Fusion proteins and uses thereof

Abstract

The present disclosure relates to fusion proteins that are highly useful for the generation of virus-like particles for the display of membrane spanning proteins. Related embodiments, methods and uses are disclosed.

Claims

1. A fusion protein comprising a membrane protein N-terminally fused to a retroviral major capsid protein selected from the Gag protein of Moloney murine leukemia virus and the Gag protein of Human immunodeficiency virus.

2. The fusion protein according to claim 1, wherein said fusion protein is capable of being incorporated or encapsulated into virus-like particles.

3. The fusion protein according to claim 1, wherein said membrane protein is a G-protein coupled receptor.

4. The fusion protein according to claim 1, wherein said G-protein coupled receptor is selected from CCR1, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, motilin, ghrelin, PAR1 and PAR2.

5. The fusion protein according to claim 1, wherein said fusion protein comprises a linker peptide between said membrane protein and said retroviral major capsid protein.

6. A nucleic acid molecule encoding a fusion protein of claim 1.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A host cell containing the nucleic acid molecule of claim 6.

9. A host cell containing the vector of claim 7.

10. A virus-like particle comprising a fusion protein according to claim 1.

11. A virus-like particle according to claim 10, wherein said fusion protein is displayed on the surface of said virus-like particle.

12. A method to identify a binding moiety binding to a membrane protein, said method comprising the steps: (a) providing a fusion protein according to claim 1, (b) generating virus-like particles (VLPs) comprising the fusion protein of step (a), (c) contacting the VLPs of step (b) with an antibody library, (d) washing the VLPs to remove those antibodies that did not bind the VLP, and (d) selecting an antibody which is reactive with the membrane protein part of said fusion protein.

Description

FIGURE LEGENDS

(1) FIG. 1 shows the results of a Western Blot analyses. Panel A shows the results of Experiment A, panel B shows the result of Experiment B. For details see Example 3. The supernatants were probed with an anti-GAG antibody or an anti-GPCR antibody as indicated in the Figure. The right lanes in the blots shown in panel A comprise a pre-stained size marker. The same is true for the left lanes in the blots shown in panel B.

(2) FIG. 2 shows a SDS-PAGE of the VLPs of Experiment B.

(3) FIG. 3 shows an ELISA experiments with three different GPCR-GAG fusion proteins. Details are given in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

(4) In certain embodiments the present disclosure relates to a fusion protein comprising a membrane protein N-terminally fused to a viral major capsid protein.

(5) The term fusion protein refers to a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes or two nucleic acid molecules that do not naturally occur together.

(6) The term membrane protein refers to a protein that is attached to or associated with a membrane of a cell or an organelle. If a membrane protein spans the cell membrane then said protein may also be referred to as integral membrane protein or transmembrane protein. Therefore in certain embodiments of the present disclosure the membrane protein which is N-terminally fused to the viral major capsid protein is an integral membrane protein. In other embodiments of the present disclosure the membrane protein which is N-terminally fused to the viral major capsid protein is a transmembrane protein.

(7) The term N-terminally fused refers to a genetic fusion of a first and a second polypeptide/protein, wherein the first polypeptide/protein forms the N-terminal portion of the fusion protein. In certain embodiments of the present disclosure the membrane protein forms the N-terminal portion of the fusion protein. GPCR or G-protein coupled receptor are membrane proteins. The term refers to a large family of cell surface receptors with an assortment of ligands and diverse biological actions. The importance of GPCRs in cellular function, their diversity, and their accessibility to exogenous agents make them an important focus of research into disease processes and drug discovery. GPCR activation events are communicated to cell signaling pathways via GTP-binding I proteins (G proteins) associated with the intracellular domain of the receptor. GPCRs constitute the largest group of drug targets today, highlighting their importance in biological research and in disease pathways. However, GPCRs are structurally complex, spanning the cell membrane seven times. Removal from the cell membrane usually destroys the receptor's native structure in which it is maintained by the environment of the lipid bilayer. GPCRs are thus extremely difficult to purify and manipulate experimentally, and their study relies on whole cells or isolated cell membranes. GPCRs include, without limitation, serotonin and olfactory receptors, glycoprotein hormone receptors, chemokine receptors, adenosine receptors, biogenic amine receptors, melanocortin receptors, neuropeptide receptors, chemotactic receptors, somatostatin receptors, opioid receptors, melatonin receptors, calcitonin receptors, PTH/PTHrP receptors, glucagon receptors, secretin receptors, latrotoxin receptors, metabotropic glutamate receptors, calcium receptors, GABA-B receptors, pheromone receptors, histamine receptors, protease-activated receptors, rhodopsins and other G-protein coupled seven transmembrane segment receptors. GPCRs also include these GPCR receptors associated with each other as homomeric or heteromeric dimers or as higher-order oligomers.

