TECHNICAL FIELD OF THE INVENTION

The invention is in the field of immunity and provides a poxviral polypeptide (referred here as M2 polypeptide or poxviral M2 polypeptide) having the ability to bind B7-1 (CD80) and B7-2 (CD86) costimulatory molecules and modify their interactions with T cell surface receptors CD28, CTLA4 (for cytotoxic T-lymphocyte-associated antigen 4) and/or PD-L1 (for programmed death-ligand 1). The present invention also relates to vector for expression of said poxviral M2 polypeptide and composition comprising said M2 polypeptide or expression vector for use as a new immunosuppressive drug as well as methods for treating diseases or disorders associated with non-adequate immune responses such as autoimmune diseases and transplant rejections that comprise the administration of such a M2 polypeptide, M2- expressing vector or composition thereof (active agent). The present invention also relates to methods for producing such a M2 polypeptide.

BACKGROUND OF THE INVENTION

When the immune system functions normally, it produces a response intended to protect against harmful or foreign substances like virus, bacteria, parasites and cancerous cells involving specific and nonspecific components. Non-specific innate immunity acts as a barrier to eliminate a wide range of pathogens irrespective of their antigenic make-up whereas specific adaptative immunity is triggered when a pathogen evades the innate immune system. Like the innate system, adaptive immunity includes both humoral (antibody response) and cell-mediated components that are carried out by two distinct lymphocytes. Humoral immunity takes place in the body fluids (humour) and is mediated by B cells whereas cell-mediated immunity is dependent upon T-lymphocytes that originate in the bone marrow as stem cells and become sensitized by a first contact with a specific foreign antigen. Exposure to the antigen stimulates a variety of chemical and mechanical activities leading to destroy or inactivate the offending foreign antigen. Unlike B cells, T cells cannot recognize foreign antigens on their own. They need to be presented by antigen-presenting cells (APCs) such as macrophages which engulf them and display part of the antigens on the APC’s surface next to histocompatibility markers. It is now well known that T cells play a central role in a number of defence mechanisms, e.g., host’s defence against slowly developing bacterial diseases that result from intracellular infection, delayed hypersensitivity reactions and recognition and rejection of self-cells undergoing alteration (e.g. cancer cells that have tumour-specific antigens at their surfaces).

Naive T cells require 2 signals for their full activation. The first signal is antigen-specific and is provided by the T cell receptor interacting with the MHC and antigenic peptide complex on APC. The second signal, or costimulatory signal, is provided by the interactions between molecules such as CD80 and CD86 at the APC’s surface with surface receptors on naive T cells. The presence of cytokines including IL-2 stimulates the process of activation resulting in T cell proliferation. If the T cell does not receive a co-stimulation signal because of blockade of this pathway, it becomes anergic and undergoes apoptosis. (Gandhi et al., 2008, Curr Opin Organ Transplant, 13: 622-6).

