WO2021239666A1 - Procédés thérapeutiques - Google Patents

Procédés thérapeutiques Download PDF

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WO2021239666A1
WO2021239666A1 PCT/EP2021/063764 EP2021063764W WO2021239666A1 WO 2021239666 A1 WO2021239666 A1 WO 2021239666A1 EP 2021063764 W EP2021063764 W EP 2021063764W WO 2021239666 A1 WO2021239666 A1 WO 2021239666A1
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protein
seq
antibody
gib
pla2
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PCT/EP2021/063764
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Julien Pothlichet
Philippe Pouletty
Jacques Theze
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Diaccurate
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01004—Phospholipase A2 (3.1.1.4)

Definitions

  • the present invention relates to novel compounds, compositions, uses and methods for treating or detecting RNA virus infections in mammals, particularly in human subjects.
  • the invention may be in used in a preventive or curative approach, alone or in combination with other treatments.
  • sPLA2-GIB is involved in the inactivation of CD4 T cells in HIV infected patients, and that sPLA2-GIB inhibitors are effective for treating disorders associated with an immune deficiency (see WO2015/097140).
  • pathogens produce or activate cofactors which bind gC1qR and cause a sensitization of CD4 T cells to inactivation by sPLA2-GIB.
  • modulating such cofactors is an effective approach for treating such diseases (WO2019/166412).
  • SARS-Cov2 infection can also utilize the sPLA2-GIB pathway.
  • the inventors have identified that SARS-Cov2 can act as a sPLA2-GIB cofactor, causing T cell anergy, especially mediated by the spike protein.
  • the invention thus provides novel efficient molecules, compositions, methods and uses for detecting and/or treating coronavirus infections as well as other RNA virus infections, particularly SARS-Cov2 infection.
  • An object of the invention relates to methods for treating a Group IV RNA vims infection, particularly a coronavirus infection, in a mammal, particularly a human, comprising inhibiting the sPLA2-GIB pathway in said mammal.
  • a further object of the invention relates to the use of an inhibitor of the sPLA2-GIB pathway for the manufacture of a medicament for treating a Group IV RNA vims infection, particularly a coronavirus infection.
  • Another object of the invention concerns an inhibitor of the sPLA2-GIB pathway for use for treating a Group IV RNA vims infection, particularly a coronavirus infection.
  • a further object of the invention relates to any methods for inhibiting a sPLA2-GIB cofactor effect of SARS-Cov2 in a mammal.
  • a further object of the invention is a polypeptide comprising a sequence selected from anyone of SEQ ID Nos: 2-208 or an immunogenic-fragment thereof.
  • the polypeptide is preferably a polypeptide of less than 50 amino acids, more preferably less than 40 amino acids.
  • the polypeptide may consist or consist essentially of anyone of SEQ ID Nos: 2- 208.
  • the polypeptides of the invention may be conjugated to any molecule, such as a protein, carrier, surface, device, adjuvant, bead, column, etc.
  • a further object of the invention relates to a molecule which binds a Group IV RNA virus, particularly a coronavirus, in a binding domain comprising at least one amino acid residue of any one of SEQ ID Nos: 2-208.
  • a further object of the invention relates to an antibody (or a fragment or derivative thereof) which binds a Group IV RNA virus, particularly a coronavirus, wherein said antibody binds an epitope comprising at least one amino acid residue of any one of SEQ ID Nos: 2-208.
  • a further object of the invention relates to a vaccine composition
  • a vaccine composition comprising an immunogen, wherein the immunogen comprises a sequence selected from anyone of SEQ ID Nos: 2-208 or a fragment thereof.
  • a further object of the invention relates to a vaccine composition
  • a vaccine composition comprising an immunogen, wherein the immunogen comprises a viral envelope protein with a modified gC1qR binding motif, preferably a deleted and/or mutated gC1qR binding motif.
  • the immunogen comprises a viral envelope protein with a modified gC1qR binding motif, preferably a deleted and/or mutated gC1qR binding motif.
  • a specific example is a SARS-Cov-2 spike protein having at least one amino acid modification in a domain selected from SEQ ID NO: 156-185, even more preferably in a domain selected from aa 348-362 or 431-445 (by reference to SEQ ID NO: 209).
  • the invention also relates to a combination therapy or therapeutic regimen for treating a Group IV RNA virus infection, particularly a coronavirus, comprising (i) an inhibitor of PLA2-GIB pathway, in combination with (ii) at least one further active agent.
  • the further active agent may be an antiviral agent, an antibiotic, an anti-inflammatory agent, etc.
  • the agents in a combination therapy of the invention may be formulated together or separately, for combined, separate or sequential administration(s).
  • the invention also relates to the use of a PLA2-GIB cofactor, or an agonist, fragment, derivative, or mimotope thereof, for the manufacture of a medicament to induce immunosuppression in a subject in need thereof, by increasing the effect of PLA2-GIB on T cells.
  • the invention may be used in any mammal, particularly in human subjects. It is suitable to treat Group IV RNA vims infections, particularly coronavirus infections, at any stage of infection, either alone or in combination with other agents/therapies.
  • FIG. 1 Position Specific Scoring Matrix (PSSM) defining peptide binding motif to gClqR.
  • PSSM Position Specific Scoring Matrix
  • Figure 2 gClqR:PSSM motifs across the SARS-CoV-2 proteome (SEQ ID NO: 1).
  • the graphic reports gC1qR:PSSM scores for every given 15 a.a. window scanning the SARS-CoV-2 proteome (SEQ ID Nol).
  • the gC1qR:PSSM score is assigned to the N- terminal amino acid position of every given 15 a.a. window (2A).
  • the location of the different open-reading frames within the SARS-CoV-2 proteome are presented below the x-axis.
  • FIG. 3 High scoring gClqR:PSSM motifs across the spike protein sequence from SARS-CoV-2 (SEQ ID NO: 209).
  • the graphic reports gC1qR:PSSM scores for every given 15 a.a. window scanning the sequence of spike from SARS-CoV-2.
  • gC1qR:PSSM scores assigned to 15 a.a. motifs are represented by bars.
  • Figure 4 Co-localization of high scoring gClqR:PSSM motifs with solvent accessible domains within the spike protein sequence.
  • the graphic reports gC1qR:PSSM scores for every given 15 a.a. window scanning the sequence of spike from SARS-CoV-2.
  • gC1qR:PSSM scores assigned to 15 a.a. motifs are represented by bars.
  • Solvent accessible scores were calculated according to the method presented in example 3. The location of the different open- reading frames within the SARS-CoV-2 proteome are presented below the x-axis.
  • FIG. 5 Co-localization of high scoring gC1qR:PSSM motifs with predicted B cell epitopes (Discotope) within the spike protein sequence.
  • the graphic reports gC1qR:PSSM scores for every given 15 a.a. window scanning the sequence of spike from SARS-CoV-2.
  • gC1qR:PSSM scores assigned to 15 a.a. motifs are represented by bars.
  • Discotope scores were calculated according to the method presented in example 3. The location of the different open- reading frames within the SARS-CoV-2 proteome are presented below the x-axis.
  • Figure 6 Co-localization of high scoring gC1qR:PSSM motifs with predicted HLA class II binding motif-rich domains within the spike protein sequence.
  • the graphic reports gC1qR:PSSM scores for every given 15 a.a. window scanning the sequence of spike from SARS-CoV-2.
  • gC1qR:PSSM scores assigned to 15 a.a. motifs are represented by bars.
  • the invention generally relates to novel molecules, compositions, uses and methods for treating or detecting a subject infected by a Group IV RNA virus, particularly SARS- Cov2.
  • SARS-Cov-2 exerts PLA2-G1B cofactor effect.
  • Applicant identified various peptides in SARS-Cov2 proteins that can bind to gC1qR and cause CD4 T cell anergy.
  • Particularly relevant peptides are located in SI protein, such as in the N-terminal domain (NTD) and in the receptor binding domain (RBD).
  • NTD N-terminal domain
  • RBD receptor binding domain
  • These peptides have remarkable immunogenic character (e.g., they overlap with predicted B cell epitopes) and some are present in the RBD and thus represent efficient molecules for inhibiting Group IV RNA virus infections, particularly coronavirus infections, as well as for producing protective antibodies.
  • Vaccines against these regions would also be a very potent strategy to protect against SARS-Cov2 infection by inhibiting both PLA2G1B deleterious effect and viral infection.
  • PLA2-GIB designates group IB pancreatic phospholipase A2.
  • PLA2-GIB has been identified and cloned from various mammalian species. The human PLA2-GIB protein is disclosed, for instance, in Lambeau and Gelb (2008). The sequence is available on Genbank No. NP_000919.
  • the amino acid sequence of an exemplary human PLA2-GIB is shown below (SEQ ID NO: 210).
  • PLA2-GIB designates preferably human PLA2-GIB.
  • the human PLA2-GIB protein may be present under two distinct forms: a pro form (pro- sPLA2-GIB), which is activated by proteolytic cleavage of a pro-peptide, leading to the mature secreted form (sPLA2-GIB).
  • the term PLA2-GIB includes any form of the protein, such as the pro-form and/or the mature form.
  • the mature secreted form comprises the sequence of amino acid residues 23-148 of SEQ ID NO: 210, or any natural variants thereof.
  • Natural variants of a protein include variants resulting e.g., from polymorphism or splicing. Natural variants may also include any protein comprising the sequence of SEQ ID NO: 210, or the sequence of amino acid residues 23-148 of SEQ ID NO: 210, with one or more amino acid substitution(s), addition(s) and/or deletion(s) of one or several (typically 1, 2 or 3) amino acid residues. Variants include naturally-occurring variants having e.g., at least 90% amino acid sequence identity to SEQ ID NO: 210.
  • PLA2-GIB has at least one activity selected from induction of formation of membrane microdomains (MMD) in CD4 T cells from healthy subjects, or rendering CD4 T cells of healthy subjects refractory to interleukin signaling, such as refractory to IL-2 signaling or refractory to IL-7 signaling or refractory to IL-4 signaling.
  • MMD membrane microdomains
  • rendering CD4 T cells of healthy subjects refractory to interleukin-7 signaling comprises a reduction of STAT5A and/or B phosphorylation in said cells by at least about 10%, at least about 20%, at least about 30%, or at least about 40%. In some embodiments rendering CD4 T cells of healthy subjects refractory to interleukin-7 signaling comprises reducing the rate of nuclear translocation of phospho-STAT5A and/or phospho-STAT5B by at least about 20%, at least about 30%, at least about 40%, or at least about 50%.
  • sequence identity refers to the quantification (usually percentage) of nucleotide or amino acid residue matches between at least two sequences aligned using a standardized algorithm such as Smith- Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402).
  • BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them.
  • inactivation indicates, in relation to CD4 T cells, that such cells lose at least part of their ability to contribute to the development of an effective immune response. Inactivation may be partial or complete, transient or permanent. Inactivation designates preferably reducing by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more a function of CD4 T cells, particularly pSTAT5 nuclear translocation and/or CD4 T cell’s immunostimulatory activity. Typically, inactive CD4 T cells have no effective pSTAT5 nuclear translocation. In a particular embodiment, an inactive CD4 T cell is an anergic CD4 T cell.
  • the term “resistance” (or “insensitivity”) of CD4 T cells to inactivation by sPLA2-GIB indicates, within the context of this invention, that CD4 T cells are essentially not inactivated in vitro when incubated in the presence of 5nM of sPLA2-GIB. Resistance indicates, for instance, that CD4 T cells retain an active nuclear translocation of pSTAT5 when incubated in vitro in the presence of 5nM sPLA2-GIB and interleukin-7. Resistance (or insensitivity) of CD4 T cells to sPLA2-GIB may also indicate that CD4 T cells incubated in vitro with 5nM PLA2-GIB remain immunologically functional, e.g., do not become anergic.
  • sPLA2-GIB Pathway designates any component or molecule involved in sPLA2-G IB -mediated T cell anergy. This includes SPLA2-G1B, as well as any cofactor of SPLA2-G1B.
  • treatment includes preventive and curative treatments.
  • the treatment includes prevention of vims infection, such as reduction of the infection, as well as treatment of an existing infection (e.g., reduction of viral load, reduction of immune deficiency, reduction of CD4 T cell anergy) and related disease(s) or symptom(s) (e.g., pain, fever, cough, respiratory disorders, etc.).
  • treatment includes a reduction of mortality caused by or associated with coronavirus infection.
  • Group IV RNA virus refers to the Baltimore classification and includes, without limitation, coronaviruses, such as particularly Sars-cov-2, Dengue vims, Chikungunya vims, and Polio vims.
  • SARS Cov-2 utilizes the sPLA2-GIB pathway
  • SARS-CoV-2 vims (a.k.a 2019-nCoV), is the causative agent of covid-19. There are no approved treatments against SARS-CoV-2 vims infection, and vaccines being developed are not expected to reach patients promptly.
  • SARS-CoV-2 vims is a coronavirus, with a fully sequenced single- stranded RNA genome.
