Engineered virus-like particles for targeted capture of membrane proteins

Abstract

Engineered virus-like particles (VLPs) and uses thereof are disclosed. In the VPL, the integrated plasma membrane protein is captured and displayed in native conformation on the surface of the VLP based on the interaction between the viral scaffold protein fused to the PDZ domain and the synthetic polypeptide in the C-terminal tail of the cytoplasm of the target membrane protein.

Description

[0001] Sequence Listing

[0002] The Sequence Listing, created on December 13, 2023, and named 11641-002WO1.XML with a file size of 8,104 bytes, is hereby incorporated by reference in accordance with 37 C.F.R. § 1.52(e)(5). Technical Field

[0003] The present invention relates to engineered virus-like particles (VLPs) in which integral membrane proteins are captured and displayed in their native conformation on the surface of the VLPs based on the interaction between a viral scaffold protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of a target membrane protein. The present invention further relates to, but is not limited to, the use of such engineered pseudotyped VLPs for antibody discovery. Background Art

[0004] Biomolecules that modulate targeted membrane proteins have emerged as one of the most promising therapeutic classes (1). Antibodies as drug candidates have several advantages over small molecules, including better specificity, lower dosing frequency, and limitations from the central nervous system. There are two basic methods for antibody development: harnessing the animal immune system or synthetic molecular display methods (e.g., phage display-based methods) (2).

[0005] To harness the immune systems of various organisms to produce antibodies, the target antigen is presented to be recognized by the host humoral immune system, leading to the activation of B cells and the secretion of antibodies that recognize the presented antigenic epitopes. Subsequently, individual monoclonal antibodies can be isolated and produced by recombinant or hybridoma techniques.

[0006] Phage display of synthetic antibody fragments has proven to be an effective molecular display tool for antibody development, which allows the selection of antibodies against a large number of biological and non-biological targets and provides the opportunity to link antibody phenotype and genotype (3)(4).

[0007] In addition, the recombinant in vitro nature of phage display instead uses alternative depletion steps, epitope-directed, binding to protein-protein interaction complexes, and binding using composite component antigens.

[0008] G protein-coupled receptors (GPCRs) are one of the largest protein superfamilies with over 800 members (5). Ion channel proteins are the second largest class of membrane proteins and are involved in regulating a variety of physiological processes, which is an important class of multi-transmembrane proteins expressed in almost all living cells (6). Aberrant signaling of GPCRs and ion channel proteins is associated with many immune, neurological, and metabolic disorders as well as cancer.

[0009] Approximately 35% of all approved drugs target GPCRs, making them the largest protein family targeted for drug development to date (7). However, in terms of antibody development strategies, multi-transmembrane proteins are a challenging group of targets. They are often expressed at low density on the cell surface and are unstable and misfolded when removed from the lipid bilayer. Therefore, obtaining sufficient amounts of relevant antigens for antibody discovery efforts through antibody display techniques or by animal immunization remains one of the limiting factors, thus making the development of specific antibodies very challenging.

[0010] In addition, the structure of GPCR and ion channel targets themselves presents another limiting factor, namely the minimal epitopes available for potential antibody binding displayed on the cell surface.

[0011] To date, most anti-GPCR antibodies have been generated against the larger N-terminal extracellular domain or linear peptides of GPCRs. Generating appropriate full-length GPCR antigens remains the biggest bottleneck (8).

[0012] Since developing immune-derived antibody libraries against evolutionarily conserved membrane proteins has proven difficult, synthetic antibody fragments using phage display have been an effective molecular tool for antibody development, which allows the selection of antibodies against a large number of biological and non-biological targets (9).

[0013] Virus-like particles (VLPs) formed by assembled capsid proteins surrounded by a cell membrane represent a suitable model for membrane-bound protein expression (10)(11). VLPs have been developed for many different viruses and are non-replicating macromolecular protein assemblies formed from minimal individual viral proteins (12). By co-overexpressing the target membrane protein in host cells, pseudotyped VLPs can be produced (11). At the same time, using VLPs as a "snapshot" tool for capturing the highly transient overexpressed cell membrane state enables these particles to be used as antigens selected in molecular display techniques and has been successfully used to develop antibodies against multi-transmembrane proteins that are not feasible with traditional recombinant expression systems (13).

