To generate functional anti-protease-activated receptor-4 (PAR4), a G protein-coupled receptor, antibodies through PAR4-mRNA-LNP immunization

To generate functional anti-protease-activated receptor-4 (PAR4), a G protein-coupled receptor, antibodies through PAR4-mRNA-LNP immunization | 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 Article To generate functional anti-protease-activated receptor-4 (PAR4), a G protein-coupled receptor, antibodies through PAR4-mRNA-LNP immunization En-Shuo Liu, Kai-Wen Ho, Chin-Chung Wu, Hsiao-Li Fan, Ting-Yu Wang, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6763159/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2025 Read the published version in npj Vaccines → Version 1 posted 10 You are reading this latest preprint version Abstract G protein-coupled receptors (GPCRs) are key therapeutic targets for various diseases, such as the thrombin receptor protease-activated receptor 4 (PAR4) in thrombotic cardiovascular disorders. The structural complexity of native GPCRs limits recombinant protein production. Traditional GPCR antibody development relies on GPCR peptide fragments for animal immunization. These peptides poorly mimic the native structure and affect the quality of the antibodies. Therefore, it is necessary to develop a strategy for immunizing native GPCRs to generate potent anti-GPCR antibodies. Here, we developed a PAR4-mRNA-LNP to immunize mice, which promotes the surface expression of the native PAR4 structure in vivo to induce highly specific and functional anti-PAR4 antibodies. These anti-PAR4 antibodies effectively inhibit platelet aggregation and provide a long-term therapeutic approach for cardiovascular disease. In result, PAR4-mRNA-LNP was synthesized with an encapsulation efficiency of 93.44%, mean size of 127.5 nm, and polydispersity index (PDI) of 0.1033. We confirmed the 55-kDa native PAR4 structure with complete glycosylation expressing on cell surface. We immunized mice with PAR4-mRNA-LNP and detected high anti-PAR4 antibody titers with ELISA. This assay used 293T cells that stably express the native PAR4 structure, enabling us to screen for high-quality anti-PAR4 antibodies. We established 15 hybridoma cell lines, which resulted in 13 including 10 IgG and 3 IgM anti-PAR4 antibodies. Five clones significantly inhibited PAR4-mediated platelet aggregation. We confirmed that PAR4-mRNA-LNP expresses the native PAR4 structure on the cell surface and the immunization produced highly functional anti-PAR4 antibodies. mRNA-LNP technology may be widely used to produce anti-GPCR antagonists and agonists for therapy. Biological sciences/Biotechnology Biological sciences/Biotechnology/Applied immunology mRNA-LNP immunization GPCRs native structure anti-PAR4 antagonist antibody platelet aggregation thrombotic cardiovascular disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction G protein-coupled receptors (GPCRs) play important roles in various physiological processes, including sensory perception 1 , neurotransmitter 2 , and hormone regulation 3 . Dysregulation or abnormal GPCR signaling is associated with various diseases, such as cancer 4 , cardiovascular disorders 5 , and neurological conditions 6 . Because of their role in disease progression, GPCRs are important therapeutic targets. For example, protease-activated receptor 4 (PAR4) is a GPCR required for platelet activation and has a significant role in cardiovascular diseases, such as myocardial infarction and stroke. Therefore, targeting PAR4 with therapeutic antibodies represents a promising approach. Developing antibodies against GPCRs is challenging because of their structural complexity, which limits recombinant protein production 7 . Peptides are often used as substitutes, but their poor mimicry of the GPCR structure reduces binding specificity and antibody effectiveness 8 – 10 . Innovative strategies are essential to preserve the native structure of GPCRs. Huang et al . used murine 3T3 cells expressing human CXCR2 with membrane-bound GM-CSF as an adjuvant to enhance antibody production by stimulating local inflammation and splenocyte proliferation 11 . However, this approach may increase background noise, resulting in a weakening of the immune response and complicating the identification of specific antibodies. Therefore, a strategy to enhance GPCR antibody generation is needed that can maintain the native GPCR structure, reduce background noise, and act as a good adjuvant to produce a strong antibody titer. mRNA-lipid nanoparticle (LNP) technology 12 – 14 enables the direct expression of PAR4 on the cell surface, thus preserving its native structure. The mRNA also serves as a potent adjuvant 15 , activating toll-like receptors 7 and 8 (TLR7/8) to induce a strong immune response by eliciting the production of antibodies specifically recognizing native PAR4. Therefore, we immunized Balb/c mice with PAR4-expressing mRNA-LNPs and utilized a hybridoma strategy 16 to produce and isolate anti-PAR4 antibodies (Fig. 1 ). Initially, the structural integrity and synthesis efficiency of PAR4-mRNA-LNP will be confirmed via gel electrophoresis and particle characterization. Subsequently, we will verify molecular weight and surface expression of PAR4 on 293T cells after PAR4-mRNA-LNP using western blotting and flow cytometry, respectively. Throughout the immunization timeline in BALB/c mice, antibody titers will be quantified by PAR4-expressing cell-based ELISA, monitoring response escalation following sequential boosts. To establish sustained antibody production, hybridoma clones will be generated and screened, confirming anti-PAR4 IgG/IgM titers and isotypes. Functional inhibition assays will then evaluate the antagonistic capability of anti-PAR4 antibodies on platelet aggregation. This mRNA-LNP approach enhanced antibody specificity and efficacy by preserving the native GPCR structure and resulted in the production of high-quality, functional antibodies. 2. Materials and Methods 2.1. Cells and reagents We used 293-T and FO cells for this study. We established 293T-Flag-PAR4 cells that stably expressed Flag-PAR4 on the 293-T cell surface by lentivirus transduction. 293T cells were seeded at 2 × 10 5 cells/well in a 6-well plate and cotransfected with 2 µg of pLKO-AS3W-Flag-human PAR4-puro, 0.2 µg pMD.G, and 1.8 µg pCMV 8.91 using Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher, Inc). After 7 days, the culture medium was collected and filtered through a 0.22-µm syringe filter, mixed with 8 µg/mL polybrene, and added to 1 × 10 5 293T cells for viral infection. The cells were selected with 2 µg/mL puromycin. High-expressing Flag-PAR4 293T cells were detected using mouse anti-human PAR4 antibody (clone 5F10; MABS1298) (1:50 dilution) and FITC-conjugated goat anti-mouse IgG Fcγ (Jackson Immuno-Research Inc. #115-095-008) (1:20 dilution) and sorted using a MoFlo™ XDP Cell Sorter System (Beckman Colter) to generate 293T-Flag-PAR4 cells. The cells were cultured in DMEM (Sigma-Aldrich) containing 10% heat-inactivated bovine calf serum (GE Healthcare Life Science) and 1% penicillin combined with streptomycin (Thermo Fisher Scientific) at 37°C in a humidified atmosphere containing 5% CO 2 . 2.2. Design of the PAR4-mRNA-LNP The PAR4 DNA sequence was designed based on the NCBI Reference Sequence NM_003950.4. Dr. Ching-Jen Yang from the Development Center of Biotechnology in Taiwan assisted in the construction of the PAR4-mRNA. The PAR4-mRNA was encapsulated in lipid nanoparticles (LNP) using a formulation similar to that of Moderna. 17 Dr. Yang evaluated the encapsulation efficiency, particle size, and polydispersity index (PDI) using a dynamic light scattering system. 2.3. Evaluate the PAR4 surface expression of PAR4-mRNA-LNP by western blot analysis and flow cytometry 293-T cells were seeded at 2 × 10 5 /mL in a 12-well growth plate and incubated overnight. The medium was replaced with 750 µL of fresh medium. After 30 min, we added 250 µL of medium containing 0, 0.5, 1, or 2 µg of PAR4-mRNA-LNP to the cells and incubated for 48 h. PAR4 expression was detected by western blot analysis and flow cytometry. PAR4 expression in 293-T cells transfected with PAR4-mRNA-LNP was assessed by western blot analysis. The primary antibody was DYKDDDDK Flag-Tag (9A3) mouse mAb (Cell Signaling Technology, Inc; #8146) and the secondary antibody was Goat anti-mouse IgG Fcγ fragment specific-HRP (Jackson Immuno-Research Inc. #115-035-008). After developing the blot by chemiluminescence, the bands were quantitated using a MultiGel-21 UVP imaging system. The surface expression of PAR4 in 293T cells by flow cytometry following transfection with PAR4-mRNA-LNP and staining the cells with mouse anti-human PAR4 antibody (clone 5F10; MABS1298), followed by incubation with FITC-conjugated goat anti-mouse IgG Fcγ (Jackson Immuno-Research Inc. #115-095-008). The surface fluorescence intensity of viable cells was measured using a Merck Guava easyCyte System flow cytometer and the data were analyzed using FlowJo v10.8.1. 2.4. PAR4 antibody titers after immunization with PAR4-mRNA-LNP All procedures involving animals were performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee at Kaohsiung Medical University in Taiwan. BALB/c mice (n = 6), aged 6–8 weeks, were immunized with three intramuscular (i.m) injections of 10 µg PAR4-mRNA-LNP, administered once every 2 weeks for 6 weeks. At 8 weeks, a final (4th ) intramuscular boost of 3 µg PAR4-mRNA-LNP was administered. Tail vein blood was collected and the serum was stored at − 80°C one week after each booster. Anti-human PAR4 antibody titers from mouse serum were screened using a cell-based ELISA. 293T-Flag-PAR4 cells (1 × 10 5 cells/well) were seeded in 96-well plates (Thermo Fisher Scientific Inc. #167008) coated with 50 µg/mL poly-D-lysine and incubated overnight. The cells were fixed in 1% paraformaldehyde in PBS for 5 min at room temperature, followed by the addition of 0.1 M glycine to neutralize the paraformaldehyde for 30 min at room temperature. The plates were blocked with 5% milk and diluted anti-human PAR4 antibody from mouse serum (in 2% skim milk) was added to each well and incubated for 50 min at room temperature. