Bioparticles for the expression of multimeric proteins

Abstract

The present invention pertains to specific bioparticules at the surface of which are expressed multimeric protein. The bioparticles according to the present invention comprise an envelope consisting of a plasma membrane; and at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising successively a) a first monomer of a multimeric protein of interest, b) a coiled-coil domain or oligomerization sequence; and c) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment. Fragments a) and b) are exposed at the surface of the bioparticle, and fragment a) is bound to a second monomer of said multimeric protein by means of a bond which is not a peptide bond. The bioparticles according to the present invention can be used in therapy such as immunotherapy. The present invention also pertains to methods for producing such bioparticles.

Description

Title: BIOPARTICLES FOR THE EXPRESSION OF MULTIMERIC PROTEINSTechnical Field

[0001] The present invention pertains to novel bioparticles that are capable of expressing multimeric proteins onto their surfaces. The bioparticles according to the present invention can advantageously be used as an immunotherapy for the treatment of various diseases.Background Art

[0002] It is now well-established that bioparticles (BP), and in particular virus-like particles (VLPs), can be used for the presentation of target antigens to the immune system. There is now accumulating evidence that delivery of antigens in the form of bioparticles has a profound impact on the skewing of the immune response (De Souza RebouQas et al. BioMed research international 2012 (2012); Anzaghe, Martina, Stefan Schulke, & Stephan Scheurer. Current allergy and asthma reports 18 (2018): 1 -12; Klimek et al. Allergo journal international 27 (2018): 245-255). Most bioparticles contain repetitive displays of conformational epitopes that can elicit strong T cell and B cell responses, effectively making them high potency immunization ‘devices’ (Singha et al. ACS nano 12.11 (2018): 10621-10635).

[0003] Successful nanoparticle-based vaccines have e.g. been developed against human papilloma virus (HPV) and hepatitis B virus (HBV). Both vaccines are considered to be virus-like particles (VLPs), a specific class of bioparticles in which the structural and immunogenic constituents are of viral origin. The HPV- VLPs results from self-assembly of recombinant HPV capsid L1 proteins. The HBV- VLPs is an example of a so-called ‘enveloped’ VLP (eVLP) in which a target antigen — in this case the HBV surface antigen — is embedded in the envelope of the host cell in which this antigen is expressed. This latter type of VLP is more complex but more flexible than the capsid-type’ VLP since there is no requirement for self-assembly. However, assembly and budding of eVLPs require oligomerization and palmitoylation of the structural transmembrane domains of surface proteins in the endoplasmic reticulum (Chlanda et al. Journal of virology 91.21 (2017): 10-1128; Barman et al. Journal of virology 78. 0 (2004): 5258-5269; Chen, Benjamin J., Makoto Takeda, and Robert A. Lamb. Journal of virology 79.21 (2005): 13673-13684). In most virus-derived bioparticles, this oligomerization is carried out by one key domain of surface protein monomers, the stalk, through disulfide bonding, which results in oligomerization of the protein, including its transmembrane section.

[0004] Enveloped VLPs have an additional potential benefit, compared to capsid VLPs, for the presentation of antigens. It appears that membrane components of the host cell in which these VLPs are produced (eg: mammalian, insect, plant) may have immunostimulatory effects. Although the mechanisms of action have not yet been clearly defined, it has been shown repeatedly that the immunostimulatory effect of eVLPs does not require the addition of adjuvants, and eVLPs are said to be self-adjuvanted (Dai, Shiyu, Hualin Wang, and Fei Deng. Journal of Immunological Sciences 2.2 (2018)).

[0005] Recently, synthetic biology has been used to produce artificial modular bioparticles, in which supra-molecular assemblies are made of elements from heterogeneous biological sources. This is a new approach to the design of antigen presentation devices.

[0006] This approach has been used to link small immunogenic peptides to self-assembling viral and bacteriophage capsid components (Pattenden et al. Trends in biotechnology 23.10 (2005): 523- 529; Roy, Polly, and Rob Noad. Pharmaceutical biotechnology (2009): 145-158). Many of these engineered bioparticles require the presence of viral matrix proteins, in particular the M1 matrix protein, to assemble (Quan et al., J Virol. 2007 Apr;81(7):3514-24); Gomez-Puertas et al, 2000. J. Virol. 74:11538-11547; Kang et al., Expert review of vaccines 11.8 (2012): 995-1007).

[0007] Recently, novel bioparticles having the ability to self-assemble without requiring the presence of viral matrix proteins have been developed (Gomord et al. PloS one 15.12 (2020): e0242867; international patent application published under reference WO2018 / 020195). These bioparticles are based on fusion proteins comprising a coiled-coil domain which was fused at the C- terminus to the transmembrane domain and the cytosolic tail (TM / CT) of hemagglutinin and at the N-terminus to a target protein such as the allergen Der p 2. They were shown to be extremely efficient for presenting antigens and activating immune system cells: they stimulate the production of allergen specific IgGs while at the same time minimizing accessibility to basophils and having extremely low reactivity towards human IgEs thereby reducing the risk anaphylactic reaction.

[0008] The presence of the coiled-coil domain allows for the presentation of target proteins that are normally expressed as monomers in polymerized forms (particularly as trimers). However, such bioparticles do not allow for the presentation of more complex protein that are naturally in the form of polymers, in particular heteropolymers.

[0009] Therefore, there is still a need for the development of novel bioparticles that allow for the presentation of complex proteins such as polymers. The polymers thereby presented must be recognizable by the immune system so as to be able to elicit an immune reaction that would efficiently target said polymer in its natural / soluble form.Summary of the Invention

[0010] The invention is defined by the claims.

[0011] Surprisingly, the present inventors have shown that it is possible to produce bioparticles that allow for the presentation and recognition of proteins that are in the form of polymers. These bioparticles are based on fusion proteins that comprise a monomer of a polymeric protein. The present inventors have shown that said monomer is capable of interacting and forming covalent bonds with other monomers that would be expressed in a soluble form, so as to mimic the conformation of the natural polymeric protein at the surface of the bioparticle. The inventors have demonstrated that said bioparticles therefore allow for the efficient presentation of polymeric proteins to immune cells.

[0012] The present inventors have particularly shown that a bioparticle according to the present invention can be detected by polyclonal antibodies generated in an animal model, but also by IgEmonoclonal antibodies from patients, whereas the corresponding natural multimeric protein was not detected by mAbs from patients, and the corresponding recombinantly expressed multimeric protein was poorly detected. The data presented herein demonstrate that the bioparticles according to the present invention show an optimal immunological profile, while triggering a hypoallergic response. Without wishing to be bound by theory, the inventors show that these bioparticle can be presented to antigen presenting cells of the immune system and are likely to activate a stronger immune response leading to a better therapy.