[0014] There is a continuing need to develop novel methods for antibody development and to provide novel systems that will allow high yields of full-length membrane proteins for the purpose of developing and screening novel antibodies for novel drugs and vaccines. In addition, there is a need to provide methods for VLPs to provide effective immune responses for the development of new vaccines and therapies. SUMMARY OF THE INVENTION

[0015] The present invention aims to provide solutions to the above problems and more. The present invention provides constructs and methods for enhancing the capture and display of membrane proteins, preferably integral membrane proteins, in particular G protein-coupled receptors (GPCRs), ion channels, tetraspanins, and single-span membrane proteins, but not limited to these groups.

[0016] The present invention provides a virus-like particle (VLP)-based membrane protein expression system capable of expressing / capturing membrane proteins on the surface of VLPs at significantly enhanced levels compared to currently available methods.

[0017] Inspired by the fact that several viral capsid scaffolding proteins interact intracellularly with the cytoplasmic tails of viral envelope glycoproteins for their efficient incorporation into the surface of VLPs (14)(15), we developed a membrane protein expression system based on HIV-1 gag and Ebola VP40, in which the corresponding viral scaffolding gag and VP40 proteins will interact with the cytoplasmic tails of target membrane proteins, thus promoting increased capture and display of the target membrane proteins.

[0018] To achieve this, we fused the viral gag and VP40 proteins with the Erbin PDZ domain, which is known to interact tightly and specifically with a synthetic peptide of 7 amino acids (16). The Ebola VP40-PDZ amino acid sequence is shown in SEQ ID NO:1; HIV-1 GAG-PDZ is shown in SEQ ID NO:2. The Erbin PDZ domain is SEQ ID NO:3. PDZ domains are known to interact with short C-terminal amino acid motifs and are thus suitable for developing such interactions. In addition, PDZ domains generally show interaction with the C-terminal tails of GPCRs and play a role in the trafficking and recycling of GPCRs to the plasma membrane (17). The ability of PDZ domains to promote recycling and stabilize interacting membrane proteins further supports the rationale for exploiting such interactions (18). More specifically, Erbin has been shown to interact with and stabilize membrane-bound Erbb2 (19).

[0019] We surprisingly found that, based on the above system, the capture and display of class A, B, and F GPCRs, ion channels, tetraspanins, and single-span membrane proteins can be significantly increased. The system according to the present disclosure can provide expression of GPCRs, ion channels, tetraspanins, and single-span membrane proteins at levels 3 to 5 times higher compared to using wild-type VLP expression constructs.

[0020] In summary, we report the development of a robust membrane protein expression system based on the interaction between a viral scaffolding protein and a cytoplasmic protein motif of a target membrane protein, which enables high-density display of membrane proteins, can be used to develop a variety of assay systems, and more importantly, represents an effective antigen system for antibody development against therapeutically relevant classes of membrane proteins.

[0021] Accordingly, an object of the present invention is to provide a virus-like particle (VLP) that expresses one or more membrane-embedded proteins on its surface and comprises a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of one or more target membrane proteins, and optionally a signal peptide incorporated at the N-terminus of one or more target membrane proteins.

[0022] In one aspect of the present invention, the optional signal peptide incorporated at the N-terminus of the target membrane protein can be the signal peptide of albumin, melittin, Pr-MCH, Gaussia luciferase, hemagglutinin, H1V-glycoprotein, LRRCP32, growth hormone receptor, protease-activated R1, tissue-type plasminogen activator, secretin, AcMNPVEnv, growth hormone, IL-2, MCHR-1, but is not limited to these.

[0023] According to certain aspects of the present invention, the PDZ domain fused to the scaffolding protein is according to SEQ ID NO:3.

[0024] According to certain aspects of the present invention, the viral scaffolding protein can be a member of the Filoviridae family, such as Ebola virus VP40, or a member of the Retroviridae family of HIV-1 or MLV viral gag proteins, but is not limited to these.

[0025] According to certain aspects of the present invention, the VLP expresses one or more membrane proteins selected from the group consisting of G protein-coupled receptors (GPCRs), ion channels, tetraspanins, and single-span membrane proteins.