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Fcγ fragment specific-HRP (Jackson Immuno-Research Inc. #115-035-008) (1:2000) was added and incubated for 50 min at room temperature. Following three washes with PBS, ABTS, and H 2 O 2 were added. The binding capacities of each serum sample were evaluated using an ELISA reader and the data were analyzed using GraphPad Prism software (v10.0). 2.5. Establishing a hybridoma using the ECM-2001 Hybridoma System for electrofusion Splenocytes (1.6 × 10 7 /ml) and FO myeloma cells (4 × 10 6 /ml) were mixed at a 4:1 ratio and washed twice with 20 ml electrofusion medium (centrifuged at 1,400 rpm for 7 min). The pellets were resuspended with 2 mL of room temperature Cytofusion Medium C and the pellet was transferred to a 2 mL coaxial chamber. We immediately performed electrofusion (within 30 seconds) using the recommended parameters (Step-1. Pre-AC: V0 = 40 V, VF = 40 V, T = 15 s, F = 1.4 MHz; Step-2. Pre-AC: V0 = 70V, VF = 70V, T = 20 sec, F = 1.4 MHz; DC-PULSE: V = 800V, T = 40 µs, N = 1, I = 0; Post-AC: V0 = 70V, VF = 5V, T = 30 sec, F = 1.4 MHz). Finally, the fusion mixture was plated into twelve 96-well plates (1,152 total wells) and cultured in HAT medium (Sigma-Aldrich) to screen for hybridomas. 2.6. Evaluation of anti-PAR4 antibody secreted from hybridoma cells After establishing hybridoma cells and observing their growth to approximately 50% confluency in 96-well plates over 1–2 weeks, the supernatants were collected and the PAR4 antibody titers were determined using a cell-based ELISA. t293T-Flag-PAR4 cells (1 × 10 5 cells/well) were seeded into 96-well plates (Thermo Fisher Scientific Inc. #167008) coated with 50 µg/mL poly-D-lysine and incubated overnight. The cells were fixed in 1% paraformaldehyde in PBS for 5 min at room temperature, followed by the addition of 0.1 M glycine to neutralize the paraformaldehyde for 30 min at room temperature. The plates were blocked in 5% milk and the supernatants were added to each well for 50 min at room temperature. After three washes with PBS, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG + IgM (H + L) antibody (Jackson Immuno-Research Inc. #115-035-044) (1:1,000) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Fc fragment antibody (Jackson Immuno-Research Inc. #115-035-008) (1:1,000) were added and incubated for 50 min at room temperature. After three washes with PBS, ABTS and H 2 O 2 were added. The binding capacities of each serum were evaluated using an ELISA reader and the data were analyzed using GraphPad Prism software (v10.0). 2.7. Screening for functional anti-PAR4 antagonist antibodies The protocol for this study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital. Human blood anticoagulated with acid citrate dextrose was obtained from healthy human volunteers, who had not taken any drugs during the previous two weeks. The blood was centrifuged at 450 × g for 15 min and the supernatant was obtained as platelet-rich plasma. The platelet-rich plasma was further centrifuged at 1,800 × g for 15 min in the presence of prostaglandin E1 (0.5 µM). The platelet pellet was washed with Tyrode’s solution containing 2 mM Ca 2+ , 11.1 mM glucose, and 3.5 mg/ml bovine serum albumin and centrifuged at 1,200 × g for 7 min in the presence of prostaglandin E1 (0.5 µM) and apyrase (0.15 U/ml). The platelet pellet was washed again as described above except for the omission of prostaglandin E1. The washed platelets were suspended in Tyrode’s solution at 3 × 10 8 platelets/mL unless otherwise specified. Platelet aggregation was measured turbidimetrically with a light-transmission aggregometer (Chrono-Log Co., Havertown, PA, USA). The platelet suspension was pretreated with anti-PAR4 antibodies in the supernatant at 37°C for 3 min while stirring (1,200 rpm) and 100 µM of PAR4-agonist peptide (PAR4-AP, sequence: AYPGKF-NH 2 ) or 5 µM PAR1-agonist peptide (PAR1-AP, sequence: SFLLRN-NH 2 ) was added to induce platelet aggregation. The extent of platelet aggregation was measured as the maximal increase in light transmission within 5 min following the addition of inducers. 2.8. Identification of the mouse IgG isotype of the PAR4 antagonist antibodies The mouse IgG isotype of the PAR4 antagonist antibodies was determined using the Mouse Monoclonal Antibody Isotyping Test Kit (Bio-Rad). Freshly collected supernatant (150 µL) was pipetted into each development tube and incubated at room temperature for 30 s. The tube was briefly vortexed to ensure that the colored microparticle solution was completely resuspended. An isotypic strip, with a solid red end at the bottom, was placed into each development tube. The results were interpreted after 5–10 min once the positive flow control bands had appeared. 2.9. Statistical analyses Statistical analyses were performed using GraphPad Prism software (v10.0) and FlowJo v10.8.1. The results are expressed as the means ± standard error of the mean (S.E.M.). Data were analyzed by a one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison post-hoc test. p < 0.05 was considered statistically significant. 3. Results 3.1. Characterization of the PAR4-mRNA-LNP To express native PAR4 on the cell surface in vivo, we designed a human PAR4 (a type of GPCR) mRNA that contained the PAR4 signal peptide along with the full-length human PAR4 gene to facilitate surface expression and a Flag-tag for detection. In addition, the 5' cap, UTR, and poly (A) tail were synthesized on the mRNA of PAR4 to optimize its stability and transcription efficiency 18 , 19 (Fig. 2 A). To confirm that the PAR4 mRNA was successfully constructed, 1,182 base pairs were detected using agarose gel electrophoresis (Fig. 2 B). Next, we encapsulated the mRNA of PAR4 into lipid nanoparticles (LNP) to form PAR4-mRNA-LNP. The encapsulation efficiency of PAR4-mRNA-LNP was 93.44%, the mean size was 127.5 nm, and the PDI was 0.1033, which complies with FDA guidelines (PDI should remain below 0.3) 20 . The results indicated that all nanoparticles were of similar size and demonstrated a monodispersed size distribution (Fig. 2 C). Thus, the PAR4-mRNA-LNP was successfully developed and ready for cell transfection and PAR4 surface expression. 3.2. PAR4 surface expression following PAR4-mRNA-LNP transfection To confirm PAR4-mRNA-LNP-expressing native PAR4 on the cell surface, different doses (0.5, 1, 2 µg) of PAR4-mRNA-LNP were transfected into 293T cells, and the native structure and surface expression of PAR4 were evaluated by western blot analysis and flow cytometry. For western blot analysis, we used an anti-Flag antibody (clone: 9A3) to verify the correct molecular weight of Flag-PAR4. Two bands were observed at 55 kDa and 40 kDa in the PAR4-expressing cells (293T-Flag-PAR4 cells, used as a positive control), which corresponded to the presence or absence of native PAR4 glycosylation. PAR4-mRNA-LNP effectively expressed a 55 kDa band representing PAR4 with complete glycosylation in a dose-dependent manner (Fig. 3 A). To further assess the surface expression of PAR4, the 293T-Flag-PAR4 group and different transfection doses of PAR4-mRNA-LNP were analyzed using a commercial anti-PAR4 antibody (clone: 5F10) and evaluated by flow cytometry. The results indicated that both 293T-Flag-PAR4 cells and different transfection amounts of PAR4-mRNA-LNP increased the intensity of FITC fluorescence, which confirmed surface PAR4 expression in a dose-dependent manner (Fig. 3 B). The results indicated that PAR4-mRNA-LNP results in complete glycosylation of native PAR4 on the cell surface. 3.3. Anti-human PAR4 antibody titer following PAR4-mRNA-LNP immunization BALB/c mice (n = 6) were administered three intramuscular injections of 10 µg PAR4-mRNA-LNP, followed by a final (4th ) 3 µg boost. Serum was collected during the 2nd, 3rd, and final boosts to measure anti-PAR4 antibody titer. PAR4-expressing cells were seeded into a 96-well plate and various dilutions of mouse serum were added to screen for anti-PAR4 antibody titers using horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody. The results indicated that anti-PAR4 antibodies were produced in two mice (No. mouse-2 and mouse-5) after the 2nd boost (Fig. 4 A). Following the 3rd boost, five mice produced anti-PAR4 antibody titers (Fig. 4 B). After the final boost (4th ), all mice exhibited anti-PAR4 antibody titers. Notably, 5 of 6 mice produced high antibody titers at a 30,000-fold dilution (absorbance 405 nm > 1), with the exception of mouse-3 (absorbance 405 nm < 1) (Fig. 4 C). The results indicate that PAR4-mRNA-LNP effectively induces anti-human PAR4 antibodies following immunization. 