Engineered virus-like particles for targeted capture of membrane proteins

Abstract

Disclosed are engineered virus-like particles (VLPs) and uses thereof, in which integral plasma membrane proteins are captured and displayed in their native conformation on the surface of the VLPs based on interactions between a viral scaffolding protein fused to a PDZ domain and a synthetic polypeptide of the cytoplasmic C-terminal tail of the target membrane protein.

Description

[Technical Field]

[0001] Sequence Listing The Sequence Listing entitled 11641-002WO1.XML, created on December 13, 2023, and having a file size of 8,104 bytes, is incorporated herein by reference pursuant to 37 C.FR Section 1.52(e)(5).

[0002] The present invention relates to engineered virus-like particles (VLPs) in which integral plasma membrane proteins are captured and displayed on the surface of the VLP in a native conformation based on interactions between a viral scaffold protein fused to a PDZ domain and a synthetic polypeptide of the cytoplasmic C-terminal tail of the 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 technology]

[0003] Modulating biological molecules that target membrane proteins has become one of the most promising therapeutic areas (1). Antibodies as drug candidates have several advantages over small molecules, including better specificity, lower dosing frequency, and restriction from the central nervous system. There are two basic approaches to antibody generation: utilizing the animal immune system or synthetic molecular display methods (e.g., based on phage display) (2).

[0004] To harness the immune systems of various organisms to produce antibodies, target antigens are presented for recognition by the host humoral immune system, resulting in the activation of B cells and the secretion of antibodies that recognize the presented epitopes of the antigen. Individual monoclonal antibodies can then be isolated and produced by recombinant or hybridoma technology.

[0005] Phage display of synthetic antibody fragments has proven to be an effective molecular display tool for antibody generation, allowing the selection of antibodies against a vast number of biological and nonbiological targets and providing the opportunity to correlate antibody phenotype with genotype (3)(4).

[0006] Furthermore, the recombinant in vitro nature of phage display has been supplanted by the use of alternative depletion steps, epitope steering, binding to protein-protein interaction complexes, and the use of antigens of the complex composition for binding.

[0007] G protein-coupled receptors (GPCRs) are one of the largest protein superfamilies, with over 800 members (5). Ion channel proteins are the second largest membrane protein class, an important class of multitransmembrane proteins expressed in nearly all living cells and involved in the regulation of various physiological processes (6). Aberrant signaling of GPCRs and ion channel proteins has been linked to numerous immunological, neurological, and metabolic disorders, as well as cancer.

[0008] Approximately 35% of all approved drugs target GPCRs, making GPCRs the largest family of proteins targeted for drug development to date. (7) However, from the perspective of antibody generation strategies, multi-transmembrane proteins represent a challenging group of targets. They are frequently expressed at low density on the cell surface and are unstable and misfold when removed from the lipid bilayer. As a result, obtaining sufficient amounts of relevant antigen for antibody discovery efforts, either by antibody display technology or animal immunization, remains one of the limiting factors, thus making the generation of specific antibodies extremely difficult.

[0009] Furthermore, the structure of GPCR and ion channel targets poses another limiting factor, with the minimal epitope displayed on the cell surface for potential antibody binding.

[0010] To date, most anti-GPCR antibodies have been raised against larger N-terminal extracellular domains or linear peptides containing GPCRs. The production of suitable full-length GPCR antigens remains the biggest bottleneck (8).

[0011] Because developing immune-derived antibody libraries against evolutionarily conserved membrane proteins has proven difficult, the use of synthetic antibody fragments displayed on phage displays is an effective molecular tool for antibody generation, allowing the selection of antibodies against a vast number of biological and nonbiological targets (9).

[0012] Virus-like particles (VLPs), formed by assembled capsid proteins surrounded by a cellular membrane, represent a suitable model for the expression of membrane-associated proteins (10)(11). VLPs, which are replication-incompetent macromolecular protein assemblies formed by a minimal number of single viral proteins, have been developed for a number of different viruses (12). It is possible to generate pseudotyped VLPs by co-overexpressing target membrane proteins in host cells (11). At the same time, using VLPs as a "snapshot" tool to capture highly transiently overexpressed states of the cellular membrane allows these particles to be used as antigens for selection in molecular display technology, and they have been successfully used to generate antibodies against multi-transmembrane proteins for which traditional recombinant expression systems are not feasible (13).

[0013] There is a continuing need for methods to generate novel antibodies and a need to provide new systems that allow high yields of full-length membrane proteins for the purpose of generating and screening novel antibodies for new drugs and vaccines. Furthermore, there is a need for methods that provide VLPs to provide an efficient immune response for the development of new vaccines and therapeutics. Summary of the Invention

[0014] The present invention aims to provide a solution to the above problems and others. The present invention provides constructs and methods for enhancing the capture and presentation of membrane proteins, preferably integral membrane proteins, particularly, but not limited to, G protein-coupled receptors (GPCRs), ion channels, tetraspanins, and single-pass transmembrane proteins.

