"order_by\":3,\"name\":\"Young-Jin Seo\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Young-Jin\",\"middleName\":\"\",\"lastName\":\"Seo\",\"suffix\":\"\"},{\"id\":368878495,\"identity\":\"570039db-4b10-4d62-a07b-13c690d8bfa7\",\"order_by\":4,\"name\":\"Peter Hinterdorfer\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Johannes Kepler University Linz\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Peter\",\"middleName\":\"\",\"lastName\":\"Hinterdorfer\",\"suffix\":\"\"},{\"id\":368878496,\"identity\":\"85b9159d-e7bf-4fe6-ac35-d03e95d0c563\",\"order_by\":5,\"name\":\"Kisung Ko\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACCQY2BoYPUA5jA7FaGGeQrIWZhyQtkv3HEh/b1NglNrAffsA4cw8RWqQl0g4b5xxLTmzgSTNg3PCMCC1yEuxt0rkNzIkNDDkMjA8OEKOF/3ibtGVDfWID/xsitUgzpB2TZmw4nNggAbRlAzFaJGekJRv2HDtu3CbxzODgDGK0SJw/ZvjgR021bD9/8sOHPcRogQNgImAgScMoGAWjYBSMAjwAAEXtMyJA1j5OAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Kisung\",\"middleName\":\"\",\"lastName\":\"Ko\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-10-18 04:08:12\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-5286242/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-5286242/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s11248-025-00433-0\",\"type\":\"published\",\"date\":\"2025-03-26T15:57:12+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":67884408,\"identity\":\"18f8985c-ba76-4aa8-ab10-06f6c02a2a55\",\"added_by\":\"auto\",\"created_at\":\"2024-10-30 17:50:59\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":366232,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSchematic diagram of the plant expression vectors and expected recombinant protein and glycan structures. \\u003c/strong\\u003eEach gene expression cassette of PAP-Fc and PAP-FcK fusion proteins was incorporated into the plant expression vector for Agrobacterium-mediated plant transformation. The components include: E/35S-P, the Cauliflower mosaic virus (CaMV) 35S promoter with upstream enhancer duplication; A, untranslated leader sequence of Alfalfa Mosaic Virus RNA4; K; the ER retention signal peptide, (KDEL, Lys-Asp-Glu-Leu); NOST, terminator of CaMV 35S gene. \\u003cem\\u003eIn vitro\\u003c/em\\u003e plant tissue (Top) and transgenic plants (Bottom) expressing PAP-Fc and PAP-FcK proteins.\\u003cem\\u003e N\\u003c/em\\u003e-glycan structures of PAP-Fc and PAP-FcK recombinant proteins. The expected protein structures of the PAP-Fc and PAP-FcK fusion proteins are described to highlight their configurations and functionalities. Glycan structures are represented as: black square, \\u003cem\\u003eN\\u003c/em\\u003e-acetylglucosamine; white circle, mannose; diamond with a dot inside, a(1,3)-fucose; and white triangle, b(1,2)-xylose.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5286242/v1/7a18078cc381c9e6f83d71cf.png\"},{\"id\":67884401,\"identity\":\"8dbd51f7-a853-4866-98a6-d49da2f41d30\",\"added_by\":\"auto\",\"created_at\":\"2024-10-30 17:50:59\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":376840,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePolymerase chain reaction (PCR), reverse transcription (RT-PCR), and Western blot analyses of PAP-Fc, and PAP-FcK recombinant proteins in transgenic plants. (A)\\u003c/strong\\u003e PCR amplification of PAP-Fc (1,905 bp) and PAP-FcK (1,917 bp) from genomic DNA of transgenic plants. +\\u003csub\\u003e1\\u003c/sub\\u003e, PAP-Fc expression vector as a positive control; +\\u003csub\\u003e2\\u003c/sub\\u003e, pBI PAP-FcK expression vector as a positive control. (B) RT-PCR of recombinant genes in plant leaf. Total mRNA was isolated from leaves of three randomly selected PAP-Fc and PAP-FcK transgenic plants. -, non-transgenic tobacco plant as a negative control. The elongation factor 1-α (EF-1α) was used as a housekeeping gene. (C and D) Western blot analysis to confirm expression of PAP-Fc and PAP-FcK recombinant proteins in transgenic tobacco plants. A protein band of 75 kDa was obtained in both PAP-Fc and PAP-FcK transgenic plants. +\\u003csub\\u003e1\\u003c/sub\\u003e, 1\\u003csup\\u003est\\u003c/sup\\u003e positive control human cell-derived PAP; +\\u003csub\\u003e2\\u003c/sub\\u003e, 2\\u003csup\\u003end\\u003c/sup\\u003e positive control human immunoglobulin G (IgG); -, non-transgenic plant as a negative control; Soluble proteins were detected using (C) rabbit anti-human PAP antibody and (D) mouse anti-human IgG Fc gamma (Fcγ) antibody. Twenty microlitres of recombinant PAP-Fc and PAP-FcK proteins extracted from leaf samples were loaded for western blot analysis. SDS-PAGE of purified PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e (E) and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e (F) recombinant proteins from transgenic plants. F1 to F8, fraction sample numbers; TSP, total soluble protein.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5286242/v1/44bff4752b5b5ad5aabb66bb.png\"},{\"id\":67884407,\"identity\":\"76a1df5d-c951-4b26-a460-9d4c7ffdd937\",\"added_by\":\"auto\",\"created_at\":\"2024-10-30 17:50:59\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":605004,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSize exclusion-high-performance liquid chromatography (SEC-HPLC) and Bio-transmission electron microscopy (Bio-TEM) of plant-derived recombinant PAP\\u003c/strong\\u003e\\u003csup\\u003e\\u003cstrong\\u003eP\\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003e, PAPK\\u003c/strong\\u003e\\u003csup\\u003e\\u003cstrong\\u003eP\\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003e, PAP-Fc\\u003c/strong\\u003e\\u003csup\\u003eP\\u003c/sup\\u003e\\u003cstrong\\u003e, and PAP-FcK\\u003c/strong\\u003e\\u003csup\\u003eP\\u003c/sup\\u003e\\u003cstrong\\u003e proteins. \\u003c/strong\\u003eThe protein molecular weights was estimated using indicated retention times based on AdvanceBio SEC 300Å Protein Standard (A). SEC-HPLC chromatography was performed to determine the molecular weight of recombinant proteins (B) PAP\\u003csup\\u003eP\\u003c/sup\\u003e, (C) PAPK\\u003csup\\u003eP\\u003c/sup\\u003e, (D) PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, and (E) PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e. The PAP\\u003csup\\u003eP\\u003c/sup\\u003e and PAPK\\u003csup\\u003eP\\u003c/sup\\u003e proteins were used to compare the molecular weights of PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e fusion proteins. Bio-transmission electron microscopy (Bio-TEM) images of recombinant proteins (F) PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and (G) PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e. Bio-TEM was performed to analyze the protein structures, and the white dot circle with the number randomly indicated the protein structure as Y-shape monomers.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5286242/v1/298425d23d76e88f9f1553d3.png\"},{\"id\":67884405,\"identity\":\"4ac0c145-3b92-464c-8b6e-b62f41167e6b\",\"added_by\":\"auto\",\"created_at\":\"2024-10-30 17:50:59\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":336185,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSurface plasmon resonance analysis to confirm interaction between plant-derived PAP-Fc and PAP-FcK proteins, and human Fc gamma receptor.\\u003c/strong\\u003e Binding capacity (avidity) [1,000-1,500 relative units (RU)] of PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e to immobilized (A) FcγRⅠ (CD64), (B) FcγRⅡa (CD32a), (C) FcγRⅡb (CD32b), (D) FcγRⅢa (CD16a), and (E) FcγRⅢb (CD16b) receptors.