(8) Exemplary GPCRs include: 5-HT1A, 5-HT1B, 5-HT1 D, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT4, 5-HT5A, 5-HT6, 5-HT7, MI, M2, M3, M4, M5, AI, A2A, A2B, A3, aIA, aIB, aID, a2A, a2B, a2C, bI, b2, b3, ATI, AT2, BBI, BB2, BB3, BI, B2, CBI, CB2, CXCRI, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRIO, CX3CR1, XCRI, CCKI, CCK2, DI, D2, D3, D4, D5, ETA, ETB, GALI, GAL2, GAL3, motilin, ghrelin, HI, H2, H3, H4, CysLTI, CysLT2, BLTI, BLT2, OXE, ALX, LPAI, LPA2, LPA3, SIPI, S1P2, S1P3, S1P4, S1P5, MCHI, MCH2, MCI, MC2, MC3, MC4, MC5, NMUI, NMU2, YI, Y2, Y4, Y5, NTSI, NTS2, d, k, m, NOP, OXI, 0X2, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, PAF, PKRI, PKR2, PRRP, DP, EPI, EP2, EP3, EP4, FP, IPI, TP, PAR1, PAR2, PAR3, PAR4, sst2, sst5, sst3, sstI, sst4, NKI, NK2, NK3, TRH, UT, OT, VIA, V2, VIB, APJ, FFAI, FFA2, FFA3, GPBA, TSH, LH, FSH, GnRH, KiSSI, MTI, MT2, NPFFI, NPFF2, NPS, NPBWI, NPBW2, P2Y12, P2Y13, QRFP, RXFPI, RXFP2, RXFP3, RXFP4, TAI, TA3, TA4 and TA5.

(9) In certain embodiments of the present disclosure the membrane protein which is N-terminally fused to the viral major capsid protein is a GPCR. In other embodiments of the present disclosure said GPCR is selected from the list of CCR1, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, motilin, ghrelin, PAR1 and PAR2. In other embodiments of the present disclosure said GPCR is CXCR2.

(10) Ion channels are membrane proteins. The term refers to a protein crossing the lipid bilayer of a cell, which, in a regulated manner, transports solutes and/or water across cell membranes. Channels are responsible for generating and propagating electrical impulses in excitable tissues in the brain, heart, and muscle, and for setting the membrane potential of excitable and non-excitable cells. Exemplary ion channels include sodium channels, potassium channels, and calcium channels, as well as ligand gated ion channels such as serotonin, glutamate, and -aminobutyric acid (GABA) channels.

(11) In certain embodiments of the present disclosure the membrane protein which is N-terminally fused to the viral major capsid protein is an ion channel.

(12) The term major capsid protein or capsid protein refers to a viral protein or a functional equivalent thereof, which directs the assembly and the release of virus particles from the infected host cell. In certain embodiments of the present disclosure the major capsid protein or capsid protein is a retroviral capsid protein. In a preferred embodiment the major capsid protein or capsid protein is a Gag protein or a functional equivalent thereof.

(13) The capsid protein may be a Gag protein. The term Gag protein, GAG protein or group-specific antigen refers to a family of glycoproteins that form the capsid of certain viruses.

(14) Two specific Gag proteins that may be used in accordance with the present disclosure include:

(15) The Gag protein of Moloney murine leukemia virus (MoMLV)-(UniPort: P03332):

(16) TABLE-US-00001 (SEQIDNO.:1) MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTFNVGW PRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPF VHPKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDS GGPLIDLLTEDPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRG RREPPVADSTTSQAFPLRAGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPG KLTALIESVLITHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGDDGR PTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYRQLLLAGLQNAGRSPTN LAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETNVSMSFIWQ SAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLD RDQCAYCKEKGHWAKDCPKKPRGPRGPRPQTSLLTLDD
The Gag protein of Human immunodeficiency virus (HIVB1)-(UniPort: P03347):

(17) TABLE-US-00002 (SEQIDNO.:2) MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGL LETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEA LDKIEEEQNKSKKKAQQAAADTGHSSQVSQNYPIVQNIQGQMVHQAISPR TLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQM LKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWM TNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRF YKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTAC QGVGGPGHKARVLAEAMSQVTNTATIMMQRGNFRNQRKMVKCFNCGKEGH TARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQ SRPEPTAPPFLQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTS LRSLFGNDPSSQ

(18) In certain embodiments the present disclosure relates to a fusion protein comprising a membrane protein N-terminally fused to a viral major capsid protein, wherein said viral major capsid protein is a GAG protein. In certain embodiments said Gag protein is a Gag protein of Moloney murine leukemia virus. In certain embodiments said Gag protein is a Gag protein of Human immunodeficiency virus.