[0015] The present invention provides a virus-like particle (VLP)-based membrane protein expression system that allows for the expression / entrapment of membrane proteins on the surface of VLPs at levels significantly higher than currently available methods.

[0016] Inspired by the fact that some viral capsid scaffolding proteins interact with the cytoplasmic tails of viral envelope glycoproteins intracellularly to efficiently incorporate them onto the surface of VLPs (14)(15), we developed a membrane protein expression system based on HIV-1 gag and Ebola VP40, in which the respective viral scaffolding gag and VP40 proteins interact with the cytoplasmic tails of target membrane proteins, thereby promoting increased capture and presentation of target membrane proteins.

[0017] To achieve this, we fused the viral gag and VP40 proteins with the Erbin PDZ domain, which is known to interact strongly and specifically with a seven-amino acid synthetic peptide (16). The Ebola VP40-PDZ amino acid sequence is shown as SEQ ID NO:1, and the HIV-1 GAG-PDZ is shown as 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, making them well-suited for such interactions. Furthermore, PDZ domains have often been shown to interact with the C-terminal tails of GPCRs and play a role in their transport and recycling to the plasma membrane (17). The ability of PDZ domains to promote recycling and stabilize interacting membrane proteins further supported the rationale for exploiting such interactions (18). More specifically, Erbin has been shown to interact with and stabilize membrane-bound Erbb2 (19).

[0018] The present inventors have surprisingly found that, based on the above system, the capture and presentation of class A, B, and F GPCRs, ion channels, tetraspanins, and single-pass transmembrane proteins can be significantly increased. The system according to the present disclosure can provide expression of GPCRs, ion channels, tetraspanins, and single-pass transmembrane proteins at levels 3-5 times higher than using wild-type VLP expression constructs.

[0019] In summary, we report the development of a robust membrane protein expression system based on the interaction between a viral scaffold protein and a cytoplasmic protein motif of a target membrane protein, which allows for high-density display of membrane proteins that can be used for the development of various assay systems and, more importantly, represents an efficient antigen system for antibody generation against therapeutically relevant membrane protein classes.

[0020] It is therefore an object of the present invention to provide a virus-like particle (VLP) that expresses one or more membrane-integrating proteins on its surface and comprises a viral scaffold 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 into the N-terminus of one or more target membrane proteins.

[0021] In one embodiment of the present invention, the optional signal peptide incorporated into the N-terminus of the target membrane protein may be, but is not limited to, the signal peptides of albumin, melittin, Pr-MCH, Gaussia luciferase, hemagglutinin, H1V-glycoprotein, LRRCP32, growth hormone receptor, proteinase-activated R1, tissue-type plasminogen activator, secreton, AcMNPVEnv, somatotopin, IL-2, and MCHR-1.

[0022] According to one particular embodiment of the invention, the PDZ domain fused to the scaffold protein is according to SEQ ID NO:3.

[0023] According to certain embodiments of the invention, the viral scaffold protein can be, but is not limited to, a member of the Filoviridae family, such as Ebola virus VP40, or the gag protein of a member of the Retroviridae family of HIV-1 or MLV viruses.

[0024] According to certain aspects of the invention, the VLPs express one or more membrane proteins selected from the group consisting of G protein-coupled receptors (GPCRs), ion channels, tetraspanins and single-pass transmembrane proteins.

[0025] According to one particular embodiment of the invention, in the VLP, the synthetic polypeptide fused to the pseudotyped membrane protein is according to SEQ ID NO:6.

[0026] Another object of the present invention is to provide a VLP vaccine or VLP preparation consisting of or comprising a VLP expressing one or more membrane-integrating proteins on its surface and comprising a viral scaffold 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 into the N-terminus of one or more target membrane proteins.

[0027] According to certain aspects of the invention, the VLP vaccine or preparation is injectable, intranasal or inhalable.

[0028] It is yet another object of the present invention to provide a method for generating an immune response comprising administering to an animal a vaccine or preparation consisting of or comprising VLPs expressing one or more membrane-integrated proteins on their surface and comprising a viral scaffold 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 into the N-terminus of one or more target membrane proteins.

[0029] According to certain aspects, the present invention comprises a method for developing antibodies or antibody preparations or membrane protein binding affinity molecules through immunization of an animal by administering to the animal a vaccine or preparation consisting of or comprising a VLP that expresses one or more membrane-integrated proteins on its surface and comprises a viral scaffold 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 into the N-terminus of one or more target membrane proteins.

[0030] In certain aspects, the present invention includes antibodies obtained from an animal immunized by administering to the animal a vaccine or preparation, wherein the vaccine or preparation consists of or comprises a VLP expressing one or more membrane-integrating proteins on its surface and comprising a viral scaffold 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 into the N-terminus of one or more target membrane proteins.