[0013] Accordingly, according to a first embodiment, the present invention pertains to a bioparticle comprising:

[0014] - an envelope consisting of a plasma membrane; and

[0015] - at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising the following fragments, successively:

[0016] a) a first monomer of a multimeric protein of interest ;

[0017] b) a coiled-coil domain or oligomerization sequence; and

[0018] c) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment;

[0019] fragments a) and b) being exposed at the surface of the bioparticle;

[0020] wherein said first monomer of a multimeric protein of interest exposed at the surface of the bioparticle is bound to a second monomer of said multimeric protein by means of a bond which is not a peptide bond.

[0021] The first and second monomer can advantageously be bound by a bond selected from the group consisting of disulfide bridge, an ionic or hydrogen electrostatic bond and a van Der Waals bond.

[0022] The bioparticles according to the present invention are obtained by means of a method wherein the fusion protein as defined above and the second monomer of said multimeric protein are co-expressed in a eukaryotic cell.

[0023] The bioparticles according to the present invention are of particular use in therapy such as immunotherapy.Brief Description of Drawings

[0024] Figure 1: Schematic representation of cDNA (A) or protein (B) of soluble Fel d 1 (sol Feld 1 ) compared to the Fel d 1 - BP protein. A cDNA encoding carrier was fused at the C-terminal end of the Feld 1-CH2. This carrier anchors the dimer in the lipidic membrane when it is expressed in heterogenous expression system.

[0025] Figure 2: Expression and purification of soluble Fel d1 or Fel d1-BP by coexpression of soluble Fel d1-CH1 and soluble Fel d1-CH2 (A) or by coexpression of soluble Feld 1-CH1 and Fel d1-CH2-BP (B) in plant cells. Protein extracts from leaves were analyzed in absence or in presenceof reducing agent. These extracts producing monomers (lanes 1-4 and lanes 7-8) or dimers (lanes 5-6 and 9-10) were separated by SDS-PAGE and immunodetected by immunoserum containing antibodies directed against CH1 and CH2.

[0026] Figure 3: Allergenicity of the soluble dimers or dimers-BP using biolayer interferometry. The interactions between the soluble Fel d1 or the Fel d1-BP and characterized IgE directed against Fel d1 were analyzed.

[0027] Figure 4: Schematic representation of the immunogenicity study.

[0028] Figure 5: Schematic representation of cDNA (A) or protein (B) of two examples of dimer formation at the surface of the BP.

[0029] Figure 6: Western blot of different Feld-1 protein using 3 different human anti-lgE monoclonal and a Rabbit polyclonal antibody raised against natural Feld-1 extracted from cat hair. 1) 250 ng of non-reduced Feld-1 BP; 2) 250 ng of non-reduced natural Feld-1; 3) 250 ng of nonreduced recombinant Feld-1; 4) 250 ng of reduced Feld-1 BP; 5) 250 ng of reduced natural Feld-1; 6) 250 ng of reduced recombinant Feld-1. Exposure 8 seconds.

[0030] Figure 7: Western blot of different Feld-1 protein using 3 different human anti-lgE monoclonal. 1) 250 ng of non-reduced Feld-1 BP; 2) 250 ng of non-reduced natural Feld-1; 3) 250 ng of non-reduced recombinant Feld-1; 4)250 ng of reduced Feld-1 BP; 5)250 ng of reduced natural Feld-1 ; 6) 250 ng of reduced recombinant Feld-1. Exposure 50 seconds.Detailed description of the invention

[0031] The present invention pertains to a bioparticle comprising:

[0032] - an envelope consisting of a plasma membrane; and

[0033] - at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising the following fragments, successively:

[0034] a) a first monomer of a multimeric protein of interest ;

[0035] b) a coiled-coil domain or oligomerization sequence; and

[0036] c) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment ;

[0037] fragments a) and b) being exposed at the surface of the bioparticle;

[0038] wherein said first monomer of a multimeric protein of interest exposed at the surface of the bioparticle is bound to a second monomer of said multimeric protein by means of a bond which is not a peptide bond.

[0039] The fusion protein behaves like a viral surface protein when it is expressed in eukaryotic cells. This protein is synthesized in the endoplasmic reticulum and then transported, via the Golgi apparatus, to the plasma membrane. Once it reaches the plasma membrane, this transmembrane fusion protein causes curving of said membrane, which finally forms a bud which separates from thecell membrane and is released into the extracellular space. During its synthesis in the endoplasmic reticulum and then transportation to the plasma membrane, the first monomer comprised in the fusion protein interacts with the second monomer, which is expressed in a soluble form, and binds with it by means of a bond which is not a peptide bond. During the budding, the first monomer carried by the coiled-coil domain (or oligomerization sequence), and bound to said second monomer, is exposed at the outer surface of the newly formed particle.

[0040] The transmembrane domain remains anchored in the membrane and is not exposed at the surface. A bioparticle is thus obtained, comprising a plasma membrane in which the fusion proteins are attached at the level of their anchoring domain, and which exposes at its surface the monomers of the multimeric protein of interest bound by means of bonds which are not peptide bonds, thereby mimicking the quaternary structure of the wild-type multimeric protein.

[0041] A “bioparticle” or “BP” refers to a biological nanoparticle consisting of a plasma membrane envelope in which one or more proteins are anchored, which contains no genetic material, which is non-infectious and incapable of multiplying, and which self-assembles to mimic the original structure of a virus. “Virus-like particles” or “VLP” are a specific class of bioparticles in which the structural and immunogenic constituents are of viral origin.

[0042] The bioparticles according to the invention typically have a diameter of between 120 and 200 nm.

[0043] The bioparticles according to the present invention are made of a plasma membrane into which specific type I or II fusion proteins are anchored.

[0044] A “plasma membrane” is a structure well known by the skilled person. A plasma membrane is a lipid bilayer made up of two layers of phospholipids. According to a specific embodiment, a portion of the plasma membrane used in the bioparticles is typical of lipid rafts. The expression "portion of plasma membrane typical of lipid rafts" is intended to mean a phospholipid bilayer (i.e. plasma membrane) found in the microdomains of lipid rafts. Such a bilayer is rich in cholesterol and in phospholipids, preferably in phosphatidylcholine and in phosphatidylethanolamine, and in sphingolipids, such as sphingomyelin, but poor in docosahexaenoic acid. In addition, it has a low density, and is insoluble in mild detergents (for example polysorbates).