More specifically, CD80 (also designated B7-1) is the ligand for three T cell surface receptors, respectively CD28, PD-L1 (CD274 or B7-H1) and CTLA-4 (CD152). Under normal circumstances, ligation of CD80 with CD28 delivers a positive stimulatory signal to T cells that have engaged the T cell receptor which results in T cell proliferation, release of IL-2, and inhibition of apoptosis through increased expression of Bcl-XL (Chen, 2004, Nat. Rev. Immunol. 4: 336-347). In contrast, ligation of CD80 to CTLA-4 or PD-L1 is thought to deliver a negative (i.e. immunosuppressive) signal that inhibits T cell proliferation, IL-2 production, and cell cycle progression, serving to dampen the immune response in order to prevent damages to healthy tissues. Furthermore, CD86 (also designated B7-2) is the ligand for CD28 and CTLA-4 receptors. As with CD80, ligation of CD86 with CD28 delivers a positive stimulatory signal to T cells whereas ligation with CTLA-4 delivers a negative immunosuppressive signal. CTLA-4 binds with higher avidity to the CD80 and CD86 ligands than CD28 and competes with CD28 to downregulate T cell activity. There are a large number of diseases or disorders that occur as a result of non- adequate immune responses (e.g. autoimmunity diseases, chronic inflammatory diseases, and some allergic reactions). In autoimmunity, the patient's immune system is activated against the body's own constituents (e.g. protein, cell, tissue, organ, etc.,) whereas in chronic inflammatory diseases, neutrophils and other leukocytes are constitutively recruited by cytokines and chemokines, leading to tissue damage. Graft rejections have also an immune basis, arising when the normally functioning immune system of the transplanted recipient recognizes the transplanted organ as "non-self” and mounts a vigorous immune response against the graft, ultimately resulting in loss of biological functioning or death of the transplanted organ. The National Institutes of Health (NIH) estimates about 24 million (7%) people in the United States are affected by an autoimmune disease. Nearly any body part can be affected. Some common diseases that are generally considered autoimmune include celiac disease, type 1 diabetes, inflammatory bowel diseases, multiple sclerosis, rheumatoid arthritis, psoriasis and systemic lupus erythematosus. The causes are generally unknown although some autoimmune diseases have hereditary roots, and others may be triggered by infections or other environmental factors. Treatment of inflammatory and autoimmune diseases typically involves nonsteroidal anti-inflammatory (NSAIDs) and immunosuppressive drugs. The success of organ or tissue transplantation is largely dependent on the ability of the clinician to modulate and control the immune response of the transplanted recipient against the transplanted graft, also requiring immunosuppression to permit the graft to survive and function (Pillai et al., 2009, World J Gastroenterol, 15(34): 4225-33). Current immunosuppressive agents include calcineurin inhibitors such as cyclosporine and tacrolimus and mTOR inhibitors such as sirolimus. However, these agents inhibit signal transduction pathways which is not specific to T cells and, thus, cause side effects in other tissues (Emamaullee at al., 2009, Expert Opin. Biol Ther. 9(6): 789-96).

Compounds that block immune responses through the B7-mediated co-stimulatory signals or other pathway (e.g. the CD40-mediated pathway) constitute a new generation of immunosuppressive drugs and a significant body of work has demonstrated their immunomodulatory effects in preclinical models of transplantation and autoimmunity. CD80 and CD86 are targets of for current immunosuppressive drugs. For example, Abatacept® (developed by Bristol-Myers Squibb (BMS) under brand name Orencia) is now on the market for the treatment of rheumatoid polyarthritis and Belatacept® (also developed by BMS under brand name Nulojix) is approved for preventing organ rejection after kidney transplant. Abatacept® and Belatacept® are both fusion proteins combining the extracellular portion of human CTLA4 with the constant-region fragment (Fc) of human lgG1 (CTLA4lg). They bind to co-stimulatory CD80 and CD86 and inhibit their ligation to CD28 but also to the immunosuppressive receptors CTLA-4 and PD-L1 (see for example Vincenti et al., 2014, J Allergy Clin Immunol, 121 (2): 299-306; Butte et al. 2007, Immunity 27, 11 1-122). The benefits of such agents are often tempered by the apparition of resistant rejections (Khiew et al., 2017, JCI Insight 2(9): pii : 92033 doi : 10.1172) which limit their clinical use as well as susceptibility to opportunistic infections, long-term risk of malignancy, toxicity and other unfavourable side effects.

There is still a need for developing new immunosuppressive therapies especially for treating diseases or disorders associated with non-adequate immune responses and the subject invention provides a new immunosuppressive drug based on a poxviral protein binding both CD80 and CD86.

TECHNICAL PROBLEM AND PROPOSED SOLUTION

The present invention accordingly provides a composition comprising a poxviral M2 polypeptide or an expression vector thereof.

The inventors have identified that supernatants of cells infected with vaccinia virus (VV) interact with CD80 and CD86 whereas supernatants of cells infected with the attenuated Modified Vaccinia Ankara (MVA) lack this property. The inventors have assigned the CD80 and CD86 binding properties to the M2 protein encoded by the VV M2L gene which is not present in MVA genome. Before the invention, M2 was reported as a protein retained in endoplasmic reticulum acting as an inhibitor of the NfKb pathway (Hinthong et al. , 2008, Virology 373(2): 248-62) and involved in uncoating of the virus (Baoming Liu et al., 2018, J. Virol. 92(7) e02152-17). Further to VV, the inventors have identified the existence of M2 orthologs in numerous replicative poxviruses.