[0031] It is a further object of the present invention to provide a method for generating monoclonal antibodies in a phage display system by using a VLP that expresses one or more membrane-integrating proteins on its surface and comprises a viral scaffold 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 into the N-terminus of one or more target membrane proteins.

[0032] A further object of the present invention is an antibody obtained in a phage display system by using a VLP, which expresses one or more membrane-integrating proteins on its surface and comprises a viral scaffold 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 into the N-terminus of one or more target membrane proteins.

[0033] It is yet another object of the present invention to provide antibodies for disease treatment and / or drug discovery.

[0034] It is a further object of the present invention to provide a membrane protein expression system that expresses one or more membrane-integrating proteins on its surface and comprises a viral scaffold 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 into the N-terminus of one or more target membrane proteins, which expression system allows for the expression and capture of membrane proteins on the VLP surface at enhanced levels compared to systems comprising wild-type VLP constructs. [Brief explanation of the drawings]

[0035] [Figure 1A] A. Schematic diagram of HIV-1 gag / Ebola VP40 viral particle formation. Viral gag and VP40 oligomerize beneath the plasma membrane to form viral particles that emerge from the cell and capture the host cell 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 images of HIV-1 gag and Ebola VP40-derived VLPs. HIV-1 gag VLPs consist of spherical particles with diameters of 100-200 nm. Ebola VP40 forms filamentous particles with widths of approximately 80-100 nm but with variable lengths. [Figure 1B] A. Schematic diagram of HIV-1 gag / Ebola VP40 viral particle formation. Viral gag and VP40 oligomerize beneath the plasma membrane to form viral particles that emerge from the cell and capture the host cell 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 images of HIV-1 gag and Ebola VP40-derived VLPs. HIV-1 gag VLPs consist of spherical particles with diameters of 100-200 nm. Ebola VP40 forms filamentous particles with widths of approximately 80-100 nm but with variable lengths. [Figure 1C]A. Schematic diagram of HIV-1 gag / Ebola VP40 viral particle formation. Viral gag and VP40 oligomerize beneath the plasma membrane to form viral particles that emerge from the cell and capture the host cell 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 images of HIV-1 gag and Ebola VP40-derived VLPs. HIV-1 gag VLPs consist of spherical particles with diameters of 100-200 nm. Ebola VP40 forms filamentous particles with widths of approximately 80-100 nm but with variable lengths. [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, involved in oligomerization of gag 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. [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, involved in oligomerization and particle formation, is shown in cyan. The C-terminal domain of VP40, involved in plasma membrane targeting, is shown in orange. [Figure 3A]A. Schematic of plasma membrane-integrated GPCRs bearing synthetic intracellularly localized C-terminal Erbin PDZ domain-binding peptides that interact with the HIV-1 gag-Erbin PDZ protein. B. Effect of heterologous signal peptides on cell surface expression of the class A GPCR MCHR1. The Y-axis represents relative median fluorescence intensity, and the X-axis represents the origin of each signal peptide sequence introduced at the N-terminus of MCHR1. C. Normalized VLP-based Flag-ELISA assay of HIV-1 gag VLPs expressed in various combinations (PDZ-gag MCHR1wt, gag MCHR1wt, gag SP-MCHR1wt), representing no interaction between MCHR1 and the viral scaffold protein, or mutants (PDZ-gag MCHR1 C-term pep (SEQ ID NO: 4) and PDZ-gag SP-MCHR1 C-term pep (SEQ ID NO: 5)) representing interaction between MCHR1 and the viral scaffold gag molecule. A fivefold increase in MCHR1 expression was detected upon PDZ-gag-MCHR1 interaction. Furthermore, the addition of a signal peptide to the N-terminus of MCHR1 has a further synergistic effect on MCHR1-VLP expression levels, based on the detection of the N-terminal Flag tag of MCHR1. The 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C-termini of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. The respective expression levels based on Gag-normalized Flag-ELISA are shown. Interaction-specific increases in target membrane protein capture on VLPs were observed for all selected target proteins except for HIV-1 Env. The E 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C terminus of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. Expression levels of each were measured using VP40-normalized Flag-ELISA.Interaction-specific increases in target membrane protein capture on VLPs are observed for all selected target proteins except for HIV-1 Env protein. [Figure 3B]A. Schematic of plasma membrane-integrated GPCRs bearing synthetic intracellularly localized C-terminal Erbin PDZ domain-binding peptides that interact with the HIV-1 gag-Erbin PDZ protein. B. Effect of heterologous signal peptides on cell surface expression of the class A GPCR MCHR1. The Y-axis represents relative median fluorescence intensity, and the X-axis represents the origin of each signal peptide sequence introduced at the N-terminus of MCHR1. C. Normalized VLP-based Flag-ELISA assay of HIV-1 gag VLPs expressed in various combinations (PDZ-gag MCHR1wt, gag MCHR1wt, gag SP-MCHR1wt), representing no interaction between MCHR1 and the viral scaffold protein, or mutants (PDZ-gag MCHR1 C-term pep (SEQ ID NO: 4) and PDZ-gag SP-MCHR1 C-term pep (SEQ ID NO: 5)) representing interaction between MCHR1 and the viral scaffold gag molecule. A fivefold increase in MCHR1 expression was detected upon PDZ-gag-MCHR1 interaction. Furthermore, the addition of a signal peptide to the N-terminus of MCHR1 has a further synergistic effect on MCHR1-VLP expression levels, based on the detection of the N-terminal Flag tag of MCHR1. The 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C-termini of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. The respective expression levels based on Gag-normalized Flag-ELISA are shown. Interaction-specific increases in target membrane protein capture on VLPs were observed for all selected target proteins except for HIV-1 Env. The E 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C terminus of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. Expression levels of each were measured using VP40-normalized