(19) It is also possible to utilize only a part of a viral major capsid protein, such as a GAG protein. In such cases the membrane protein is N-terminally fused to a part of a viral major capsid protein or to a part of a GAG protein.

(20) Therefore, in certain embodiments the present disclosure provides a fusion protein comprising a membrane protein N-terminally fused to a part of a viral major capsid protein. In other embodiments the present disclosure provides a fusion protein comprising a membrane protein N-terminally fused to a functional part of a viral major capsid protein. In other embodiments the present disclosure provides a fusion protein comprising a membrane protein N-terminally fused to a part of a GAG protein. In other embodiments the present disclosure provides a fusion protein comprising a membrane protein N-terminally fused to a functional part of a GAG protein. In certain embodiments the present disclosure relates to a fusion protein comprising a membrane protein N-terminally fused to a viral major capsid protein. In other embodiments the fusion protein comprises a linker peptide between said membrane protein and said viral major capsid protein.

(21) In certain embodiments the fusion protein of the present disclosure is capable of being incorporated or encapsulated into virus-like particles. In other embodiments the fusion protein of the present disclosure are incorporated or encapsulated into virus-like particles.

(22) In certain embodiments the present disclosure relates to nucleic acid molecules encoding the fusion proteins of the present invention.

(23) In certain embodiments the present disclosure relates to a vector comprising the nucleic acid molecule encoding the fusion proteins of the present invention.

(24) The term vector refers to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. One type of vector is a plasmid, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian vectors). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Vectors may be compatible with prokaryotic or eukaryotic cells. Prokaryotic vectors typically include a prokaryotic replicon which may include a prokaryotic promoter capable of directing the expression (transcription and translation) of the peptide in a bacterial host cell, such as Escherichia coli transformed therewith. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment. Preferred vector are mammalian vectors. Other preferred vectors are mammalian vectors comprising a CMV promoter.

(25) In certain embodiments the present disclosure relates to a host cell comprising the nucleic acid molecules or the vector encoding the fusion proteins of the present invention. In certain embodiments of the present disclosure the host cell is a mammalian host cell. In other embodiments of the present disclosure the host cell is an HKB11 cell or a HEK cell. In yet other embodiments of the present disclosure the fusion protein expressed in said host cell is under the control of a CMV promoter.

(26) The term recombinant host cell or host cell refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term host cell as used herein. Typical host cells are eukaryotic host cells, such as mammalian host cells. Preferred eukaryotic host cells include yeast and mammalian cells including murine and rodents, preferably vertebrate cells such as a mouse, rat, monkey or human cell line, for example HKB11 cells, PER.C6 cells, HEK cells or CHO cells.

(27) In certain embodiments the present disclosure relates to a virus-like-particle comprising a fusion protein of the present invention. In other embodiments the present disclosure relates to a virus-like-particle comprising a fusion protein wherein said fusion protein is displayed on the surface of said virus-like-particle.

(28) The term Virus-like particle or VLP refers to a structure resembling a retrovirus particle or a retrovirus-like particle with an envelope composed of a lipid bilayer and membrane proteins. Typically the envelope of the virus-like particle contains plasma membrane and membrane proteins obtained from the eukaryotic host cell. In certain embodiments of the present disclosure the virus-like particles are non-replicative or non-infectious, preferably non-replicative and non-infectious. The term non-replicative, as used herein, refers to being incapable of replicating the genome comprised by the VLP. The term non-infectious, as used herein, refers to being incapable of entering the host cell. Preferably a virus-like particle in accordance with the invention is non-replicative and/or non-infectious since it lacks all or part of the viral genome or genome function. Typically, a virus-like particle lacks all or part of the replicative and infectious components of the viral genome. A virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome. A typical and preferred embodiment of a virus-like particle in accordance with the present invention is a viral capsid such as the viral capsid of the corresponding virus coated with a lipid membrane known as the viral envelope. The terms viral capsid or capsid, refer to a macromolecular assembly composed of viral protein subunits. Typically, there are 60, 120, 180, 240, 300, 360 and more than 360 viral protein subunits. Typically and preferably, the interactions of these subunits lead to the formation of a viral capsid or a viral-capsid like structure with an inherent repetitive organization, wherein said structure is, typically, spherical. For example, the capsids of retroviruses have a spherical form.

(29) In certain embodiments the present disclosure relates to a virus-like-particle comprising a fusion protein of the present invention, wherein the virus-like-particle was produced from a eukaryotic host cell.

(30) In certain embodiments the present disclosure relates to a method for the generation of virus-like-particles comprising a fusion protein of the present invention. In another embodiment the present disclosure relates to a method for the generation of virus-like-particles said method comprising the steps:

(31) (a) providing a vector encoding a fusion protein of the present invention,

(32) (b) transfecting a eukaryotic host cell with a vector of step (a)

(33) (c) purifying the VLPs form the supernatant.