3.4. Establishment of hybridoma cells producing monoclonal anti-PAR4 antibodies To stably generate monoclonal anti-PAR4 antibodies, we collected splenocytes from high-titer mice and fused them with FO myeloma cells at a 4:1 ratio to establish hybridomas using the ECM-2001 Hybridoma System. The fused cells were distributed into 1,152 wells in twelve 96-well plates and incubated in HAT medium to select the surviving hybridoma cells and establish single clones. A total of 92 single clones were screened for anti-PAR4 antibodies using a cell-based ELISA and 293T-Flag-PAR4 cells. Fifteen candidate clones were identified. After incubating the clones in a 6-well plate, they were tested with horseradish peroxidase (HRP)-conjugated IgG + IgM antibody to confirm the production of anti-PAR4 antibodies. The results indicated that 13 of the 15 hybridoma clones produced anti-PAR4 IgG or IgM (Fig. 5 A). To identify the isotypes of the anti-PAR4 antibodies, an HRP-conjugated anti-mouse IgG antibody was used to determine the presence of anti-PAR4 mouse IgG. Consequently, 10 of the 15 hybridoma clones generated anti-PAR4 IgG (Fig. 5 B). Both assays showed no nonspecific binding in 293-T cells, confirming the successful establishment of hybridoma cells that stably express specific anti-PAR4 IgG and IgM. 3.5. Screening of the functional anti-PAR4 antagonist antibody and identification of its isotype To determine whether the 13 anti-PAR4 IgG and IgM antibodies from Fig. 5 act as PAR4 antagonists to inhibit PAR4-mediated platelet aggregation, we pretreated human platelet suspensions with the antibodies followed by stimulation with 100 µM protease-activated receptor 4 agonist peptide (PAR4-AP). If the anti-PAR4 antibodies act as antagonists, they may compete with PAR4-AP to inhibit platelet aggregation. The results indicated that five functional anti-PAR4 IgG antibodies (8-9B, 1F-2B, 12A-6D, 12A-9B, and 12A-10E) inhibited platelet aggregation in the presence of PAR4-AP. Notably, clones 8–9 B and 12A-10E completely inhibited platelet aggregation and inhibited PAR4-mediated aggregation (Fig. 6 A). PAR1-AP was used to confirm the specificity of the five anti-PAR4 IgG antibodies for PAR4 compared with other platelet-agonist receptors. The antibodies had no significant effect on inhibiting PAR1-AP (5 µM)-induced platelet aggregation (Fig. 6 B). Finally, we determined the mouse IgG isotype of the PAR4 antagonist antibodies (8-9B, 1F-2B, 12A-6D, 12A-9B, and 12A-10E) using a Mouse Monoclonal Antibody Isotyping Test Kit. Clone 8-9B exhibited an IgG2a isotype with a kappa light chain, whereas the other clones (1F-2B, 12A-6D, 12A-9B, and 12A-10E) were of the IgG2b isotype, also with kappa light chains (Table 1 , Supplementary Fig. 1). Taken together, these findings suggest that PAR4-mRNA-LNP can immunize mice and induce high titers of PAR4 antagonist IgG antibodies. 4. Discussion We describe a PAR4-mRNA-LNP approach that efficiently generates high-titer, functional anti-PAR4 antibodies, and overcomes the structural and specificity challenges of GPCRs. The adjuvant properties of mRNA-LNP further enhance the immune response and mRNA can be used to express any complex protein (like GPCRs). for antibody development through in vivo expression. This simple and convenient mRNA-LNP strategy will accelerate the development of any targeted antibody, particularly against complex proteins. The mRNA of mRNA-LNP acts as an adjuvant and is potentially recognized by toll-like receptors 7 and 8 (TLR7/8) in the endosomes, which triggers an immune response and affects vaccine effectiveness 21 – 25 . Upon activation, TLR7/8 recruits the adaptor protein MyD88, which leads to the activation of the transcription factor NF-κB 26 – 29 and plays an important role in B cell activation by promoting B cell proliferation, differentiation, and antibody production, thereby driving an effective immune response 30 – 33 . Therefore, mRNA-LNP technology is widely used for vaccines and is effective against COVID-19 and other infectious diseases, such as Zika, HIV, and influenza 34 – 38 by triggering a strong immune response. Our results show that following the 4th booster, antibody titers remained significantly strong and maintained effectiveness even when diluted 30,000-fold. This highlights the potential of our approach to generate high antibody titers, which are well-suited for efficient hybridoma screening during subsequent stages. Overall, the PAR4-mRNA-LNP platform streamlines antibody development and leverages the adjuvant properties of mRNA-LNPs to activate TLR7/8, enhancing the immune response, and enabling the production of high-titer, specific antibodies against complex GPCR proteins. mRNA-LNP technology provides a convenient and versatile alternative to traditional GPCR protein production methods and bypasses the need for detergent solubilization and complex purification steps 39 , 40 . Traditional approaches face significant challenges, such as turkey β1-adrenergic receptor (β1AR), human adenosine A2A receptor (A2AR), and rat neurotensin receptor (NTSR1), which must be solubilized from the cell surface using detergents, a process that can disrupt the protein’s native structure and reduce its functionality 39 – 42 . Affinity purification is required to isolate the GPCR; however, this step can be complicated by the presence of contaminants or improperly folded proteins 43 , 44 . Our PAR4-mRNA-LNP strategy addresses traditional challenges by delivering mRNA directly into cells, where PAR4 is naturally synthesized, properly folded, and fully glycosylated on the cell surface. This approach preserves the structural integrity of PAR4, ensures it remains in its native conformation, and significantly streamlines the production of antibodies against complex GPCR targets. In addition, mRNA-LNP technology has promise for generating antibodies against challenging targets, including unstable multi-pass membrane proteins and glycosylated proteins 44 , 45 , as well as low-immunogenic, low-abundance proteins, such as viral antigens 46 – 48 . Using the native structure of GPCR as an antigen is essential for accurately screening therapeutic anti-GPCR agonists and antagonists. Traditional selection strategies often involve the use of GPCR peptides or GPCR-expressing virus-like particles (VLPs). For example, Tohidkia et al . used biotinylated peptides from the second extracellular loop of the GPCR cholecystokinin-B receptor (CCK-BR) to screen an anti-GPCR scFv phage library and isolate scFvs that recognized the native receptor 49 . Similarly, Huang et al . synthesized a biotinylated N-terminal peptide of GPCR C5aR to screen an anti-GPCR Fab phage library to produce IgG antibodies with strong binding to C5aR-expressing cells 50 . However, GPCR peptides often fail to mimic the complex native structure of the target protein, which results in antibodies that recognize linear rather than functional conformational epitopes, reducing their specificity and therapeutic efficacy 51 , 52 . VLP displays GPCRs in a membrane-like environment that closely mimics their natural structure. Ho et al. developed a VLP system in HEK293 cells to efficiently screen anti-GPCR antibodies by analyzing 210 GPCR GALR3 variants for yield, stability, and functionality 53 . However, membrane protein impurities, such as misfolded proteins, present significant challenges in VLP systems and complicate the isolation of highly specific antibodies. Furthermore, the process of isolating VLPs is inherently difficult and complex, which further reduces the yield of therapeutically effective antibodies. To address these limitations, our strategy involves using lentiviruses to stably express GPCRs on the cell surface. This method preserves the native structure of GPCRs, ensuring proper folding, post-translational modifications, and membrane embedding. Figure 5 shows the use of a PAR4-expressing, cell-based ELISA to evaluate anti-PAR4 antibody titers produced by hybridoma cells. This GPCR-expressing, cell-based system enhances the selection of specific and functional anti-GPCR antibodies, making the process more accessible and efficient for research and therapeutic development. PAR4 has an important role in platelet activation and thrombo inflammation, and it has emerged as a potential target for anti-thrombotic therapy. Unlike PAR1, which mediates rapid but transient platelet activation, PAR4 is associated with a slower, more sustained response that is essential for stable thrombus formation 54 – 56 . Although PAR1 inhibitors, such as Vorapaxar, prevent thrombotic events, they carry significant risks, including hemorrhage, particularly intracranial bleeding 57 , 58 . This necessitates careful patient selection and monitoring and highlights the need for safer therapeutics that target PAR4. In addition to its distinct signaling characteristics, PAR4 expression is significantly upregulated in various diseased tissues 59 – 61 , including those associated with cardiovascular and thrombo-inflammatory disorders 62 – 64 . This increased expression underscores the role of PAR4 in disease pathology and reinforces its potential as an effective therapeutic target. Our anti-PAR4 antibody specifically inhibits PAR4-mediated platelet activation, offering an effective approach for the safer management of thromboembolic events and cardiovascular conditions. In conclusion, our mRNA-LNP strategy provides several key advantages for the development of anti-GPCR antibodies. mRNA-LNP serves as a potent adjuvant that stimulates a strong immune response. This approach is also highly convenient as it only requires in vivo injection to efficiently express the target GPCR. This method preserves the native structure of GPCRs on the cell surface, ensuring proper folding and functionality, which is necessary for generating specific antibodies. In addition, the system maintains the native structure of GPCRs, making it ideal for the efficient screening and selection of therapeutic antibodies. Targeting PAR4, which is a promising candidate for diseases like cardiovascular and inflammatory conditions, further emphasizes the relevance of this approach. Taken together, we believe our strategy has the potential to accelerate the development of GPCR-targeted antibodies for various diseases, such as myocardial infarction, stroke, and cancer-associated thrombosis. Declarations 5. Funding This work was supported by grants from the Ministry of Science and Technology, Taipei, Taiwan (MOST 111-2314-B-037-051-MY3); the National Science and Technology Council, Taipei, Taiwan (NSTC 112-2320-B-037 -011 -MY3 and NSTC 112-2124-M-037-001); and the KMU-KMUH Co-Project of Key Research (KMU-DK(B)112001-3) from Kaohsiung Medical University, Taiwan. 6. Acknowledgements We also thank the Drug Development and Value Creation Research Center and the Center for Laboratory Animals, Kaohsiung Medical University, Taiwan for the instrumentation and equipment support. 7. Authors’ contributions ESL and KWH designed and performed experiments reported in the paper analyzed data, and wrote the manuscript; CCW and HLF helped with the platelet aggregation experiments data analysis; TYW and YCH helped with the other experiments and data analysis; BCH and STH contributed to the manuscript editing; TYL helped with designing human PAR4 plasmid construct; YLL provided the information of mRNA-LNP immunization; YTC and CCL provided the information of establishing hybridoma cells; CYC provided the concept and contributed to manuscript writing and editing; TLC and CLL provided the concept, experimental design and contributed to manuscript writing and editing. All authors read and approved the final manuscript. 