Flag-ELISA.Interaction-specific increases in target membrane protein capture on VLPs are observed for all selected target proteins except for HIV-1 Env protein. [Figure 3C]A. Schematic of plasma membrane-integrated GPCRs bearing synthetic intracellularly localized C-terminal Erbin PDZ domain-binding peptides that interact with the HIV-1 gag-Erbin PDZ protein. B. Effect of heterologous signal peptides on cell surface expression of the class A GPCR MCHR1. The Y-axis represents relative median fluorescence intensity, and the X-axis represents the origin of each signal peptide sequence introduced at the N-terminus of MCHR1. C. Normalized VLP-based Flag-ELISA assay of HIV-1 gag VLPs expressed in various combinations (PDZ-gag MCHR1wt, gag MCHR1wt, gag SP-MCHR1wt), representing no interaction between MCHR1 and the viral scaffold protein, or mutants (PDZ-gag MCHR1 C-term pep (SEQ ID NO: 4) and PDZ-gag SP-MCHR1 C-term pep (SEQ ID NO: 5)) representing interaction between MCHR1 and the viral scaffold gag molecule. A fivefold increase in MCHR1 expression was detected upon PDZ-gag-MCHR1 interaction. Furthermore, the addition of a signal peptide to the N-terminus of MCHR1 has a further synergistic effect on MCHR1-VLP expression levels, based on the detection of the N-terminal Flag tag of MCHR1. The 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C-termini of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. The respective expression levels based on Gag-normalized Flag-ELISA are shown. Interaction-specific increases in target membrane protein capture on VLPs were observed for all selected target proteins except for HIV-1 Env. The E 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C terminus of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. Expression levels of each were measured using VP40-normalized Flag-ELISA.Interaction-specific increases in target membrane protein capture on VLPs are observed for all selected target proteins except for HIV-1 Env protein. [Figure 3D]A. Schematic of plasma membrane-integrated GPCRs bearing synthetic intracellularly localized C-terminal Erbin PDZ domain-binding peptides that interact with the HIV-1 gag-Erbin PDZ protein. B. Effect of heterologous signal peptides on cell surface expression of the class A GPCR MCHR1. The Y-axis represents relative median fluorescence intensity, and the X-axis represents the origin of each signal peptide sequence introduced at the N-terminus of MCHR1. C. Normalized VLP-based Flag-ELISA assay of HIV-1 gag VLPs expressed in various combinations (PDZ-gag MCHR1wt, gag MCHR1wt, gag SP-MCHR1wt), representing no interaction between MCHR1 and the viral scaffold protein, or mutants (PDZ-gag MCHR1 C-term pep (SEQ ID NO: 4) and PDZ-gag SP-MCHR1 C-term pep (SEQ ID NO: 5)) representing interaction between MCHR1 and the viral scaffold gag molecule. A fivefold increase in MCHR1 expression was detected upon PDZ-gag-MCHR1 interaction. Furthermore, the addition of a signal peptide to the N-terminus of MCHR1 has a further synergistic effect on MCHR1-VLP expression levels, based on the detection of the N-terminal Flag tag of MCHR1. The 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C-termini of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. The respective expression levels based on Gag-normalized Flag-ELISA are shown. Interaction-specific increases in target membrane protein capture on VLPs were observed for all selected target proteins except for HIV-1 Env. The E 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C terminus of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. Expression levels of each were measured using VP40-normalized Flag-ELISA.Interaction-specific increases in target membrane protein capture on VLPs are observed for all selected target proteins except for HIV-1 Env protein. [Figure 3E]A. Schematic of plasma membrane-integrated GPCRs bearing synthetic intracellularly localized C-terminal Erbin PDZ domain-binding peptides that interact with the HIV-1 gag-Erbin PDZ protein. B. Effect of heterologous signal peptides on cell surface expression of the class A GPCR MCHR1. The Y-axis represents relative median fluorescence intensity, and the X-axis represents the origin of each signal peptide sequence introduced at the N-terminus of MCHR1. C. Normalized VLP-based Flag-ELISA assay of HIV-1 gag VLPs expressed in various combinations (PDZ-gag MCHR1wt, gag MCHR1wt, gag SP-MCHR1wt), representing no interaction between MCHR1 and the viral scaffold protein, or mutants (PDZ-gag MCHR1 C-term pep (SEQ ID NO: 4) and PDZ-gag SP-MCHR1 C-term pep (SEQ ID NO: 5)) representing interaction between MCHR1 and the viral scaffold gag molecule. A fivefold increase in MCHR1 expression was detected upon PDZ-gag-MCHR1 interaction. Furthermore, the addition of a signal peptide to the N-terminus of MCHR1 has a further synergistic effect on MCHR1-VLP expression levels, based on the detection of the N-terminal Flag tag of MCHR1. The 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C-termini of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. The respective expression levels based on Gag-normalized Flag-ELISA are shown. Interaction-specific increases in target membrane protein capture on VLPs were observed for all selected target proteins except for HIV-1 Env. The E 7aa Erbin PDZ domain-binding sequence was fused to the intracellular proximal C terminus of Flag-tagged class F GPCRs Fzd5 and Fzd3, class B GPCR GLP1R, ion channel P2RX3, single-pass transmembrane protein GFRAL, and HIV-1 Env. Expression levels of each were measured using VP40-normalized Flag-ELISA.Interaction-specific increases in target membrane protein capture on VLPs are observed for all selected target proteins except for HIV-1 Env protein. [Figure 4A] LC-MS / MS analysis of VLPs derived from HIV-1 Gag-Erbin PDZ and Ebola VP40-Erbin PDZ pseudotyped with MCHR1. Venn diagrams showing either the differences in the total proteome or the differences in the VLP surface proteome of HIV-1 and Ebola-derived VLPs. Bar graphs show the total number of detected spectra per surface protein on each VLP. [Figure 4B] Complete list of VLP-specific and shared surfaceome proteins from the Ebola VP40-Erbin PDZ and HIV-1 gag-Erbin PDZ. [Figure 5A]A. Phage display antibody selection scheme based on PDZ-VLPs. VLPs were coated onto MaxiSorp solid surface plates, and an antibody Fab fragment-displaying phage library was introduced to bind VLPs overexpressing target membrane proteins. In each round, VLPs were alternated between HIV-1 and Ebola. After a preliminary clearing step against target-negative VLPs, they were introduced into target-positive VLPs. After five rounds of selection, target-positive clonal phages were determined by phage ELISA. B. Phage