[0045] The term “fusion protein” is intended to mean a protein comprising the various fragments a) to c), wherein said fragments are of different origin. In other words, fragments a) to c) are never present fused in the way they exist naturally.

[0046] The expression “type I transmembrane protein anchored in a membrane” is intended to mean a transmembrane protein of which the N-terminal end is extracellular and the C-terminal end is cytosolic. Consequently, the type I transmembrane protein comprises, from the N-terminal to C-terminal end the first monomer of a multimeric protein of interest a), then the coiled-coil domain b) and, finally, the anchoring domain c).

[0047] The expression “type II transmembrane protein anchored in a membrane” is intended to mean a transmembrane protein of which the C-terminal end is extracellular, and the N-terminalend is cytosolic. Consequently, the type II transmembrane protein comprises, from the N-terminal to C-terminal end, the anchoring domain c), then the coiled-coil domain b) and, finally, the first monomer of a multimeric protein of interest a).

[0048] Preferably, the fusion protein according to the invention is a type I transmembrane protein.

[0049] As mentioned above, the fusion protein according to the present invention comprises the following fragments, successively:

[0050] a) a first monomer of a multimeric protein of interest;

[0051] b) a coiled-coil domain or oligomerization sequence; and

[0052] c) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment.

[0053] The term “successively” is intended to mean that fragments a) to c) are present in the order a)-b)-c) (or c)-b)-a)). These various fragments can be directly fused to one another, or else fused to one another via one or more linker(s). Preferably, the fusion protein according to the invention comprises a linker present between the sequences a) and b), and / or between the sequences b) and c).

[0054] Segment a): a first monomer of a multimeric protein of interest a)

[0055] As mentioned above, the bioparticle according to the present invention allows presenting multimeric proteins in a conformation that mimics that of the natural (wild type) protein. The fusion protein anchored in the plasma membrane of the bioparticle according to the present invention comprises a first monomer of a multimeric protein of interest which will associate with a second monomer of said multimeric protein, said second monomer being in a soluble form (i.e. expressed as a peptide), by means of a bond which is not a peptide bond. Said monomers will then be presented at the surface of the bioparticle, thereby mimicking the quaternary structure of the natural multimeric protein.

[0056] The multimeric protein of interest according to the present invention can be any protein which is of therapeutic or prophylactic interest, which would benefit from being exposed at the surface of a bioparticle particle, and which is capable of being recognized by immune cells, and / or of triggering a biological reaction.

[0057] The expression “multimeric protein” or “oligomeric protein” refers to proteins that are made of more than one polypeptide / protein chain, each polypeptide / protein chain representing a protein subunit, also referred to as a 'monomer' or “protomer”. The number and arrangement of the protein subunits with respect to one another correspond to the quaternary structure of the multimeric protein. Multimeric proteins can be divided into two classes: homo-oligomers and heterooligomers; the former are composed of identical subunits while the latter are composed of nonidentical subunits (Yu et al. BMC bioinformatics ? (2006): 1-6). Multimeric proteins can be made up of two monomers, and in such a case be referred to as a dimer (homodimer when it is made of two identical subunits or heterodimer when it is made of two non-identical subunits), but also of three (inthe case of a trimer), four (in the case of a tetramer), five (in the case of a pentamer), six (in the case of an hexamer), seven (in the case of an heptamer), eight (in the case of an octamer), nine (in the case of a nonamer), ten (in the case of a decamer) etc. In the context of the present invention the multimeric protein is preferably made of between 2 to 8 monomers, preferably 2 to 5 monomers. In the context of the present invention, the multimeric protein can be any protein having a quaternary structure. The protein can thus be a protein that is naturally expressed as a multimer, i.e. that naturally has a quaternary structure, or a protein that has been artificially engineered in the form of a multimer.

[0058] Many proteins have a quaternary structure and are in the form of a multimer. Multimeric proteins of interest that can be used in the context of the present invention e.g. include allergens immunoglobulins and immunoglobulin fragments.

[0059] Many allergens are in the form of multimeric proteins (Hasan-Abad et al. Clinical and Molecular Allergy 20.1 (2022): 5). Therefore, according to a specific embodiment, the multimeric protein of interest is an allergen. The major application of a bioparticles containing such a multimeric protein is immunotherapy. Preferably, the allergen in the form of a multimeric protein is chosen from allergens responsible for respiratory allergies resulting from domestic mites, such as Dermatophagoides farinae, Dermatophagoides pteronyssinus or Euroglyphus manei, allergens from storage mites such as Blomia tropicalis, allergens from mites of Acarus siro type (otherwise known as Tyroglyphus farinae), cockroach allergens, tree or grass pollen allergens (including ragweed allergens), animal (cat, dog, horse) allergens, mold allergens, allergens responsible for contact allergies, such as those of hevea latex, or else allergens responsible for food allergies (milk, eggs, fish, fruit).

[0060] Allergens having a quaternary structure e.g. include cat (Felis domesticus) allergens such as Fel d 1 and Fel d 1 .0101. The sequences ofthese allergens are known, in particular in the Uniprot base.

[0061] According to a preferred embodiment, the multimeric protein of interest is the allergen Fel d 1. Fel d 1 , a well-known cat allergen, is naturally expressed in the form of a heterodimer. The two monomers forming this heterodimer are referred to as the CH1 chain and the CH2 chain. According to this specific embodiment, the first monomer of the multimeric protein of interest can therefore be the CH1 or the CH2 chain.

[0062] According to a preferred embodiment, the first monomer of Fel d 1 comprised in the fusion protein is the CH2 chain. According to this specific embodiment, the CH2 chain exposed at the surface of the bioparticle associates with the second monomer of Fel d 1 , i.e. the CH1 chain, which would be expressed in a soluble form. The exact description of such a bioparticle is provided in the experimental section of the present invention. According to this specific embodiment the first monomer of interest comprised in the fusion protein can advantageously have the amino acid sequence as set forth in SEQ ID No. 1: VKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGR (SEQ ID No.1). This amino acid sequence corresponds to the sequence of the mature wild type CH2 chain of Fel d 1 (i.e. without its native signal peptide).

[0063] According to this specific embodiment, the second monomer can advantageously have the amino acid sequence as set forth in SEQ ID No. 2: EICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSLLDKIYTS PLC (SEQ ID No. 2). This amino acid sequence corresponds to the sequence of the mature wild type CH1 chain of Fel d 1 (i.e. without its native signal peptide).