[0026] According to certain aspects of the present invention, in the VLP, the synthetic polypeptide fused to the pseudotyped membrane protein is according to SEQ IDNO:6.

[0027] Another object of the present invention is to provide a VLP vaccine or VLP preparation, which consists of or contains a VLP that expresses one or more membrane-embedded proteins on its surface and comprises a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of one or more target membrane proteins, and optionally a signal peptide incorporated at the N-terminus of one or more target membrane proteins.

[0028] According to certain aspects of the present invention, the VLP vaccine or preparation is injectable, intranasal, or inhalable.

[0029] Another object of the present invention is to provide a method of generating an immune response, the method comprising administering to an animal a vaccine or preparation comprising or consisting of VLPs, the VLPs expressing one or more membrane-embedded proteins on their surface and comprising a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of one or more target membrane proteins, and optionally a signal peptide incorporated at the N-terminus of one or more target membrane proteins.

[0030] According to certain aspects, the present invention includes a method of immunizing an animal to develop an antibody or antibody preparation or a membrane protein-binding affinity molecule by administering to the animal a vaccine or preparation comprising or consisting of VLPs, the VLPs expressing one or more membrane-embedded proteins on their surface and comprising a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of one or more target membrane proteins, and optionally a signal peptide incorporated at the N-terminus of one or more target membrane proteins.

[0031] According to certain aspects, the present invention includes an antibody obtained from an animal immunized by administering to the animal a vaccine or preparation comprising or consisting of VLPs, the VLPs expressing one or more membrane-embedded proteins on their surface and comprising a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of one or more target membrane proteins, and optionally a signal peptide incorporated at the N-terminus of one or more target membrane proteins.

[0032] Another object of the present invention is to provide a method of developing monoclonal antibodies in a phage display system using VLPs, the VLPs expressing one or more membrane-embedded proteins on their surface and comprising a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of one or more target membrane proteins, and optionally a signal peptide incorporated at the N-terminus of one or more target membrane proteins.

[0033] Another object of the present invention is an antibody obtained using VLPs in a phage display system, the VLPs expressing one or more membrane-embedded proteins on their surface and comprising a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of one or more target membrane proteins, and optionally a signal peptide incorporated at the N-terminus of one or more target membrane proteins.

[0034] Another object of the present invention is to provide antibodies for disease treatment and / or drug discovery.

[0035] Another object of the present invention is to provide a membrane protein expression system comprising virus-like particles that express one or more membrane-embedded proteins on their surface and contain a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of one or more target membrane proteins, and optionally a signal peptide incorporated at the N-terminus of one or more target membrane proteins, wherein the expression system is capable of expressing and capturing membrane proteins on the surface of VLPs at enhanced levels compared to a system comprising a wild-type VLP construct. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A- Figure 1C : Schematic diagram of the formation of HIV-1 gag / Ebola VP40 virus particles. The viral gag and VP40 oligomerize under the plasma membrane to form virus particles that bud from the cell and capture the host plasma membrane as an envelope. B Schematic flow chart of the expression and purification of HIV-1 gag and Ebola VP40-derived VLPs. C Transmission electron microscopy image of HIV-1 gag and Ebola VP40-derived VLPs. The HIV-1 gag VLPs are composed of spherical particles with a diameter of 100 nm - 200 nm. The Ebola VP40 forms filamentous particles with a width of approximately 80 nm - 100 nm but with variable lengths.

[0037] Figure 2A : Schematic diagram of the HIV-1 gag-Erbin PDZ fusion protein (SEQ ID NO:2). The plasma membrane-targeting matrix-associated MA domain is highlighted in orange. The CA domain responsible for gag oligomerization into particles is highlighted in blue. The nucleocapsid domain (green) is then linked to the Erbin PDZ domain (SEQ ID NO:3) shown in blue. The TGWETWV binding peptide (SEQ ID NO:6) is shown in red.

[0038] Figure 2B : Schematic diagram of the Ebola VP40-Erbin PDZ fusion protein (SEQ ID NO:1). The Erbin PDZ domain (SEQ ID NO:3) shown in blue is fused to the N-terminus of VP40. The N-terminal domain of VP40 responsible for oligomerization and particle formation is shown in cyan. The C-terminal domain of VP40 responsible for plasma membrane targeting is shown in orange.