ELISA results of antibody selection performed against Fzd5-VLPs. Of 180 clones analyzed by phage ELISA, 175 were positive for binding to Fzd5-VLPs compared to target-negative VLPs. A total of 44 unique antibody clones were identified based on the CDR H3 sequence. C. Biolayer interferometry (BLI)-based specificity analysis of antibodies derived from seven Fzd5-VLPs in IgG1 format. D. Fzd5 TopFlash assay to determine the functional ability of Fzd5-VLP-derived anti-Fzd5 IgG to block canonical Wnt signaling. Luciferase signal is expressed relative to untreated cells. E. Cell proliferation assay of HPAF-II pancreatic cancer cell line in response to treatment with anti-Fzd5 13080, 13082, and isotype control 4275 antibodies. Cell viability was determined 6 days after treatment of cells with each antibody using the Alamar Blue assay. [Figure 5B]A. Phage display antibody selection scheme based on PDZ-VLPs. VLPs were coated onto MaxiSorp solid surface plates, and an antibody Fab fragment-displaying phage library was introduced to bind VLPs overexpressing target membrane proteins. In each round, VLPs were alternated between HIV-1 and Ebola. After a preliminary clearing step against target-negative VLPs, they were introduced into target-positive VLPs. After five rounds of selection, target-positive clonal phages were determined by phage ELISA. B. Phage ELISA results of antibody selection performed against Fzd5-VLPs. Of 180 clones analyzed by phage ELISA, 175 were positive for binding to Fzd5-VLPs compared to target-negative VLPs. A total of 44 unique antibody clones were identified based on the CDR H3 sequence. C. Biolayer interferometry (BLI)-based specificity analysis of antibodies derived from seven Fzd5-VLPs in IgG1 format. D. Fzd5 TopFlash assay to determine the functional ability of Fzd5-VLP-derived anti-Fzd5 IgG to block canonical Wnt signaling. Luciferase signal is expressed relative to untreated cells. E. Cell proliferation assay of HPAF-II pancreatic cancer cell line in response to treatment with anti-Fzd5 13080, 13082, and isotype control 4275 antibodies. Cell viability was determined 6 days after treatment of cells with each antibody using the Alamar Blue assay. [Figure 5C]A. Phage display antibody selection scheme based on PDZ-VLPs. VLPs were coated onto MaxiSorp solid surface plates, and an antibody Fab fragment-displaying phage library was introduced to bind VLPs overexpressing target membrane proteins. In each round, VLPs were alternated between HIV-1 and Ebola. After a preliminary clearing step against target-negative VLPs, they were introduced into target-positive VLPs. After five rounds of selection, target-positive clonal phages were determined by phage ELISA. B. Phage ELISA results of antibody selection performed against Fzd5-VLPs. Of 180 clones analyzed by phage ELISA, 175 were positive for binding to Fzd5-VLPs compared to target-negative VLPs. A total of 44 unique antibody clones were identified based on the CDR H3 sequence. C. Biolayer interferometry (BLI)-based specificity analysis of antibodies derived from seven Fzd5-VLPs in IgG1 format. D. Fzd5 TopFlash assay to determine the functional ability of Fzd5-VLP-derived anti-Fzd5 IgG to block canonical Wnt signaling. Luciferase signal is expressed relative to untreated cells. E. Cell proliferation assay of HPAF-II pancreatic cancer cell line in response to treatment with anti-Fzd5 13080, 13082, and isotype control 4275 antibodies. Cell viability was determined 6 days after treatment of cells with each antibody using the Alamar Blue assay. [Figure 5D]A. Phage display antibody selection scheme based on PDZ-VLPs. VLPs were coated onto MaxiSorp solid surface plates, and an antibody Fab fragment-displaying phage library was introduced to bind VLPs overexpressing target membrane proteins. In each round, VLPs were alternated between HIV-1 and Ebola. After a preliminary clearing step against target-negative VLPs, they were introduced into target-positive VLPs. After five rounds of selection, target-positive clonal phages were determined by phage ELISA. B. Phage ELISA results of antibody selection performed against Fzd5-VLPs. Of 180 clones analyzed by phage ELISA, 175 were positive for binding to Fzd5-VLPs compared to target-negative VLPs. A total of 44 unique antibody clones were identified based on the CDR H3 sequence. C. Biolayer interferometry (BLI)-based specificity analysis of antibodies derived from seven Fzd5-VLPs in IgG1 format. D. Fzd5 TopFlash assay to determine the functional ability of Fzd5-VLP-derived anti-Fzd5 IgG to block canonical Wnt signaling. Luciferase signal is expressed relative to untreated cells. E. Cell proliferation assay of HPAF-II pancreatic cancer cell line in response to treatment with anti-Fzd5 13080, 13082, and isotype control 4275 antibodies. Cell viability was determined 6 days after treatment of cells with each antibody using the Alamar Blue assay. [Figure 5E]A. Phage display antibody selection scheme based on PDZ-VLPs. VLPs were coated onto MaxiSorp solid surface plates, and an antibody Fab fragment-displaying phage library was introduced to bind VLPs overexpressing target membrane proteins. In each round, VLPs were alternated between HIV-1 and Ebola. After a preliminary clearing step against target-negative VLPs, they were introduced into target-positive VLPs. After five rounds of selection, target-positive clonal phages were determined by phage ELISA. B. Phage ELISA results of antibody selection performed against Fzd5-VLPs. Of 180 clones analyzed by phage ELISA, 175 were positive for binding to Fzd5-VLPs compared to target-negative VLPs. A total of 44 unique antibody clones were identified based on the CDR H3 sequence. C. Biolayer interferometry (BLI)-based specificity analysis of antibodies derived from seven Fzd5-VLPs in IgG1 format. D. Fzd5 TopFlash assay to determine the functional ability of Fzd5-VLP-derived anti-Fzd5 IgG to block canonical Wnt signaling. Luciferase signal is expressed relative to untreated cells. E. Cell proliferation assay of HPAF-II pancreatic cancer cell line in response to treatment with anti-Fzd5 13080, 13082, and isotype control 4275 antibodies. Cell viability was determined 6 days after treatment of cells with each antibody using the Alamar Blue assay. [Figure 6] Figure 1 shows a comparison of ELISA-based membrane protein expression levels of different membrane proteins expressed on HIV-1 gag VLPs using the EPEP peptide fused to the membrane protein C-terminus, and either using gag-ERBIN PDZ fusion protein-based VLPs or as the wt sequence. Throughout all examples, the ERBIN-PDZ and EPEP interaction provides enhanced presentation of the target membrane protein on the surface of the VLPs. DETAILED DESCRIPTION OF THE INVENTION