The present invention illustrates the capacity of the M2 of binding to CD80 and CD86 and impacting three immunosuppressive pathways; respectively i) it blocks the CD80 and CD86 interactions with CD28; ii) it promotes the interaction of CD80 with PD-L1 ; and iii) it triggers a reverse signalling to the CD80/CD86 positive cells. The inventors have also found that the IC50 of M2 for CD80/CD86-CTLA4/CD28 interactions is lower than that observed with CTLA4-Fc fusions.

The poxviral M2 protein presents several advantages over the other CD80/CD86- targeting drugs existing in the prior art (e.g., antibodies, CTLA4-Fc fusions). Importantly and contrary to antibodies, M2 is capable of acting on both CD80 and CD86 co-stimulatory antigens and of cross-reacting with at least two different species (human and mouse), thus facilitating preclinical studies. In addition, M2 affinity for CD80 and CD86 is on the same order as affinity of an antibody for its target (whereas CTLA4-Fc fusions have lower affinity). Unexpectedly and contrary to CTLA-4-Fc fusions, M2 also potentiates the interaction of CD80 with PD-L1 which is thought important to generate a tolerogenic signal. For these reasons, M2 provides a wider immunosuppressive spectrum compared to conventional immunosuppressive drugs such as CTLA4-Fc fusions.

Based on the inventor’s observations, a recombinant or vectorized M2 polypeptide is useful as a new immunosuppressive drug. This technical problem is solved by the provision of the embodiments as defined in the claims. Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure. SUMMARY OF THE INVENTION

According to a first aspect, there is provided a composition comprising a poxviral M2 polypeptide or an expression vector thereof. Said composition is for use as a medicament, and more specifically as an immunosuppressive drug.

In an embodiment of the invention, the poxviral M2 polypeptide binds to CD80 or CD86 or both CD80 and CD86 co-stimulatory antigen(s). In another embodiment, the poxviral M2 polypeptide is able (i) to block the CD80/CD86 interaction with CD28; and/or ii) to promote the interaction of CD80 with PD-L1 ; and/or iii) to trigger a reverse signalling to the CD80/CD86 positive cells. Said M2 polypeptide can be identified by its signature in specialized databank. It is assigned in Uniprot a PFAM motif n°PF04887 or an Interpro motif n° IPR006971 signature. Said M2 polypeptide preferably comprises 150 to 250 amino acids, preferably 180 to 220 amino acid residues, more preferably 190 to 210 amino acid residues and even more preferably 195 to 208 amino acid residues. It comprises at least four Cys residues, preferably at least six, or seven Cys residues and even more preferably about 8 Cys residues.

In still another embodiment, the M2 polypeptide comprises an amino acid sequence displaying at least 40%, at least 50%, desirably at least 60%, preferably, at least 70%, more preferably at least 80% and as an absolute preference at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO 1 or SEQ ID NO: 16 or a part thereof containing at least 60 contiguous amino acids.

In a further embodiment, the M2 polypeptide is generated from or encoded by a Chordopoxvirinae, preferably selected from the group of genus consisting of Avipoxvirus, Capripoxvirus, Lepori poxvirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, Cervidpoxvirus and Yatapoxvirus.