  • the inventors have surprisingly found that SARS-Cov-2 utilizes the sPLA2-GIB pathway. In particular, the inventors have found that SARS-Cov-2 can act by rendering CD4 T cells sensitive to inactivation by PLA2-GIB.
  • Such mechanism involves the binding of a molecule of (or induced by) SARS-Cov-2 to gC1qR at the surface of CD4 T cells, causing sensitization of CD4 T cells to inactivation by physiological concentrations of PLA2-GIB.
  • various proteins of SARS-Cov-2 such as the spike protein, contain sequences or regions that can bind to gC1qR and may act as cofactor increasing the sPLA2-GIB inhibitory activity on CD4 T cells.
  • Applicant thus identified a novel general approach for treating coronavirus infection, through a modulation of the SPLA2-G1B pathway.
  • One aspect of the invention resides in the treatment of a Group IV RNA virus infection, particularly coronavirus infections using an inhibitor of PLA2-G1B.
  • PLA2-GIB inhibitors suitable for use in the invention may be any compound that inhibits or neutralizes the expression or activity of PLA2-GIB, such as expression inhibitors, antagonists, or sequestrators.
  • Preferred types of inhibitors include PLA2-GIB ligands (covalent or non-covalent), anti-PLA2-GIB antibodies (and fragments and derivatives thereof), nucleic acids encoding anti-PLA2-GIB antibodies (or fragments and derivatives thereof), inhibitory nucleic acids, peptides, or small drugs, soluble receptors, or combination(s) thereof.
  • the PLA2-GIB inhibitor can be a PLA2-GIB antigen which, upon administration to the subject, induces the production of anti-PLA2GIB antibodies.
  • Inhibiting PLA2-GIB designates typically reducing by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more PLA2-GIB level or activity, as well as completely blocking or suppressing PLA2-GIB level or activity. Depending on the situation, inhibition may be transient, sustained or permanent.
  • PLA2-GIB inhibitors are anti-PLA2-GIB antibodies, e.g., antibodies that bind to PLA2-GIB and/or have been generated by immunization of a mammal with a PLA2-GIB antigen.
  • Antibodies can be synthetic, monoclonal, or polyclonal and can be made by techniques well known in the art. Such antibodies specifically bind via the antigen-binding sites of the antibody (as opposed to non-specific binding).
  • PLA2-GIB polypeptides, fragments, variants, fusion proteins, etc. can be employed as immunogens in producing antibodies immunoreactive therewith. More specifically, the polypeptides, fragments, variants, fusion proteins, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies.
  • antibodies is meant to include polyclonal antibodies, monoclonal antibodies, fragments thereof, such as F(ab')2 and Fab fragments, single-chain variable fragments (scFvs), single-domain antibody fragments (VHHs or Nanobodies), bivalent antibody fragments (diabodies), as well as any recombinantly and synthetically produced binding partners, human antibodies or humanized antibodies.
  • Antibodies are defined to be specifically binding preferably if they bind to PLA2-GIB with a Ka of greater than or equal to about 10 7 M-l. Affinities of antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).
  • Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, donkeys, goats, sheep, dogs, chickens, rabbits, mice, or rats, using procedures that are well known in the art.
  • purified PLA2-GIB or a peptide based on the amino acid sequence of PLA2-GIB that is appropriately conjugated is administered to the host animal typically through parenteral injection.
  • the immunogenicity of PLA2-GIB can be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to PLA2-GIB polypeptide.
  • Examples of various assays useful for such determination include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures, such as countercurrent Immuno- electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays. See U.S. Pat. Nos. 4,376, 110 and 4,486,530.
  • Monoclonal antibodies can be readily prepared using well known procedures. See, for example, the procedures described in U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980.
  • the host animals such as mice
  • Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse.
  • ABSC antibody capture
  • mice are given an intravenous boost of protein or peptide.
  • Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols.
  • ATCC Ag8.653
  • the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell.
  • the fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig. Following washes, a label, such as a labeled PFA2-GIB polypeptide, is added to each well followed by incubation. Positive wells can be subsequently detected. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia).
  • PEG polyethylene glycol
  • the monoclonal antibodies of the disclosure can be produced using alternative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody Expression Fibraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3:1-9 (1990), which is incorporated herein by reference.
  • binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Farrick et al., Biotechnology, 7:394 (1989).
  • Antigen-binding fragments of such antibodies which can be produced by conventional techniques, are also encompassed by the present invention.
  • fragments include, but are not limited to, Fab and F(ab')2 fragments.
  • Antibody fragments and derivatives produced by genetic engineering techniques are also provided.
  • the monoclonal antibodies include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans.
  • a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
  • a humanized antibody fragment can comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al.
  • Antibodies produced by genetic engineering methods such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, can be used.
  • Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art, for example using methods described in Robinson et al. International Publication No. WO 87/02671; Akira, et al. European Patent Application 0184187; Taniguchi, M., European Patent Application 0171496; Morrison et al. European Patent Application 0173494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No.
  • antibodies In connection with synthetic and semi-synthetic antibodies, such terms are intended to cover but are not limited to antibody fragments, isotype switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse), hybrids, antibodies having plural specificities, and fully synthetic antibody-like molecules.
  • Human monoclonal antibodies having human constant and variable regions can be generated by immunizing transgenic animals which contain human immunoglobulin genes. See Jakobovits et al. Ann NY Acad Sci 764:525-535 (1995). Human monoclonal antibodies against PLA2-GIB polypeptides can also be prepared by constructing a combinatorial immunoglobulin library, such as a Fab phage display library or a scFv phage display library, using immunoglobulin light chain and heavy chain cDNAs prepared from mRNA derived from lymphocytes of a subject. See, e.g., McCafferty et al. PCT publication WO 92/01047; Marks et al. (1991) J. Mol.
  • a combinatorial library of antibody variable regions can be generated by mutating a known human antibody.
  • a variable region of a human antibody known to bind PLA2-GIB can be mutated by, for example, using randomly altered mutagenized oligonucleotides, to generate a library of mutated variable regions which can then be screened to bind to PLA2-GIB.
  • Methods of inducing random mutagenesis within the CDR regions of immunoglobin heavy and/or light chains, methods of crossing randomized heavy and light chains to form pairings and screening methods can be found in, for example, Barbas et al. PCT publication WO 96/07754; Barbas et al. (1992) Proc. Nat'l Acad. Sci. USA 89:4457 4461.
  • An immunoglobulin library can be expressed by a population of display packages, preferably derived from filamentous phage, to form an antibody display library.
  • Examples of methods and reagents particularly amenable for use in generating antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT publication WO 92/18619; Dower et al. PCT publication WO 91/17271; Winter et al. PCT publication WO 92/20791; Markland et al. PCT publication WO 92/15679; Breitling et al. PCT publication WO 93/01288; McCafferty et al.
  • the antibody library is screened to identify and isolate packages that express an antibody that binds a PLA2-GIB polypeptide.
  • a display package e.g., filamentous phage
  • the primary screening of the library involves panning with an immobilized PLA2-GIB polypeptide and display packages expressing antibodies that bind immobilized PLA2-GIB polypeptide are selected.
  • Preferred antibodies for use in the invention are directed to a PLA2-GIB epitope, and/or have been generated by immunization with a polypeptide comprising a PLA2-GIB epitope selected from: the mature PLA2-GIB protein, a fragment of PLA2-GIB comprising at least 8 consecutive amino acid residues of SEQ ID NO: 210 (or the corresponding residues of a natural variant of SEQ ID NO: 210), said fragment preferably comprising at least amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof (numbering by reference to SEQ ID NO: 210).
  • Particular anti-PLA2-GIB antibodies for use in the invention bind mature human PLA2- GIB, even more preferably an epitope comprised in a domain of PLA2-GIB comprising an amino acid residue selected from amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof (numbering by reference to SEQ ID NO: 210).
  • Particular antibodies for use in the invention bind an epitope comprised between amino acid residues 50-71 of mature human PLA2-GIB (by reference to SEQ ID NO: 210) or the corresponding residues of a natural variant of SEQ ID NO: 210.
  • Examples of anti-PLA2-GIB antibodies suitable for use in the invention have been disclosed in W02015/097140.
  • anti-PLA2-GIB antibodies for use in the present invention bind an epitope comprising at least one amino acid residue selected from W3, R6, K7, K10, C77, Y75, G79 and S80 of human mature PLA2-GIB (numbering by reference to SEQ ID NO: 210), more preferably an epitope comprising at least 2 or at least 3 amino acid residues selected from W3, R6, K7, K10, C77, Y75, G79 and S80 of human mature PLA2-GIB, further more preferably an epitope comprising at least 4, at least 5, at least 6 or at least 7 amino acid residues selected from W3, R6, K7, K10, C77, Y75, G79 and S80 of human mature PLA2-GIB.
  • antibodies for use in the invention bind an epitope comprising an amino acid residue comprised between amino acids 1-10 or 75-80 of mature human PLA2-GIB (by reference to SEQ ID NO: 210) or the corresponding residues of a natural variant of SEQ ID NO: 210.
  • Such antibodies exhibit potent neutralizing activity and represent valuable therapeutic agents for use in the invention.
  • anti-PLA2-GIB antibodies suitable for use in the invention have been disclosed in WO2019/166665, incorporated therein by reference.
  • the antibodies or derivatives for use in the invention is monoclonal antibody 14G9 as disclosed in WO2019/166665, or an anti-PLA2-GIB antibody that competitively inhibits binding of monoclonal antibody 14G9 to human PLA2-GIB.
  • the antibody may be human or humanized.
  • the antibodies or derivatives for use in the invention is monoclonal antibody #2B as disclosed in WO2019/166665, or an anti-PLA2-GIB antibody that competitively inhibits binding of monoclonal antibody #2B to human SPLA2-GIB.
  • the antibodies or derivatives for use in the invention is monoclonal antibody #2B1 as disclosed in WO2019/166665, or an anti-PLA2-GIB antibody that competitively inhibits binding of monoclonal antibody #2B1 to human SPLA2-GIB.
  • the antibodies or derivatives for use in the invention is monoclonal antibody #2B2 as disclosed in WO2019/166665, or an anti-PLA2-GIB antibody that competitively inhibits binding of monoclonal antibody #2B2 to human SPLA2-GIB.
  • competitive inhibits indicates that the antibody can reduce or inhibit or displace the binding of a reference antibody to sPLA2-GIB.
  • Competition assays can be performed using standard techniques such as, for instance, competitive ELISA or other binding assays.
  • a competitive binding assay involves a purified target antigen, generally bound either to a solid substrate or cells, an unlabeled test antibody and a labeled reference antibody.
  • Competitive inhibition is measured by determining the amount of labeled antibody bound in the presence of the test antibody.
  • the test antibody is present in excess, such as about 5 to 500 times the amount of reference antibody.
  • the test antibody is in 100X excess, and for enzymatic methods, the test antibody in in 10X excess.
  • test antibody present in excess inhibits or displaces at least 70% of the binding of the reference antibody to the antigen, it is considered as competitively inhibiting said reference antibody.
  • test antibody present in 100X excess inhibits or displaces at least 70%, more preferably at least 80% of the binding of the reference antibody to the antigen in ELISA, it is considered as competitively inhibiting said reference antibody.
  • Preferred competing antibodies bind epitopes that share common amino acid residues.
  • the inhibitor is a monoclonal antibody comprising: (i) a light chain variable region comprising a CDR-L1, a CDR-L2, a CDR-L3 and a FR- L, wherein the CDR-L1, CDR-L2 and/or CDR-L3 consists, or consists essentially, of the CDR-L1, CDR-L2 and CDR-L3, respectively, of the light chain variable region of SEQ ID NO: 3 or 5 as disclosed in WO2019/166665, and wherein a FR-L is of a human immunoglobulin sequence; and
  • a heavy chain variable region comprising a CDR-H1, a CDR-H2, a CDR-H3 and a FR-H, wherein the CDR-H1, CDR-H2 and/or CDR-H3 consists, or consists essentially, of the CDR-H1, CDR-H2 and CDR-H3, respectively, of the heavy chain variable region of SEQ ID NO: 4, 6, 7 or 8 as disclosed in WO2019/166665, and wherein a FR-H is of a human immunoglobulin sequence.
  • variable region refers to the amino-terminal domains of the heavy or light chain (“VH” or “VL”), which contain the antigen-binding sites.
  • VL or VH generally consists of a framework region (“FR”) interrupted by three hypervariable regions referred to as "complementarity determining regions" or "CDRs".
  • FR framework region
  • CDRs complementarity determining regions
  • CDR-L1 amino acid residues QDVSTA (residues 27-31 of SEQ ID NO: 3),
  • CDR-L2 amino acid residues WAS (residues 50-52 of SEQ ID NO: 3),
  • CDR-L3 amino acid residues QQDYSTPPT (residues 89-97 of SEQ ID NO: 3).
  • CDR-H1 amino acid residues GYTFTNYW (residues 26-33 of SEQ ID NO: 4),
  • CDR-H2 amino acid residues IDPSDTRT (residues 51-58 of SEQ ID NO: 4),
  • CDR-H3 amino acid residues ARQTLYYEALDY (residues 97-108 of SEQ ID NO: 4).