[0036] definition By membrane protein herein is meant a protein that is attached to or associated with the cell membrane. A membrane protein may be an integral membrane protein that spans the cell membrane, a peripheral membrane protein integrated into one side of the cell membrane, or a surface protein. A membrane protein may be a single-pass transmembrane protein or a multi-pass transmembrane protein. A membrane protein may be a transport protein. A membrane protein may be a carrier protein or a channel protein. A membrane protein may be a receptor protein.

[0037] Development of PDZ-VLP system A key step for the efficient generation of regulatory antibodies against membrane proteins is preserving and presenting the antigenic epitope in a relevant, native context. Removing multi-transmembrane proteins, such as GPCRs and ion channels, from the lipid bilayer can alter the protein's displayed fold and result in non-functional antibodies. The use of VLPs to capture membrane proteins is relatively well established, but its efficiency is determined by the target protein's expression level and membrane microdomain localization in the host cell. Expression of the retroviral HIV-1 gag protein and the filoviral Ebola VP40 protein leads to the assembly and release of VLPs from host cells (20) (21). As viral particles emerge from the cell, they capture the host cell membrane as an envelope, thereby enabling the capture of transiently co-overexpressed membrane proteins. The development of an efficient transient expression system under serum-free conditions has enabled the development of a direct expression and purification pipeline by extracting VLPs from the expression supernatant (22) (Figure 1). To further develop a robust system that allows for efficient capture and purification of membrane proteins from different classes on VLPs, we engineered the interaction between viral scaffolding proteins and target integral membrane proteins. PDZ domains are known to interact with short C-terminal and internal peptide motifs and have been shown to stabilize and recycle membrane proteins on the plasma membrane, making them suitable functional domains for such engineering strategies. ERBIN is a member of the LAP (leucine-rich repeat and PDZ domain) protein family that has been shown to strongly bind to a synthetic phage-display-derived seven-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). We fused the 7 aa TGWETWV ERBIN PDZ-binding sequence to the C-terminal tail of the target membrane protein (Figure 3A). Only approximately 10% of GPCRs are known to contain cleavable N-terminal signal peptides (23).Because incorporation of a signal peptide into the N-terminus of a membrane protein has been shown to increase cell surface expression yield, 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 via the N-terminal Flag tag. As shown in Figure 3B, all of the introduced signal peptides resulted in increased cell surface expression of Flag-MCHR1 (SEQ ID NO: 4), with the albumin signal peptide resulting in a maximum three-fold increase.