In a preferred embodiment, the poxviral M2 polypeptide is generated from or encoded by an Orthopoxvirus genome, with a specific preference for a vaccinia virus, a cowpoxvirus (CPXV), a raccoon poxvirus (RCN), a Monkey poxvirus, a Horsepox virus, a Volepox virus, a Skunkpox virus, a variola virus (or smallpox), a Tateropox and a Camelpox. The vaccinia virus is suitably selected from the group consisting of Elstree, Lister, Wyeth, Copenhagen, Tian Tan and Western Reserve. A suitable M2 polypeptide originates from a vaccinia virus and preferably comprises an amino acid sequence as disclosed in SEQ ID NO: 2 starting either at the M residue in position 1 (VV M2 polypeptide with peptide signal) or at the V residue in position 18 (mature VV M2 polypeptide) with one or more of the following amino acid variations: X1 (position 36) being either F or L, X2 (position 39) being E or V, X3 (position 99) being D or N, X4 (position 143) being I or F, X5 (position 189) being G or S and X6 (position 211) being E or K. A preferred M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 starting at the M residue in position 1 or at the V residue in position 18.

In another preferred embodiment, the poxviral M2 polypeptide is generated from or encoded by a Leporipoxvirus, with a preference for Myxoma virus, Rabbit Fibroma Virus and Squirrel Fibroma Virus. A preferred myxoma virus M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 16; starting either at the M residue in position 1 or at the A residue in position 19.

In still a further embodiment, the poxviral M2 polypeptide is fused to at least one fusion partner. Said fusion partner is preferably one or more tag peptide(s) and preferably a tag comprising the amino acid sequence disclosed in SEQ ID NO: 29 (HHHHHHDYKDDDDKLVPRGS) or SEQ ID NO: 30 (DYKDDDDK). Exemplary tagged M2 polypeptides comprise the amino acid sequence shown in SEQ ID NO: 31 and 32.

In an additional embodiment, the composition comprises a therapeutically effective amount of the poxviral M2 polypeptide or the M2 expression vector described herein and a pharmaceutically acceptable carrier. Said therapeutically effective amount of the poxviral M2 polypeptide is suitably from about is 0.05 to about 100 mg/kg relative to the body weight of the patient, desirably from 1 to 50 mg/kg, preferably from 2 to 40mg/kg, more preferably from 5 to 30mg/kg, and even more preferably 7 to 20mg/kg. The composition is preferably formulated for parenteral administration, and preferably by intravenous, intravascular, intra- arterial, intradermal, subcutaneous, intramuscular or intraperitoneal route.

In an aspect of the invention, there is provided a nucleic acid molecule encoding the poxviral M2 polypeptide described herein.

In an aspect of the invention, there is provided a vector comprising the M2 polypeptide encoding nucleic acid molecule placed under the control of suitable regulatory elements for its expression in a host cell or organism. The vector is suitably selected from the group of vectors consisting of plasmids for expression in prokaryotic, yeast and mammalian hosts and viral vectors. In an aspect of the invention, there is provided a process for producing the M2 polypeptide described herein, comprising (a) introducing the vector described into a suitable producer host cell to produce a transfected or infected producer host cell, (b) culturing in-vitro said transfected or infected producer host cell under conditions suitable for its growth, (c) recovering the M2 polypeptide from the cell culture, and (d) optionally, purifying the recovered M2 polypeptide. Preferably, the recovered or purified M2 polypeptide is oligomeric.

In an aspect of the invention, the composition is for use for delivering an immunosuppressive signal to a subject in need thereof.

In an aspect of the invention, the composition is for use in many clinical situations, as described herein. In one embodiment, the composition is for use for downregulating an immune response or for inducing immunosuppressive responses or immunotolerance. In another embodiment, the composition is for use for treating autoimmune diseases and graft- related diseases.

Suitably, said autoimmune disease is selected from the group consisting of autoimmune rheumatologic disorders, lupus, autoimmune gastrointestinal and liver disorders, vasculitis, autoimmune neurological disorders, renal disorders, autoimmune dermatologic disorders, hematologic disorders, atherosclerosis, uveitis, autoimmune hearing diseases, Behcet's disease, Raynaud's syndrome, autoimmune endocrine disorders, Addison's disease, autoimmune thyroid disease and allergic condition. Said autoimmune endocrine disorders is preferably diabetes and notably insulin-dependent diabetes mellitus (IDDM). Said autoimmune rheumatologic disorders is preferably rheumatoid arthritis (RA). The composition is for use for treating autoimmune inflammatory responses, particularly those resulting from infiltration of T cells.