  • the invention uses a monoclonal antibody selected from:
  • monoclonal antibody #2B 1 monoclonal antibody #2B 1 ;
  • antibody derivative refers to an antibody which retains the antigenic specificity of a reference antibody but wherein one or more amino acid residues are (chemically, or biologically) modified to improve its properties. Examples of such chemical modifications include, e.g. by alkylation, PEGylation, acylation, ester or amide formation, and the like.
  • a derivative is an antibody as disclosed herein that is modified to contain one or more additional non-pro teinaceous moieties such as water-soluble polymers.
  • water-soluble polymers include, but are not limited to, PEG, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran and polyvinyl alcohol.
  • Derivatives may also be generated to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (see e.g., Wright et al. TIBTECH, 1997, 15:26-32).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%).
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies.
  • Examples of publications related to "defucosylated” or “fucose-deficient" antibody variants include, but are not limited to, Okazaki et al. J. Mol. Biol. 336: 1239- 1249 (2004) and Yamane-Ohnuki N, Satoh M. mAbs. 2009;1:230-236.
  • Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986)), and knockout cell lines, such as alpha- 1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)).
  • cysteine engineered antibodies e.g., "thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541.
  • the term derivative also includes immunoconjugates comprising an anti-sPLA2-GIB antibody as defined above conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent, a detectable moiety such as a fluorescent moiety, a diagnostic radioisotope or an imaging agent; or to a solid support, such as agarose beads or the like.
  • cytotoxic agents include, but are not limited to chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents well known by the skilled person.
  • the linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell.
  • an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 (1992)) may be used.
  • the antibodies for use in the invention are typically “isolated”, e.g., have been separated from at least one component of their natural environment.
  • the antibodies may be purified to greater e.g., at least 95%, at least 96%; at least 97%, at least 98% or at least 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) techniques.
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • Preferred antibodies of the invention are essentially neutralizing antibodies, i.e., they are able to at least partially inhibit an activity of PLA2-GIB .
  • sPLA2-GIB catalyzes the hydrolysis of the sn-2 fatty acyl bond of phospholipids to release free fatty acids and lysophospholipids.
  • Particular antibodies of the invention inhibit an enzymatic activity of sPLA2-GIB, such as the hydrolysis of the sn-2 fatty acyl bond of phospholipids. Methods for testing such a property are disclosed in detail in the experimental section.
  • Particular antibodies for use in the invention inhibit binding of sPLA2-GIB to a substrate thereof.
  • Further particular antibodies for use in the invention inhibit sPLA2-GIB -mediated inhibition of IF-7-induced phospho-STAT5 nuclear translocation in CD4 T cells. Methods for testing such a property are disclosed in detail in the experimental section.
  • the neutralizing activity of the antibody or derivative can be determined in vitro or in vivo using e.g., binding or biological assays, such as tests as described in the experimental section. Inhibition/neutralization may be complete or partial. In particular, the antibodies may inhibit 10% or more of the tested activity, preferably 20% or more, 30% or more, 40% or more, 50% or more.
  • the antibodies are IgG, e.g., gGl, IgG2, IgG3 or IgG4.
  • the antibodies or derivatives may be isolated and preserved using conventional methods and media. They may be lyophilized. They may also be frozen.
  • the PLA2-GIB inhibitor is or comprises or consists of a nucleic acid molecule encoding an anti-PLA2-GIB antibody, or a light or heavy chain thereof, or a variable domain thereof, or a nucleic acid complementary to said encoding sequence.
  • the nucleic acid can be DNA (cDNA or gDNA), RNA, or a mixture thereof. It can be in single stranded form or in duplex form or a mixture of the two. It can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to one skilled in the art, including chemical synthesis, recombination, and mutagenesis.
  • the nucleic acid according to the invention may be deduced from the sequence of the antibody according to the invention and codon usage may be adapted according to the host cell in which the nucleic acid shall be transcribed. These steps may be carried out according to methods well known to one of skill in the art and some of which are described in the reference manual Sambrook et al. (Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, Third Edition Cold Spring Harbor).
  • the nucleic acid may encode an amino acid sequence comprising the light chain and/or an amino acid sequence comprising the heavy chain of the antibody, or may be complementary to such encoding sequence.
  • the present invention further provides a vector comprising a nucleic acid of the invention.
  • the vector may comprise several nucleic acids of the invention.
  • the vector may comprise a nucleic acid of the invention operably linked to a regulatory region, i.e. a region comprising one or more control sequences.
  • the vector may comprise several nucleic acids of the invention operably linked to several regulatory regions.
  • control sequences means nucleic acid sequences necessary for expression of a coding region. Control sequences may be endogenous or heterologous. Well-known control sequences and currently used by the person skilled in the art will be preferred. Such control sequences include, but are not limited to, promoter, signal peptide sequence and transcription terminator.
  • operably linked means a configuration in which a control sequence is placed at an appropriate position relative to a coding sequence, in such a way that the control sequence directs expression of the coding region.
  • the present invention further relates to the use of a nucleic acid or vector according to the invention to transform, transfect or transduce a host cell.
  • the present invention also provides a host cell comprising one or several nucleic acids of the invention and/or one or several vectors of the invention.
  • host cell also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication.
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells such as bacteria, yeasts, insect cells, mammalian cells, etc.
  • Inhibitory Nucleic acids include prokaryotic or eukaryotic cells such as bacteria, yeasts, insect cells, mammalian cells, etc.
  • the PLA2-GIB inhibitor is an inhibitory nucleic acid, i.e., any nucleic acid molecule which inhibits PLA2-GIB gene or protein expression.
  • Preferred inhibitory nucleic acids include antisense nucleic acids, short interfering RNAs (siRNAs), small hairpin RNAs (shRNA), microRNAs, aptamers, or ribozymes.
  • the inhibitory nucleic acid is a small interfering RNA that prevents translation of PLA2-GIB mRNA.
  • the inhibitory nucleic acid is an antisense oligonucleotide that prevents translation of PLA2-GIB mRNA.
  • the inhibitory nucleic acid is a small hairpin RNA that prevents translation of PLA2-GIB mRNA.
  • siRNA comprise a sense nucleic acid sequence and an anti-sense nucleic acid sequence of the polynucleotide of interest.
  • siRNA are constructed such that a single transcript (double stranded RNA) have both the sense and complementary antisense sequences from the target gene.
  • the nucleotide sequence of siRNAs may be designed using an siRNA design computer program available from, for example, the Ambion website on the world wide web.
  • the length of the antisense oligonucleotide or siRNAs is less than or equal to 10 nucleotides. In some embodiments, the length of the antisense oligonucleotides and siRNAs is as long as the naturally occurring transcript. In some embodiments, the antisense oligonucleotides and siRNAs have 18-30 nucleotides. In some embodiments, the antisense oligonucleotides and siRNAs are less than 25 nucleotides in length.
  • Preferred inhibitory nucleic acid molecules comprise a domain having a nucleotide sequence that is perfectly complementary to a region of a PLA2-GIB gene or RNA. Such a domain contains typically from 4 to 20 nucleotides, allowing specific hybridization and optimal inhibition of the gene transcription or RNA translation.
  • the sequence of the inhibitory nucleic acids may be derived directly from the sequence of a gene encoding PLA2-GIB. Alternatively, or in addition, inhibitory nucleic acids may hybridize to a regulatory element in a PLA2-GIB gene or RNA, such as a promoter, a splicing site, etc., and prevent effective regulation thereof.
  • inhibitory nucleic acid molecules of the present invention include isolated single strand nucleic acid molecules consisting of from 10 to 50 consecutive nucleotides of a sequence encoding SEQ ID NO: 210.
  • inhibitory nucleic acid molecules of the invention are antisense nucleic acids consisting of the following nucleotide sequence or the perfectly complementary strand thereof:
  • the PLA2-GIB inhibitor is a peptide or small drug that inhibits the activity of PLA2-GIB.
  • the peptide or small drug is typically a molecule that selectively binds PLA2-GIB, or a substrate of PLA2-GIB, or a co-factor of PLA2-GIB, or a degradation product or metabolite of PLA2-GIB pathway.
  • Peptides preferably contain from 3 to 20 amino acid residues, and their sequence may be identical to a domain of PLA2-GIB (bait peptide) or to a domain of a PLA2-GIB substrate, co-factor, degradation product or metabolite.
  • Preferred peptides of the invention contain from 4 to 30 consecutive amino acid residues of SEQ ID NO: 210 (or of a corresponding sequence of a natural variant of SEQ ID NO: 210).
  • Most preferred peptides of the invention comprise from 5 to 25 consecutive amino acid residues of SEQ ID NO: 210 (or of a corresponding sequence of a natural variant of SEQ ID NO: 210) and further comprise at least one of the following amino acid residues of SEQ ID NO: 210 (or of a corresponding sequence of a natural variant of SEQ ID NO: 210): amino acid 3, amino acid 6, amino acid 7, amino acid 10, amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, amino acid 75, amino acid 77, amino acid 79, amino acid 80, or a combination thereof.
  • Specific examples of peptides of the invention are peptides of less than 25 amino acids comprising anyone of the following sequences:
  • peptides for use in the invention include a pentapeptide as disclosed in WO20 17/060405, incorporated therein by reference.
  • the compound is a cyclic peptide selected from FLSYK (SEQ ID NO: 231), FLSYR (SEQ ID NO: 232) and (2NapA)LS(2NapA)R (SEQ ID NO: 233).
  • the peptides of the invention can comprise peptide, non-peptide and/or modified peptide bonds.
  • the peptides comprise at least one peptidomimetic bond selected from intercalation of a methylene (-CH2-) or phosphate (-PO2-) group, secondary amine (-NH-) or oxygen (-0-), alpha-azapeptides, alpha-alkylpeptides, N- alkylpeptides, phosphonamidates, depsipeptides, hydroxymethylenes, hydroxyethylenes, dihydroxyethylenes, hydroxyethylamines, retro-inverso peptides, methyleneoxy, cetomethylene, esters, phosphinates, phosphinics, or phosphonamides.
  • the peptides may comprise a protected N-ter and/or C-ter function, for example, by acylation, and/or amidation and/or esterification.
  • the peptides of the invention may be produced by techniques known per se in the art such as chemical, biological, and/or genetic synthesis.
  • Preferred small drugs are hydrocarbon compounds that selectively bind PLA2-GIB.
  • small drugs include indole compounds, such as those disclosed in W02017/037041, incorporated therein by reference.
  • the compound is 3-(2-amino-l,2-dioxoethyl)-2-ethyl-l-(phenylmethyl)-lH-indol-4- yl)oxy)acetic acid or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, such as a sodium salt thereof (Varespladib).
  • Small drugs and peptides are preferably obtainable by a method comprising: (i) contacting a test compound with PLA2-GIB or a fragment thereof, (ii) selecting a test compound which binds PLA2-GIB or said fragment thereof, and (iii) selecting a compound of (ii) which inhibits an activity of PLA2-GIB.
  • a method comprising: (i) contacting a test compound with PLA2-GIB or a fragment thereof, (ii) selecting a test compound which binds PLA2-GIB or said fragment thereof, and (iii) selecting a compound of (ii) which inhibits an activity of PLA2-GIB.
  • Small drugs and peptides are also obtainable by a method comprising: (i) contacting a test compound with a PLA2-GIB substrate, co-factor, or degradation product, or a fragment thereof, (ii) selecting a test compound which binds to said PLA2-GIB substrate, co-factor, or degradation product, or a fragment thereof, and (iii) selecting a compound of (ii) which inhibits an activity of PLA2-GIB.
  • a method represents a particular object of the invention.
  • the PLA2-GIB inhibitor is a PLA2-GIB antigen.
  • the subject produces antibodies (or cells) which inhibit PLA2-GIB .
  • injection(s) of a PLA2-GIB antigen e.g., an immunogenic PLA2-GIB essentially devoid of biological activity
  • a PLA2-GIB antigen e.g., an immunogenic PLA2-GIB essentially devoid of biological activity
  • An object of the invention thus resides in a method of treating a solid cancer in a subject having solid cancer, comprising administering to the subject a PLA2-GIB antigen.
  • a further object of the invention relates to a PLA2-GIB antigen for use to treat a solid cancer in a subject in need thereof.
  • the PLA2-GIB antigen is an inactivated immunogenic molecule that induces an immune response against PLA2-GIB in a subject.
  • Inactivation may be obtained e.g., by chemically or physically altering PLA2-GIB or by mutating or truncating the protein, or both; and immunogenicity may be obtained as a result of the inactivation and/or by further conjugating the protein to a suitable carrier or hapten, such as KLH, HSA, polylysine, a viral anatoxin, or the like, and/or by polymerization, or the like.
  • the antigen may thus be chemically or physically modified, e.g., to improve its immunogenicity .
  • the PLA2-GIB antigen comprises PLA2-GIB or an epitope- containing fragment or mimotope thereof.
  • the PLA2-GIB antigen comprises a full length PLA2-GIB protein.
  • the PLA2-GIB antigen comprises a protein comprising SEQ ID NO: 210, or a sequence having at least 90% identity to SEQ ID NO: 210.