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5286242/v1/9178226ec51f713209b15436.png\"},{\"id\":67884402,\"identity\":\"9e1a6349-efcf-4034-a6a0-e4af90791b46\",\"added_by\":\"auto\",\"created_at\":\"2024-10-30 17:50:59\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":452898,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePAP-specific IgG immune response and T cell responses of mice injected with recombinant PAP-Fc\\u003c/strong\\u003e\\u003csup\\u003eP\\u003c/sup\\u003e\\u003cstrong\\u003e and PAP-FcK\\u003c/strong\\u003e\\u003csup\\u003e\\u003cstrong\\u003eP \\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003eproteins\\u003c/strong\\u003e. (A) Animal experimental design for mouse immunization of 1X PBS, PAP\\u003csup\\u003eH\\u003c/sup\\u003e, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e. Eight-week-old female BALB/c mice were immunized intraperitoneally (i.p.) at two-week intervals with 10 μg of PAP\\u003csup\\u003eH\\u003c/sup\\u003e, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e, along with a control group receiving 1X PBS. Blood samples were collected via retro-orbital bleeding: the first set 10 days after the second immunization and the second set 10 days after the third immunization. Spleens were harvested concurrently with the second blood sample collection. (B) Kinetics of anti-PAP IgG response in the immunized mice. The anti-PAP IgG response kinetics in the immunized mice are shown as mean ± standard error (SE) for each group. (C) Endpoint Titer for anti-PAP IgG. Endpoint titers for anti-PAP IgG were determined from pooled sera of five immunized mice, combined in equal ratios. (D-G) Expression of CD69 in T Cells. CD4+ and CD8+ T cells were isolated from the spleens of mice immunized with 1X PBS, PAP\\u003csup\\u003eH\\u003c/sup\\u003e, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e. Mean fluorescence intensity (MFI) values for CD69 expression on CD4\\u003csup\\u003e+\\u003c/sup\\u003e (D and E) and CD8\\u003csup\\u003e+\\u003c/sup\\u003e (F and G) T cells were measured using flow cytometry histograms. Statistical significance was evaluated by Student’s t-test comparing each group with the control group. Asterisks denote the following significance levels: *p \\u0026lt; 0.05 and **p \\u0026lt; 0.01.\\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5286242/v1/0626f03a40900eedef70c7fc.png\"},{\"id\":79604933,\"identity\":\"fad27461-8534-47c8-80e8-4b83c9fb18e7\",\"added_by\":\"auto\",\"created_at\":\"2025-03-31 16:09:18\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3629220,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5286242/v1/845c6c04-b8c2-4a2d-b3a4-f191d93a0bf4.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"\\u003cp\\u003e\\u003cstrong\\u003ePlant-Derived Recombinant Macromolecular PAP-IgG Fc as A Novel Prostate Cancer Vaccine Candidate Eliciting Robust Immune Responses\\u003c/strong\\u003e\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003ePlants have been considered an efficient expression platform for the production of recombinant pharmaceutical macromolecular proteins (MPs) (Lee et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Jin et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Kang et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2023b\\u003c/span\\u003e; Park et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Lee et al. \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). They make a convenient system that offers advantages such as no human pathogen contamination, cost-effective large-scale production with correct folding, and glycosylation patterns similar to eukaryotes. Improvements in expression levels using stable and transient plant expression methods make the plant-based production system promising for producing a variety of recombinant biological proteins (Kang et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2023a\\u003c/span\\u003e; Oh et al. \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Twyman et al. \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eVaccination with antigens provides a possible advantage in monitoring specific vaccine-induced immune responses (J\\u0026auml;ger et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e). Immunization with cancer-associated antigens is a potential approach to cancer prevention and treatment (J\\u0026auml;ger et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Tagliamonte et al. \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). Prostate cancer is commonly diagnosed in males in developed countries, and its incidence is rising among men under 50 years of age (Saif et al. \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Jemal et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). It has been reported that prostatic acid phosphatase (PAP), prostate-specific antigen, and prostate-specific membrane antigen express in both normal and cancerous prostatic tissues (Fujio et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Roos et al. \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Fong et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e; Fong et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e1997\\u003c/span\\u003e). A number of targeted antigens have been clinically tested for safety and immunotherapeutic effectiveness (Tagliamonte et al. \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Geary et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Tarassoff et al. \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e). PAP is a potential target antigen for immunotherapy; it is a prostate-specific protein overexpressed in 95% of prostate tumors. Prostate cancer therapeutic approaches based on PAP protein include DNA vaccines (McNeel et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e), cell-based medicine (Di Lorenzo et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Kawalec et al. \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e), and peptide antigen vaccines (Matsueda et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Machlenkin et al. \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eIn previous studies, the fusion of immunoglobulin G (IgG) Fc to a GA733 vaccine candidate against colorectal cancer was successfully expressed in \\u003cem\\u003eNicotiana tabacum\\u003c/em\\u003e transgenic tobacco plants (Park et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Lim et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Lu et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). The plant-derived recombinant protein GA733-Fc fused to the endoplasmic reticulum (ER) retention signal (KDEL motif) GA733-Fc\\u003csup\\u003eP\\u003c/sup\\u003e showed immune response efficacy in animals (Lim et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Lu et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). Recombinant IgG Fc fusion proteins may have considerable potential, including enhanced antigen uptake and vaccination processing. This occurs by targeting Fc receptors on antigen-presenting cells and enhancing their plasma half-life. Furthermore, to avoid β(1,2)-xylose (Xyl) and α(1,3)-fucose (Fuc) plant specific glycan residues, an oligomannose glycan structure of the recombinant proteins was generated by retaining the protein in the ER. Fusion of IgG Fc to KDEL (Munro and Pelham \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e1987\\u003c/span\\u003e; Lu et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e)could retain the protein inside the ER and reduce the degradation of recombinant fusion protein in plant cells, eventually enhancing the production level.\\u003c/p\\u003e \\u003cp\\u003eThis study investigated the functional expression of a PAP-IgG Fc fusion macromolecular protein as a recombinant vaccine for prostate cancer in \\u003cem\\u003eN. tabacum\\u003c/em\\u003e transgenic tobacco plants.