[0038] To evaluate the effect of including an N-terminal signal peptide and a 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 via the N-terminal Flag tag on MCHR1 (Figure 3C). A five-fold increase in MCHR1 expression was detected with the PDZ-gag-MCHR1 interaction based on normalized MCHR1-VLP Flag-ELISA. Furthermore, the addition of a signal peptide to the N-terminus of MCHR1 has a further synergistic effect on MCHR1-VLP expression levels based on the detection of the N-terminal Flag tag of MCHR1.

[0039] To further demonstrate the robustness of this system in promoting capture of different membrane proteins, we expressed VLPs with overexpressed class F GPCRs (Fzd5 and Fzd3), class B GPCRs (GLP1R), an ion channel (P2RX3), and single-pass transmembrane proteins (GFRAL and HIV-1 Env) (Figure 3D,E). When analyzing the effect of interactions on the uptake of different membrane proteins into both HIV-1 and Ebola VP40-derived VLPs, we observed increased uptake for all membrane proteins except for the HIV-1 Env protein, where the interactions had a negative effect, resulting in a two-fold decrease in Env presentation compared to Env-wt in a normalized VLP ELISA. In Figure 6, we further demonstrate the increased expression of target membrane proteins on the surface of VLPs due to the engineered interactions in a concentration-dependent ELISA-based assay.

[0040] Proteomic analysis of pseudotyped VLPs Retroviruses and filoviruses are also known to capture host cell membranes as envelopes, thereby incorporating host proteins into viral particles. In addition to integral membrane proteins displayed on the particle surface, many cytoplasmic proteins are captured as interaction partners with the intracellular domains of integral membrane proteins and the viral Gag and VP40 proteins (11) (24). A study analyzing the protein composition of HIV-1 core expressed in different cell types showed that only 42 of 202 proteins could be detected in all VLP samples, indicating a high degree of host cell dependence for VLP proteome composition (25).