8. Data availability Data is provided within the manuscript or supplementary information files. References Ahmad, R. & Dalziel, J. E. G Protein-Coupled Receptors in Taste Physiology and Pharmacology. 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Discovery of a novel, orally active himbacine-based thrombin receptor antagonist (SCH 530348) with potent antiplatelet activity. J Med Chem 51 , 3061-3064, doi:10.1021/jm800180e (2008). Heuberger, D. M. & Schuepbach, R. A. Protease-activated receptors (PARs): mechanisms of action and potential therapeutic modulators in PAR-driven inflammatory diseases. Thromb J 17 , 4, doi:10.1186/s12959-019-0194-8 (2019). Mitrugno, A. et al. The role of coagulation and platelets in colon cancer-associated thrombosis. Am J Physiol Cell Physiol 316 , C264-C273, doi:10.1152/ajpcell.00367.2018 (2019). Pavic, G. et al. Thrombin receptor protease-activated receptor 4 is a key regulator of exaggerated intimal thickening in diabetes mellitus. Circulation 130 , 1700-1711, doi:10.1161/CIRCULATIONAHA.113.007590 (2014). Yu, G. et al. Increased expression of protease-activated receptor 4 and Trefoil factor 2 in human colorectal cancer. PLoS One 10 , e0122678, doi:10.1371/journal.pone.0122678 (2015). Mao, Y., Zhang, M., Tuma, R. F. & Kunapuli, S. P. Deficiency of PAR4 attenuates cerebral ischemia/reperfusion injury in mice. J Cereb Blood Flow Metab 30 , 1044-1052, doi:10.1038/jcbfm.2009.283 (2010). Edelstein, L. C. et al. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race. Blood 124 , 3450-3458, doi:10.1182/blood-2014-04-572479 (2014). Lee, R. H. et al. Investigating the Roles of Platelet PAR4 in Hemostasis, Thrombosis and Viral Infection Using a Newly Generated PAR4 Floxed Mouse. Blood 138 , doi:10.1182/blood-2021-151121 (2021). Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files supplementaryfigure1.pdf Supplementary Figure 1. The mouse IgG isotypes of the functional anti-PAR4 antagonist antibody. The IgG isotypes of five hybridoma clones that produced functional anti-PAR4 antagonist antibodies were identified using a Mouse Monoclonal Antibody Isotyping Test Kit. Clone 8-9B was a mouse IgG2a with a kappa light chain. Clones 12A-10E, 12A-9B, 12A-6D, and 1F-2B produced mouse IgG2b isotypes with a kappa light chain. PC: mouse IgG2b with a kappa light chain. Table1.docx Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2025 Read the published version in npj Vaccines → Version 1 posted Editorial decision: Revision requested 07 Jul, 2025 Reviews received at journal 02 Jul, 2025 Reviews received at journal 24 Jun, 2025 Reviewers agreed at journal 20 Jun, 2025 Reviewers agreed at journal 18 Jun, 2025 Reviewers agreed at journal 18 Jun, 2025 Reviewers invited by journal 18 Jun, 2025 Editor assigned by journal 12 Jun, 2025 Submission checks completed at journal 29 May, 2025 First submitted to journal 27 May, 2025 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. 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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-6763159","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":473306471,"identity":"c4a42649-4f8c-48c7-b3fb-e34526163282","order_by":0,"name":"En-Shuo Liu","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"En-Shuo","middleName":"","lastName":"Liu","suffix":""},{"id":473306472,"identity":"8e292798-d67b-40eb-9183-f8934eaee355","order_by":1,"name":"Kai-Wen Ho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYDACZvYDBxJ4bOQkwDw2YrSw9yQ++CCTZgzXwkNQC88BY8MZNocTZxCtxXxGQpo0Tw5z+sxpZwwYPpQdZrCXSMCvReZG4jFpnjNsubOlcwwYZ5w7zMBDSIuEBNAW3h6e3HlALcy8bUAt0oS1mEnz/pNIlwNp+UuUFrD3eQwSpEFaGInSAg5kngTDmbPTCg72nEvn4bn/gIAWSFT+l5e4nbzxwY8yazn2ngP4taAAkFrCMTkKRsEoGAWjgDAAAICfPWFBRkDqAAAAAElFTkSuQmCC","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":true,"prefix":"","firstName":"Kai-Wen","middleName":"","lastName":"Ho","suffix":""},{"id":473306473,"identity":"f328ef03-dbf4-4491-972d-9cba0469b6e7","order_by":2,"name":"Chin-Chung Wu","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chin-Chung","middleName":"","lastName":"Wu","suffix":""},{"id":473306474,"identity":"34f0453c-f275-4e3b-a177-bc4374faebe9","order_by":3,"name":"Hsiao-Li Fan","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hsiao-Li","middleName":"","lastName":"Fan","suffix":""},{"id":473306475,"identity":"4b9a4351-736c-44ab-bde7-3afb23916b12","order_by":4,"name":"Ting-Yu Wang","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ting-Yu","middleName":"","lastName":"Wang","suffix":""},{"id":473306476,"identity":"e36824fd-09c6-4e0a-ae89-c60fb7000203","order_by":5,"name":"Yuan-Chin Hsieh","email":"","orcid":"","institution":"I‐Shou University","correspondingAuthor":false,"prefix":"","firstName":"Yuan-Chin","middleName":"","lastName":"Hsieh","suffix":""},{"id":473306477,"identity":"0c659ce9-beb6-4c0b-9934-b4de192166a6","order_by":6,"name":"Bo-Cheng Huang","email":"","orcid":"","institution":"Kaohsiung Medical 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University","correspondingAuthor":false,"prefix":"","firstName":"Chiao-Yun","middleName":"","lastName":"Chen","suffix":""},{"id":473306484,"identity":"e9ce222d-33b3-4f35-a529-4c2af14dcab7","order_by":13,"name":"Chih-Lung Lin","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chih-Lung","middleName":"","lastName":"Lin","suffix":""},{"id":473306485,"identity":"ca55b1ec-290b-4bd1-b43b-8532432fb8f0","order_by":14,"name":"Tian-Lu Cheng","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tian-Lu","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2025-05-28 01:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6763159/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6763159/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41541-025-01283-x","type":"published","date":"2025-11-20T15:58:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85067545,"identity":"4f3f37ef-09c3-41d8-8a2e-098caa16675b","added_by":"auto","created_at":"2025-06-20 15:14:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":154094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePAR4-mRNA-LNP immunization can generate functional anti-PAR4 antibodies to prevent platelet aggregation.\u003c/strong\u003e We developed human PAR4-mRNA-LNPs to immunize mice via intramuscular injections, enabling the transfection of PAR4-mRNA into mouse cells in vivo. This resulted in the surface expression of the native human PAR4 structure, effectively inducing an antibody response and leading to the generation of various anti-human PAR4 antibodies. We selected the most effective functional anti-PAR4 antibody to serve as a PAR4 antagonist, which was capable of inhibiting platelet aggregation.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6763159/v1/462ef9587e43befb1ae426e0.png"},{"id":85066511,"identity":"ffdfcad5-c211-484a-8069-8941083061df","added_by":"auto","created_at":"2025-06-20 14:58:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of PAR4-mRNA-LNP. \u003c/strong\u003e(A) Schematic of the PAR4 mRNA sequence design, which consists of the full-length human PAR4 gene, the PAR4 signal peptide, and a Flag-tag for PAR4 surface expression. The 5′ cap, 5′ UTR, 3' UTR, and poly(A) tail were optimized for stability and transcription efficiency. (B) The mRNA of PAR4 (1,182 base pairs, bp) was detected using agarose gel electrophoresis. M: RNA 1kb ladder. (C) The parameters of PAR4-mRNA-LNP included encapsulation efficiency, size average mean, and polydispersity index (PDI).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6763159/v1/c6a3ddba5a4204eab45b656f.png"},{"id":85066513,"identity":"d6206a81-5afb-4e83-a307-b80eb66284a2","added_by":"auto","created_at":"2025-06-20 14:58:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":252917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePAR4 surface expression following PAR4-mRNA-LNP transfection. \u003c/strong\u003eWe transfected 0.5, 1, and 2 µg of PAR4-mRNA-LNP into 293T cells. (A) To assess the molecular weight of PAR4, an anti-Flag-tag antibody (clone: 9A3) was used to detect Flag-PAR4. In 293T-Flag-PAR4, which served as a positive control, bands were observed at 40 kDa (representing native PAR4 without glycosylation) and 55 kDa (representing native PAR4 with glycosylation). The PAR4-mRNA-LNP transfection groups exhibited a major band at 55 kDa, indicating the presence of the native PAR4 structure with complete glycosylation. The mock group, which did not receive PAR4-mRNA-LNP, served as a negative control. M: Protein ladder, 10–180 kDa. (B) Surface expression of PAR4 was assessed by flow cytometry using an anti-PAR4 antibody (clone: 5F10, MABS1298). The FITC fluorescence intensity indicated the surface expression of PAR4, which increased in a dose-dependent manner with PAR4-mRNA-LNP transfection. The 293-T cell group acted as a negative control, whereas the 293T-Flag-PAR4 group served as a positive control.\u003c/p\u003e","description":"","filename":"floatimage31.png","url":"https://assets-eu.researchsquare.com/files/rs-6763159/v1/1e7309f467a291e461227e8b.png"},{"id":85067546,"identity":"7e0feb2e-fd7c-4856-932b-083ec4347494","added_by":"auto","created_at":"2025-06-20 15:14:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":113256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-human PAR4 antibody titers following PAR4-mRNA-LNP immunization.\u003c/strong\u003e After immunizing BALB/c mice (n = 6) with PAR4-mRNA-LNP through intramuscular injection, a PAR4-expressing cell-based ELISA was used to measure the anti-PAR4 antibody titers with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody for detection. (A) Following the 2\u003csup\u003end\u003c/sup\u003e boost, two mice exhibited anti-PAR4 antibody titers. (B) After the 3\u003csup\u003erd\u003c/sup\u003e boost, five mice produced anti-PAR4 antibody titers. (C) Following the final boost, all mice exhibited anti-PAR4 antibody titers; specifically, mice 1, 2, 4, 5, and 6 displayed extremely high PAR4 antibody titers, even at a 30,000-fold dilution.