\\u003c/p\\u003e\"},{\"header\":\"Materials \\u0026 Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eConstruction of plant expression vectors for recombinant PAP fusion proteins\\u003c/h2\\u003e \\u003cp\\u003eThe PAP cDNA (GenBank accession no. M34840.1) was synthesized to fuse the Fc region of human IgG\\u003csub\\u003e1\\u003c/sub\\u003e (GenBank accession No. AY172957.1) to construct the PAP-Fc fusion protein, which was further tagged with the ER retention signal KDEL to generate PAP-FcK. These fusion proteins were cloned under the control of an enhanced cauliflower mosaic virus (CaMV) 35S promoter (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The PAP-Fc and PAP-FcK expression cassettes were cloned into the plant binary vector pBI121 using \\u003cem\\u003eHind\\u003c/em\\u003eIII and \\u003cem\\u003eEco\\u003c/em\\u003eRI restriction enzyme sites to generate pBI PAP-Fc and pBI PAP-FcK, respectively, and transformed in competent \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e DH5α cells for amplification (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The synthetic PAP complementary DNA (cDNA) fragment sequence comprised a 30 amino acid plant ER signal peptide from \\u003cem\\u003eN. plumbaginifolia\\u003c/em\\u003e (Lu et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; So et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Ko et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eTransformation of tobacco plant\\u003c/h3\\u003e\\n\\u003cp\\u003eThe pBI PAP-Fc and pBI PAP-FcK plant expression vectors were transferred into \\u003cem\\u003eAgrobacterium tumefaciens\\u003c/em\\u003e strain LBA4404 using electroporation. Transgenic tobacco (\\u003cem\\u003eN. tabacum L. cv.\\u003c/em\\u003e Xanthi) plants were generated using \\u003cem\\u003eAgrobacterium\\u003c/em\\u003e-mediated transformation [30, 31]. The \\u003cem\\u003eA. tumefaciens\\u003c/em\\u003e strain LBA4404 carrying pBI PAP-Fc and pBI PAP-FcK vectors and the tobacco leaf explants were cultivated in co-cultivation media [Murashige and Skoog (MS) including B5 vitamin (4.8 g\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e), sucrose (30 g\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e), plant agar (8 g\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) (Duchefa Biochemie, Haarlem, Netherlands), 6-benzylaminopurine solution (6-BAP) (1 mg\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e), 1-naphthylacetic acid (NAA) (100 \\u0026micro;g\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e), and acetosyringone (100 \\u0026micro;M)] at 25\\u0026deg;C in dark for 3 days. The tobacco leaf explants were then transferred to a regeneration medium [MS medium (4.8 g\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e), 6-BAP (1 mg\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e), NAA (100 \\u0026micro;g\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e), acetosyringone (100 \\u0026micro;M), cefotaxime (250 mg\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e), and kanamycin (100 mg\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e)] to induce shoot and callus formation. \\u003cem\\u003eN. tabacum\\u003c/em\\u003e transgenic tobacco plant lines were selected on MS medium containing 100 mg\\u0026middot;L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e kanamycin.\\u003c/p\\u003e\\n\\u003ch3\\u003eConfirmation of PAP-Fc and PAP-FcK transgenic lines by polymerase chain reaction (PCR)\\u003c/h3\\u003e\\n\\u003cp\\u003eGenomic DNA was isolated from the leaves (100 mg) of transgenic and non-transgenic plants using HiYield\\u0026trade; Genomic DNA Mini Kit (Plant) (RBC, Taipei, Taiwan). Genomic DNA was amplified using PCR to confirm the presence of recombinant PAP-Fc (1,905 bp) and PAP-FcK (1,917 bp) genes using forward and reverse primers: forward (5\\u0026prime;-GGGGTACCATGGCTA CTCAACGAAGGGC-3\\u0026prime;) and reverse (5\\u0026prime;- GGACTAGTATCTGTACTGTCCTCAG-3\\u0026prime;) primer sets were used for PAP fragment of PAP-Fc and PAP-FcK; forward (5\\u0026prime;-CCTACTCTGGCAGCCCATC-3\\u0026prime;) and reverse (5\\u0026prime;-CCATTGCTCTCCCACTCCAC-3\\u0026prime;) primer sets were used for IgG Fc fragment of PAP-Fc and PAP-FcK; forward (5\\u0026prime;-CCTACTCTGGCAGCCCATC-3\\u0026prime;) and reverse (5\\u0026prime;-TCAGAGTTCATCTTTACCCGG-3\\u0026prime;) primer sets were used for IgG Fc fragment tagged to KDEL of PAP-FcK. The PCR was conducted as follows: 94\\u0026deg;C for 120s, 28 cycles 94\\u0026deg;C for 20s, 62\\u0026deg;C for 20s, 72\\u0026deg;C for 70s, and 72\\u0026deg;C for 5 min. A non-transgenic (NT) plant was used as a negative control, and pBI 121 vectors containing the PAP-Fc and PAP-FcK genes were used as positive controls.\\u003c/p\\u003e\\n\\u003ch3\\u003eReverse transcription (RT)-PCR amplification\\u003c/h3\\u003e\\n\\u003cp\\u003eRT-PCR was conducted to confirm the presence of PAP-Fc and PAP-FcK mRNA transcripts in transgenic plants. Total RNA was extracted from leaf samples of transgenic plants containing PAP-Fc and PAP-FcK according to an RNeasy Plant Mini Kit protocol (Qiagen, Valencia, CA, USA). Genomic DNA was removed from the isolated total RNA, and cDNA was synthesized according to the QuantiTect RT kit protocol (Qiagen, Valencia, CA, USA). Each RNA sample was used as a template for RT-PCR analysis, which was performed using a Maxime PCR Premix Kit (Intron Biotechnology, Seoul, Korea). cDNA was PCR-amplified to confirm the presence of recombinant PAP-Fc and PAP-FcK genes, using the genomic DNA PCR primer sets as described above. The RT-PCR was conducted as follows: 94\\u0026deg;C for 120s; 30 cycles at 94\\u0026deg;C for 20s, 56.5 ℃ for 10s, 72\\u0026deg;C for 22s, and 72\\u0026deg;C for 120s. The elongation factor 1-α gene (\\u003cem\\u003eEF-1α\\u003c/em\\u003e) for plant growth was used as a reference gene. A NT plant leaf was used as a negative control.\\u003c/p\\u003e\\n\\u003ch3\\u003eWestern blot analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eTo confirm the expression of PAP-Fc and PAP-FcK by western blotting, plant leaf (100 mg) was homogenized to extract total soluble protein in 300 \\u0026micro;L 1\\u0026times; phosphate-buffered saline (PBS). Twenty microliters of the plant leaf extract samples were mixed with protein loading buffer [bromophenol blue (0.1%), glycerol (50%), 2-mercaptoethanol (5%), SDS (10%), and Tris-HCl (1 M)], run using 10% SDS-PAGE. The separated total proteins on the gel were transferred to a nitrocellulose botting membrane. Skim milk (5%) was used to block the membrane in 1\\u0026times;TBS-T buffer (0.005% Tween 20 1\\u0026times;TBS; v/v) for 2 h at room temperature (RT). The blots were incubated with either rabbit anti-human PAP antibody (Abcam, Cambridge, MA, USA) (1:5,000) or murine anti-human Fc\\u003cem\\u003eγ\\u003c/em\\u003e antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch)(1:5,000). The blots were further incubated for 2 h at RT with goat anti-rabbit IgG antibody conjugated to horseradish peroxidase as a secondary antibody (Bethyl Laboratories, Montgomery, TX, USA) diluted in blocking buffer at 1:5,000. The specific protein was detected on an X-ray film using Clarity\\u0026trade; Western enhanced chemiluminescence substrate (Bio-Rad, Hercules, CA, USA). Non-transgenic tobacco plants and human recombinant PAP (PAP\\u003csup\\u003eH\\u003c/sup\\u003e) (Sino Biological, Beijing, China) were used as negative and positive controls, respectively. Western blotting was performed more than three times to confirm the results.