[0039] Figure 3A- Figure 3E:A protocol for a plasma membrane-embedded GPCR having a synthetic intracellularly-localized C-terminal Erbin-PDZ domain-binding peptide that interacts with the HIV-1 gag-Erbin PDZ protein. B Effect of heterologous signal peptides on cell surface expression of class A GPCR MCHR1. The Y-axis represents relative median fluorescence intensity and the X-axis represents the source of the corresponding signal peptide sequence introduced at the N-terminus of MCHR1. C Standardized VLP-based Flag-ELISA assays of HIV-1 gag VLPs expressed with various combinations representing no interaction between MCHR1 and the viral scaffolding protein (PDZ-gag MCHR1wt, gagMCHR1wt, gag SP-MCHR1wt) or variants in which an interaction occurs between MCHR1 and the viral scaffolding gag molecule (PDZ-gag MCHR1 C-terminal peptide (SEQ ID NO:4) and PDZ-gag SP-MCHR1 C-terminal peptide (SEQ ID NO:5)). A 5-fold increase in MCHR1 expression was detected with the PDZ-gag-MCHR1 interaction. Additionally, based on detection of the N-terminal Flag tag on MCHR1, addition of a signal peptide to the N-terminus of MCHR1 had an additional synergistic effect on the MCHR1-VLP expression level. D Intracellular proximal C-terminal fusions of the 7aa Erbin PDZ domain-binding sequence with Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single transmembrane protein GFRAL, and HIV-1 Env. Corresponding expression levels based on Gag-normalized Flag-ELISA were demonstrated. An increase in interaction specificity of target membrane protein capture on VLPs was observed for all selected target proteins except the HIV-1 Env protein. E Intracellular proximal C-terminal fusions of the 7aa Erbin PDZ domain-binding sequence with Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single transmembrane protein GFRAL, and HIV-1 Env. Corresponding expression levels based on VP40-normalized Flag-ELISA were demonstrated. An increase in interaction specificity of target membrane protein capture on VLPs was observed for all selected target proteins except the HIV-1 Env protein.

[0040] Figure 4A: LC-MS / MS analysis of HIV-1 Gag-Erbin PDZ- and Ebola VP40-Erbin PDZ-derived VLPs pseudotyped with MCHR1. Venn diagrams indicate differences in the total proteomes of HIV-1- and Ebola-derived VLPs or differences in the VLP surface proteomes. Bar graphs indicate the number of total detected spectra for each surface protein on the corresponding VLPs.

[0041] Figure 4B : Complete list of VLP-specific and shared surface proteome proteins derived from Ebola VP40-Erbin PDZ and HIV-1 gag-Erbin PDZ.

[0042] Figure 5A- Figure 5E : A Protocol for PDZ-VLP-based phage display antibody selection. VLPs are coated on a MaxiSorp solid surface plate, and a phage library displaying antibody Fab fragments is introduced to bind to VLPs overexpressing the target membrane protein. In each round, the VLPs are alternated between HIV-1 and Ebola, and a pre-clearing step against target-negative VLPs is performed before introducing the target-positive VLPs. After 5 rounds of selection, target-positive clone phages are determined by phage-ELISA. B Phage-ELISA results of antibody selection against Fzd5-VLPs. Among 180 clones analyzed by phage ELISA, 175 clones showed positive binding to Fzd5-VLPs compared to target-negative VLPs. A total of 44 unique antibody clones were identified based on CDR H3 sequences. C Specificity analysis of 7 Fzd5-VLP-derived antibodies in IgG1 form based on biolayer interferometry (BLI). D Fzd5TopFlash assay to determine the functional ability of Fzd5-VLP-derived anti-Fzd5 IgG to block canonical Wnt signaling. Luciferase signals are represented relative to cells treated without antibody. E Cell proliferation assay of the HPAF-II pancreatic cancer cell line in response to treatment with anti-Fzd5 13080, 13082, and isotype control 4275 antibodies. After treating cells with the corresponding antibodies for 6 days, an Alamar Blue assay was used to determine cell viability.