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6763159/v1/aedcaf63a14f8c021d9b39e1.png"},{"id":85066515,"identity":"bc6f3553-d2dd-4cb2-a2cb-b5d4b18e2633","added_by":"auto","created_at":"2025-06-20 14:58:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":99275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePAR4-expressing cell-based ELISA to evaluate anti-PAR4 antibody titers from hybridoma cells. \u003c/strong\u003eFifteen clones were screened using a PAR4-expressing and 293T alone cell-based ELISA. (A) The anti-PAR4 antibody titers were measured using HRP-conjugated anti-mouse IgG + IgM. (B) The HRP-conjugated anti-mouse IgG Fc fragment identified the isotypes of the anti-PAR4 antibody titers in the supernatants from each hybridoma cell. Thirteen hybridoma clones produced high levels of specific anti-PAR4 IgG (10 clones) or IgM (3 clones). Anti-PAR4 Ab served as a positive control.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6763159/v1/28f589c31eb4b89cbbefaf51.png"},{"id":85066519,"identity":"eb7b5b91-8aa8-495e-b4c6-5b076a27291a","added_by":"auto","created_at":"2025-06-20 14:58:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":269092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelective inhibition of 13 anti-PAR4 IgG and IgM antibodies on PAR4-mediated human platelet aggregation. \u003c/strong\u003eTo determine whether the functional anti-PAR4 antibodies can inhibit platelet aggregation, (A) human platelet suspensions were pretreated with 13 supernatants from the hybridoma cells for 3 min and stimulated with PAR4-AP (100 μM). Five clones (8-9B, 1F-2B, 12A-6D, 12A-9B, and 12A-10E) were identified as capable of inhibiting platelet aggregation and acting as PAR4 antagonists. (B) To confirm the specificity of the five clones for PAR4 versus other platelet-agonist receptors, PAR1-AP was used. They all showed an inhibitory effect of less than 20% or did not affect PAR1-AP-induced platelet aggregation. Control: FO cell culture medium. Values are presented as the mean ± S.E.M. (n ≥ 3). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, compared with the control.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6763159/v1/20fbf1e3ca8ae6acf7cec451.png"},{"id":96651412,"identity":"f89c8216-4205-40b3-9b27-aeb831666431","added_by":"auto","created_at":"2025-11-24 16:14:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2211550,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6763159/v1/b3c56eea-9d33-428e-a3ed-73eb0e3075a7.pdf"},{"id":85066765,"identity":"b8b95ca2-a0cd-48cc-93b0-e71936ad7501","added_by":"auto","created_at":"2025-06-20 15:06:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":135672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. The mouse IgG isotypes of the functional anti-PAR4 antagonist antibody. \u003c/strong\u003eThe IgG isotypes of five hybridoma clones that produced functional anti-PAR4 antagonist antibodies were identified using a Mouse Monoclonal Antibody Isotyping Test Kit. Clone 8-9B was a mouse IgG2a with a kappa light chain. Clones 12A-10E, 12A-9B, 12A-6D, and 1F-2B produced mouse IgG2b isotypes with a kappa light chain. PC: mouse IgG2b with a kappa light chain.\u003c/p\u003e","description":"","filename":"supplementaryfigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6763159/v1/6b9615122833cde2406cb5c2.pdf"},{"id":85066767,"identity":"88c35e6b-2b10-4680-8cfc-0f8d3a3080b9","added_by":"auto","created_at":"2025-06-20 15:06:29","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":39531,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6763159/v1/3afa311aa082f066e299624d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"To generate functional anti-protease-activated receptor-4 (PAR4), a G protein-coupled receptor, antibodies through PAR4-mRNA-LNP immunization","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eG protein-coupled receptors (GPCRs) play important roles in various physiological processes, including sensory perception\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, neurotransmitter\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and hormone regulation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Dysregulation or abnormal GPCR signaling is associated with various diseases, such as cancer\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, cardiovascular disorders\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, and neurological conditions\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Because of their role in disease progression, GPCRs are important therapeutic targets. For example, protease-activated receptor 4 (PAR4) is a GPCR required for platelet activation and has a significant role in cardiovascular diseases, such as myocardial infarction and stroke. Therefore, targeting PAR4 with therapeutic antibodies represents a promising approach. Developing antibodies against GPCRs is challenging because of their structural complexity, which limits recombinant protein production\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Peptides are often used as substitutes, but their poor mimicry of the GPCR structure reduces binding specificity and antibody effectiveness\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Innovative strategies are essential to preserve the native structure of GPCRs. Huang \u003cem\u003eet al\u003c/em\u003e. used murine 3T3 cells expressing human CXCR2 with membrane-bound GM-CSF as an adjuvant to enhance antibody production by stimulating local inflammation and splenocyte proliferation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, this approach may increase background noise, resulting in a weakening of the immune response and complicating the identification of specific antibodies. Therefore, a strategy to enhance GPCR antibody generation is needed that can maintain the native GPCR structure, reduce background noise, and act as a good adjuvant to produce a strong antibody titer.\u003c/p\u003e \u003cp\u003emRNA-lipid nanoparticle (LNP) technology\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e enables the direct expression of PAR4 on the cell surface, thus preserving its native structure. The mRNA also serves as a potent adjuvant\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, activating toll-like receptors 7 and 8 (TLR7/8) to induce a strong immune response by eliciting the production of antibodies specifically recognizing native PAR4. Therefore, we immunized Balb/c mice with PAR4-expressing mRNA-LNPs and utilized a hybridoma strategy\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e to produce and isolate anti-PAR4 antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Initially, the structural integrity and synthesis efficiency of PAR4-mRNA-LNP will be confirmed via gel electrophoresis and particle characterization. Subsequently, we will verify molecular weight and surface expression of PAR4 on 293T cells after PAR4-mRNA-LNP using western blotting and flow cytometry, respectively. Throughout the immunization timeline in BALB/c mice, antibody titers will be quantified by PAR4-expressing cell-based ELISA, monitoring response escalation following sequential boosts. To establish sustained antibody production, hybridoma clones will be generated and screened, confirming anti-PAR4 IgG/IgM titers and isotypes. Functional inhibition assays will then evaluate the antagonistic capability of anti-PAR4 antibodies on platelet aggregation. This mRNA-LNP approach enhanced antibody specificity and efficacy by preserving the native GPCR structure and resulted in the production of high-quality, functional antibodies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cells and reagents\u003c/h2\u003e \u003cp\u003eWe used 293-T and FO cells for this study. We established 293T-Flag-PAR4 cells that stably expressed Flag-PAR4 on the 293-T cell surface by lentivirus transduction. 293T cells were seeded at 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in a 6-well plate and cotransfected with 2 \u0026micro;g of pLKO-AS3W-Flag-human PAR4-puro, 0.2 \u0026micro;g pMD.G, and 1.8 \u0026micro;g pCMV 8.91 using Lipofectamine\u0026trade; 2000 Transfection Reagent (Thermo Fisher, Inc). After 7 days, the culture medium was collected and filtered through a 0.22-\u0026micro;m syringe filter, mixed with 8 \u0026micro;g/mL polybrene, and added to 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e 293T cells for viral infection. The cells were selected with 2 \u0026micro;g/mL puromycin. High-expressing Flag-PAR4 293T cells were detected using mouse anti-human PAR4 antibody (clone 5F10; MABS1298) (1:50 dilution) and FITC-conjugated goat anti-mouse IgG Fcγ (Jackson Immuno-Research Inc. #115-095-008) (1:20 dilution) and sorted using a MoFlo\u0026trade; XDP Cell Sorter System (Beckman Colter) to generate 293T-Flag-PAR4 cells. The cells were cultured in DMEM (Sigma-Aldrich) containing 10% heat-inactivated bovine calf serum (GE Healthcare Life Science) and 1% penicillin combined with streptomycin (Thermo Fisher Scientific) at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Design of the PAR4-mRNA-LNP\u003c/h2\u003e \u003cp\u003eThe PAR4 DNA sequence was designed based on the NCBI Reference Sequence NM_003950.4. Dr. Ching-Jen Yang from the Development Center of Biotechnology in Taiwan assisted in the construction of the PAR4-mRNA. The PAR4-mRNA was encapsulated in lipid nanoparticles (LNP) using a formulation similar to that of Moderna.