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePurification of recombinant fusion proteins from transgenic plant leaves\\u003c/h2\\u003e \\u003cp\\u003ePurification of PAP-Fc and PAP-FcK recombinant protein was carried out as previously described (Park et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Song et al. \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e) The tobacco leaves were homogenized in extraction buffer [15 mM EDTA (pH 8.0), 50 mM NaCl, 75 mM sodium citrate, (pH 6.7), 0.2% sodium thiosulfate, and 37.5 mM Tris-HCl (pH 7.5)] using an HMF-3250S aluminum blender (Hanil, Seoul, Korea). The leaf extracts were centrifuged (8,800 \\u0026times;g, 30 min, 4\\u0026deg;C). The supernatant was applied to Miracloth (Calbiotech, Sandiego, CA). The filtered solution was adjusted to pH 5.1 using 17.4 M glacial acetic acid (Duksan, Seoul, Korea), then centrifuged (10,200 \\u0026times;g, 30 min, 4\\u0026deg;C). The supernatant was adjusted to pH 7.0 using 3 M Tris-HCl. Subsequently, the supernatant was mixed with ammonium sulfate (Duchefa Biochemie) (8%) and incubated for 2 h at 4\\u0026deg;C. After centrifugation (8,800 \\u0026times;g, 30 min, 4\\u0026deg;C), the supernatant was mixed with ammonium sulfate again at 40% saturation, followed by overnight incubation at 4\\u0026deg;C and centrifugation (8,800 \\u0026times;g, 30 min, 4\\u0026deg;C). The protein pellet was dissolved in extraction buffer (1/10 of the original solution volume), and then centrifuged (10,200 \\u0026times;g, 30 min, 4\\u0026deg;C) (Park et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Lim et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). The supernatant was filtered using a hydrophilic polyvinylidene difluoride 0.45 \\u0026micro;m filter (Millipore, Billerica, MA, USA). PAP-Fc and PAP-FcK proteins were purified using HiTrap Protein A HP (GE Healthcare, Sweden). Elutes of plant-derived PAP-Fc and PAP-FcK proteins were dialyzed with 1\\u0026times;PBS (pH 7.4). The protein concentration was determined by an Epoch microplate spectrophotometer (Biotech, Winooski, VT, USA), and the purified proteins were visualized by SDS-PAGE. Aliquots of the purified proteins were stored at -80 ℃.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eSize-exclusion chromatography (SEC)-high-performance liquid chromatography (HPLC)\\u003c/h3\\u003e\\n\\u003cp\\u003eSEC-HPLC was conducted to measure the molecular weight of the recombinant PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e fusion proteins by Agilent 1260 Infinity Quaternary LC (Agilent Technologies Inc., Santa Clara, CA, USA). PAP\\u003csup\\u003eP\\u003c/sup\\u003e, PAPK\\u003csup\\u003eP\\u003c/sup\\u003e, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e fusion proteins were separated using an Agilent Bio SEC-5 300 \\u0026Aring; column (7.8 \\u0026times; 300 mm) (Agilent Technologies Inc.). 150 mM sodium phosphate buffer (pH 7.0) was applied to equilibrate the HPLC system and SEC column at a flow rate of 1 mL\\u0026middot;min\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e at 25\\u0026deg;C. Plant-derived proteins were detected by monitoring the absorbance at 280 nm. AdvanceBio SEC 300 \\u0026Aring; protein (Agilent Technologies Inc.) was used as the standard.\\u003c/p\\u003e\\n\\u003ch3\\u003eBio-transmission electron microscopy (Bio-TEM)\\u003c/h3\\u003e\\n\\u003cp\\u003eBio-TEM analysis was performed to analyze the structures of the purified PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e fusion proteins (dialyzed with 1\\u0026times;PBS). The negative staining of proteins was conducted at the Korea Basic Science Institute (KBSI), Korea. The protein solution (5 \\u0026micro;L) was applied on to glow-discharged carbon-coated copper grids. The proteins were absorbed for 2 min and blotted off on Whatman filter paper (Sigma Aldrich, St. Louis, MO, USA). Then, uranyl acetate [2% (w/v)] ware treated to stain the proteins on the grids. The plant-derived recombinant proteins were imaged by a Tecnai G2 Spirit Twin fitted with an anti-contaminator (FEI, Hillsboro, OR, USA) operating at 120 kV. Images were recorded a CCD camera (4K94K Ultrascan 895) (Gatan Inc., Pleasanton, CA, USA) was used to record images at a magnification of 52,000.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eSurface plasmon resonance (SPR) analysis to confirm Fc gamma (Fcγ) receptor binding activity of PAP-Fc\\u003c/b\\u003e \\u003csup\\u003e \\u003cb\\u003eP\\u003c/b\\u003e \\u003c/sup\\u003e \\u003cb\\u003eand PAP-FcK\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003eP\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe affinity of PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e toward the Fcγ receptor (FcγR) was confirmed by SPR analysis at 25 ℃ using a general layer compact (GLC) chip on a ProteOn XPR36 SPR biosensor (Bio-Rad Labs, Hercules, CA, USA). For measuring affinity, the GCL sensor chip was used to immobilize 1,000\\u0026ndash;1,500 relative units (RU) of FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), and FcγRIIIb (CD16b) (Sino Biological, Beijing, China) in sodium acetate buffer (pH 5.0\\u0026ndash;5.5). The purified PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins (1 \\u0026micro;g) were dissolved in 300 \\u0026micro;L of 1\\u0026times;PBS and applied to the immobilized Fcγ receptors at a flow rate of 30 \\u0026micro;L\\u0026middot;min\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e at 25\\u0026deg;C and pH 6.0. After each measurement, phosphoric acid buffer (pH 6.0) and 1\\u0026times;PBST buffer (0.135 mM KCl, 0.075 mM KH\\u003csub\\u003e2\\u003c/sub\\u003ePO\\u003csub\\u003e4\\u003c/sub\\u003e, 7 mM NaCl, 0.325 mM Na\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e.2H\\u003csub\\u003e2\\u003c/sub\\u003eO, and 0.05% Tween 20) (pH 7.4) were applied to the surface of the GLC sensor chip for its regeneration.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunization of mice with PAP\\u003csup\\u003eH\\u003c/sup\\u003e, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins\\u003c/h2\\u003e \\u003cp\\u003eEight-week-old male BALB/c mice (five per group) (Japan SLC, Inc. Hamamatsu, Shizuoka, Japan) were intraperitoneally (i.p.) injected with PAP\\u003csup\\u003eH\\u003c/sup\\u003e (Sino Biological, Beijing, China), PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e in the presence of an adjuvant (aluminum hydroxide, Sigma, St. Louis, MO, USA). PAP fusion proteins (10 \\u0026micro;g) were thrice injected to mice at two-week intervals for six weeks. Retro-orbital sinus bleeding method was used to collect blood samples before injection and 10 days after the second immunization. Retro-orbital plexus bleeding method was used for blood collection at 10 days after the third immunization [26]. After bleeding, the spleens were harvested (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). All animal experiments for this were conducted by the Animal Ethics Committee (ACE) (#2017-00087) of Chung-Ang University, Korea.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEnzyme-linked immunosorbent assay to quantify PAP-specific IgG\\u003c/h2\\u003e \\u003cp\\u003ePAP-specific IgG antibodies induced by PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins were evaluated using ELISA. ELISA plates were coated with 0.5 \\u0026micro;g\\u0026middot;mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e of PAP\\u003csup\\u003eH\\u003c/sup\\u003e antigen (Sino Biological, Beijing, China) and treated with 4\\u0026ndash;5 fold serial dilutions of mouse sera for the endpoint of the experiment. Goat anti-murine antibodies conjugated to horseradish peroxidase was used to detect PAP-specific whole IgG and IgG\\u003csub\\u003e1\\u003c/sub\\u003e responses (Jackson ImmunoResearch).