[0043] Figure 6 : ELISA-based comparison of membrane protein expression levels of different membrane proteins expressed on HIV-1 gag VLPs, with EPEP peptides fused to the C-terminus of the membrane proteins and expressed using VLPs based on gag-ERBIN PDZ fusion proteins or as wt sequences. In all examples, ERBIN-PDZ and EPEP interaction provides enhanced display of the target membrane protein on the surface of the VLPs. Detailed Description

[0044] Definition

[0045] A membrane protein herein refers to a protein attached to or associated with a cell membrane. A membrane protein can be an integral membrane protein penetrating the cell membrane, or a peripheral membrane protein integrated on one side of the cell membrane, or a surface protein. A membrane protein can be a single-pass transmembrane protein or a multi-pass transmembrane protein. A membrane protein can be a transporter protein. A membrane protein can be a carrier protein or a channel protein. A membrane protein can be a receptor protein.

[0046] Development of the PDZ-VLP system

[0047] A key step in the efficient generation of regulatory antibodies against membrane proteins is to preserve and display the antigenic epitope in the relevant native environment. Removal of multi-transmembrane proteins such as GPCRs and ion channels from the lipid bilayer can introduce changes in the folded presentation of the protein and generate non-functional antibodies. The use of VLPs for capturing membrane proteins has been relatively well established, but its efficiency is determined by the target protein expression level and membrane microdomain localization in the host cell. Expression of the retroviral HIV-1 gag protein and the filoviral Ebola VP40 protein results in the assembly and budding of VLPs from host cells (20)(21). The viral particles capture the host cell membrane as an envelope upon budding from the cell and are thus also able to capture transiently co-overexpressed membrane proteins. Development of an efficient transient expression system under serum-free conditions has enabled the development of a direct expression and purification process by extracting VLPs from the expression supernatant (22)(Figure 1). To further develop a powerful system that allows efficient capture and purification of different classes of membrane proteins from VLPs, we engineered the interaction between a viral scaffolding protein and the target integral membrane protein. Since PDZ domains are known to interact with short C-terminal and internal peptidic motifs and have been shown to stabilize and recycle membrane proteins at the plasma membrane, they represent suitable functional domains for such engineering strategies. ERBIN is a member of the LAP (leucine-rich repeat and PDZ domain) protein family, which has been shown to bind tightly to a synthetic phage display-derived 7-amino acid peptide (16). We fused the ERBIN PDZ domain to the C-terminus of HIV-1 gag (Figure 2a) and the N-terminus of Ebola VP40 (Figure 2b). The 7aa TGWETWV ERBIN PDZ-binding sequence was fused to the C-terminal tail of the target membrane protein (Figure 3A). Only about 10% of GPCRs are known to contain a cleavable N-terminal signal peptide (23). Since incorporation of a signal peptide at the N-terminus of a membrane protein has been shown to increase cell surface expression yields, we tested the effect of 17 different signal peptide sequences on the cell surface expression of the class A GPCR MCHR1 based on flow cytometry analysis with an N-terminal Flag tag. As shown in Figure 3B, all introduced signal peptides resulted in increased cell surface expression of Flag-MCHR1 (SEQ ID NO:4), with a maximum increase of 3-fold in the case of the albumin signal peptide.

[0048] When evaluating the effect of the interaction between the N-terminal signal peptide and the C-terminal PDZ domain interaction tag on the pseudotyping level of MCHR1 on HIV-1 gag and gag-PDZ-derived VLPs, we expressed and purified MCHR1 pseudotyped VLPs with different modifications and determined the display level by VLP-ELISA through the N-terminal Flag tag on MCHR1 (Figure 3C). Based on the standardized MCHR1-VLP Flag-ELISA, a 5-fold increase in MCHR1 expression was detected with PDZ-gag-MCHR1 interaction. In addition, based on the detection of the N-terminal Flag tag of MCHR1, adding a signal peptide to the N-terminus of MCHR1 had an additional synergistic effect on the MCHR1-VLP expression level.