[0064] Bioparticles expressing Fel d 1 can advantageously be used in immunotherapy for treating / preventing cat allergies.

[0065] Other multimeric proteins of interest according to the present invention include immunoglobulins such as antibodies and immunoglobulin fragments. Biopartides presenting immunoglobulins and immunoglobulin fragments as the multimeric protein of interest are particularly useful for reconstituting natural-like multimeric proteins on the surface of bioparticles.

[0066] In the context of the present invention the “immunoglobulin” can be an antibody of any origin such as human, murine, primate, or camel antibodies. In most mammals including humans, antibodies are made of four monomers or polypeptide chains: two identical heavy chains and two identical light chains connected by disulfide bonds. Immunoglobulin heavy chains include four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). Immunoglobulin light chains consistof one variable domain (VL) and one constant domain (CL). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). Structurally an antibody can also be partitioned into two arms corresponding to two antigen-binding fragments (Fab), containing one VL, VH, CL, and CH1 domain each, as well as the crystallisable fragment (Fc), forming the trunk of the Y shape (Putnam, Frank W., Y. S. Liu, and T. L. Low. "Primary structure of a human lgA1 immunoglobulin. IV. Streptococcal lgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain." Journal of Biological Chemistry 254.8 (1979): 2865-2874). The variable domains can also be referred to as the Fv region. It is the subregion of Fab that binds to an antigen.

[0067] According to a specific embodiment, the multimeric protein of interest presented at the surface of the bioparticle according to the present invention can therefore be an antibody. Said antibody may be a chimeric or humanised antibody. It may be an antibody of any isotype or subclass thereof, in particular an IgG, IgM, IgA, IgE or IgD.

[0068] According to a further embodiment, the multimeric protein of interest presented at the surface of the bioparticle according to the present invention can be an antibody fragment which would be in a multimeric form. Such a fragment includes antigen-binding fragments (Fab) or crystallisable fragment (Fc) of an antibody, the variable domain of an antibody (Fv), a single arm of an antibodyincluding one heavy chain and one light chain or a monovalent fragment consisting of the VL, VH, CL and CH1 domains.

[0069] One of the main advantages of using an antibody or an antibody fragment as the multimeric protein of interest according to the present invention is that the monomers thereof can be used as vehicles, as carriers, to attach various proteins / peptides, herein referred to as “second proteins of interest”, so as to express them at the surface of the bioparticle according to the invention. Such a conformation makes it possible to rapidly and easily present multiple proteins of interest at the surface of bioparticles without having to design a new fusion protein. Indeed, according to such an embodiment, the fusion protein includes as fragment a) a first monomer of an antibody / antibody fragment. As explained above, this first monomer will associate with second monomer of said antibody / antibody fragment expressed in a soluble form. According to this embodiment, the second monomer of said antibody / antibody fragment is attached to a second protein of interest. It is therefore possible to change this second protein of interest without having to synthesize and express a whole new fusion protein. According to such an embodiment, the first monomer can e.g. be the Fc region (CH2 + CH3) of a first arm of an antibody, and the second monomer be the Fc region (CH2 + CH3) of the second arm of said antibody. Alternatively, the first monomer can be the heavy chain of one arm of an antibody (VH+CH1+CH2+CH3 domains) and the second monomer be the light chain of said arm of said antibody (VL+CL domains). According to another embodiment, the first and second monomers can be Fab regions of the antibody. According to such an embodiment, one of the monomers can be made of the VL+CL domains and the other monomer can be made of the VH+CH domains.

[0070] In the context of the present invention, the “second protein of interest” can be any protein or peptide of therapeutic or prophylactic interest. Said second protein is typically chosen from antigens including allergens and fragments thereof, viral proteins and fragments thereof, cell surface proteins and fragments thereof, cytokines and fragments thereof, proteins and peptides associated with proliferative disorders such as cancer, with chronic or neurodegenerative diseases.

[0071] According to a specific embodiment, the second protein of interest is a tumor antigen. The notion of "tumor proteins / antigens" is well known to those skilled in the art. They are proteins and / or molecules expressed specifically by the tumor cells, and which can be recognized by T and B lymphocytes. According to this embodiment, tumor antigens from patients can be easily expressed on the surface of a universal bioparticle, made of one single fusion protein construct. Expressing tumor antigens at the surface of a bioparticle has a particular interest in the treatment of cancer.

[0072] According to a further embodiment, the multimeric proteins of interest can be a viral protein. The main advantage of a bioparticle containing such a protein of interest is vaccination. Viral proteins expressed in the form of a multimer that can be used in the context of the present invention are typically viral capsid proteins.

[0073] At the surface of the bioparticle, the first and second monomers of the multimeric protein of interest are bound by means of a bond which is not a peptide bond.

[0074] A “peptide bond” has a general meaning in the art. A peptide bond is a covalent chemical bond linking two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino acid and N2 (nitrogen number two) of another, along a peptide or protein chain. Therefore, according to the present invention, the first and second monomers of the multimeric protein of interest are not fused: they are not part of the same peptide chain.

[0075] The first and second monomers of the multimeric protein of interest can be bound by means of any bond naturally occurring in the formation of the multimeric protein of interest when expressed in a soluble form. The skilled person is familiar with the quaternary structure of multimeric proteins and knows which type of bonds can be used for bounding the various subunits of such proteins. Such bonds can e.g. include disulfide bridges, electrostatic bonds (ionic or hydrogen) or Van Der Waals bonds. The first and second monomers of the multimeric protein of interest can be bound by one or by multiple bonds, as long as they do not comprise a peptide bond.

[0076] The bond by which the first and second monomers are bound allows maintaining these two monomers extremely close from one another, within a distance of between 2 and 4 Angstrom (A).

[0077] According to a preferred embodiment, the bound by which the first and second monomers are bound is a disulfide bridge.

[0078] Segment b): the coiled-coil domain (or oligomerization sequence) b)

[0079] The coiled-coil domain, or oligomerization sequence, comprises several sense or antisense alpha-helix motifs which are parallel to one another and form an organized matrix that has several well-characterized biological functions. These domains are omnipresent and are found as specific domains for many types of proteins in most organisms. Coiled-coil domains from various sources can assemble to form forms which range from a dimer to a heptamer; some coiled-coil domains will adopt different polymerization levels depending on the point mutations of their amino acid sequence.