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIsolation of splenocytes\\u003c/h2\\u003e \\u003cp\\u003eThe immunized mice were sacrificed to collect spleens for splenocytes. The homogenized spleen was passed through 70 \\u0026micro;m cell strainer (SPL Life Sciences Co. Ltd., Seoul, South Korea). The spleen was treated with ammonium-chloride-potassium lysis buffer (1 mL). The spleen was homogenized using a 1 mL syringe plunger. To eliminate red blood cells, the released cells were incubated with ACK lysis buffer for 5 min at RT and washed twice with 10 mL 1\\u0026times;PBS. The released cells were centrifuged to collect pellets (680 \\u0026times;g, 5 min, 4\\u0026deg;C). The pellets were dissolved using Hyclone RPMI 1640 media (GE Healthcare, Little Chalfont, UK). Fat was removed by filtration using a strainer. 100 \\u0026micro;L cell suspensions (2\\u0026times;10\\u003csup\\u003e6\\u003c/sup\\u003e cells/well) were placed on 96-well round bottom plates.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSurface staining and flow cytometry\\u003c/h2\\u003e \\u003cp\\u003eCell suspensions isolated from the spleen were centrifuged at 680 \\u0026times;g for 5 min at RT and then resuspended in 200 \\u0026micro;L/well of fluorescence-activated cell sorting buffer [1\\u0026times;PBS with 1% bovine serum albumin (BSA), 2 mM EDTA, and 10% sodium azide]. Cell suspensions were stained using FACS buffer containing murine-specific antibodies (1:200) directly conjugated to CD4 (PerCP/Cyanine7, Tonbo Bioscience, San Diego, CA, USA) and CD8a (APC, Tonbo Bioscience, San Diego, CA, USA) for 20 min at 4\\u0026deg;C. Cell suspensions were then washed twice with 200 \\u0026micro;L/well FACS buffer, resuspended in the same buffer, and transferred to a 1.5 mL micro tube for flow cytometry analysis.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGeneration of transgenic tobacco plants expressing PAP-Fc and PAP-FcK\\u003c/h2\\u003e \\u003cp\\u003e \\u003cem\\u003eAgrobacterium\\u003c/em\\u003e-mediated transformation was applied to obtain transgenic plants using the expression vectors pBI PAP-Fc and pBI PAP-FcK (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. Left and Right). The PAP-Fc and PAP-FcK fusion genes placed downstream of the untranslated leader sequence from the alfalfa mosaic virus were controlled by the enhanced CaMV 35S promoter (E/35S-P) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Among the regenerated plants, genomic DNA was isolated from three randomly selected transgenic lines for each type of expression vector (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). The existence of PAP, IgG Fc fragment, and KDEL DNA sequences in PAP-Fc and PAP-FcK in the transgenic tobacco plant leaf tissues was confirmed by PCR using specific primers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). The transgenic plant lines with PAP-Fc and PAP-FcK showed the expected amplified bands at 1,173 bp (PAP), 1,185 bp (IgG Fc), and 1,905 bp (KDEL), respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). Likewise, semi-quantitative RT-PCR confirmed the mRNA expression of PAP, IgG Fc fragment, and KDEL of PAP-Fc and PAP-FcK in three randomly selected transgenic tobacco plants, and RT-PCR products of the expected sizes were detected at 199 bp (PAP), 224 bp (IgG Fc), and 166 bp (KDEL), respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). None of the PAP-Fc or PAP-FcK transgenes were detected in non-transgenic plants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). The EF-1α band as a internal control was observed at 67 bp (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eExpression and purification of PAP-Fc and PAP-FcK in transgenic tobacco plants\\u003c/h2\\u003e \\u003cp\\u003eThe expression of PAP-Fc and PAP-FcK in transgenic tobacco plants was confirmed by a protein band of 75 kDa in western blotting, (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eCD). PAP\\u003csup\\u003eH\\u003c/sup\\u003e (45 kDa) as a positive control was detected using the anti-PAP antibody (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC), and the heavy chain of human IgG antibody as a positive control for IgG Fc was detected as a 50 kDa band with the anti-human Fcγ antibody (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). In PAP-Fc and PAP-FcK proteins, protein bands of 45, 75, and 240 kDa were detected by anti-human PAP antibody (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). Similarly, after treatment with anti-human Fcγ antibody, protein bands at 75 kDa and \\u0026gt;\\u0026thinsp;240 kDa were detected; however, protein band of 45 kDa was not observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). PAP-Fc and PAP-FcK were expressed in tobacco plants and purified by protein A chromatography. The yields of PAP-Fc and PAP-FcK proteins were 10 and 15 mg\\u0026middot;kg\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e of fresh weight of tobacco plant leaves, respectively. In addition, SDS-PAGE showed expected molecular weights of 70.7 kDa and 71.2 kDa for PAP-Fc and PAP-FcK, respectively (Fig.\\u0026nbsp;2EF, respectively)\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMolecular weight profiling and Bio-TEM images of plant-derived recombinant fusion proteins\\u003c/h2\\u003e \\u003cp\\u003eThe PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e fusion proteins were analyzed by SEC-HPLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). PAP\\u003csup\\u003eP\\u003c/sup\\u003e and PAPK\\u003csup\\u003eP\\u003c/sup\\u003e showed one protein peak at ~\\u0026thinsp;5.4 min (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB and C, PAP), whereas PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e showed two protein peaks (~\\u0026thinsp;5.4 and 7.45 min) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD and E). The retention time for PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e was compared to those of the standard molecular weight proteins (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA), which indicated an increased molecular weight of PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e than expected. Bio-TEM analysis of plant-derived PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e showed that both the proteins were observed as 'Y' shape particles, similar to the antibodies (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eFG, respectively).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBinding affinity of PAP fusion proteins to Fcγ receptors\\u003c/h2\\u003e \\u003cp\\u003eThe binding affinity activities of plant-derived PAP-Fc and PAP-FcK fusion proteins to Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) were observed by SPR analysis. The assay was performed with the receptors (1000\\u0026ndash;1500 RU) immobilized on the chip, and the proteins were injected into the fluid phase at 50 nM (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). PAP-Fc and PAP-FcK showed high affinity for FcγRI (CD64), and neither protein dissociated on the surface during injection (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). FcγRIIIa (CD16) showed a relatively high affinity at 250s after injection, with little dissociation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD). In contrast, the fusion proteins bound weakly to FcγRIIa (CD32a) and could not bind to both FcγRIIb and FcγRIIIb (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB, C, and E).