(34) The fusion proteins and the virus-like particles of the present disclosure have numerous uses. For example, the VLPs can be used to display the respective fusion protein. Such a display system can be used, for example, for screening or panning of antibody libraries. This may lead to the identification of antibodies which may not be generated by other means due to the absence of appropriate antigen material.

(35) In other embodiments the present disclosure therefore relates to the use of the fusion proteins or the VLPs of the present invention for the selection of a moiety, such as an antibody, that is reactive with the GPCR- or ion-channel part of the fusion protein of the present invention.

(36) In other embodiments the present disclosure provides a method to identify a binding moiety binding to a membrane protein, said method comprising the steps:

(37) (a) providing a fusion protein of the present invention,

(38) (b) generating VLPs comprising the fusion protein of step (a),

(39) (c) contacting the VLPs of step (b) with an antibody library,

(40) (d) washing the VLPs to remove those antibodies that did not bind the VLP, and

(41) (d) selecting an antibody which is reactive with the membrane protein part of said fusion protein.

EXAMPLES

Example 1: Selection of GAG Proteins and Membrane Proteins

(42) First, two exemplary GAG proteins were selected for the proof of concept experiments. The Gag proteins chosen are the Gag protein of Moloney murine leukemia virus (UniPort: P03332; SEQ ID NO.: 1) and the Gag protein of Human immunodeficiency virus (UniPort: P03347; SEQ ID NO.: 2).

(43) As exemplary integral membrane proteins three GPCRs were chosen: GPCR 1, GPCR 2 and GPCR 3. GPCR 1 is CXCR2 (UniPort: P03347; SEQ ID NO.: 3):

(44) TABLE-US-00003 (SEQIDNO.:3) MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYF VVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALT LPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVH ATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDM GNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAM RVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATE ILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVG SSSGHTSTTL.

Example 2: Generation of Constructs and Expression of Proteins

(45) All cloning experiments were performed using standard technologies. Proteins were cloned in pMAX vectors for the expression in mammalian cells. Expression in these vectors is under the control of the CMV promoter. The constructs that were generated produced either:

(46) (a) the GAG protein,

(47) (b) the GPCR protein, or

(48) a fusion protein, in which the GPCR protein is fused N-terminal to the GAG-protein.

(49) Expression of the proteins and production of the VLPs was done under standard conditions in suspension cultures. Host cells used in the present experiments are HKB11 cells (ATCC; CRL-12568) and HEK cells (Life Technologies). In one set of experiments (Experiment A) the host cells were transfected with the following two vectors: one vector expressing GAG and one vector expressing the GPCR. In another set of experiments (Experiment B) the host cells were also transfected with two vectors. However, one vector expressed GAG and the other vector expresses the GPCR-GAG fusion protein.

Example 3: Purification of VLPs and Analyses

(50) Three days post transfection the supernatants containing the VLPs were harvested and purified using standard procedures (including precipitation and ion exchange chromatography). The proteins isolated were then subjected to Western Blot analysis and SDS-PAGE chromatography.

(51) The results of the Western Blot analyses are shown in FIG. 1. Panel A shows the results of Experiment A, panel B shows the result of Experiment B. The supernatants were probed with an anti-GAG antibody or an anti-GPCR antibody as indicated in FIG. 1.

(52) Panel A of FIG. 1 shows that the co-expression of GAG and the GPCR in a host cell transfected with both vectors leads to the excretion of VLPs, as indicated by the presence of a positive GAG band on the Western blot. However, essentially no GPCR is detectable in the VLPs. This confirms that the co-expression of the individual molecules leads to a scenario in which the integration of the GPCR into the VLP only occurs by chance and at a low frequency.

(53) In contrast, Panel B of FIG. 1 shows that the co-expression of GAG and a GPCR-GAG fusion protein leads to a high expression level of GAG and GPCR-GAG in the VLPs. This confirms that the GPCR are efficiently integrated into the VLPs, and that the GPCRs are detectable with antibodies. This means that the GPCR is displayed in a manner that makes it accessible for further molecular manipulation.

(54) FIG. 2 shows a SDS-PAGE of the VLPs of Experiment B. As can be seen, the VLPs essentially consist of two proteins: GAG and the GPCR-GAG fusion protein.

Example 4: Repetition with Additional Integral Membrane Proteins

(55) Examples 1-3 were repeated with two additional GPCR molecules. All results could be confirmed with these additional molecules.

(56) FIG. 3 shows a comparative analysis of the three GPCRs tested in the present disclosure. As can be seen all three GPCR could be expressed efficiently in the form as GPCR-GAG fusion proteins. Display rates were high enough for standard ELISA measurements.