[0049] To further demonstrate the robustness of this system for promoting the capture of different membrane proteins, we expressed VLPs with overexpressed class F GPCRs (Fzd5 and Fzd3), class B GPCR (GLP1R), ion channels (P2RX3), and single-pass transmembrane proteins (GFRAL and HIV-1 Env) ( Figure 3D, Figure 3E ). When analyzing the effect of the interaction of the incorporation of different membrane proteins on HIV-1 and Ebola VP40-derived VLPs, we were able to see an increase in the incorporation of all membrane proteins except the HIV-1 Env protein, where the interaction had a negative effect, resulting in a 2-fold decrease in Env display compared to Env-wt in the standardized VLP ELISA. In Figure 6 , we further demonstrated the increased expression of the target membrane protein on the surface of VLPs due to engineered interactions in a concentration-dependent ELISA-based assay.

[0050] Proteomic analysis of pseudotyped VLPs

[0051] Retroviruses and filoviruses are known to capture host cell membranes as envelopes and thus also incorporate host proteins into virus particles. In addition to the integral membrane proteins displayed on the surface of the particles, many cytoplasmic proteins are captured as interaction partners with the intracellular domains of integral membrane proteins as well as the viral Gag and VP40 proteins (11)(24). Studies analyzing the protein composition of the HIV-1 core expressed in different cell types have shown that only 42 out of 202 proteins could be detected in all VLP samples, indicating a high host cell dependence of the VLP proteome composition (25).

[0052] To determine the total proteome composition and surface proteome composition of Gag- and Vp40-derived VLPs expressed in the Expi HEK293 expression system, we performed LC-MS / MS analysis on PDZ-VLPs overexpressing MCHR1. We were surprised to find that, as shown in Figure 4, the total proteome composition between HIV- and Ebola-derived VLPs was significantly different, and compared to HIV-1, Ebola VP40 captured 143 additional host proteins. This can be explained by the relatively large size of Ebola VP40-derived filamentous virus particles with a diameter of approximately 80 nm - 100 nm and a length of several μm, enabling the recruitment of a larger host plasma membrane surface as an envelope and thus embedding more host proteins. Since understanding the composition of the surface proteome of VLPs is of great interest in antibody discovery-based assays and potential vaccine development, we performed a cross-reference analysis of the obtained VLP proteome with a list of human surface proteome proteins (26). From this analysis, we were able to characterize the obtained VLP surface proteome and demonstrate that only the single-cell surface protein CD44 is unique to HIV-1, and the remaining 18 host surface proteins derived from HIV-1 VLPs are shared with Ebola VP40-derived VLPs that contain a total of 40 cell surface proteins. As seen from Figure 4, based on the observed peptide spectra, the most abundant detected cell surface protein was MCHR1, which was overexpressed to be captured on the VLPs. The complete list of cell surface proteins is presented in Table 1( Figure 4B ).

[0053] Development of Wnt signaling modulating antibodies by VLPs

[0054] To demonstrate the ability to develop antibodies by using PDZ-VLPs, we expressed Frizzled-5 of class F GPCR on HIV-1 gag-PDZ and Ebola VP40-PDZ VLPs and used them as antigens for synthetic antibody discovery by phage display. The VLPs were coated at 20 μg / ml on a solid surface MaxiSorp immunoassay plate and introduced with the synthetic Fab-displaying antibody library F(27). Unbound phages were washed away, and the bound phages were amplified overnight in Omnimax Escherichia coli cells. As shown in Fig. 5A, a total of 5 rounds of selection were performed, alternating between HIV-1- and Ebola-derived Fzd5-VLPs in each round. Each round of selection additionally included a negative selection step, in which the phage antibody library was exposed to Fzd5-negative VLPs to remove any background-binding phages. A total of 180 individual clones were analyzed for specific binding to Fzd5-VLPs compared to the negative VLPs. As shown in Fig. 5B, a total of 175 clones were positive for binding to Fzd5-VLPs, with the ratio of Fzd5 signal compared to the negative signal exceeding 10. Sequencing of all the obtained phage clones identified a total of 44 unique antibody clones based on the CDRH3 sequences. Seven unique antibody clones not obtained by previous antibody selection campaigns with the recombinant Fzd5 extracellular CRD domain were further characterized. As seen from the biolayer interferometry assay in Fig. 5C, the obtained antibody clones were all positive for binding to the Fzd5-CRD domain but had additional cross-reactive binding specificities to other Frizzled isoforms. In addition to having variable cross-reactivities to other Fzd isoforms in different clones, all the obtained antibody clones were confirmed to be cross-reactive with Frizzled 8. Antibody 4275, which recognizes Gaussian luciferase, was used as a negative control.