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIgG response in mice immunized with PAP-Fc and PAP-FcK fusion proteins\\u003c/h2\\u003e \\u003cp\\u003eThe immunogenicity of the PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins was investigated in BALB/c mice, with blood samples collected to analyze the serum IgG response following immunization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). To confirm the induction of anti-EpCAM IgGs in the mice immunized with PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e recombinant proteins, ELISA analysis was performed on sera collected 10 days after the second and third immunizations (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA, B, and C). In the sample from the first blood draw after the second injection of PAP\\u003csup\\u003eH\\u003c/sup\\u003e, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e, the signal was undetectable (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB \\u0026lsquo;1st\\u0026rsquo;). However, in the second blood draw after the third immunization, increased optical density (OD) values were observed in all groups of mice, except those immunized with 1\\u0026times;PBS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB \\u0026lsquo;2nd\\u0026rsquo;). Notably, both PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins induced IgG responses similar to those elicited by PAP\\u003csup\\u003eH\\u003c/sup\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC). Overall, the plant-derived PAP-Fc fusion proteins demonstrated comparable induction of anti-PAP IgG responses, similar to PAP\\u003csup\\u003eH\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eT-cell responses in mice immunized with PAP-Fc fusion proteins\\u003c/h2\\u003e \\u003cp\\u003eFlow cytometry analysis was conducted to confirm activation of the CD8 and CD4\\u003csup\\u003e+\\u003c/sup\\u003e T To investigate CD4\\u003csup\\u003e+\\u003c/sup\\u003e helper and CD8\\u003csup\\u003e+\\u003c/sup\\u003e cytotoxic T cell responses to PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e or PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e fusion proteins, we examined the activation state of splenic CD4\\u003csup\\u003e+\\u003c/sup\\u003e and CD8\\u003csup\\u003e+\\u003c/sup\\u003e T lymphocytes in lymphocytes isolated from splenocytes of the mice immunized with PAP\\u003csup\\u003eH\\u003c/sup\\u003e, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e at 10 days after the third immunization (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD, E, F, and G). The expression level of CD69, a T-cell activation marker, on CD4\\u003csup\\u003e+\\u003c/sup\\u003e and CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells was analyzed using the gating strategy. Immunization with commercial PAP\\u003csup\\u003eH\\u003c/sup\\u003e slightly increased the expression of CD69 on CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells [mean fluorescence intensity (MFI): 135] compared to that on the negative control (1\\u0026times;PBS-treated group; MFI: 131) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD and E). However, immunization with PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e (MFI: 181) or PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e (MFI: 201) markedly increased the expression of CD69 in CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD and E). In CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells, a slight increase in CD69 expression was observed in all mouse groups (immunized with PAP\\u003csup\\u003eH\\u003c/sup\\u003e, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e) than in the PBS-treated group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eF and G). Collectively, these results suggest that immunization with PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e or PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e fusion proteins efficiently induces CD4\\u003csup\\u003e+\\u003c/sup\\u003e and CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cell-mediated immune responses.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eWe demonstrated the potential of a vaccination approach against prostate cancer using the PAP antigen-IgG Fc fusion protein expressed in transgenic plants. In previous studies, the cancer vaccine candidates fused to the Fc fragment have been successfully expressed and assembled to induce immunogenicity in mice (Lim et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Lu et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Shin et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Kang et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Kang et al. \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). In the current study, PAP-Fc and PAP-FcK fusion proteins were expressed in transgenic \\u003cem\\u003eN. tabaccum\\u003c/em\\u003e plants and their purified forms were demonstrated as immunogenic candidate cancer vaccines in mouse experiments.\\u003c/p\\u003e \\u003cp\\u003eThe feasible expression and immune function of the recombinant protein are essential for the development of a plant production system for vaccine candidates (Lee and Ko \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Kamo et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e) In plant expression systems, recombinant protein expression in the extracellular space involves initial protein entry into the ER by a signal peptide and passing through the Golgi complex in plant cells. In the ER, the recombinant proteins are folded, assembled, and glycosylated. The glycosylated protein them moves to the Golgi apparatus and secreted outside (Pagny et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e; Kermode \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e). To enhance the accumulation of recombinant proteins in plants, subcellular targeting to ER is effective in retaining their biological characteristics (So et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Song et al. \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Pires et al. \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). In this study, two different transgenic tobacco plant lines expressing PAP-Fc and PAP-FcK were generated to investigate the effect of the Fc fragment and KDEL ER retention signal on PAP expression and its biological activity. Each transgenic plant with PAP-Fc and PAP-FcK was grown \\u003cem\\u003ein vivo\\u003c/em\\u003e to investigate the transgene insertion and protein expression. PCR and RT-PCR revealed that all randomly tested plants had PAP-Fc and PAP-FcK transgenes insertion and mRNA transcripts of the transgenes. Western blot analysis confirmed the protein expression of PAP-Fc and PAP-FcK in transgenic plants. Protein band of 75 kDa was detected, and the molecular weight was similar to that predicted for the fusion of PAP (50 kDa) and IgG Fc fragment (25 kDa). PAP-FcK transgenic plants showed higher fusion protein expression than PAP-Fc transgenic plants. In PAP-FcK protein fused to the ER retention signal peptide KDEL, the protein was retained in the ER resulting in increased accumulation. This result is consistent with previous studies where the expression of recombinant proteins tagged to KDEL could be enhanced in transgenic plants (Kang et al. \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Song et al. \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Gomord et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Schouten et al. \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e). Both transgenic plant lines expressing PAP-Fc and PAP-FcK showed \\u0026gt; 240 kDa and 45 kDa protein bands. The \\u0026gt; 240 kDa protein band could be due to the dimerization of the PAP-Fc and PAP-FcK monomers. The IgG Fc fragment can become dimeric (Park et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Lim et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Shin et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e) .In addition, the 45 kDa protein bands were detected with anti-PAP but not with anti-Fc, suggesting that the 45 kDa band corresponds to PAP protein. These results indicate that the PAP-Fc and PAP-FcK fusion proteins are cleaved into PAP and Fc fragments. However, it is currently difficult to explain why the anti-Fc antibody did not detect the Fc fragment in western blotting. It is speculated that the Fc fragment itself cannot be recognized by the anti-Fc antibody.