Suitably, the composition is for use for reducing the risk of graft rejection in a transplanted patient or a patient in the process of being transplanted, and notably for treating a graft versus host disease (GVHD).

In still another embodiment, the composition is for use alone or in combination therapy with one or more other therapeutic agent(s), preferably selected from the group consisting of blocking antibodies, cytokines, anti-inflammatory agents and immunosuppressive agents. DESCRIPTION OF THE FIGURES

Figure 1 illustrates CD80/CTLA4 (1A) and CD86/CTLA4 (1 B) competition ELISA assays carried out with the supernatants collected from avian DF1 cells either uninfected (dotted line) or infected with wild type VV (diamond) or Yervoy (inverted triangle). Binding of His-tagged B7-Fc proteins to immobilized CTLA4-Fc was performed using an anti-His tag- HRP conjugated antibody.

Figure 2 illustrates CD80/CTLA4 competition ELISA carried out with the supernatants collected from HeLa cells infected with MVA (MVA), vaccinia virus of Copenhagen strain (Cop VV), Western Reserve strain (WR VV), Wyeth strain (Wyeth VV), raccoonpox (RCN), rabbitpox (RPX), cowpox (CPX), fowlpox (FPV) and pseudocowpox (PCPV) and the supernatant of uninfected HeLa cells (negative control).

Figure 3 illustrates western blot performed in non-reducing SDS-PAGE with supernatants of CEF cells either uninfected (Sup. cells) or infected with MVA (Sup. MVA) or Copenhagen vaccinia virus (Sup.VV) collected directly or 20-fold concentrated (x20) and probed with fusions of human CD86 with Fc fragment (hCD86-Fc), human CD80 with Fc fragment (hCD80-Fc) and human CTLA4 with Fc fragment (hCTLA4-Fc). Detection was performed with an anti-Fc HRP conjugated antibody. Figure 4 illustrates competition ELISA testing the interaction of biotinylated-CD80 and biotinylated-CD86 with their cognate receptors, CD28/CD86, CD28/CD80, CTLA4/CD80 and PDL1/CD80 respectively. Supernatants collected from CEF cells infected with MVA (MVA) and vaccinia virus of Copenhagen strain (VV) are compared to the supernatant of uninfected CEF cells (CEF) (negative control) and Yervoy antibody (10pg/ml). Reactivity of recombinant human PD1 (hPD1), human CD80 (hCD80) and human CTLA4 (hCTLA4) all at 10pg/ml are used as positive control for competing with the PDL1/CD80 interaction. Detection of the bound biotinylated B7 proteins was performed using HRP conjugated streptavidin.

Figure 5A illustrates the experimental approach used to identify the“interference factor (IF)” by affinity chromatography with immobilized biotinylated-CD86-Fc fusion and Figure 5B provides the sequence coverage of the IF captured in the supernatant of VV- infected CEF cells.

Figure 6 illustrates CD80/CTLA4 competition ELISA carried out with the supernatants collected from uninfected HeLa or DF1 cells (HeLa or DF1) as negative controls or infected with a double deleted (tk- rr-) Copenhagen vaccinia virus (VVTG18277) or a triple deleted (tk- rr- m2-) Copenhagen vaccinia virus (COPTG19289). Binding of his-tagged CD80-Fc proteins to immobilized CTLA4-Fc was monitored using an anti-His tag- HRP conjugated antibody. Figure 7 illustrates a SDS-PAGE of 7.5 pg of purified recombinant M2 performed under non-reducing conditions (OmM DTT; first lane after the molecular weight ladder) and in the presence of increasing amounts of reducing agent (DTT) from 1 mM to 16 mM loaded respectively in lanes 2 to 6). The seventh lane correspond to the protein completely reduced by pmercapto-ethanol. Detection was performed with an anti-Flag monoclonal antibody coupled to HRP enzyme.