[0080] A coiled-coil domain typically consists of a repeat motif of 7 amino acids, of "hxxhcxc" type, wherein "h" is a hydrophobic amino acid, ”c" is a charged amino acid, and ”x“ is any amino acid.

[0081] According to a specific embodiment, the coiled-coil domain used in the context of the invention does not originate from a virus; it is not viral.

[0082] Among the coiled-coil domains that can be used according to the invention, mention will preferably be made of those from cortexillin, vimentin, tetrabrachion, golgins, proteins of the "Soluble N-ethylmaleimide-sensitive factor (NSF) Attachment protein REceptor" or SNARE superfamily, or else transcription factors such as GCN4 or a variant thereof, such as GCN4-pLI or GCN4-plL

[0083] Preferably, the coiled-coil domain is that from the GCN4, GCN4-pLI or GCN4-pll transcription factor.

[0084] According to a preferred embodiment, the coiled-coil domain is the GCNA-pll trimerization sequence of yeast GCN4 transcription factor which has the amino acid sequence as set forth in SEQ ID No. 3: LKQIEDKIEEILSKIYHIENEIARIKKLIGESAA (SEQ ID No. 3).

[0085] The coiled-coil domain can also be chosen from the amino acid sequences as set forth in SEQ ID No. 4:RMKQIEDKLEEILSKLYHIENELARIKKLLGER (GCN4-pLI tetramerization sequence of yeast GCN4 transcription factor); SEQ ID No. 5: VKQLADAVEELASANYHLANAVARLAKAVGER (GCN4-pAA heptamerization sequence of yeast GCN4 transcription factor); SEQ ID No. 6 KQIEDKIENITSKIYNITNEIARIKKLIGNRT (IZN4 glycosylated oligomerization sequence of yeast GCN4 transcription factor), SEQ ID No. 7:INETADDIVYRLTVIIDDRYESLKNL (SNARE oligomerization sequence) and SEQ ID No. 8: LKSRLDTLSQEVALLKEQQALQTVCL (synthetic sequence mimicking a coiled-coil).

[0086] Segment c): the domain for anchoring in the plasma membrane (or transmembrane domain) c)

[0087] The transmembrane domain is a short sequence of lipophilic amino acids which interacts with the specific lipids of the plasma membrane components.

[0088] According to a specific embodiment, the plasma membrane comprises at least one portion typical of lipid rafts.

[0089] These anchoring domains are common (but not through a consensus sequence) to the surface proteins of viruses, but also to proteins which are naturally integrated into the membrane of the living cells. Each transmembrane domain participates in the bending and the budding of the plasma membrane.

[0090] Among the anchoring domains that can be used according to the invention, mention will preferably be made of those from the proteins listed in table 1A:

[0091] Table 1A: Transmembrane proteins

[0092] Among the anchoring domains that can be used according to the invention, mention will preferably be made of those from the viral envelope proteins listed in table 1B below:

[0093] Preferably, the anchoring domain that can be used according to the invention is chosen from the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin corresponding to the amino acid sequence as set forth in SEQ ID No. 9: YQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI (SEQ ID No. 9) and the anchoring sequence of the PDLP1 protein (AOAOD3D8S3) corresponding to the amino acid sequence as set forth in SEQ ID No. 10:IALAVGGVAVLGFVIVCLLVLKSAMKKKSKYDSY (SEQ ID No. 10).

[0094] Linkers

[0095] The fusion protein according to the invention can advantageously comprise a linker between fragments a) and b), and / or between fragments b) and c). A linker may also be present when the second monomer as defined above is further linked to a second protein of interest.

[0096] Linkers are short sequences of amino acids (2 to 10 amino acids, preferably 2 to 6) which create a flexible arm. They may be useful for creating a flexible space between specific peptide domains, if the fact that the two domains are too close together interferes with correct assembling.

[0097] Preferably, the linker is a sequence of-(GGGS)n-type, wherein n is an integer. Preferably, the linker is chosen from SEQ ID No. 11 (n=1 ), SEQ ID No. 12 (n=2) and SEQ ID No. 13 (n=3).

[0098] Thus, according to a specific embodiment the fusion protein useful in the context of the present invention comprises successively:

[0099] a) a first monomer of a multimeric protein, said multimeric protein being preferably selected from Feld 1 and immunoglobulins;

[0100] b) a coiled-coil domain having a sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8; and

[0101] c) an anchoring domain selected from the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin and the anchoring sequence of the PDLP1 protein.

[0102] Thus, according to a very specific embodiment, the fusion protein comprises, preferably consists of, the amino acid sequence as set forth in SEQ ID No. 14: VKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTI SSSKDCMGEAVQNTVEDLKLNTLGRLKQIEDKIEEILSKIYHIENEIARIKKLIGESAAGGGSYQILSIY STVASSLALAIMMAGLSLWMCSNGSLQCRICI (SEQ ID No. 14).

[0103] Likewise, preferably, the BP according to the invention comprises:

[0104] - an envelope consisting of a plasma membrane; and

[0105] - at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising the following fragments, successively:

[0106] a) a first monomer of a multimeric protein, said multimeric protein being preferably selected from Feld 1 and immunoglobulins;

[0107] b) a coiled-coil domain having a sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8; and

[0108] c) an anchoring domain selected from the anchoring sequence of the H5N1 influenza virus H5 hemagglutinin and the anchoring sequence of the PDLP1 protein,

[0109] fragments b) and c) being exposed on the outside of the bioparticle,

[0110] wherein said first monomer exposed at the surface of the bioparticle is bound to a second monomer of said multimeric protein by means of a bond which is not a peptide bond.

[0111] Methods for producing the BP according to the present invention

[0112] According to a further aspect, the present invention also relates to a method for producing a bioparticle as described above, comprising co-expressing the fusion protein as described aboveand the second monomer of the multimeric protein of interest in eukaryotic cells, preferably in plant cells.

[0113] By “co-expressing”, it is meant that the fusion protein as described above and the second monomer of the multimeric protein are expressed in the eukaryotic cell simultaneously, so that the amino acid sequence of the fusion protein and that of the second monomer be present at the same time in the eukaryotic cell.

[0114] Typically, the nucleotide sequence coding for the fusion protein and that coding for the second monomer of the multimeric protein of interest are placed in an expression vector using conventional methods. The sequences (that coding for the fusion protein and that coding for the second monomer of the multimeric protein of interest) can be placed in one single expression vector or be placed in two distinct expression vectors.