\\u003c/p\\u003e \\u003cp\\u003eBoth PAP-Fc and PAP-FcK proteins were purified from plant leaves and analyzed using SEC-HPLC and EM. SEC-HPLC indicated the presence of a protein peak corresponding to dimers in PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e. In addition, EM analysis confirmed the dimerization of PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e. However, the polymeric molecular weight peaks of PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e at unexpected larger sizes may result from multiple glycan residues or protein aggregation in the HPLC system. In Bio-TEM, the aggregation formation was more identified in PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e compared to PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e. It is speculated that high mannose type glycans in PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e could reduce the aggregation of glycoproteins compared to PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e, which harbors more plant-specific type glycans and less high mannose type glycans (Schaefer and Plückthun \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe capacity of the PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e fusion proteins to bind complement component 1q (C1q) was analyzed using ELISA (Data not shown). The Fc fragment can bind to C1q to initiate assembly of the complement membrane attack complex (MAC) to kill target cells (complement-dependent cytotoxicity) (Yang et al. \\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Walport \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e). PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins could bind to C1q in a concentration-dependent manner, forming immune complexes. These \\u003cem\\u003ein vitro\\u003c/em\\u003e assay results demonstrated the binding activity of the PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e recombinant fusion proteins to C1q, indicating their potential for immunogenicity.\\u003c/p\\u003e \\u003cp\\u003eImmune leukocytes exhibiting Fcγ receptors (FcγRs) on their surface membrane are recruited and activated by binding the IgG Fc fragment to FcγRs. The binding triggers antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated phagocytosis to kill and remove target cells (Yang et al. \\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Woof and Burton \\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e; Ravetch and Bolland \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e). We investigated whether PAP-Fc and PAP-FcK proteins showed a stronger association level for FcγRs (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb). Both PAP-Fc and PAP-FcK bound with high association level to human FcγRI, with slight differences. In addition, the fusion proteins could bind to FcγIIIa, but showing a slightly lower association level for FcγRIIa; however, the binding was poor for both FcγRIIb and FcγRIIIb. PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e showed a higher association than PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e but similar to PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e. Further, we investigated the ability of fusion proteins to interact with neonatal Fc receptor (FcRn), which plays a role in maintaining the long half-life of antibodies in circulation. The PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e bound with a higher association level to human FcRn, and binding occurred only at a pH of 6.0, similar to the previous studies (Mekhaiel et al. \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e; Andersen et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). Overall, these results indicate that PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e exhibit a high association level for human FcγR and FcRn. Moreover, the association levels of PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e for FcRn and FcγRs were not markedly different.\\u003c/p\\u003e \\u003cp\\u003eThe recombinant PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins induced antibody responses in mice, as demonstrated by the total IgG levels. The proteins were found to have a much higher IgG\\u003csub\\u003e1\\u003c/sub\\u003e response, suggesting a Th2 bias. Therefore, plant-derived recombinant proteins were immunogenic in mice; however, IgG\\u003csub\\u003e1\\u003c/sub\\u003e responses were quantitatively different. Notably, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins induced relatively similar responses to PAP\\u003csup\\u003eH\\u003c/sup\\u003e in mice, which possibly explained by Fc receptors or cross-reactivity to immune complex-capturing mechanisms in mice. In addition, these proteins elicited CD4\\u003csup\\u003e+\\u003c/sup\\u003e and CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cell responses in mice. Indeed, CD4\\u003csup\\u003e+\\u003c/sup\\u003e helper T cells regulate B cells to produce antibodies, including anti-tumor antibodies (Kim and Cantor \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Lai et al. \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). An effective therapeutic cancer vaccine should generate T-cell responses (Kartikasari et al. \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Westdorp et al. \\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). The activities of CD4\\u003csup\\u003e+\\u003c/sup\\u003e and CD8\\u003csup\\u003e+\\u003c/sup\\u003e cells were determined by expression of CD69, which is significantly expressed on T-cells upon (Ziegler et al. \\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e1994\\u003c/span\\u003e). PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins induced the activity of CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells rather than CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells. Specifically, the cellular immune responses were more efficiently induced in the PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e proteins than PAP\\u003csup\\u003eH\\u003c/sup\\u003e, most probably due to the efficient binding to the Fc receptors. Proliferation and activation of effector CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells are more crucial for induction of immune-mediated tumor control by cancer vaccines than CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells (Kim and Cantor \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Melssen and Slingluff \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Kumai et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells promote anti-cancer immunity through various mechanisms, including T-cell activation, T-cell homing enhanced, antigen-presentation co-stimulation, effector function (Melssen and Slingluff \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Taken together, the PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e protein was immunogenically more active than the PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e protein. These results suggest that the efficacy of the vaccine could be increased due to the oligo-mannose glycan structure of ER retained proteins in plants (Lu et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Schouten et al. \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e; Wandelt et al. \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e). Overall, plant-derived PAP-Fc and PAP-FcK proteins are immunogenic in mice and induce cellular immunity, which is required to prevent prostate cancer. Therefore, these plant-derived proteins can be potential candidates to develop vaccines for prostate cancer prevention.