[0041] To confirm the whole-proteome and surface-membrane composition of Gag- and Vp40-derived VLPs expressed in the Expi HEK293 expression system, we performed LC-MS / MS analysis of PDZ-VLPs overexpressing MCHR1. Surprisingly, we found that the whole-proteome composition between HIV- and Ebola-derived VLPs differed significantly, with Ebola VP40 capturing 143 additional host proteins compared to HIV-1, as shown in Figure 4. This can be explained by the relatively large size of the filamentous viral particles of Ebola Vp40-derived VLPs, which are approximately 80-100 nm in diameter and several micrometers in length, allowing them to recruit a larger host plasma membrane surface as an envelope and thus incorporate more host proteins. Because understanding the composition of the VLP surfaceome is of significant interest in antibody discovery-based assays and potentially vaccine development, we performed a cross-reference analysis of the resulting VLP proteome with a list of human surfaceome proteins (26). From this analysis, we were able to characterize the resulting VLP surfaceome and demonstrate that only a single cell surface protein, CD44, is exclusive to HIV-1, while the remaining 18 host surface proteins derived from HIV-1 VLPs were shared with Ebola VP40-derived VLPs, which contain a total of 40 cell surface proteins. As can be seen in Figure 4, the most abundant cell surface protein detected based on the observed peptide spectrum is MCHR1, which was overexpressed and captured on the VLPs. A complete list of cell surface proteins is presented in Table 1 (Figure 4B).

[0042] Generation of Wnt signaling-modulating antibodies by VLPs To demonstrate the ability to generate antibodies using PDZ-VLPs, we expressed the class F GPCR Frizzled-5 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 loaded onto solid-surface MaxiSorp immunoassay plates at 20 μg / ml and introduced into the synthetic Fab display antibody library F (27). Unbound phages were washed away, and bound phages were amplified overnight in Omnimax E. coli cells. As shown in Figure 5A, a total of five rounds of selection were performed, alternating between HIV-1-derived Fzd5-VLPs and Ebola-derived Fzd5-VLPs in each round. Each selection round further included a negative selection step in which the phage antibody library was exposed to Fzd5-negative VLPs to remove background-binding phages. A total of 180 individual clones were analyzed for specific binding to Fzd5-VLPs compared to negative VLPs. As shown in Figure 5B, a total of 175 clones were positive for binding to Fzd5-VLPs, with an Fzd5 signal-to-negative ratio greater than 10. Sequencing of all the resulting phage clones identified a total of 44 unique antibody clones based on their CDRH3 sequences. Seven unique antibody clones not obtained in the previous antibody selection campaign using the recombinant Fzd5 extracellular CRD domain were used for further characterization. As can be seen from the biolayer interference assay in Figure 5C, the resulting antibody clones all positively bound to the Fzd5-CRD domain but possessed additional cross-reactive binding specificity to other Frizzled isoforms. All the resulting antibody clones were confirmed to be cross-reactive to Frizzled 8, in addition to variable cross-reactivity to other Fzd isoforms across different clones. Antibody 4275, which recognizes Gaussia luciferase, was used as a negative control.

[0043] To further demonstrate the potential of the resulting antibodies to modulate Wnt-Frizzled signaling, we used the TopFlash assay in HEK293 cells to monitor beta-catenin translocation-driven activation of TCF / LEF transcription factor-activated luciferase expression upon Frizzled 5 binding to Wnt3a (28). Frizzled 5 was transiently overexpressed in HEK293 cells, and 24 hours after transfection, the cells were treated with Wnt3a and anti-Fzd5 antibodies. As shown in Figure 5D, antibodies 13080 and 13082 were effective in blocking Wnt3a-driven activation of luciferase, indicating that these antibodies act as antagonists of Fzd5-driven canonical signaling.

[0044] Since Fzd5 has been demonstrated to be essential for the growth of RNF43-mutant pancreatic cancer cells, we tested the ability of 13080 and 13082 to inhibit the growth of HPAF-II pancreatic cancer cells (29). HPAF-II cells were treated with 13080 and 13082 antibodies at concentrations ranging from 0 to 100 nM for 6 days, and cell proliferation was analyzed by Alamar-Blue assay. As seen in Figure 5E, HPAF-II cells responded to the Fzd5 antagonist antibody with a dose-dependent decrease in cell proliferation, whereas the 4275 control IgG had no effect on cell viability. This demonstrates 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-modulating antibodies.

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Claims

1. A virus-like particle (VLP) expressing one or more membrane-integrating proteins on its surface, comprising a viral scaffold 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 into the N-terminus of said one or more target membrane proteins.

2. 2. The VLP of claim 1, wherein the signal peptide is, but is not limited to, a signal peptide of albumin, melittin, Pr-MCH, Gaussia luciferase, hemagglutinin, H1V glycoprotein, LRRCP32, growth hormone receptor, proteinase-activated receptor, tissue-type plasminogen activator, secreton, AcMNPVEnv, somatotopin, IL-2, or MCHR-1.

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

3.

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

5. The VLP of 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-pass transmembrane 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 consisting of or comprising a VLP according to any one of claims 1 to 6.

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

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

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

11. A method for producing monoclonal antibodies in a phage display system by using the VLP according to any one of claims 1 to 6.

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

13. The antibody of claim 12 for the treatment of a disease.

14. A membrane protein expression system comprising a virus-like particle according to any one of claims 1 to 6, which allows expression and capture of 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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