  • the PLA2-GIB antigen comprises a fragment of a PLA2- GIB protein comprising at least 6 consecutive amino acid residues and containing an immunogenic epitope, or a mimotope thereof. In a preferred embodiment, the PLA2-GIB antigen comprises at least from 6 to 20 amino acid residues. Preferred peptides of the invention contain from 4 to 30 consecutive amino acid residues of SEQ ID NO: 210 (or of a corresponding sequence of a natural variant of SEQ ID NO: 210).
  • Most preferred peptides of the invention comprise from 5 to 25 consecutive amino acid residues of SEQ ID NO: 210 (or of a corresponding sequence of a natural variant of SEQ ID NO: 210) and further comprise at least one of the following amino acid residues of SEQ ID NO: 210 (or of a corresponding sequence of a natural variant of SEQ ID NO: 210): amino acid 3, 6, 7, 10, 70, 121, 50, 52, 54, 71, 75, 77, 79, 80, or a combination thereof.
  • Specific examples of such antigens are the peptides of SEQ ID NO: 222-230 as disclosed above.
  • the PLA2-GIB antigen may be in various forms such as in free form, polymerized, chemically or physically modified, and/or coupled (i.e., linked) to a carrier molecule. Coupling to a carrier may increase the immunogenicity and (further) suppress the biological activity of the PLA2-GIB polypeptide.
  • the carrier molecule may be any carrier molecule or protein conventionally used in immunology such as for instance KLH (Keyhole limpet hemocyanin), ovalbumin, bovine serum albumin (BSA), a viral or bacterial anatoxin such as toxoid tetanos, toxoid diphteric B cholera toxin, mutants thereof such as diphtheria toxin CRM 197, an outer membrane vesicle protein, a polylysine molecule, or a virus like particle (VLP).
  • the carrier is KLH or CRM 197 or a VLP.
  • Coupling of PLA2-GIB to a carrier may be performed by covalent chemistry using linking chemical groups or reactions, such as for instance glutaraldehyde, biotin, etc.
  • the conjugate or the PLA2-GIB protein or fragment or mimotope is submitted to treatment with formaldehyde in order to complete inactivation of PLA2-GIB.
  • the PLA2-GIB antigen comprises a full length PLA2-GIB protein, optionally coupled to a carrier protein.
  • the PLA2- GIB antigen comprises a protein comprising SEQ ID NO: 210, or a sequence having at least 90% identity to SEQ ID NO: 210, coupled to a carrier protein.
  • the PLA2-GIB antigen comprises an immunogenic peptide or mimotope of PLA2-GIB, optionally coupled to a carrier protein.
  • the PLA2-GIB antigen comprises a polypeptide of at least 10 amino acids long comprising at least one of the following amino acid residues of SEQ ID NO: 210 (or of a corresponding sequence of a natural variant of SEQ ID NO: 210): amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof, optionally coupled to a carrier molecule.
  • the immunogenicity of the PLA2-GIB antigen may be tested by various methods, such as by immunization of a non-human animal grafted with human immune cells, followed by verification of the presence of antibodies, or by sandwich ELISA using human or humanized antibodies. The lack of biological activity may be verified by any of the activity tests described in the application.
  • the PLA2-GIB antigen has less than 20%, more preferably less than 15%, 10%, 5% or even 1% of the activity of a wild-type PLA2-GIB protein in an in vitro method of (i) induction of formation of membrane microdomains (MMD) in CD4 T cells or (ii) in rendering CD4 T cells refractory to IL-2 signaling or refractory to IL-7 signaling.
  • MMD membrane microdomains
  • Such molecules and conjugates and vaccines represent potent agents for use to immunize subjects, thereby causing a sustained PLA2-GIB inhibition. Upon repetition, such methods can be used to cause a permanent PLA2-GIB inhibition.
  • the invention also relates to an inhibitor of a PLA2-GIB cofactor, for use for treating a Group IV RNA vims infection, particularly a coronavirus infection in a subject in need thereof.
  • the invention also relates to the use of an inhibitor of a PLA2-GIB cofactor, for the manufacture of a medicament for treating a Group IV RNA vims infection, particularly a coronavirus infection in a subject in need thereof.
  • SARS-Cov-2 can act as (or produce) a cofactor of PLA2-GIB that, in combination with PLA2-GIB, leads to CD4 T cell inactivation.
  • cofactor of PLA2-GIB designates any molecule or agent which potentiates or amplifies or mediates an effect of PLA2-GIB, particularly an effect of PLA2-GIB on CD4 T cells.
  • Preferred cofactors are molecules which can sensitize CD4 T cells to inactivation by low concentrations of PLA2-GIB.
  • the PLA2-GIB cofactor is a ligand of gC1qR.
  • the PLA2-GIB cofactor is an agonist of gC1qR, e.g., can induce signaling through gC1qR, more particularly can induce gC1qR- mediated exocytosis.
  • the inventors have identified various proteins which can act as cofactor of PLA2-GIB, as listed in Table 3. In particular, such proteins include ORFlab, the Spike protein, ORF3, E protein, ORF7b, ORF8, N protein and M protein.
  • Each of said proteins thus represents a relevant target for inhibition according to the invention.
  • the inventors also identified various peptides from said proteins which can bind gC1qR, as listed in Table 3. Each of said peptides represents an object of the invention.
  • Each of said proteins and peptides can act as PLA2-GIB cofactors in SARS-Cov2 infected patients.
  • Each of said peptides represents a strong inhibitor (or be used to construct a strong inhibitor) for use according to the invention
  • Each of said proteins or peptides can be used to produce relevant inhibitors, such as antibodies or derivatives thereof, or immunogens for vaccination.
  • Each of said proteins and peptides can be used as a reagent to detect the presence of anti- Cov-2 antibodies in a sample, particularly of a protective immunity.
  • the PLA2-GIB cofactor is a protein selected from ORFlab, the Spike protein, ORF3, E protein, ORF7b, ORF8, N protein and M protein of SARS-Cov-2, or a gC1qR-binding element of such proteins, such as listed in Table 3. More particularly, the cofactor may be any protein comprising anyone of SEQ ID NOs: 2-209, or any fragment or mimotope thereof.
  • fragment in relation to such cofactors, designates preferably a fragment containing a gC1qR-binding element of such a protein, and/or a fragment retaining a capacity of binding gC1qR.
  • Preferred fragments contain at least 5 consecutive amino acid residues, typically between 5 and 100, between 5-80, between 5-60, between 5-50, between 5-40, between 5-30, between 5-25, or between 5-20, or even less.
  • the PLA2-GIB cofactor is SARS-Cov2 spike (or S) protein, or a fragment or mimotope thereof.
  • the amino acid sequence of a Spike protein is depicted as SEQ ID NO: 209. Such sequence is also available as GenBank reference QHD43416.1.
  • the PLA2-GIB cofactor is a protein or peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 156-185 and 209, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is SARS-Cov2 ORFlab protein, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is a protein or peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 2-155, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is SARS-Cov2 ORF3 protein, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is a protein or peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 186-193, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is SARS-Cov2 protein E, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is a protein or peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 194, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is SARS-Cov2 protein M, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is a protein or peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 195-202, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is SARS-Cov2 ORF7b protein, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is a protein or peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 203, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is SARS-Cov2 ORF8 protein, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is a protein or peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 204, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is SARS-Cov2 protein N, or a fragment or mimotope thereof.
  • the PLA2-GIB cofactor is a protein or peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 205-208, or a fragment or mimotope thereof.
  • the invention relates to the treatment of a Group IV RNA virus infection, particularly a coronavirus infection, more particularly SARS-Cov2 infection, in subjects by modulating such a cofactor effect.
  • the invention thus provides methods and compositions for treating diseased subjects and/or for restoring/enhancing CD4 T cell activity in subjects using an inhibitor of a PLA2-GIB cofactor.
  • the term “inhibitor” of a cofactor designates any molecule or treatment which causes (directly or indirectly) an inhibition of the expression or a function of the cofactor, e.g., cofactor binding to gC1qR or cofactor ability to sensitize CD4 T cells to PLA2-GIB.
  • Inhibiting the cofactor designates preferably reducing by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more the expression or a function of the cofactor, as well as completely blocking or suppressing said expression or function. Depending on the situation, the inhibition may be transient, sustained or permanent.
  • an inhibitor of the cofactor is a gC1qR inhibitor.
  • cofactors bind gC1qR as a target receptor. Blocking or reducing or preventing binding of the cofactor to gC1qR using gC1qR inhibitors can affect the cofactor effect.
  • the term “gC1qR inhibitor” designates any molecule or treatment which causes (directly or indirectly) an inhibition of a function of gC1qR, e.g., gC1qR-mediated exocytosis.
  • gC1qR designates the receptor for complement Clq at the surface of cells, particularly of CD4 T cells, especially the human form of said receptor.
  • gC1qR is also known as Clq binding protein (C1QBP), ASF/SF2-associated protein p32 (SF2P32); Glycoprotein gClqBP; Hyaluronan-binding protein 1 (HABP1); Mitochondrial matrix protein p32; gClq-R protein; p33; ClqBP and GC1QBP.
  • C1QBP Clq binding protein
  • SF2P32 ASF/SF2-associated protein p32
  • Glycoprotein gClqBP Glycoprotein gClqBP
  • HABP1 Hyaluronan-binding protein 1
  • Mitochondrial matrix protein p32 gClq-R protein
  • p33 ClqBP and GC1QBP.
  • the amino acid sequence of the receptor was disclosed in the art.
  • An exemplary amino acid sequence of human gC1qR is reproduced below (SEQ ID NO: 211):
  • gC1qR designates any receptor of SEQ ID NO: 211 (accession number UniProtKB/Swiss-Prot: Q07021.1) above, as well as processed forms and variants thereof. Variants include naturally-occurring variants having e.g., at least 90% amino acid sequence identity to SEQ ID NO: 211.
  • gC1qR Upon binding of a cofactor, gC1qR triggers a signaling pathway that results in exocytosis of intracellular vesicles. Without being bound by theory, it is believed that the fusion of these vesicles with the cytoplasmic membrane could change the lipid composition and increase sPLA2-GIB activity on CD4 T cells membrane, resulting in an inhibition of phosphoSTAT5 signaling.
  • the fusion of these vesicles with plasma membrane can change the lipid composition and cause sPLA2-GIB activity on CD4 T cells membranes.
  • membrane fluidity is increased and cytokines receptors are aggregated in abnormal membrane domain, resulting in a dramatic decrease of cytokine signaling, and anergy of CD4 T cells.
  • gC1qR inhibitor thus includes any molecule which binds to gC1qR, or to a partner of gC1qR, and inhibits a function of gC1qR, such as gC1qR- mediated exocytosis.
  • the cofactor inhibitor is a molecule which directly inhibits an activity of the cofactor, e.g., which binds the cofactor and/or inhibits binding of the cofactor to its receptor.
  • cofactor inhibitors include, for instance, antibodies and variants thereof, synthetic specific ligands, peptides, small drugs, or inhibitory nucleic acids.
  • a cofactor inhibitor is an antibody or an antibody variant/fragment having essentially the same antigen specificity, or a nucleic acid encoding such an antibody or variant/fragment.
  • the antibody may bind a cofactor, or gC1qR, or a partner of gC1qR, or a gC1qR-binding element thereof, and preferably inhibits a function of the cognate antigen (e.g., gC1qR or the cofactor).
  • Antibodies can be synthetic, monoclonal, or polyclonal and can be made by techniques well known per se in the art.
  • antibodies is meant to include polyclonal antibodies, monoclonal antibodies, fragments thereof, such as F(ab')2 and Fab fragments, single-chain variable fragments (scFvs), single-domain antibody fragments (VHHs or Nanobodies), bivalent antibody fragments (diabodies), as well as any recombinantly and synthetically produced binding partners, human antibodies or humanized antibodies.
  • Antibodies are defined to be specifically binding, preferably if they bind to the cognate antigen with a Ka of greater than or equal to about 10 7 M-l. Affinities of antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).
  • Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, donkeys, goats, sheeps, dogs, chickens, rabbits, mice, hamsters, or rats, using procedures that are well known in the art.
  • a purified immunogen optionally appropriately conjugated, is administered to the host animal typically through parenteral injection.
  • the immunogenicity of immunogen can be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant.
  • small samples of serum are collected and tested for reactivity to the antigen polypeptide.
  • Examples of various assays useful for such determination include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures, such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays. See U.S. Pat. Nos. 4,376,110 and 4,486,530.
  • Monoclonal antibodies can be readily prepared using well known procedures. See, for example, the procedures described in U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980. Lor example, the host animals, such as mice, can be injected intraperitoneally at least once and preferably at least twice at about 3 week intervals with isolated and purified immunogen, optionally in the presence of adjuvant. Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse.
  • ABSC antibody capture
  • mice are given an intravenous boost of protein or peptide.
  • Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell.
  • the fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days.
  • Monoclonal antibodies may also be produced using alternative techniques, such as those described by Alting-Mees et al. , "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3:1-9 (1990), which is incorporated herein by reference.
  • binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et ah, Biotechnology, 7:394 (1989).
  • Antigen-binding fragments of antibodies which can be produced by conventional techniques, are also encompassed by the present invention.
  • fragments include, but are not limited to, Fab and F(ab')2 fragments.
  • Antibody fragments and derivatives produced by genetic engineering techniques are also provided.
  • the monoclonal antibodies of the invention also include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies.
  • humanized antibodies can be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans.
  • a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
  • a humanized antibody fragment can comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody.
  • Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al.
  • Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art, for example using methods described in Robinson et al. International Publication No. WO 87/02671; Akira, et al. European Patent Application 0184187; Taniguchi, M., European Patent Application 0171496; Morrison et al. European Patent Application 0173494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.
  • antibodies In connection with synthetic and semi-synthetic antibodies, such terms are intended to cover but are not limited to antibody fragments, isotype switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse), hybrids, antibodies having plural specificities, and fully synthetic antibody-like molecules.
  • Human monoclonal antibodies can also be prepared by constructing a combinatorial immunoglobulin library, such as a Fab phage display library or a scFv phage display library, using immunoglobulin light chain and heavy chain cDNAs prepared from mRNA derived from lymphocytes of a subject. See, e.g., McCafferty et al. PCT publication WO 92/01047; Marks et al. (1991) J. Mol. Biol. 222:581 597; and Griffths et al. (1993) EMBO J 12:725 734.
  • a combinatorial library of antibody variable regions can be generated by mutating a known human antibody.
  • variable region of a human antibody known to bind gC1qR can be mutated by, for example, using randomly altered mutagenized oligonucleotides, to generate a library of mutated variable regions which can then be screened to bind to gC1qR.
  • Methods of inducing random mutagenesis within the CDR regions of immunoglobin heavy and/or light chains, methods of crossing randomized heavy and light chains to form pairings and screening methods can be found in, for example, Barbas et al. PCT publication WO 96/07754; Barbas et al. (1992) Proc. Nat'l Acad. Sci. USA 89:44574461.
  • Antibodies of the invention may be directed against gC1qR, a gC1qR ligand, or a gC1qR partner, and cause an inhibition of signaling mediated by gC1qR.
  • an immunogen may be used comprising gC1qR, a gC1qR ligand, or a gC1qR partner, or a fragment, variant, or fusion molecule thereof.
  • Particular antibodies of the invention bind a gC1qR epitope, and/or have been generated by immunization with a polypeptide comprising a gC1qR epitope, selected from the mature gC1qR protein or a fragment of gC1qR comprising at least 8 consecutive amino acid residues thereof.
  • Preferred anti-gC1qR antibodies of the invention bind an epitope of a ligand-binding site within gC1qR, thereby interfering with binding of the ligand.
  • the antibodies bind an epitope comprised between amino acid residues 76-282 of SEQ ID NO: 211, which contain the gC1qR ligand bind site.
  • Clq binding to gC1qR can involve at least three different motifs on gC1qR, namely: amino acid residues 75-96, 190-202 and 144-162 (by reference to SEQ ID NO: 211).
  • HCV core protein binding to gC1qR can involve at least two different motifs on gC1qR, namely: amino acid residues 144-148 and 196-202 (by reference to SEQ ID NO: 211).
  • HIV gp41 binding to gC1qR can involve at least amino acid residues 174- 180 on gC1qR (by reference to SEQ ID NO: 211).
  • an antibody which binds an epitope containing at least one amino acid residue contained in one of said epitopes or close to one of said epitopes.
  • antibodies include antibody 60.11, which binds to residues 75-96 of gC1qR; as well as antibody 74.5.2, which binds to an epitope with the residues 204 to 218.
  • Preferred gC1qR inhibitors are therefore monoclonal antibodies against gC1qR, more preferably against an epitope of gC1qR located within amino acid residues 76-282 of the protein (by reference to SEQ ID NO: 211), even more preferably an epitope containing an amino acid residue selected from amino acids 75-96, 144-162, 174-180, and 190- 210.
  • Preferred antibodies are neutralizing (or antagonist) antibodies, i.e., they prevent or inhibit or reduce binding of a natural ligand to the receptor and/or signaling through the receptor.
  • Other particular inhibitors of the invention are antibodies that bind a PLA2-GIB cofactor and/or have been generated by immunization with a PLA2-GIB cofactor or a fragment or variant thereof, and preferably inhibit at least partially an activity of such cofactor, preferably the binding of such a cofactor to gC1qR.
  • antibodies of the invention are polyclonal antibodies or monoclonal antibodies, or variants thereof, which bind a protein selected from the proteins listed in Table 3, and inhibit at least partially the binding of said protein to gC1qR.
  • Preferred antibodies of the invention are polyclonal antibodies or monoclonal antibodies, or variants thereof, which bind a protein selected from the proteins listed in Table 3, and inhibit at least partially the binding of said protein to gC1qR.
  • the inhibitor is an antibody or a variant thereof that binds SARS-Cov-2 spike protein.
  • the antibody binds an epitope comprising an amino acid residue contained in the NTD domain or in the RBD domain of the spike protein (see example 3 for domain position).
  • Most preferred antibodies bind an epitope comprising an amino acid residue located in the RBD (amino acid residues 331-527 of SEQ ID NO: 209), or even in the RBM (amino acid residues 436-509 of SEQ ID NO: 209).
  • Particular antibodies bind an epitope comprising any amino acid residue of the spike located in anyone of SEQ ID Nos: 156-185.
  • Particularly preferred antibodies bind an epitope containing at least one amino acid residues of Spike protein located at any of the following positions (by reference to SEQ ID NO: 209): 99-113; 147-161; 253-267; 348-445, 348-362, 431-445.
  • Particular antibodies bind an epitope comprising at least one of the following amino acid residues of spike protein (by reference to SEQ ID NO: 209): 431, 432, 433, 434, 435, 436, 437, 438, 438, 440, 441, 442, 443, 444, 445.
  • Particular antibodies bind an epitope comprising at least one of the following amino acid residues of spike protein (by reference to SEQ ID NO: 209): 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362.
  • the inhibitor is an antibody or a variant thereof that binds SARS-Cov-2 ORFlab protein.
  • the antibody binds an epitope comprising an amino acid residue contained in anyone of SEQ ID Nos: 2-155.
  • the inhibitor is an antibody or a variant thereof that binds SARS-Cov-2 ORF3 protein.
  • the antibody binds an epitope comprising an amino acid residue contained in anyone of SEQ ID Nos: 186- 193.
  • the inhibitor is an antibody or a variant thereof that binds SARS-Cov-2 protein E. In a further particular embodiment, the antibody binds an epitope comprising an amino acid residue contained in SEQ ID No: 194. In another particular embodiment, the inhibitor is an antibody or a variant thereof that binds SARS-Cov-2 protein M. In a further particular embodiment, the antibody binds an epitope comprising an amino acid residue contained in anyone of SEQ ID Nos: 195-202.
  • the inhibitor is an antibody or a variant thereof that binds SARS-Cov-2 protein ORF7b.
  • the antibody binds an epitope comprising an amino acid residue contained in SEQ ID No: 203.
  • the inhibitor is an antibody or a variant thereof that binds SARS-Cov-2 protein ORF8.
  • the antibody binds an epitope comprising an amino acid residue contained in SEQ ID No: 204.
  • the inhibitor is an antibody or a variant thereof that binds SARS-Cov-2 protein N.
  • the antibody binds an epitope comprising an amino acid residue contained in anyone of SEQ ID Nos: 205-208.
  • the cofactor inhibitor is an inhibitory nucleic acid.
  • Preferred inhibitory nucleic acids include aptamers which are designed to bind the cofactor, or gC1qR, or a partner of gC1qR, and to inhibit a function thereof.
  • the cofactor inhibitor is a peptide that inhibits a function of the cofactor.
  • the peptide is typically a molecule that selectively binds a cofactor, a gC1qR, or a partner of gC1qR.
  • Peptides preferably contain from 4 to 40 amino acid residues, and their sequence may be identical to a domain of gC1qR or to a domain of a cofactor (bait peptide), or their sequence may contain a variation as compared to the sequence of a domain of gC1qR or to a domain of a cofactor (peptide antagonist).
  • Preferred peptides of the invention contain from 4 to 40 consecutive amino acid residues of SEQ ID NO: 211 (gC1qR) or of protein selected from ORFlab, Spike, ORF3, E, M, ORF7b, ORF8 or N. Examples of such peptides are or comprise the amino acid sequence of anyone of SEQ ID Nos: 2-208. Particularly preferred peptides of the invention contain from 4 to 40 consecutive amino acid residues of the spike protein of SEQ ID NO: 209, such as peptides consisting of or comprising anyone of SEQ ID NOs: 156-185.
  • Peptides of the invention may be modified to increase their properties.
  • the modification may consist of one or more amino acid substitution. Examples of such substitution includes, without limitation, replacement of a charged or reactive amino acid residue by a more neutral residue such as alanine, or conversely.
  • the modification may alternatively (or in addition) consist of a chemical modification, such as addition of a chemical group to one (or both) ends of the peptide, or to a lateral chain thereof, or to a peptide bond.
  • the peptides of the invention can comprise peptide, non-peptide and/or modified peptide bonds.
  • the peptides comprise at least one peptidomimetic bond selected from intercalation of a methylene (-CH2-) or phosphate (- PO2-) group, secondary amine (-NH-) or oxygen (-0-), alpha-azapeptides, alpha- alky lpeptides, N-alkylpeptides, phosphonamidates, depsipeptides, hydroxymethylenes, hydroxyethylenes, dihydroxyethylenes, hydroxyethylamines, retro-inverso peptides, methyleneoxy, cetomethylene, esters, phosphinates, phosphinics, or phosphonamides.
  • the peptides may comprise a protected N-ter and/or C-ter function, for example, by acylation, and/or amidation and/or esterification.
  • peptides include, for instance the peptide with amino acid residues 144- 162 of SEQ ID NO: 211 (gC1qR) and the peptide with amino acid residues 204-218 of SEQ ID NO: 211 (gC1qR).
  • peptides of the invention include peptides comprising a sequence of anyone of SEQ ID NOs: 156-185 with one amino acid substitution, more preferably with at least one amino acid selected from W, I or K replaced with an Alanine.
  • the peptides of the invention may be produced by techniques known per se in the art such as chemical, biological, and/or genetic synthesis.
  • isolated refers to molecules (e.g., nucleic or amino acid) that are removed from a component of their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • an “isolated” polypeptide (or protein) is for instance a polypeptide separated from a component of its natural environment and, preferably purified to greater than 90% or 95% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) migration.
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • inhibitors are small drug inhibitors, such as are hydrocarbon compounds that selectively bind gC1qR or a cofactor.
  • Small drugs are preferably obtainable by a method comprising: (i) contacting a test compound with a cell expressing gC1qR, (ii) selecting a test compound which binds gC1qR, and (iii) selecting a compound of (ii) which inhibits an activity of gC1qR.
  • a method comprising: (i) contacting a test compound with a cell expressing gC1qR, (ii) selecting a test compound which binds gC1qR, and (iii) selecting a compound of (ii) which inhibits an activity of gC1qR.
  • the cofactor inhibitor is a soluble form of gC1qR.
  • viruses express the peptide at the surface of the virus to ensure exposure.
  • the peptide is inserted in the envelope protein of the virus.
  • Viruses that may be used for that purpose include any safe virus for use in humans, such as adenoviruses, AAVs, retroviruses (including lentiviruses), etc. gClqR soluble receptors
  • the cofactor inhibitor is a soluble form of gC1qR.
  • inhibition of the cofactor in a subject is obtained by using (e.g., vaccinating or immunizing the subject with) an immunogen or an antigen that can induce an immune response in vivo.
  • the subject produces antibodies (or immune cells) which inhibit the cofactor (such as its interaction with gC1qR).
  • administration(s) of a cofactor immunogen e.g., any immunogenic portion of a cofactor
  • These antibodies will inhibit the cofactor effect as immunotherapy or a vaccine prophylaxis.
  • An object of the invention thus resides in a method of treating coronavirus infection by vaccinating a subject with an antigen that induces an immune response against a PLA2- GIB cofactor, particularly against SARS-Cov-2 spike antigen.
  • a further object of the invention relates to an antigen that induces an immune response against of a PLA2-GIB cofactor, particularly against SARS-Cov-2 spike antigen, for use to vaccinate a subject in need thereof.
  • the immunogen of a PLA2-GIB cofactor antigen used for vaccination is an inactivated immunogenic molecule that induces an immune response against the cofactor in a subject.
  • Inactivation may be obtained e.g., by chemically or physically altering the cofactor or by mutating or truncating the protein, or both; and immunogenicity may be obtained as a result of the inactivation and/or by further conjugating the protein to a suitable carrier or hapten, such as KLH, HSA, polylysine, a viral anatoxin, or the like, and/or by polymerization, or the like.
  • the immunogen may thus be chemically or physically modified, e.g., to improve its immunogenicity.
  • the immunogen of a PLA2-GIB cofactor of the invention comprises the entire cofactor or a variant thereof.