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Dr. Yang evaluated the encapsulation efficiency, particle size, and polydispersity index (PDI) using a dynamic light scattering system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Evaluate the PAR4 surface expression of PAR4-mRNA-LNP by western blot analysis and flow cytometry\u003c/h2\u003e \u003cp\u003e293-T cells were seeded at 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/mL in a 12-well growth plate and incubated overnight. The medium was replaced with 750 \u0026micro;L of fresh medium. After 30 min, we added 250 \u0026micro;L of medium containing 0, 0.5, 1, or 2 \u0026micro;g of PAR4-mRNA-LNP to the cells and incubated for 48 h. PAR4 expression was detected by western blot analysis and flow cytometry. PAR4 expression in 293-T cells transfected with PAR4-mRNA-LNP was assessed by western blot analysis. The primary antibody was DYKDDDDK Flag-Tag (9A3) mouse mAb (Cell Signaling Technology, Inc; #8146) and the secondary antibody was Goat anti-mouse IgG Fcγ fragment specific-HRP (Jackson Immuno-Research Inc. #115-035-008). After developing the blot by chemiluminescence, the bands were quantitated using a MultiGel-21 UVP imaging system. The surface expression of PAR4 in 293T cells by flow cytometry following transfection with PAR4-mRNA-LNP and staining the cells with mouse anti-human PAR4 antibody (clone 5F10; MABS1298), followed by incubation with FITC-conjugated goat anti-mouse IgG Fcγ (Jackson Immuno-Research Inc. #115-095-008). The surface fluorescence intensity of viable cells was measured using a Merck Guava easyCyte System flow cytometer and the data were analyzed using FlowJo v10.8.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. PAR4 antibody titers after immunization with PAR4-mRNA-LNP\u003c/h2\u003e \u003cp\u003e All procedures involving animals were performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee at Kaohsiung Medical University in Taiwan. BALB/c mice (n\u0026thinsp;=\u0026thinsp;6), aged 6\u0026ndash;8 weeks, were immunized with three intramuscular (i.m) injections of 10 \u0026micro;g PAR4-mRNA-LNP, administered once every 2 weeks for 6 weeks. At 8 weeks, a final (4th ) intramuscular boost of 3 \u0026micro;g PAR4-mRNA-LNP was administered. Tail vein blood was collected and the serum was stored at \u0026minus;\u0026thinsp;80\u0026deg;C one week after each booster. Anti-human PAR4 antibody titers from mouse serum were screened using a cell-based ELISA. 293T-Flag-PAR4 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded in 96-well plates (Thermo Fisher Scientific Inc. #167008) coated with 50 \u0026micro;g/mL poly-D-lysine and incubated overnight. The cells were fixed in 1% paraformaldehyde in PBS for 5 min at room temperature, followed by the addition of 0.1 M glycine to neutralize the paraformaldehyde for 30 min at room temperature. The plates were blocked with 5% milk and diluted anti-human PAR4 antibody from mouse serum (in 2% skim milk) was added to each well and incubated for 50 min at room temperature. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Fcγ fragment specific-HRP (Jackson Immuno-Research Inc. #115-035-008) (1:2000) was added and incubated for 50 min at room temperature. Following three washes with PBS, ABTS, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were added. The binding capacities of each serum sample were evaluated using an ELISA reader and the data were analyzed using GraphPad Prism software (v10.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Establishing a hybridoma using the ECM-2001 Hybridoma System for electrofusion\u003c/h2\u003e \u003cp\u003eSplenocytes (1.6 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e/ml) and FO myeloma cells (4 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/ml) were mixed at a 4:1 ratio and washed twice with 20 ml electrofusion medium (centrifuged at 1,400 rpm for 7 min). The pellets were resuspended with 2 mL of room temperature Cytofusion Medium C and the pellet was transferred to a 2 mL coaxial chamber. We immediately performed electrofusion (within 30 seconds) using the recommended parameters (Step-1. Pre-AC: V0\u0026thinsp;=\u0026thinsp;40 V, VF\u0026thinsp;=\u0026thinsp;40 V, T\u0026thinsp;=\u0026thinsp;15 s, F\u0026thinsp;=\u0026thinsp;1.4 MHz; Step-2. Pre-AC: V0\u0026thinsp;=\u0026thinsp;70V, VF\u0026thinsp;=\u0026thinsp;70V, T\u0026thinsp;=\u0026thinsp;20 sec, F\u0026thinsp;=\u0026thinsp;1.4 MHz; DC-PULSE: V\u0026thinsp;=\u0026thinsp;800V, T\u0026thinsp;=\u0026thinsp;40 \u0026micro;s, N\u0026thinsp;=\u0026thinsp;1, I\u0026thinsp;=\u0026thinsp;0; Post-AC: V0\u0026thinsp;=\u0026thinsp;70V, VF\u0026thinsp;=\u0026thinsp;5V, T\u0026thinsp;=\u0026thinsp;30 sec, F\u0026thinsp;=\u0026thinsp;1.4 MHz). Finally, the fusion mixture was plated into twelve 96-well plates (1,152 total wells) and cultured in HAT medium (Sigma-Aldrich) to screen for hybridomas.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Evaluation of anti-PAR4 antibody secreted from hybridoma cells\u003c/h2\u003e \u003cp\u003eAfter establishing hybridoma cells and observing their growth to approximately 50% confluency in 96-well plates over 1\u0026ndash;2 weeks, the supernatants were collected and the PAR4 antibody titers were determined using a cell-based ELISA. t293T-Flag-PAR4 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded into 96-well plates (Thermo Fisher Scientific Inc. #167008) coated with 50 \u0026micro;g/mL poly-D-lysine and incubated overnight. The cells were fixed in 1% paraformaldehyde in PBS for 5 min at room temperature, followed by the addition of 0.1 M glycine to neutralize the paraformaldehyde for 30 min at room temperature. The plates were blocked in 5% milk and the supernatants were added to each well for 50 min at room temperature. After three washes with PBS, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG\u0026thinsp;+\u0026thinsp;IgM (H\u0026thinsp;+\u0026thinsp;L) antibody (Jackson Immuno-Research Inc. #115-035-044) (1:1,000) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Fc fragment antibody (Jackson Immuno-Research Inc. #115-035-008) (1:1,000) were added and incubated for 50 min at room temperature. After three washes with PBS, ABTS and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were added. The binding capacities of each serum were evaluated using an ELISA reader and the data were analyzed using GraphPad Prism software (v10.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Screening for functional anti-PAR4 antagonist antibodies\u003c/h2\u003e \u003cp\u003e The protocol for this study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital. Human blood anticoagulated with acid citrate dextrose was obtained from healthy human volunteers, who had not taken any drugs during the previous two weeks. The blood was centrifuged at 450 \u0026times; g for 15 min and the supernatant was obtained as platelet-rich plasma. The platelet-rich plasma was further centrifuged at 1,800 \u0026times; g for 15 min in the presence of prostaglandin E1 (0.5 \u0026micro;M). The platelet pellet was washed with Tyrode\u0026rsquo;s solution containing 2 mM Ca\u003csup\u003e2+\u003c/sup\u003e, 11.1 mM glucose, and 3.5 mg/ml bovine serum albumin and centrifuged at 1,200 \u0026times; g for 7 min in the presence of prostaglandin E1 (0.5 \u0026micro;M) and apyrase (0.15 U/ml). The platelet pellet was washed again as described above except for the omission of prostaglandin E1. The washed platelets were suspended in Tyrode\u0026rsquo;s solution at 3 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e platelets/mL unless otherwise specified. Platelet aggregation was measured turbidimetrically with a light-transmission aggregometer (Chrono-Log Co., Havertown, PA, USA). The platelet suspension was pretreated with anti-PAR4 antibodies in the supernatant at 37\u0026deg;C for 3 min while stirring (1,200 rpm) and 100 \u0026micro;M of PAR4-agonist peptide (PAR4-AP, sequence: AYPGKF-NH\u003csub\u003e2\u003c/sub\u003e) or 5 \u0026micro;M PAR1-agonist peptide (PAR1-AP, sequence: SFLLRN-NH\u003csub\u003e2\u003c/sub\u003e) was added to induce platelet aggregation. The extent of platelet aggregation was measured as the maximal increase in light transmission within 5 min following the addition of inducers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Identification of the mouse IgG isotype of the PAR4 antagonist antibodies\u003c/h2\u003e \u003cp\u003eThe mouse IgG isotype of the PAR4 antagonist antibodies was determined using the Mouse Monoclonal Antibody Isotyping Test Kit (Bio-Rad). Freshly collected supernatant (150 \u0026micro;L) was pipetted into each development tube and incubated at room temperature for 30 s. The tube was briefly vortexed to ensure that the colored microparticle solution was completely resuspended. An isotypic strip, with a solid red end at the bottom, was placed into each development tube. The results were interpreted after 5\u0026ndash;10 min once the positive flow control bands had appeared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism software (v10.0) and FlowJo v10.8.1. The results are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (S.E.M.). Data were analyzed by a one-way analysis of variance (ANOVA), followed by Dunnett\u0026rsquo;s multiple comparison post-hoc test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of the PAR4-mRNA-LNP\u003c/h2\u003e \u003cp\u003eTo express native PAR4 on the cell surface in vivo, we designed a human PAR4 (a type of GPCR) mRNA that contained the PAR4 signal peptide along with the full-length human PAR4 gene to facilitate surface expression and a Flag-tag for detection. In addition, the 5' cap, UTR, and poly (A) tail were synthesized on the mRNA of PAR4 to optimize its stability and transcription efficiency\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To confirm that the PAR4 mRNA was successfully constructed, 1,182 base pairs were detected using agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, we encapsulated the mRNA of PAR4 into lipid nanoparticles (LNP) to form PAR4-mRNA-LNP. The encapsulation efficiency of PAR4-mRNA-LNP was 93.44%, the mean size was 127.5 nm, and the PDI was 0.1033, which complies with FDA guidelines (PDI should remain below 0.3)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The results indicated that all nanoparticles were of similar size and demonstrated a monodispersed size distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Thus, the PAR4-mRNA-LNP was successfully developed and ready for cell transfection and PAR4 surface expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. PAR4 surface expression following PAR4-mRNA-LNP transfection\u003c/h2\u003e \u003cp\u003eTo confirm PAR4-mRNA-LNP-expressing native PAR4 on the cell surface, different doses (0.5, 1, 2 \u0026micro;g) of PAR4-mRNA-LNP were transfected into 293T cells, and the native structure and surface expression of PAR4 were evaluated by western blot analysis and flow cytometry. For western blot analysis, we used an anti-Flag antibody (clone: 9A3) to verify the correct molecular weight of Flag-PAR4. Two bands were observed at 55 kDa and 40 kDa in the PAR4-expressing cells (293T-Flag-PAR4 cells, used as a positive control), which corresponded to the presence or absence of native PAR4 glycosylation. PAR4-mRNA-LNP effectively expressed a 55 kDa band representing PAR4 with complete glycosylation in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To further assess the surface expression of PAR4, the 293T-Flag-PAR4 group and different transfection doses of PAR4-mRNA-LNP were analyzed using a commercial anti-PAR4 antibody (clone: 5F10) and evaluated by flow cytometry. The results indicated that both 293T-Flag-PAR4 cells and different transfection amounts of PAR4-mRNA-LNP increased the intensity of FITC fluorescence, which confirmed surface PAR4 expression in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results indicated that PAR4-mRNA-LNP results in complete glycosylation of native PAR4 on the cell surface.