\\u003c/p\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eData availability \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis research was funded by the National Research Foundation of Korea grant [RS-2024-00409554].\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNone\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDepartment of Medicine, Medicine of College, Chung-Ang University, 06974, Seoul, South Korea\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eYangjoo Kang, Deuk-Su Kim, Hyunjoo Hwang \\u0026amp; Kisung Ko\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDepartment of Life Science, Chung-Ang University, 06974, Seoul, South Korea\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eYoung-Jin Seo\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDepartment of Applied Experimental Biophysics, Johannes Kepler University Linz, 4040, Linz, Austria\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePeter Hinterdorfer\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eC\\u003c/strong\\u003e\\u003cstrong\\u003eontributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization: Yangjoo Kang, Deuk-Su Kim, Young-Jin Seo, and Kisung Ko; methodology: Yangjoo Kang, Deuk-Su Kim, and Hyunjoo Hwang; validation: Yangjoo Kang, Young-Jin Seo, and Kisung Ko; formal analysis: Yangjoo Kang, Young-Jin Seo, and Kisung Ko; investigation: Yangjoo Kang, Deuk-Su Kim, and Hyunjoo Hwang; writing\\u0026mdash;original draft preparation Yangjoo Kang, Deuk-Su Kim, and Kisung Ko; writing\\u0026mdash;review and editing: Yangjoo Kang, Young-Jin Seo, and Kisung Ko; supervision, Kisung Ko; project administration, Kisung Ko; funding acquisition, Kisung Ko. All authors read and approved the manuscript.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eCorresponding author\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCorrespondence to Kisung Ko\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics declarations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing financial interest\\u003cbr\\u003e \\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAndersen JT, Daba MB, Berntzen G, Michaelsen TE, Sandlie I (2010) Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding. 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Stem Cells 12 (5):456-465. doi:10.1002/stem.5530120502\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"transgenic-research\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"trag\",\"sideBox\":\"Learn more about [Transgenic Research](http://link.springer.com/journal/11248)\",\"snPcode\":\"11248\",\"submissionUrl\":\"https://submission.nature.com/new-submission/11248/3\",\"title\":\"Transgenic Research\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Prostate acid phosphatase, Prostate cancer, Prostate specific antigen, transgenic plant, vaccine\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5286242/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5286242/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eProstatic acid phosphatase (PAP) is a specific protein that is highly expressed in prostate cancer. In this study, we constructed two recombinant PAP fusion genes: PAP fused to the immunoglobulin G (IgG) Fc fragment (designated PAP-Fc) and PAP-Fc fused to the endoplasmic reticulum retention sequence KDEL (designated PAP-FcK). Transgenic \\u003cem\\u003eNicotiana tabacum\\u003c/em\\u003e plants expressing these recombinant macromolecular proteins (MPs) were generated using Agrobacterium-mediated transformation, and the presence of both genes was confirmed through genomic PCR. Western blot analysis validated the expression of PAP-Fc and PAP-FcK MPs, which were successfully purified via protein A affinity chromatography. Size-exclusion high-performance liquid chromatography revealed dimeric peaks for PAP-Fc (PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e) and PAP-FcK (PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e). Bio-transmission electron microscopy demonstrated 'Y'-shaped protein particles resembling antibody structures. Moreover, PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e exhibited a high association rate with human FcγR and FcRn. Vaccination of mice with both PAP-Fc\\u003csup\\u003eP\\u003c/sup\\u003e and PAP-FcK\\u003csup\\u003eP\\u003c/sup\\u003e resulted in increased total IgG against PAP and enhanced activation of CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells, comparable to mice immunized with PAP, which served as a positive control. These findings indicate that both plant-derived MPs can effectively induce adaptive immunity, positioning them as promising candidates for prostate cancer vaccines. Overall, plants expressing PAP-Fc and PAP-FcK represent a viable production system for antigenic macromolecule-based prostate cancer vaccines.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Plant-Derived Recombinant Macromolecular PAP-IgG Fc as A Novel Prostate Cancer Vaccine Candidate Eliciting Robust Immune Responses\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-10-30 17:50:54\",\"doi\":\"10.21203/rs.3.rs-5286242/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-11-15T16:06:56+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-11-15T16:02:43+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"67726899614191725062677741795772479822\",\"date\":\"2024-11-15T09:16:00+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-11-04T12:22:50+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"291244598779987609736787381644966510019\",\"date\":\"2024-10-21T15:46:51+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-10-21T09:43:56+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-10-21T05:08:18+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-10-21T00:40:42+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Transgenic Research\",\"date\":\"2024-10-18T03:57:56+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"transgenic-research\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"trag\",\"sideBox\":\"Learn more about [Transgenic Research](http://link.springer.com/journal/11248)\",\"snPcode\":\"11248\",\"submissionUrl\":\"https://submission.nature.com/new-submission/11248/3\",\"title\":\"Transgenic Research\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"086e2191-7304-4b68-88aa-a8aace05e2b1\",\"owner\":[],\"postedDate\":\"October 30th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-03-31T16:04:05+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5286242\",\"link\":\"https://doi.org/10.1007/s11248-025-00433-0\",\"journal\":{\"identity\":\"transgenic-research\",\"isVorOnly\":false,\"title\":\"Transgenic Research\"},\"publishedOn\":\"2025-03-26 15:57:12\",\"publishedOnDateReadable\":\"March 26th, 2025\"},\"versionCreatedAt\":\"2024-10-30 17:50:54\",\"video\":\"\",\"vorDoi\":\"10.1007/s11248-025-00433-0\",\"vorDoiUrl\":\"https://doi.org/10.1007/s11248-025-00433-0\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5286242\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5286242\",\"identity\":\"rs-5286242\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}