  • the immunogen comprises a virus envelope protein with a modified gC1qR binding motif.
  • Such immunogen thus retains immunogenicity while having no or less PLA2-GIB cofactor effect.
  • a further object of the invention thus relates to an immunogen, wherein the immunogen comprises a viral envelope protein with a modified gC1qR binding motif, preferably a deleted and/or mutated gC1qR binding motif.
  • the immunogen comprises a viral envelope protein with a modified gC1qR binding motif, preferably a deleted and/or mutated gC1qR binding motif.
  • a specific example is a SARS-Cov-2 spike protein having, by reference to SEQ ID NO: 209, at least one amino acid modification in a residue selected from anyone of SEQ ID NOs: 156-185, even more preferably in a residue selected from aa 348-362 or 431-445 (by reference to SEQ ID NO: 209).
  • a specific embodiment of the invention is a spike protein comprising the amino acid sequence of SEQ ID NO: 209, or a natural variant thereof, wherein the sequence contains at least one amino acid substitution or deletion in a position selected from: aa99-113, aal47-161, aa253-267, aa348-362, aa431-445.
  • the sequence contains at least one deletion of anyone said residues, even more preferably of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more consecutive aa residues at said positions.
  • a particular protein of the invention is a protein comprising SEQ ID NO: 209 with a deletion, preferably of at most 50 amino acids, spanning all or part of AA431-445.
  • Another particular protein of the invention is a protein comprising SEQ ID NO: 209 with a deletion, preferably of at most 50 amino acids, spanning all or part of AA348-362.
  • Such antigens retain immunogenicity of the viral protein, such as an ability to induce immune response against the vims when administered in vivo.
  • Such antigens have a reduced cofactor effect, such as a cofactor effect reduced by at least 20% as compared to the native protein or virus.
  • the immunogen of a PLA2-GIB cofactor comprises a fragment of a cofactor comprising at least 6 consecutive amino acid residues and containing an immunogenic epitope thereof. In a preferred embodiment, the immunogen comprises at least from 6 to 20 amino acid residues.
  • Preferred immunogens of the invention comprise or consist of from 4 to 30 consecutive amino acid residues of a protein or peptide selected from anyone of SEQ ID NOs: 2-211.
  • the immunogen may be in various forms such as in free form, polymerized, chemically or physically modified, and/or coupled (i.e., linked) to a carrier molecule. Coupling to a carrier may increase the immunogenicity and (further) suppress the biological activity of the immunogen.
  • the carrier molecule may be any carrier molecule or protein conventionally used in immunology such as for instance KLH (Keyhole limpet hemocyanin), ovalbumin, bovine serum albumin (BSA), a viral or bacterial anatoxin such as toxoid tetanos, toxoid diphteric B cholera toxin, mutants thereof such as diphtheria toxin CRM 197, an outer membrane vesicle protein, a polylysine molecule, or a vims like particle (VLP).
  • the carrier is KLH or CRM 197 or a VLP.
  • Coupling of the immunogen to a carrier may be performed by covalent chemistry using linking chemical groups or reactions, such as for instance glutaraldehyde, biotin, etc.
  • the conjugate or the immunogen is submitted to treatment with formaldehyde in order to complete inactivation of the cofactor.
  • the immunogenicity of the immunogen may be tested by various methods, such as by immunization of a non-human animal grafted with human immune cells, followed by verification of the presence of antibodies, or by sandwich ELISA using human or humanized antibodies. The lack of biological activity may be verified by any of the activity tests described in the application.
  • the invention relates to an inactivated and immunogenic PLA2-GIB cofactor.
  • the invention relates to a PLA2-GIB cofactor protein or a fragment or variant thereof conjugated to a carrier molecule, preferably to KLH.
  • the invention relates to a vaccine comprising an immunogen of PLA2- GIB cofactor, a suitable excipient and, optionally, a suitable adjuvant.
  • a further object of the invention relates to a method for inducing the production of antibodies that neutralize the activity of a PLA2-GIB cofactor in a subject in need thereof, the method comprising administering to said subject an effective amount of an immunogen or vaccine as defined above.
  • Administration of an immunogen or vaccine of the invention may be by any suitable route, such as by injection, preferably intramuscular, subcutaneous, transdermal, intravenous or intraarterial; by nasal, oral, mucosal or rectal administration.
  • the inhibitor of the SPLA2G1B pathway may be used alone, or in combination with one or more other active agents, such as a cytostatic or cytotoxic agent.
  • the agent may be an antibiotic, such as any broad-spectrum antibiotic, or an antibiotic with specific spectrum towards the target bacterium.
  • antibiotics include, but are not limited to, amoxicillin, clarithromycin, cefuroxime, cephalexin ciprofloxacin, clindamycin, doxycycline, metronidazole, terbinafine, levofloxacin, nitrofurantoin, tetracycline, penicillin and azithromycin.
  • the agent may be an anti-fungal agent, such as, without limitation, clotrimazole, butenafine, butoconazole, ciclopirox, clioquinol, clioquinol, clotrimazole, econazole, fluconazole, flucytosine, griseofulvin, haloprogin, itraconazole, ketoconazole, miconazole, naftifine, nystatin, oxiconazole, sulconazole, terbinafine, terconazole, tioconazole, and tolnaftate.
  • an anti-fungal agent such as, without limitation, clotrimazole, butenafine, butoconazole, ciclopirox, clioquinol, clioquinol, clotrimazole, econazole, fluconazole, flucytosine, griseofulvin, haloprogin, itraconazole, ketoconazole, miconazole,
  • the agent may be an anti-viral agent, such as, without limitation, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, tenofovir, nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, saquinavir, amprenavir, and lopinavir.
  • the agent may be selected from other drugs such as chloroquine, hydroxychloroquine, Remdesivir, or ABX464, for instance.
  • the invention also relates to a composition
  • a composition comprising an inhibitor as defined above and, preferably, a pharmaceutically acceptable diluent, excipient or carrier.
  • a “pharmaceutical composition” refers to a formulation of a compound of the invention (active ingredient) and a medium generally accepted in the art for the delivery of biologically active compounds to the subject in need thereof.
  • a carrier includes all pharmaceutically acceptable carriers, diluents, medium or supports therefore.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to subjects, for example in unit dosage form.
  • the compounds or compositions according to the invention may be formulated in the form of ointment, gel, paste, liquid solutions, suspensions, tablets, gelatin capsules, capsules, suppository, powders, nasal drops, or aerosol, preferably in the form of an injectable solution or suspension.
  • the compounds are generally packaged in the form of liquid suspensions, which may be injected via syringes or perfusions, for example.
  • the compounds are generally dissolved in saline, physiological, isotonic or buffered solutions, compatible with pharmaceutical use and known to the person skilled in the art.
  • the compositions may contain one or more agents or excipients selected from dispersants, solubilizers, stabilizers, preservatives, etc.
  • Agents or excipients that can be used in liquid and/or injectable formulations are notably methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc.
  • the carrier can also be selected for example from methyl-beta-cyclodextrin, a polymer of acrylic acid (such as carbopol), a mixture of polyethylene glycol and polypropylene glycol, monoethanolamine and hydroxymethyl cellulose.
  • compositions generally comprise an effective amount of a compound of the invention, e.g., an amount that is effective to modulate directly or indirectly an effect of PLA2-GIB on CD4 T cells.
  • Inhibitors are typically used in an amount effective to maintain/restore resistance of CD4 T cells to inactivation by PLA2-GIB.
  • compositions according to the invention comprise from about 1 ⁇ g to 1000 mg of a cofactor or modulator, such as from 0.001-0.01, 0.01-0.1, 0.05-100, 0.05-10, 0.05-5, 0.05- 1, 0.1-100, 0.1-1.0, 0.1-5, 1.0-10, 5-10, 10-20, 20-50, and 50-100 mg, for example between 0.05 and 100 mg, preferably between 0.05 and 5 mg, for example 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4 or 5 mg.
  • a cofactor or modulator such as from 0.001-0.01, 0.01-0.1, 0.05-100, 0.05-10, 0.05-5, 0.05- 1, 0.1-100, 0.1-1.0, 0.1-5, 1.0-10, 5-10, 10-20, 20-50, and 50-100 mg, for example between 0.05 and 100 mg, preferably between 0.05 and 5 mg, for example 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4 or 5 mg.
  • compositions of the invention can further comprise one or more additional active compounds, for separate, simultaneous or sequential use.
  • additional active compounds include, but are not limited to, chemotherapeutic drug, antibiotics, antiparasitic agents, antifungal agents or antiviral agents.
  • the inhibitor is used in combination with a modulator of PLA2-GIB.
  • the methods or compositions of the invention use a combination of (i) an inhibitor of a PLA2GIB cofactor and (ii) an antibody against PLA2GIB (or a derivative or fragment thereof).
  • the inhibitor of a PLA2GIB cofactor in an antibody against the cofactor, or an antibiotic, or an antifungal agent, or an antivirus agent.
  • the method or compositions of the invention use a combination of (i) an inhibitor of a PLA2GIB cofactor and (ii) an indole-based inhibitor of PLA2GIB (such as 3-(2-amino-l,2-dioxoethyl)-2-ethyl-l-(phenylmethyl)-lH-indol-4- yl)oxy)acetic acid or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, such as a sodium salt thereof (Varespladib)).
  • the inhibitor of a PLA2GIB cofactor in an antibody against the cofactor, or an antibiotic, or an antifungal agent, or an antivirus agent.
  • the method or compositions of the invention use a combination of (i) an inhibitor of a PLA2GIB cofactor and (ii) a pentapeptide inhibitor of PLA2GIB (such as a cyclic peptide selected from FLSYK, FLSYR and (2NapA)LS(2NapA)R).
  • a pentapeptide inhibitor of PLA2GIB such as a cyclic peptide selected from FLSYK, FLSYR and (2NapA)LS(2NapA)R.
  • the inhibitor of a PLA2GIB cofactor in an antibody against the cofactor, or an antibiotic, or an antifungal agent, or an antivirus agent.
  • the method or compositions of the invention use a combination of (i) an inhibitor of a PLA2GIB cofactor and (ii) a further active agent such as an antiviral agent, an antibiotic, chloroquine or hydroxychloroquine.
  • the invention also relates to a method for preparing a pharmaceutical composition, comprising mixing a cofactor or modulator as previously described and a pharmaceutically acceptable diluent or excipient, and formulating the composition in any suitable form or container (syringe, ampoule, flask, bottle, pouch, etc.).
  • the invention also relates to a kit comprising (i) a composition comprising a cofactor or modulator as previously described, (ii) at least one container, and optionally (iii) written instructions for using the kit.
  • the compounds and compositions and methods of the invention may be used to treat a variety of diseases caused by a Group IV RNA virus infection, particularly coronavirus infection, more particularly SARS-Cov-2 infection.
  • the invention is directed to methods for stimulating an immune response in a subject infected with a Group IV RNA vims, particularly coronavirus.
  • the invention is directed to methods for treating an immunodeficiency or an associated disorder in a subject infected with a Group IV RNA virus infection, particularly a coronavirus.
  • the inhibitor may be administered by any suitable route.
  • administration is by injection, such as systemic or parenteral injection or perfusion, e.g., intramuscular, intravenous, intraarterial, subcutaneous, etc. Administration is typically repeated, or continuous.
  • the duration, dosages and frequency of administering compounds or compositions of the invention may be adapted according to the subject and disease.
  • a typical regimen comprises a single or repeated administration of an effective amount of an inhibitor over a period of one or several days, up to one year, and including between one week and about six months. It is understood that the dosage of a pharmaceutical compound or composition of the invention administered in vivo will be dependent upon the age, health, sex, and weight of the recipient (subject), kind of concurrent treatment, if any, frequency of treatment, and the nature of the pharmaceutical effect desired.
  • the ranges of effectives doses provided herein are not intended to be limiting and represent preferred dose ranges.
  • the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one skilled in the relevant arts (see, e.g., Berkowet et al., eds., The Merck Manual, 16 th edition, Merck and Co., Rahway, N.J., 1992; Goodmanetna., eds., Goodman and Oilman’s The pharmacological Basis of Therapeutics, 10 th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001)).
  • the invention may be used in any mammal, particularly any human.
  • the present invention also provides efficient methods for detecting SARS-Cov-2 infection in a sample. More particularly, the cofactors and antibodies of the inention can be used as reagent for detecting SARS-Cov-2 infection in a sample.
  • the invention relates to the use of an antibody of the invention for detecting the presence of SARS-Cov-2 in a sample.
  • the invention relates to the use of a polypeptide or molecule of the invention for detecting the presence of anti-SARS-Cov-2 antibodies in a sample.
  • the detection is preferably performed in vivo, using conventional techniques such as immunoassays or radioassays.
  • detection is through ELISA type assay.
  • the sample is contacted with either the polypeptides or antibodies of the invention, and the formation of an immune complex is detected, such as with an antibody.
  • the method is typically conducted in any suitable device, such as plate, microplate, tube, etc.
  • the reagents are typically coated on a surface of the device.