[0115] The selection of a suitable expression vector will depend on the method for introducing the expression vector into host cells. A typical expression vector contains eukaryotic DNA elements, such as a transcription initiation sequence for the exogenous gene, for instance a promoter, and DNA elements which control the processing of the transcripts, such as termination / polyadenylation sequences, and an expression cassette allowing for the expression of a silencing inhibitor. It also contains sequences such as t-DNAs which are required for the integration of a piece of DNA into the plant or into the plant cell.

[0116] Preferably, the expression vector comprises:

[0117] at least one nucleotide sequence encoding the fusion protein and / or the second monomer, preferably functionally linked to a strong promoter, preferably a 35S promoter;

[0118] an expression cassette allowing the expression of a silencing inhibitor, preferably p19; and

[0119] DNA elements which control the processing of the transcript, such as termination / polyadenylation sequences, preferably the Tnos sequence (nopaline synthase termination sequence).

[0120] The expression vector is preferably pAG01 as described in the international patent application No. WO2013 / 186495.

[0121] In order for the expressed fusion protein and second monomer to be properly routed in the endoplasmic reticulum, their amino acid sequences can advantageously comprise a signal peptide. The presence of the signal peptide enables correct trafficking of said protein into the endoplasmic reticulum. The signal peptide is then cleaved. Thus, during the budding and the formation of the BPs according to the invention, the fusion protein and the second monomer no longer contain the signal peptide. Consequently, the BPs according to the invention do not contain signal peptides.

[0122] The signal peptide is any signal peptide recognized by a eukaryotic cell.

[0123] Preferably, the signal peptide is chosen from the natural signal peptide of pectate lyase and the signal peptide of tobacco chitinase.

[0124] Preferably, the signal peptide is that of tobacco chitinase, corresponding to the amino acid sequence as set forth in SEQ ID No. 15: MKTNLFLFLIFSLLLSLSSA (SEQ ID No. 15).

[0125] The promoters used for controlling the expression of the fusion protein and of the second monomer are strong promoters, and may be plant gene promoters, such as for example the ubiquitin promoter, the ribulose-1, 5-bisphosphate carboxylase small subunit promoter, Agrobacterium tumefaciens promoters, the nopaline synthase and octopine synthase promoters, or else viral promoters such as cauliflower mosaic virus (CaMV) 19S and 35S. Preferably, the strong promoter is 35S.

[0126] The vector / s is / are then introduced into a eukaryotic host cell for recombinant expression. The host cell may be a plant cell.

[0127] The general methods for culturing plants, and also the methods for introducing expression vectors into a plant tissue, are available to those skilled in the art. They are varied and depend on the plant selected. Preferably, the plants will be cultivated according to the techniques described in the international patent application No. WO2013 / 186495. Such techniques comprise a first step of culturing the plant, under aeroponic or hydroponic conditions and under LED lighting. After this first step, the agroinfiltration of the plants is carried out under vacuum, using agrobacteria comprising DNA fragments encoding the fusion protein and the second monomer. This agroinfiltration step can be carried out by any means for producing a vacuum. Preferably, in the method used according to the invention, it is carried out under vacuum by Venturi effect. Among the agrobacteria that can be used according to the invention, mention is preferably made of the LBA4404, GV3101 , EHA 101 / 105 or C58 strains. Once the agroinfiltration step has been carried out, the plants are put back in culture, typically for 3 to 6 days, ideally while providing frequent misting of said plants for the first 6 hours of culture following the agroinfiltration. The BPs are then extracted and purified.

[0128] The BP extraction can be carried out by enzymatic extraction. This method is an adaptation of the method described in particular in the international application published under reference WO 2014 / 153674. Preferably, the enzymatic extraction of the BPs is carried out by means of the following steps:

[0129] - infiltration under vacuum (in particular as described above for the agroinfiltration) of the aerial part of plants (i.e. the leaves), in an enzymatic solution containing pectocellulosic enzymes, which does not exhibit any proteolytic activity; preferably, a mixture of pectinases and cellulases which is formulated at 4% in a medium comprising 50 mM of sodium citrate, pH 5.2, 0.5 M NaCI and 0.04% metabisulfite. Preferably, the macerozyme is formulated at 0.5% in a medium comprising 50 mM of sodium citrate, pH 5.2, 0.5 M NaCI and 0.04% metabisulfite,

[0130] - the leaves are subsequently sampled and then incubated in the enzymatic solution,

[0131] - the mixture is placed with shaking on an orbital shaker between 20 and 30 rpm at ambient temperature (i.e. approximately 20-23°C) for a period of between 30 minutes and 2 h,

[0132] - the digestate is then filtered, preferably on a 2-3 mm then 250 pm cloth, then optionally continuously centrifuged (for example at 1000 x g for 2-5 minutes), and the supernatant is recovered in order to perform a tangential filtration.

[0133] The BP according to the invention may be used in therapy. It may be used as a medicament. It may also be used in allergen immunotherapy (AIT).

[0134] The invention will now be illustrated by means of the following examples.Examples

[0135] Example 1 : Synthesis of the bioparticles according to the present invention

[0136] Multimeric protein of interest: Fel d 1

[0137] As shown in Figure 1 , a bioparticle expressing Fel d 1 was synthesized. The fusion protein comprised in this bioparticle comprises the CH2 chain of Fel d 1. The CH2 chain is bound to the CH1 chain of Fel d 1 by means of disulfide bridges. The fusion protein anchors the Fel d1 dimer in the lipidic membrane when it is expressed in heterogenous expression system.

[0138] The bioparticle was obtained by inserting a cDNA encoding the CH2-fosion protein and a cDNA encoding the Fel d1 CH1 -chain into two distinct expression pAG01 vectors and expressing them in a Nicotiana benthamiana tobacco plant expression system as described in the international patent application No. WO2013 / 186495.

[0139] The cDNA encoding the fusion protein has a nucleic acid sequence as set forth in SEQ ID No. 16: tctagaggtaccatgaagaccaacctgttcttgttcctgatcttcagcctgctgctgagcctgtcatctgctgctgtgaagatggcggagacctg ccctatcttctacgatgtgttcttcgcggttgctaacggtaacgagctacttttagatctcagcctgactaaagtgaacgctaccgagccagaa aggaccgctatgaaaaaaattcaagactgctatgttgaaaatggtctgatctctagggtgctcgacggtctagtgatgaccactattagcag cagcaaagactgcatgggcgaggctgtccaaaatacagttgaggaccttaagcttaataccctcggaaggcttaagcagattgaggata agatcgaagagatcctgagcaagatctaccacatcgagaacgagatcgctaggatcaagaagctgatcggagaatctgctgctggtggt ggtagttaccagatcctgtctatctacagcaccgtggcttcatctcttgctctggctattatgatggctggtctgtctctgtggatgtgctctaacgg ttctcttcagtgcaggatctgcatttaaactagtgtcgac (SEQ ID No. 16).