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Anti-human PAR4 antibody titer following PAR4-mRNA-LNP immunization\u003c/h2\u003e \u003cp\u003eBALB/c mice (n\u0026thinsp;=\u0026thinsp;6) were administered three intramuscular injections of 10 \u0026micro;g PAR4-mRNA-LNP, followed by a final (4th ) 3 \u0026micro;g boost. Serum was collected during the 2nd, 3rd, and final boosts to measure anti-PAR4 antibody titer. PAR4-expressing cells were seeded into a 96-well plate and various dilutions of mouse serum were added to screen for anti-PAR4 antibody titers using horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody. The results indicated that anti-PAR4 antibodies were produced in two mice (No. mouse-2 and mouse-5) after the 2nd boost (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Following the 3rd boost, five mice produced anti-PAR4 antibody titers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). After the final boost (4th ), all mice exhibited anti-PAR4 antibody titers. Notably, 5 of 6 mice produced high antibody titers at a 30,000-fold dilution (absorbance 405 nm\u0026thinsp;\u0026gt;\u0026thinsp;1), with the exception of mouse-3 (absorbance 405 nm\u0026thinsp;\u0026lt;\u0026thinsp;1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The results indicate that PAR4-mRNA-LNP effectively induces anti-human PAR4 antibodies following immunization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Establishment of hybridoma cells producing monoclonal anti-PAR4 antibodies\u003c/h2\u003e \u003cp\u003eTo stably generate monoclonal anti-PAR4 antibodies, we collected splenocytes from high-titer mice and fused them with FO myeloma cells at a 4:1 ratio to establish hybridomas using the ECM-2001 Hybridoma System. The fused cells were distributed into 1,152 wells in twelve 96-well plates and incubated in HAT medium to select the surviving hybridoma cells and establish single clones. A total of 92 single clones were screened for anti-PAR4 antibodies using a cell-based ELISA and 293T-Flag-PAR4 cells. Fifteen candidate clones were identified. After incubating the clones in a 6-well plate, they were tested with horseradish peroxidase (HRP)-conjugated IgG\u0026thinsp;+\u0026thinsp;IgM antibody to confirm the production of anti-PAR4 antibodies. The results indicated that 13 of the 15 hybridoma clones produced anti-PAR4 IgG or IgM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To identify the isotypes of the anti-PAR4 antibodies, an HRP-conjugated anti-mouse IgG antibody was used to determine the presence of anti-PAR4 mouse IgG. Consequently, 10 of the 15 hybridoma clones generated anti-PAR4 IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Both assays showed no nonspecific binding in 293-T cells, confirming the successful establishment of hybridoma cells that stably express specific anti-PAR4 IgG and IgM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Screening of the functional anti-PAR4 antagonist antibody and identification of its isotype\u003c/h2\u003e \u003cp\u003eTo determine whether the 13 anti-PAR4 IgG and IgM antibodies from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e act as PAR4 antagonists to inhibit PAR4-mediated platelet aggregation, we pretreated human platelet suspensions with the antibodies followed by stimulation with 100 \u0026micro;M protease-activated receptor 4 agonist peptide (PAR4-AP). If the anti-PAR4 antibodies act as antagonists, they may compete with PAR4-AP to inhibit platelet aggregation. The results indicated that five functional anti-PAR4 IgG antibodies (8-9B, 1F-2B, 12A-6D, 12A-9B, and 12A-10E) inhibited platelet aggregation in the presence of PAR4-AP. Notably, clones 8\u0026ndash;9 B and 12A-10E completely inhibited platelet aggregation and inhibited PAR4-mediated aggregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). PAR1-AP was used to confirm the specificity of the five anti-PAR4 IgG antibodies for PAR4 compared with other platelet-agonist receptors. The antibodies had no significant effect on inhibiting PAR1-AP (5 \u0026micro;M)-induced platelet aggregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Finally, we determined the mouse IgG isotype of the PAR4 antagonist antibodies (8-9B, 1F-2B, 12A-6D, 12A-9B, and 12A-10E) using a Mouse Monoclonal Antibody Isotyping Test Kit. Clone 8-9B exhibited an IgG2a isotype with a kappa light chain, whereas the other clones (1F-2B, 12A-6D, 12A-9B, and 12A-10E) were of the IgG2b isotype, also with kappa light chains (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Fig.\u0026nbsp;1). Taken together, these findings suggest that PAR4-mRNA-LNP can immunize mice and induce high titers of PAR4 antagonist IgG antibodies.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWe describe a PAR4-mRNA-LNP approach that efficiently generates high-titer, functional anti-PAR4 antibodies, and overcomes the structural and specificity challenges of GPCRs. The adjuvant properties of mRNA-LNP further enhance the immune response and mRNA can be used to express any complex protein (like GPCRs). for antibody development through in vivo expression. This simple and convenient mRNA-LNP strategy will accelerate the development of any targeted antibody, particularly against complex proteins.\u003c/p\u003e \u003cp\u003eThe mRNA of mRNA-LNP acts as an adjuvant and is potentially recognized by toll-like receptors 7 and 8 (TLR7/8) in the endosomes, which triggers an immune response and affects vaccine effectiveness\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Upon activation, TLR7/8 recruits the adaptor protein MyD88, which leads to the activation of the transcription factor NF-κB\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and plays an important role in B cell activation by promoting B cell proliferation, differentiation, and antibody production, thereby driving an effective immune response\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Therefore, mRNA-LNP technology is widely used for vaccines and is effective against COVID-19 and other infectious diseases, such as Zika, HIV, and influenza\u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e by triggering a strong immune response. Our results show that following the 4th booster, antibody titers remained significantly strong and maintained effectiveness even when diluted 30,000-fold. This highlights the potential of our approach to generate high antibody titers, which are well-suited for efficient hybridoma screening during subsequent stages. Overall, the PAR4-mRNA-LNP platform streamlines antibody development and leverages the adjuvant properties of mRNA-LNPs to activate TLR7/8, enhancing the immune response, and enabling the production of high-titer, specific antibodies against complex GPCR proteins.\u003c/p\u003e \u003cp\u003emRNA-LNP technology provides a convenient and versatile alternative to traditional GPCR protein production methods and bypasses the need for detergent solubilization and complex purification steps\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Traditional approaches face significant challenges, such as turkey β1-adrenergic receptor (β1AR), human adenosine A2A receptor (A2AR), and rat neurotensin receptor (NTSR1), which must be solubilized from the cell surface using detergents, a process that can disrupt the protein\u0026rsquo;s native structure and reduce its functionality\u003csup\u003e\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Affinity purification is required to isolate the GPCR; however, this step can be complicated by the presence of contaminants or improperly folded proteins\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Our PAR4-mRNA-LNP strategy addresses traditional challenges by delivering mRNA directly into cells, where PAR4 is naturally synthesized, properly folded, and fully glycosylated on the cell surface. This approach preserves the structural integrity of PAR4, ensures it remains in its native conformation, and significantly streamlines the production of antibodies against complex GPCR targets. In addition, mRNA-LNP technology has promise for generating antibodies against challenging targets, including unstable multi-pass membrane proteins and glycosylated proteins\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, as well as low-immunogenic, low-abundance proteins, such as viral antigens\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUsing the native structure of GPCR as an antigen is essential for accurately screening therapeutic anti-GPCR agonists and antagonists. Traditional selection strategies often involve the use of GPCR peptides or GPCR-expressing virus-like particles (VLPs). For example, Tohidkia \u003cem\u003eet al\u003c/em\u003e. used biotinylated peptides from the second extracellular loop of the GPCR cholecystokinin-B receptor (CCK-BR) to screen an anti-GPCR scFv phage library and isolate scFvs that recognized the native receptor\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Similarly, Huang \u003cem\u003eet al\u003c/em\u003e. synthesized a biotinylated N-terminal peptide of GPCR C5aR to screen an anti-GPCR Fab phage library to produce IgG antibodies with strong binding to C5aR-expressing cells\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, GPCR peptides often fail to mimic the complex native structure of the target protein, which results in antibodies that recognize linear rather than functional conformational epitopes, reducing their specificity and therapeutic efficacy\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. VLP displays GPCRs in a membrane-like environment that closely mimics their natural structure. Ho \u003cem\u003eet al.