  • the sample may by any biological sample or derivatives thereof (dilutions, concentrate, enriched, aliquotes, etc), such as a biological fluid (e.g., blood, serum, plasma, urine, nasal swab, etc.).
  • the invention relates to a method for detecting in vitro the presence of SARS-Cov-2 in a sample, comprising (i) contacting the sample in vitro with an antibody (or fragment or derivative thereof) of the invention, particularly an anti-Spike antibody (or fragment or derivative thereof) of the invention, and (ii) detecting the presence of antibody-antigen type complexes.
  • the invention relates to a method for detecting in vitro the presence of anti-SARS-Cov-2 antibodies in a sample, comprising (i) contacting the sample in vitro with a molecule comprising a sequence selected from anyone of SEQ ID Nos: 1-208 or an epitope-containing fragment thereof, particularly a molecule comprising a sequence selected from anyone of SEQ ID Nos: 156-185 or an epitope-containing fragment thereof, and (ii) detecting the presence of antibody-antigen type complexes.
  • the invention also relates to a kit comprising (i) an antibody (or fragment or derivative thereof) of the invention, or (ii) a molecule comprising a sequence selected from anyone of SEQ ID Nos: 1-208 or an epitope-containing fragment thereof.
  • the kit may further comprise a support and/or reagents and/or instructions for performing an immunoassay.
  • Example 1 Definition of the Position Specific Scoring Matrix (PSSM) for the identification of gClqR peptide binding motifs
  • PSSM Position Specific Scoring Matrix
  • Synthetic peptides designed by the inventors were assayed in vitro for their binding capacity to the receptor for the globular heads of Clq (gC1qR).
  • the in vitro assay is based on an enzyme-linked immunosorbent assay (ELISA), as disclosed in Pednekar et al. Mol Immunol. 2016. The sequence of the tested peptides is presented in table 1 below. Table 1:
  • Peptide sequences and binding values were used to define an optimized peptide-binding motif to gC1qR using GibbsCluster 2.0 (http://www.cbs.dtu.dk/services/GibbsCluster- 2.01. GibbsCluster 2.0 was run with default parameters except length which was set to 15 amino-acids.
  • Cluster 1 obtained from the analysis was selected to generate a Position Specific Scoring Matrix (PSSM), which is disclosed in Figure 1.
  • PSSM Position Specific Scoring Matrix
  • the matrix displays weight (or "preference") for any given amino acid at each position for a window of 15 amino acids in length.
  • Example 2 Identification of gClqR binding motifs (gC1qR:PSSM) within the SARS-CoV-2 proteome.
  • PSSM Position Specific Scoring Matrix
  • SEQ ID NO:l A SARS-CoV-2 proteome sequence (SEQ ID NO:l) was constructed by co-linear assembly of open-reading frames from the SARS-CoV-2 reference genome (NC_045512.2). Positions of the different open-reading frames (ORFlab, Spike, ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N and ORF10) are presented in table 2. Table 2: Positions of SARS-CoV-2 open-reading frames within SEQ ID NQ:1.
  • PSSM search For any selected windows of 15 amino acids, the PSSM returns a single score calculated by adding the relevant values at each position.
  • the PSSM score is assigned to the N- terminal amino acid position of the window unless otherwise stated.
  • Figure 2A provides the PSSM scores across the SARS-CoV-2 proteome (SEQ ID NO: 1).
  • gC1qR-binding motifs were thus identified in the SARS-CoV-2 proteins.
  • binding domains were identified in the following proteins: ORFlab, Spike, ORF3, E, ORF7b, ORF8, N and M.
  • Each of said proteins thus represents a relevant target for inhibition according to the invention.
  • Each of said peptides sequence represents an object of the invention.
  • Each of said proteins and peptides can act as PLA2-GIB cofactors in SARS-Cov2 infected patients.
  • Each of said peptides represents a strong inhibitor (or be used to construct a strong inhibitor) for use according to the invention
  • Each of said proteins or peptides can be used to produce relevant inhibitors, such as antibodies or derivatives thereof.
  • Each of said proteins and peptides can be used as a reagent to detect the presence of anti- Cov-2 antibodies in a sample, particularly of a protective immunity.
  • Example 3 Analysis of binding peptides from Spike antigen Spike is the principal antigen exposed at the surface of the SARS-CoV-2 virus. It is an ideal target for antibody as it is expressed on the surface of the enveloped virus. Also, the spike antigen interacts with the receptor ACE-2.
  • the amino acid sequence of the Spike protein is represented as SEQ ID NO: 209.
  • the spike antigen comprises several functional domains, which are disclosed in the following Table: Thirty (30) gC1qR-binding peptides derived from the spike antigen were identified (see example 2). Each of said peptides was further analyzed for selection/optimization.
  • Figure 3 provides the PSSM score across the spike antigen (SEQ ID NO: 209). In this figure, PSSM scores are assigned to a 15aa peptide represented by a bar. The figure shows most peptides are located within the following sub-domains of the spike antigen: NTD, RBD, S2 and TM. Each peptide was analyzed for solvent accessibility, co-localization with B cell epitopes and/or with HLA class II binding motif-rich domains. The results are presented below. Said further analysis confirmed the relevance of the claimed therapeutic approach and further led to the selection of most preferred peptides and domains. 3.1. Co-localization of peptides with solvent accessible domains.
  • gC1qR binding motifs identified in example 2 were aligned with surface accessible domains within the spike antigen (SEQ ID NO: 209).
  • Solvent accessible structure was measured from PDB structure using the “Dictionary of Secondary Structure of Proteins” (DSSP) developed by Kabsch and Sander (Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen- bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577-637.).
  • the DSSP program defines secondary structure, geometrical features and solvent exposure of proteins, given atomic coordinates in Protein Data Bank format (PDB) or macromolecular Crystallographic Information File format.
  • Solvent exposure is given as the number of water molecules in possible contact with a residue (residue water exposed surface in Angstrom 2 ).
  • DSSP software package can be obtained from https://swift.cmbi.umcn.nl/gv/dssp/DSSP 3.html.
  • the structure information for the spike antigen from SARS-CoV-2 used for solvent accessibility analysis was obtained from SWISS-MODEL (https://swissmodel.expasy.org/) based on a model number 4 (https://swissmodel.expasv.org/assess/7dVLxC/04) derived the cryo-EM structure of spike (PDB accession number: 6vsb) as established by Wrapp et al (Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, McLellan JS. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020 Mar 13;367(6483):1260-1263).
  • the 3D model for spike SARS-CoV-2 is covering a sequence corresponding to residues 27 to 1146 from SEQ ID NO:209. For this analysis, only information from chain (a) - monomeric chain in the “up” conformation - was
  • Figure 4 presents the co-localization of high scoring gC1qR:PSSM motifs with solvent accessible domains within the spike protein sequence from SARS-CoV-2. It can be shown that most peptides in NTD, RBD and S2 domains are solvent accessible, and thus exposed and suitable for use as medicament or target.
  • B cell epitopes were determined in the sequence of the Spike protein and then aligned with the peptide position.
  • B cell epitopes are regions of an antigen protein surface that are preferentially recognized by antibodies. B cell epitopes interact with an antibody based on the conformation adopted by the epitope, which is determined by the surface features of the involved epitope residues and the shape or tertiary structure of other segments of the antigen. Based on spatial structure, B-cell epitopes can be categorized as a continuous and discontinuous (nonlinear or conformational) epitopes. In the latter case, amino acid residues are in close contact due to the three-dimensional conformation.
  • DiscoTope 2.0 http ://tool s.iedb.org/discotope/
  • DiscoTope method incorporating a spatial neighborhood definition and half-sphere exposure as surface measure. J. V. Kringelum, C. Lundegaard, O. Lund, M. Nielsen. 2012. Reliable B cell epitope predictions: impacts of method development and improved benchmarking. PLoS Comput Biol. 8:(12):el002829.
  • the method utilizes calculation of surface accessibility (estimated in terms of contact numbers) and an epitope propensity amino acid score.
  • the final scores are calculated by combining the propensity scores of residues in spatial proximity and the contact numbers.
  • the structure information for the spike antigen from SARS-CoV-2 used for DiscoTope 2.0 analysis was obtained from SWISS-MODEL (https://swissmodel.expasy.org/) based on a model number 4 (https://swissmodel.expasv.org/assess/7dVLxC/04) derived the cryo-EM structure of spike (PDB accession number: 6vsb) as established by Wrapp et al (Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, McLellan JS.
  • Figure 5 presents co-localization of high scoring gC1qR-binding peptides with predicted B cell epitopes (Discotope) within the spike protein sequence from SARS-CoV-2.
  • peptides in NTD, RBD and S2 domains colocalize with predicted B cell epitopes, thus confirming their relevance as targets for antibody development.
  • HLA class II binding motif-rich domains of the SARS-CoV-2 virus spike antigen were determined. Such domains were then aligned with the identified binding peptides.
  • HLA Human leukocyte antigen
  • HLA -DR Human leukocyte antigen
  • -DP Human leukocyte antigen
  • -DQ Human leukocyte antigen
  • Each locus is comprised of many different allelic variants, which makes the HLA class II molecule highly polymorphic across the human population.
  • Peptides presented by HLA class II molecules bind to a binding groove formed by residues of the HLA a- and the b-chains and a 9 amino-acid long core binding motif contained within the peptide antigen sequences.
  • HLA class II binding is a prerequisite for the stimulation of CD4+ T-cells.
  • One additional challenge is to identify peptide that can bind a broad number of different HLA class II alleles in order to provide the ability of the peptide antigen to be immunogenic in a large proportion of the population.
  • the phenotypic frequency of HLA class II molecules is highly variable in the human population.
  • HLA class II binding motifs are not evenly distributed along an antigen sequence and tend to form hotspots of overlapping motifs.
  • Many different methods to predict the ability of peptide to bind to MHC class II molecules have been developed, including TEPITOPE, TEPITOPEpan, PROPRED, RANKPEP, SVRMHC, NetMHCIIpan, NetMHCII.
  • NetMHCII 2.3 has been shown to be among the best methods for predicting binding affinities to a large number of HLA class II molecules (Jensen KK, Andreatta M, Marcatili P, Buus S, Greenbaum JA, Yan Z, Sette A, Peters B, Nielsen M. Improved methods for predicting peptide binding affinity to MHC class II molecules. Immunology. 2018 Jul;154(3):394-406.).
  • NetMHCII is based on ensembles of artificial neural networks that are trained on quantitative peptide binding affinity data from the Immune Epitope Database (IEDB).
  • IEDB Immune Epitope Database
  • NetMHCII 2.3 server predicts binding of peptides to HLA class II molecules providing a range of information including the %-Rank and the core binding motif corresponding to a sequence of 9 amino-acids. Positive binding peptides are considered to have a %-rank below 10.
  • NetMHCII 2.3 analysis was performed with a set of 20 HLA-DR alleles, 20 HLA-DQ, 9 HLA-DP: DRal*0101-DR ⁇ 1*0101, DRal*0101-DR ⁇ 1*0301, DRal*0101-DR ⁇ 1*0401, DRal*0101-DR ⁇ 1*0701, DRal*0101-DR ⁇ 1*0801,
  • DRal*0101-DR ⁇ 1*0802 DRal*0101-DR ⁇ 1*0901, DRal*0101-DR ⁇ 1*1001, DRal*0101-DR ⁇ 1*1101, DRal*0101-DR ⁇ 1*1201, DRal*0101-DR ⁇ 1*1301, DRal*0101-DR ⁇ 1*1302,, DRal*0101-DR ⁇ 1*150, DRal *0101 -DR ⁇ 1*1602, DRal*0101-DR ⁇ 3*0101, DRal*0101-DR ⁇ 3*0202, DRal*0101-DR ⁇ 3*0301, DRal*0101-DR ⁇ 4*0101, DRal*0101-DR ⁇ 4*0103, DRal*0101-DR ⁇ 5*0101, DQal *0101-DQ ⁇ 1*0501, DQal*0102-DQ ⁇ 1*0501, DQal *0102-DQ ⁇ 1*0502, DQal *0
  • Figure 6 presents co-localization results between the peptides and the predicted HLA class II binding motif-rich domains within the spike protein sequence from SARS-CoV- 2.
  • Example 4 Detection of anti-SARS-CoV-2 immunity in a sample
  • Recombinant proteins and peptides- SARS-Cov-2 SI (Genbank accession #YP_009724390.1, which is identical to QHD43416) recombinant protein was purified from HEK293 cell culture and purchased from antibodies online (SARS-CoV-2 Spike Subunit SI (SARS-CoV-2 SI) (Active) protein, ABIN6952739, a.a. 11-683, purity >90%).
  • GCVIAWNSNNLDSKV-COOH were ordered from Covalab (purity >98%).
  • Detection of anti-SARS-CoV-2 immunity in a sample- Detection of anti-SARS-Cov-2 SI IgG and IgM antibodies in SARS-Cov-2 patient plasma/serum is performed by ELISA on coated SARS-Cov-2 SI peptides or SI protein (Antibodies online, ABIN6952739).