[0055] To further demonstrate the potential ability of the obtained antibodies to modulate Wnt-Frizzled signaling, we used the TopFlash assay in HEK293 cells to monitor luciferase expression driven by β-catenin translocation-mediated activation of TCF / LEF transcription factors upon binding of Frizzled 5 to Wnt3a (28). Frizzled 5 was transiently overexpressed in HEK293 cells, and 24 h after transfection, the cells were treated with Wnt3a and anti-Fzd5 antibodies. As demonstrated in Fig. 5D, antibodies 13080 and 13082 effectively blocked Wnt3a-driven luciferase activation, indicating that these antibodies act as antagonists of Fzd5-driven canonical signaling.

[0056] Since Fzd5 has been shown to be essential for the proliferation of RNF43 mutant pancreatic cancer cells, we tested the ability of 13080 and 13082 to inhibit the proliferation of HPAF-II pancreatic cancer cells (29). HPAF-II cells were treated with 13080 and 13082 antibodies at concentrations of 0 nM - 100 nM for 6 days, and cell proliferation was analyzed by Alamar-Blue assay. As Figure 5E seen, HPAF-II cells responded to the Fzd5 antagonist antibody, and cell proliferation in response to the antagonistic Fzd5 antibody decreased in a dose-dependent manner, while the 4275 control IgG had no effect on cell viability. This indicates that HIV-1 and Ebola PDZ-VLPs can be used for synthetic antibody discovery by phage display, and we successfully obtained functional Wnt-Fzd signaling regulatory antibodies.

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Claims

1. A virus-like particle (VLP), said virus-like particle expressing one or more membrane-embedded proteins on its surface and comprising a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide in the cytoplasmic C-terminal tail of one or more target membrane proteins, and optionally a signal peptide incorporated at the N-terminus of said one or more target membrane proteins.

2. The VLP according to claim 1, wherein the signal peptide is the signal peptide of albumin, melittin, Pro-MCH, Gaussia luciferase, hemagglutinin, HIV glycoprotein, LRRCP32, growth hormone receptor, protease-activated receptor, tissue plasminogen activator, secretin, AcMNPV, growth hormone, IL-2 or MCHR-1, but is not limited to these.

3. The VLP according to claim 1 or 2, wherein the PDZ domain is according to SEQ ID NO:

3.

4. The VLP according to any one of claims 1 to 3, wherein the viral scaffolding protein is Ebola virus VP40 or HIV-1 virus gag.

5. The VLP according to any one of claims 1 to 4, wherein the one or more membrane proteins are selected from the group consisting of G protein-coupled receptors (GPCRs), ion channels, tetraspanins, and single-span membrane proteins.

6. The VLP according to any one of claims 1 to 5, wherein the synthetic polypeptide fused to the pseudotyped membrane protein is according to SEQ ID NO:

6.

7. A VLP vaccine or VLP preparation, said VLP vaccine or VLP preparation consisting of or comprising the VLP according to any one of claims 1 to 6.

8. The VLP vaccine or VLP preparation according to claim 7, wherein the vaccine or preparation is an injectable, intranasal, or inhalable vaccine.

9. A method of generating an immune response, said method comprising administering to an animal a vaccine or preparation comprising the VLP according to any one of claims 1 to 6 or consisting of said VLP.

10. The method according to claim 9, wherein the method further comprises obtaining antibodies from the VLP-immunized animal or using the VLP according to any one of claims 1 to 6 in an antibody screening and discovery process.

11. A method of developing monoclonal antibodies in a phage display system, said method being carried out by using the VLP according to any one of claims 1 to 6.

12. An antibody obtained by the method according to claim 10 or 11.

13. The antibody according to claim 12, said antibody being used for treating a disease.

14. A membrane protein expression system, said membrane protein expression system comprising the virus-like particle according to any one of claims 1 to 6, wherein the expression system is capable of expressing and capturing the membrane protein on the VLP surface at an enhanced level compared to a system comprising a wild-type VLP construct.

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