\u003c/em\u003e developed a VLP system in HEK293 cells to efficiently screen anti-GPCR antibodies by analyzing 210 GPCR GALR3 variants for yield, stability, and functionality\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. However, membrane protein impurities, such as misfolded proteins, present significant challenges in VLP systems and complicate the isolation of highly specific antibodies. Furthermore, the process of isolating VLPs is inherently difficult and complex, which further reduces the yield of therapeutically effective antibodies. To address these limitations, our strategy involves using lentiviruses to stably express GPCRs on the cell surface. This method preserves the native structure of GPCRs, ensuring proper folding, post-translational modifications, and membrane embedding. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the use of a PAR4-expressing, cell-based ELISA to evaluate anti-PAR4 antibody titers produced by hybridoma cells. This GPCR-expressing, cell-based system enhances the selection of specific and functional anti-GPCR antibodies, making the process more accessible and efficient for research and therapeutic development.\u003c/p\u003e \u003cp\u003ePAR4 has an important role in platelet activation and thrombo inflammation, and it has emerged as a potential target for anti-thrombotic therapy. Unlike PAR1, which mediates rapid but transient platelet activation, PAR4 is associated with a slower, more sustained response that is essential for stable thrombus formation\u003csup\u003e\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Although PAR1 inhibitors, such as Vorapaxar, prevent thrombotic events, they carry significant risks, including hemorrhage, particularly intracranial bleeding\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. This necessitates careful patient selection and monitoring and highlights the need for safer therapeutics that target PAR4. In addition to its distinct signaling characteristics, PAR4 expression is significantly upregulated in various diseased tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, including those associated with cardiovascular and thrombo-inflammatory disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. This increased expression underscores the role of PAR4 in disease pathology and reinforces its potential as an effective therapeutic target. Our anti-PAR4 antibody specifically inhibits PAR4-mediated platelet activation, offering an effective approach for the safer management of thromboembolic events and cardiovascular conditions.\u003c/p\u003e \u003cp\u003eIn conclusion, our mRNA-LNP strategy provides several key advantages for the development of anti-GPCR antibodies. mRNA-LNP serves as a potent adjuvant that stimulates a strong immune response. This approach is also highly convenient as it only requires in vivo injection to efficiently express the target GPCR. This method preserves the native structure of GPCRs on the cell surface, ensuring proper folding and functionality, which is necessary for generating specific antibodies. In addition, the system maintains the native structure of GPCRs, making it ideal for the efficient screening and selection of therapeutic antibodies. Targeting PAR4, which is a promising candidate for diseases like cardiovascular and inflammatory conditions, further emphasizes the relevance of this approach. Taken together, we believe our strategy has the potential to accelerate the development of GPCR-targeted antibodies for various diseases, such as myocardial infarction, stroke, and cancer-associated thrombosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e5. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Ministry of Science and Technology, Taipei, Taiwan (MOST 111-2314-B-037-051-MY3); the National Science and Technology Council, Taipei, Taiwan (NSTC 112-2320-B-037 -011 -MY3 and NSTC 112-2124-M-037-001); and the KMU-KMUH Co-Project of Key Research (KMU-DK(B)112001-3) from Kaohsiung Medical University, Taiwan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also thank the Drug Development and Value Creation Research Center and the Center for Laboratory Animals, Kaohsiung Medical University, Taiwan for the instrumentation and equipment support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Authors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eESL and KWH designed and performed experiments reported in the paper analyzed data, and wrote the manuscript; CCW and HLF helped with the platelet aggregation experiments data analysis; TYW and YCH helped with the other experiments and data analysis; BCH and STH contributed to the manuscript editing; TYL helped with designing human PAR4 plasmid construct; YLL provided the information of mRNA-LNP immunization; YTC and CCL provided the information of establishing hybridoma cells; CYC provided the concept and contributed to manuscript writing and editing; TLC and CLL provided the concept, experimental design and contributed to manuscript writing and editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Data availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmad, R. \u0026amp; Dalziel, J. E. G Protein-Coupled Receptors in Taste Physiology and Pharmacology. \u003cem\u003eFront Pharmacol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 587664, doi:10.3389/fphar.2020.587664 (2020).\u003c/li\u003e\n\u003cli\u003eKumar, A. \u0026amp; Pluckthun, A. In vivo assembly and large-scale purification of a GPCR - Galpha fusion with Gbetagamma, and characterization of the active complex. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, e0210131, doi:10.1371/journal.pone.0210131 (2019).\u003c/li\u003e\n\u003cli\u003eFeng, Z., Sun, R., Cong, Y. \u0026amp; Liu, Z. 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The structural complexity of native GPCRs limits recombinant protein production. Traditional GPCR antibody development relies on GPCR peptide fragments for animal immunization. These peptides poorly mimic the native structure and affect the quality of the antibodies. Therefore, it is necessary to develop a strategy for immunizing native GPCRs to generate potent anti-GPCR antibodies. Here, we developed a PAR4-mRNA-LNP to immunize mice, which promotes the surface expression of the native PAR4 structure \u003cem\u003ein vivo\u003c/em\u003e to induce highly specific and functional anti-PAR4 antibodies. These anti-PAR4 antibodies effectively inhibit platelet aggregation and provide a long-term therapeutic approach for cardiovascular disease. In result, PAR4-mRNA-LNP was synthesized with an encapsulation efficiency of 93.44%, mean size of 127.5 nm, and polydispersity index (PDI) of 0.1033. We confirmed the 55-kDa native PAR4 structure with complete glycosylation expressing on cell surface. We immunized mice with PAR4-mRNA-LNP and detected high anti-PAR4 antibody titers with ELISA. This assay used 293T cells that stably express the native PAR4 structure, enabling us to screen for high-quality anti-PAR4 antibodies. We established 15 hybridoma cell lines, which resulted in 13 including 10 IgG and 3 IgM anti-PAR4 antibodies. Five clones significantly inhibited PAR4-mediated platelet aggregation. We confirmed that PAR4-mRNA-LNP expresses the native PAR4 structure on the cell surface and the immunization produced highly functional anti-PAR4 antibodies. mRNA-LNP technology may be widely used to produce anti-GPCR antagonists and agonists for therapy.\u003c/p\u003e","manuscriptTitle":"To generate functional anti-protease-activated receptor-4 (PAR4), a G protein-coupled receptor, antibodies through PAR4-mRNA-LNP immunization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 14:58:24","doi":"10.21203/rs.3.rs-6763159/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-08T00:30:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-02T17:32:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T10:44:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139276372417216362711124673996387330643","date":"2025-06-20T16:43:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37959459204251819422528973761234062410","date":"2025-06-18T16:21:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59889634151739053341798619659790611423","date":"2025-06-18T16:16:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-18T16:11:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-12T12:41:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-29T10:20:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-05-28T01:36:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"519f9ecc-79f9-4669-b917-2b31f3565a9f","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50266054,"name":"Biological sciences/Biotechnology"},{"id":50266055,"name":"Biological sciences/Biotechnology/Applied immunology"}],"tags":[],"updatedAt":"2025-11-24T16:14:29+00:00","versionOfRecord":{"articleIdentity":"rs-6763159","link":"https://doi.org/10.1038/s41541-025-01283-x","journal":{"identity":"npj-vaccines","isVorOnly":false,"title":"npj Vaccines"},"publishedOn":"2025-11-20 15:58:37","publishedOnDateReadable":"November 20th, 2025"},"versionCreatedAt":"2025-06-20 14:58:24","video":"","vorDoi":"10.1038/s41541-025-01283-x","vorDoiUrl":"https://doi.org/10.1038/s41541-025-01283-x","workflowStages":[]},"version":"v1","identity":"rs-6763159","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6763159","identity":"rs-6763159","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}