[0140] The fusion protein has the amino acid sequence as set forth in SEQ ID No. 17: MKTNLFLFLI FSLLLSLSSA AVKMAETCPI FYDVFFAVAN GNELLLDLSL TKVNATEPER TAMKKIQDCY VENGLISRVL DGLVMTTISS SKDCMGEAVQ NTVEDLKLNT LGRLKQIEDK IEEILSKIYH IENEIARIKK LIGESAAGGG SYQILSIYST VASSLALAIM MAGLSLWMCS NGSLQCRICI (SEQ ID No. 17).

[0141] The cDNA encoding the soluble Fel d1 CH 1 -chain has a nucleic acid sequence as set forth in SEQ ID No. 18: tctagaggtaccatgaagaccaacctgttcttgttcctgatcttcagcctgctgctgagcctgtcatctgctgattacaaggacgatgatgataa agtgcatcaccaccaccatcacgagatctgcccagctgttaagagggacgtcgatctatttcttacgggaactcctgatgagtacgtcgaac aagttgcacagtacaaagcactaccagtagtactcgagaacgctaggatacttaagaattgtgtggacgcaaaaatgaccgaggaaga caaggaaaacgcattgagcttgcttgataaaatatacacctctccactttgctaaggatcc (SEQ ID No. 18).

[0142] The soluble Fel d1 CH 1 -chain has the amino acid sequence as set forth in SEQ ID No. 19: MKTNLFLFLIFSLLLSLSSADYKDDDDKVHHHHHHEICPAVKRDVDLFLTGTPDEYVEQVAQYKALP WLENARILKNCVDAKMTEEDKENALSLLDKIYTSPLC (SEQ ID No. 19).

[0143] A soluble Fel d1 CH2-chain, a soluble CH1 / CH2 fusion (wherein the Feld 1 CH1 and CH2 chains are expressed in a soluble form, but fused together by means of a peptide bond) and a soluble CH1 / CH2 fusion bioparticle (expressing a CH1 / CH2 fusion onto its surface), were also synthesized.

[0144] The proteins thereby produced were purified and analyzed. For soluble allergens, samples were heated at 90" C for 5 min in denaturation buffer A (Tris 62.5 mM pH 6.8, 10% glycerol, 1% SDS and 2% p-mercaptoethanol). For bioparticles, samples were heated at 90" C for 5 min in denaturation buffer B (Tris 62.5 mM pH 6.8, 10% glycerol, 2.5% SDS and 5% p-mercaptoethanol). Both preparations were centrifuged at 8000 g for 15 min before loading on gels. SDS-PAGE was performed on 18% polyacrylamide gels. Following electrophoretic separation, gels were either silver- stained or transferred onto a nitrocellulose membrane (Amersham™ Protran™ 0.45pm NC) for immunodetection. The primary antibody used for immunodetection was a polyclonal rabbit-antisera directed against nDer p 2 at a 1:5,000 dilution followed by a secondary goat anti-rabbit IgG antibody coupled to horseradish peroxidase (Bio-Rad, Hercules, CA, USA) at a 1 : 30,000- dilution. Western blots were visualized with Amersham™ ECL™ Western Blotting Detection Reagents.

[0145] The results are shown in Figure 2. Protein extracts from leaves were analyzed in absence or in presence of a reducing agent. These extracts producing monomers (lanes 1-4 and lanes 7-8 or dimers (lanes 5-6 and 9-10) were separated by SDS-PAGE and immunodetected par specific immunoserum containing antibodies directed against CH1 and CH2. Figure 2 shows the presence of two polypeptides in presence of the reducing agent (lane 5) when soluble CH 1 and soluble CH2 were co-expressed and only one polypeptide in the absence of reducing agent (lane 6), illustrating the association of the polypeptides in plants as it was observed in cat. This association is clearly shown when CH2 is expressed onto the surface of a bioparticle and CH1 is expressed in a soluble form (lanes 9 and 10). These results show that CH2 and CH1 can successfully assemble by means of disulfide bridges when the CH2 monomer is expressed on the surface of a BP and CH1 is expressed in a soluble form.

[0146] Allergenicity of the soluble dimers or dimers-BP was analyzed using biolayer interferometry. Bio-layer interferometry (BLI) is an optical biosensing technology that analyzes biomolecular interactions in real-time without the need for fluorescent labeling. The interactions between the soluble Fel d1 or the Feld 1-BP and characterized IgE directed Feld 1 were analyzed accordingly (Figure 3). Ag-Ac association measurements are carried out using an “Octet® R8 Protein Analysis System” (Sartorius) equipped with Octet® Streptavidin (SA) Biosensors, for the first tests, or Octet® High Precision Streptavidin 2.0 (SAX2), for actual dosages. The Ag-Ac association reactions are carried out in black ELISA plates, in order to avoid the diffusion of light during the measurement (Greiner plates, ref: 655209). The interferometry data are analyzed using Octet® Analysis studio software.

[0147] The constructs will then be tested in vivo to evaluate the production of IgGs in mice (tested with either soluble Fel d 1-CH1 + Feld 1- CH2 -BP; Fusion FD1-BP or Feld 1-CH2-BP).

[0148] As shown in Figure 4, mice are acclimated for seven days, during which they have a physical examination, body weight measurement, blood collection (hematology and clinical chemistry), and daily clinical observations. At the end of this acclimation phase, mice (n=6) are selected for the testing phase. The mice receive three subcutaneous injections of 5 pg of enveloped bioparticles generated by tobacco expressing Feld 1-BP, two weeks apart. Blood are collected on days 0, 14, 28, 41 and 56.

[0149] Multimeric protein of interest: an immunoglobulin.

[0150] As shown in Figure 5, a bioparticle expressing immunoglobulins is synthesized. The fusion protein comprised in this bioparticle comprises either an Fc domain or a Fc+Fab domain

[0151] Example 2: Detection of the bioparticles according to the invention by the immune system

[0152] The bioparticle approach proposed in the present invention was designed to improve the presentation of antigens and of their immunogenic epitopes for obtaining an improved therapeutic protein that is efficient at low dose.