  • Microtiter plates (Nunc Maxisorp flat-bottom microplate, 44-2404-21, Thermofisher Scientific) are coated overnight at 4°C with 100 ⁇ l of a solution at 5 ⁇ g/ml of SI recombinant protein or SARS-Cov-2 peptides in carbonate buffer pH 9.6.
  • Casein blocking buffer (37528, Pierce Blocker Casein in PBS, Thermofisher Scientific). After washing, various dilutions in Casein blocking buffer of the anti-Sl specific Ab as positive control for IgM and IgG (Human IgGl or IgM ANTI SARS-COV-2 SPIKE (SI) ANTIBODY (CR3022), IgM: MAB 12423-50, IgG: MAB 12422-100, the native antigen company) at 125ng/ml and plasma/serum from SARS-Cov-2 patients or healthy donors (negative control) at 1:20 and gradually diluted 1:3 are incubated in quadruplicate for 2h at RT in 100 ⁇ l.
  • IgM and IgG Human IgGl or IgM ANTI SARS-COV-2 SPIKE (SI) ANTIBODY (CR3022)
  • IgM MAB 12423-50
  • IgG MAB 12422-100, the native
  • Bound antibody are then detected by interaction with biotinylated rabbit anti-human IgG Ab (Anti-Human IgG antibody [EPR12700] (Biotin), ab201842, abeam) for IgG detection or biotinylated F(ab')2-Goat anti-Human IgM (F(ab')2-Goat anti-Human IgM (Heavy chain) Secondary Antibody, Biotin, A24486, Thermofish scientific), both at 1:10,000 dilution, IOOmI/well for 2h, and is revealed by sequential reaction with HRP-Streptavidin at 1:10,000 (Peroxidase labeled Streptavidin ( SA-HRP) UP395888, Interchim, lh, room temp.) followed by the addition of 100 ⁇ l/well TMB substrate (UP664781, Interchim).
  • SA-HRP Peroxidase labeled Streptavidin
  • GC VIA WN S NNLDS KV in SARS-Cov-2 SI (Genbank accession #YP_009724390.1) are generated by immunization of OF1 mice with the peptide NH2-C- KNNKS WMESEFRVY S -COOH, NH2-C-DS S S GWT AGAAA YYV -COOH or NH2-C- GCVIAWNSNNLDSKV-COOH conjugated to KLH (KLH-SARS-Cov-2_147-161, KLH-SARS-Cov-2_253-267, KLH-SARS-Cov-2_431-445, respectively). Mice are immunized 5 times every 3 weeks.
  • First immunization is subcutaneous and intraperitoneal with 50 ⁇ g of peptides and complete freund’s adjuvant. Then mice are intraperitoneally injected three times with 50 ⁇ g of peptides and incomplete freund’s adjuvant.
  • the antibody titer in serum is evaluated by EFISA on SARS-Cov-2_147-161, SARS-Cov-2_253-267, SARS-Cov-2_431-445 peptides and SARS-Cov-2 SI protein (ABIN6952739).
  • the mouse with the best titer on protein and peptide is selected and injected intravenously with 5 ⁇ g of SARS-Cov-2 peptide (KLH-SARS-Cov-2_147-161,KLH-SARS-Cov-2_253-267 or KLH-SARS-Cov-2_431-445) alone.
  • SARS-Cov-2 peptide KLH-SARS-Cov-2_147-161,KLH-SARS-Cov-2_253-267 or KLH-SARS-Cov-2_431-445
  • spleen Three days later the spleen is collected and cells are fused with myeloma (Sp2/0-Agl4) cells to generate hybridoma.
  • the best 120 positive on BSA-SARS-Cov- 2 peptides are selected and tested another time on BS A-S ARS-Cov-2 peptide and S 1.
  • the isotypes of the best clones are determined and they are frozen including 15 clones that are maintained in cell culture to obtain stable clone, 5 clones per peptide. These 15 clones, are seeded in High Yielding Performance Flask (hyperflask) for cell culture to produce 560 ml of supernatant with 10% Low IgG fetal bovine serum (FBS) in DMEM medium. IgG are purified from supernatant by affinity chromatography on protein A in low endotoxin condition ( ⁇ 10 EU / mg, FAF test). Antibodies binding to BSA-SARS-Cov-2 and S 1 is verified and the cDNA sequence coding the variable regions of these antibodies is determined.
  • High Yielding Performance Flask hyperflask
  • FBS Low IgG fetal bovine serum
  • ELISA to screen new anti-Sl antibodies produced- EFISA on peptides or proteins is performed on microtiter plates coated overnight at room temperature (RT) with 50 pi of a solution in PBS (pH7.4) of BSA-SARS-Cov-2_147-161, BSA-SARS-Cov- 2_253-267, BSA-SARS-Cov-2_431-445, SI protein at 2 ⁇ g/ml. After washing, wells are then blocked lh at RT with 150 pi of PBS/2.5% skimmed milk. After washing, various dilutions in PBS/0.05%Tween 20/0.5%BSA of Ab from 100-0.0001 ⁇ g/ml are incubated for 2h at RT in 50 pi.
  • Example 6 Inhibition of sPLA2-GIB affects SARS-CoV-2 plasma inhibitor effect
  • CD4 T -lymphocytes Purification of Human CD4 T -lymphocytes- Venous blood was obtained from healthy volunteers through the EFS (Etableau Francais du Sang, Centre Necker-Cabanel, Paris). CD4 T-cells were purified from whole blood using RosetteSep Human CD4+ T cell Enrichment Cocktail (Stem Cell, 15062). This cocktail contains mouse and rat monoclonal antibodies purified from mouse ascites fluid or hybridoma culture supernatant, by affinity chromatography using protein A or Protein G sepharose.
  • bispecific tetrameric antibody complexes which are directed against cell surface antigens on human hematopoietic cells (CD8, CD16, CD 19, CD36, CD56 CD66b, TCR ⁇ /d) and glycophorin A on red blood cells.
  • the rosetteSep antibody cocktail crosslinks unwanted cells in human whole blood to multiple red blood cells, forming immunorosettes . This increases the density of unwanted cells, such that they pellet along with the free red blood cells when centrifuged over a buoyant density medium such as lymphocytes separation medium (Eurobio, CMSMSLOl-01).
  • Cells are subsequently resuspended in RPMI 1640 medium (Lonza) supplemented with 5% FBS, 50 mM HEPES pH 7.4, glutamine, penicillin, streptomycin and fungizone (complete medium), counted with a Moxi Z mini automated cell counter (ORFLO, MXZ000). Cells suspension is adjusted at 7xl0 6 cells/ml and equilibrated at least 2 h at 37°C in a 5% CO2 humidified atmosphere.
  • the enriched CD4-T cell population is controlled by flow cytometry on a cytoflex (Beckman coulter). The quiescence of recovered CD4 T-cells is controlled by the low level of IL-2Ro ⁇ (CD25).
  • CD4 T cells are labeled with anti-Human CD3 eFluor780 (eBioscience, clone UCHT1, 47-0038-42), anti-Human CD25-PE (Biolegend, clone BC96, 302605) and anti-human CD4-PerCP (BD, clone SK3, 345770).
  • the enriched CD4-T cell population contains >95% CD3+CD4+ and less than 8% of CD25+.
  • PLA2-G1B and SARS-Cov-2 SI protein immunodepletion to study plasma cofactor activity-
  • PLA2-G1B and SARS-Cov-2 SI protein role in SARS-Cov-2 patient plasma inhibitory activity plasma are immunodepleted with anti-PLA2GlB antibodies and anti-Sl antibodies.
  • 1 ml VP or HD plasma is incubated with 100 ⁇ g of rabbit anti-PLA2GlB pAb (produced at BIOTEM) or control isotype (Rabbit Gamma Globulin, 31887, Invitrogen) to immunodeplete PLA2-G1B, or with 100 ⁇ g of anti-Sl specific Ab (Human IgGl ANTI SARS-COV-2 SPIKE (SI) ANTIBODY (CR3022), MAB 12422- 100, the native antigen company) or the control goat polyclonal antibody (Ultra-LEAFTM Purified Human IgGl Isotype Control Recombinant Antibody, 403502, Biolegends) to immunodeplete SI, in 1.5-ml Eppendorf tubes overnight (Test tube-rotor, 34528, Snijders, Netherland) at 4°C.
  • CD4 T- cells are loaded (3.5x10 5 cells/50 ⁇ l in complete medium) on poly-L-Lysine-coated (Sigma, P8920) round coverslips (14mm-diameter, Marienfeld) in 24-well polystyrene plates at 37°C in a thermo-regulated water and mixed with 50pl of a suspension in PBS BSA1% containing peptides, recombinant proteins together with recombinant PLA2-GIB or not or containing SARS-Cov-2 patient plasma (1 or 3%), PLA2-GlB-depleted or Sl- depleted or not plasma (1 or 3%), or healthy donor plasma.
  • the cells suspension was either pretreated with 40pl of peptides, recombinant protein dilutions in PBS BSA1% for 15 minutes with subsequent addition of IOmI PLA2-GIB (5nM at the end) for 30 minutes or directly treated with 50m1 of dilution in PBS BSA 1% with peptides or recombinant protein together with PLA2-GIB (5nM at the end) for 45 minutes.
  • Cells were activated for 15 minutes with 2 nM recombinant glycosylated human IL-7 (Accrobio System).
  • cDNA was obtained from 1 ⁇ g total RNA as follows: total RNA was extracted using QIAshredder (79654, Qiagen) and the RNeasy Kit (74104, Qiagen). Extracted RNA was treated with DNase I (RNase-Free DNase Set, 79254, Qiagen) and reverse transcribed using Superscript III reverse transcriptase (18080-044, Invitrogen) according to the manufacturer’s instructions.
  • DNase I RNase-Free DNase Set, 79254, Qiagen
  • Superscript III reverse transcriptase 18080-044, Invitrogen
  • Example 7 Peptide SEQ ID NOs: 164 and 168 affect SARS-CoV-2
  • Purified SARS-Cov-2 particles were purified from African green monkey kidney Vero E6 (ATCC ® CRL-1586) cell culture.
  • Vero E6 cells were seeded (60,000/well) in 500- ⁇ l 24-well plates in DMEM 10% FBS. 24h later cells were treated for lh with SARS-Cov-2_348-362 and SARS-Cov-2_431-445 or a control scrambled peptide.
  • Percent of [3H] arachidonic acid in CD4 T cells is the (1 minus ratio of [3H] arachidonic acid in the supernatant of CD4 T cells without cells (cpm/ml) on total [3H] arachidonic acid in supernatant and cells (cpm/ml).
  • Jurkat E6.1 T cells (ECACC 88042803) or gC1qR KO Jurkat E6.1 T cells were incubated for 17h at 5x10 5 cells/ml with 1 ⁇ Ci/ml of arachidonic acid [5,6,8,9,11,14,15- 3 H(N)] (Perkin Elmer, NET298Z250UC) in RPMI 1640 medium (Lonza) supplemented with 10% FBS, 50 mM HEPES pH 7.4, glutamine, penicillin, streptomycin and fungizone at 2 ml/well in 6-well plates at 37°C in a 5% C02 humidified atmosphere.
  • arachidonic acid [5,6,8,9,11,14,15- 3 H(N)]
  • Percent of [3H] arachidonic acid in CD4 T cells is the (1 minus ratio of [3H] arachidonic acid in the supernatant of CD4 T cells without cells (cpm/ml) on total [3H] arachidonic acid in supernatant and cells (cpm/ml).
  • Results are expressed as PLA2GIB activity (release of [3H] arachidonic acid in the supernatant of cells treated with peptide together with PLA2G1B minus spontaneous release of [3H] arachidonic acid by cells in cpm/ml) or ⁇ PLA2G1B activity with peptides minus activity with Scrambled peptide (release of [3H] arachidonic acid in the supernatant of cells treated with peptide minus release of [3H] arachidonic acid by cells treated with Scrambled in cpm/ml).
  • SEQ ID NO:l SARS-CoV-2 proteome (9744 amino acids)
  • MFVFF VFFPFV S S QC VNFTTRTQFPP A YTN S FTRG V Y YPDKVFRS S VFHS T QDF FFPFFSNVTWFHAIHVSGTNGTKRFDNPVFPFNDGVYFASTEKSNIIRGWIFGTT FDSKTQSFFIVNNATNVVIKVCEFQFCNDPFFGVYYHKNNKSWMESEFRVYSS ANNCTFEY V S QPFFMDFEGKQGNFKNFREFVFKNIDGYFKIY S KHTPINFVRDF PQGF S AFEPFVDFPIGINITRFQTFF AFHRS YFTPGDS S S GWT AG A A A Y Y V G YFQ PRTFFFKYNENGTITDAVDCAFDPFSETKCTFKSFTVEKGIYQTSNFRVQPTESI VRFPNITNFCPF GE VFN ATRFAS V Y A WNRKRIS NC V AD Y S VF YN S AS FS

Abstract

La présente invention concerne de nouvelles approches thérapeutiques pour le traitement ou la prévention de maladies chez des mammifères, en particulier chez des sujets humains, à l'aide d'un cofacteur PLA2-GIB, ou d'un modulateur d'un cofacteur PLA2-GIB.
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