[0153] To support this hypothesis, the inventors compared the recognition of three different preparations of purified Feld-1 by different antibodies:

[0154] - Natural Feld-1: extracted from cat hair and purified to ~95% purity (Source: Indoor biotechnology, product code: NA-FD1-2)

[0155] - Recombinant deglycosylated Feld-1: Recombinantly produced in Pichia pastoris, with mutation N 103Q to abolish glycosylation, containing a 19 amino acid linker to fuse together CH 1 and CH2 peptides, and purified to >95% by a series of chromatography steps (source: Inbio, product code: RP-FD1 D-1 )

[0156] - Feld-1 BP as described in Example 1: Produced in plants by recombinant expression of Feld-1 containing a trimerization coil-coil domain and membrane-anchoring sequence to produce bioparticles and purified by a series of chromatography steps.

[0157] Four individual SDS-PAGE were migrated and detected by Western blot using each of the antibody reagents listed above using the following procedure: 250 ng of each preparation of Feld-1 listed above were loaded side-by-side on SDS-PAGE. All samples were heated at 85°C for 10min in denaturation buffer (Tris 62.5 mM pH 6.8, 10% glycerol, 2.5% SDS) with or without 5% |3- mercaptoethanol). Both preparations were centrifuged at 8000 g for 15 min before loading on gels. SDS-PAGE (Novex 16% Tris-Glycine Plus Wedge Well gel) was migrated for 1 h10 at 150V. Following electrophoretic separation, the gels were transferred on nitrocellulose membrane (Amersham™ Protran™ 0.45pm NC) for and block with Blotto 1% overnight at 4°C.

[0158] The Feld-1 proteins were detected by Western blot using four different primary antibody reagents:

[0159] - Rabbit antisera 395.3 raised against Natural Feld 1 (Indoor Biotechnology; product code NA-FD1-2),

[0160] - 1B7Anti-Feld-1 human IgE mAb derived from patient allergic to cat (Inbio; product code E-1B7) 50,000 lll / ml based on ImmunoCAP,

[0161] - 11A12 Anti-Feld-1 Human IgE mAb derived from patient allergic to cat (Inbio; product code E-11A12) 50,000 lll / ml based on ImmunoCAP,

[0162] - 6A1 Anti-Feld-1 Human IgE mAb derived from patient allergic to cat (Inbio; product code E-11A12) 50,000 lU / ml based on ImmunoCAP

[0163] at a dilution 1 / 2500 and revealed with their respective secondary antibody coupled to horseradish peroxidase, (goat anti-rabbit, Immunoreagent, GTxRb-003-DHRPX) or anti-human (Goat anti-human, Thermo Fisher Scientific, A18793) both used at a dilution 1 / 30 000. Primary and secondary antibodies were both incubated for 2 hours at room temperature. Western blots were visualized with Amersham™ ECL™ Western Blotting Detection Reagents. The results are presented in Figure 6. A second picture was taken with 50 seconds exposure in order to better detect signals from Human IgEs detection antibody reagents. These results are presented in Figure 7.

[0164] As explained in the certificate of analysis of the natural and recombinant preparation of Feld-1 , the protein shows 2 bands on SDS-PAGE, the monomeric and dimeric form of Feld-1 , while the same multimerization is observed for the recombinant Feld-1 from Pichia pastoris, but exhibiting lower molecular weight due to elimination of the glycosylation site. Feld-1 BP shows similar multimerization profile, but to a greater molecular weight has the fusion protein contains approximately 60 additional amino acids from the coil-coil trimeric, and transmembrane domains.

[0165] Those immunodetection results suggest that Feld-1 displayed at the surface of a bioparticle maintains a structural presentation that has the greatest immunological potential as it is detected by 3 different IgE monoclonal antibodies from allergic patients and by the rabbit polyclonal antibody raised against the natural Feld-1 obtained from cat hair. The natural Feld-1 is not detected by any of those 3 antibodies but the polyclonal antibody and appears poorly immunogenic. The recombinant Feld-1 , which is loaded to the same quantity (250 ng) is barely detected by two out of three IgE mAb reagents, suggesting a sub-optimal epitope presentation.

[0166] Feld-1 presented as a bioparticle according to the present invention therefore shows an optimal immunological profile, while demonstrating an hypoallergic response. The BP is presented to the antigen presenting cells of the immune system and is likely to activate a stronger immune response leading to a better therapy.

Claims

Claims

1. A bioparticle comprising:- an envelope consisting of a plasma membrane; and- at least one type I or II transmembrane fusion protein anchored in said membrane, said fusion protein comprising the following fragments, successively: a) a first monomer of a multimeric protein of interest ; b) a coiled-coil domain or oligomerization sequence; and c) a domain for anchoring in the plasma membrane, consisting of a transmembrane segment and a cytosolic segment; fragments a) and b) being exposed at the surface of the bioparticle; wherein said first monomer of a multimeric protein of interest exposed at the surface of the bioparticle is bound to a second monomer of said multimeric protein by means of a bond which is not a peptide bond.

2. The bioparticle according to claim 1 , wherein said bond is selected from the group consisting of disulfide bridge, an ionic or hydrogen electrostatic bond and a van Der Waals bond.

3. The bioparticle according to claim 1 or 2, wherein said multimeric protein of interest is an immunoglobulin or a fragment thereof.

4. The bioparticle according to any one of claims 1 to 3, wherein said first and second monomers are a fragment crystallizable (Fc) region of an immunoglobulin or a Fc fragment with antigen-binding (Fab).

5. The bioparticle according to any one of claims 1 to 4, wherein said second monomer is further linked to a second protein of interest.

6. The bioparticle according to claim 5, wherein said second protein of interest is a tumor antigen.

7. The bioparticle according to claim 1 or 2, wherein said multimeric protein of interest is a heterodimer.

8. The bioparticle according to claim 1, 2 or 7, wherein said multimeric protein of interest is Fel dl .

9. The bioparticle according to any one of claims 1-2 and 7-8, wherein said first monomer is the CH2 chain of Fel d1 and said second monomer is the CH1 chain of Fel d1.

10. The bioparticle according to any one of claims 1 to 9, for use in therapy.

11. The bioparticle according to any one of claims 1 to 9, for use in immunotherapy.

12. The bioparticle according to any one of claims 1 to 6, for use in the treatment of cancer.

13. The bioparticle according to any one of claims 7 to 9, for use in the treatment of cat allergy.

14. A method for producing bioparticle according to any one of claims 1 to 9, wherein said fusion protein and said second monomer are co-expressed in a eukaryotic cell.

15. The method according to claim 14, wherein said eukaryotic cell is a plant cell.

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