Figure 8 illustrates competition ELISA for PDL-1/CD80 (A), CD80/CD28 (B), CD86/CD28 (C), CD80/CTLA4 (D) and CD86/CTLA4 (E) interactions. The inhibitory effect of the purified M2 protein (M2), was compared to Yervoy, recombinant CTLA4, anti-hCD80 monoclonal antibody (MAB B7-1) and anti-hCD86 monoclonal antibody (MAB B7-2) as well as with the supernatant collected from CEF cells infected with Copenhagen vaccinia virus (VVTG18058). Binding of his-tagged B7-Fc proteins to immobilized CTLA4-Fc, CD28-Fc or PD-LI-Fc was monitored using an anti-His tag- HRP conjugated antibody.

Figure 9 illustrates ELISA for testing the binding activity of the tagged M2 protein; Figure 9A: a series of recombinant proteins from the human B7 family, respectively human CD80 (hCD80), mouse CD80 (mCD80), human CD86 (hCD86), mouse CD86 (mCD86), human CTLA4 (hCTLA4), mouse CTLA4 (mCTLA4), human CD28 (hCD28), mouse CD28 (mCD28), human B7-H2 (hB7-H2), human B7-H3 (hB7-H3) and human PDL-1 were immobilized on ELISA plate (0.5pg/ml) before adding undiluted supernatants of transfected cells with pTG19262 expressing the Flag-tagged M2 or undiluted supernatants of transfected cells with GFP-expressing pTG15839 (as negative control). Detection was performed by adding an anti-Flag-HPR antibody. Figure 9B: human CD80 (hCD80), mouse CD80 (mCD80), human CD86 (hCD86), mouse CD86 (mCD86) and human CTLA-4 (hCTLA4) were coated on ELISA plate (1 pg/ml) before adding different concentrations of purified Flag-tagged M2 protein. Bound M2 was detected by adding an anti-Flag-HPR antibody.

Figure 10 illustrates competition ELISA for interactions of hCTLA4 with human CD80 (hCD80) and human CD86 (hCD86). The inhibitory effect of the supernatants obtained from cells transfected with pTG19348 plasmid expressing a tagged Gp120LP myxoma virus protein (pM154L) was compared to supernatants obtained from cells transfected with the pTG19262 plasmid expressing a tagged VV M2 protein (pM2) and purified M2 protein (M2 1 pg/ml). Supernatants of non-transfected cells (Cells) and supernatant of cells transfected with an irrelevant plasmid (pGFP) were used as negative controls. Binding of his-tagged B7- Fc proteins to immobilized CTLA4-Fc was performed using an anti-His tag- HRP conjugated antibody. Figure 11 illustrates an immunoblot performed after a SDS-PAGE in non-reducing (without pmercapto-ethanol) and reducing (with pmercapto-ethanol) conditions of various volumes (1 OmI, 5mI, 2.5mI of non-diluted supernatants or 20mI diluted 100 times) of supernatants of cells transfected with either pTG19262 expressing the VV M2 protein or pTG19348 expressing the myxoma virus gp120 like protein. Detection was performed by an anti-Flag-HPR antibody. MW represents the molecular weight ladder.

Figure 12 illustrates M2 effect on mixed lymphocyte reaction (MLR). PBMC were purified from two different donors and cultured in the presence of purified recombinant M2 protein or human CTLA4-Fc fusion protein (R&D Systems) at final concentrations ranging from 0.01 to 10 pg/mL or buffer (mock treatment) as negative control and the amount of IL-2 secreted in the culture supernatants was measured by ELISA. IL-2 measurement was made in triplicate for each sample tested. The measures were normalized by dividing the mean of IL-2 concentration of the three replicates of a given sample by the mean of IL-2 concentration of the three replicates of PBMC incubated with buffer. GENERAL DEFINITIONS

As used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

The term“one or more” refers to either one or a number above one (e.g. 2, 3, 4, 5, etc).

The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

As used herein to define products, compositions and methods, the term "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are open-ended and do not exclude additional, unrecited elements or method steps. Thus, a polypeptide "comprises" an amino acid sequence when the amino acid sequence might be part of the final amino acid sequence of the polypeptide. Such a polypeptide can have up to several hundred additional amino acids residues. "Consisting essentially of" means excluding other components or steps of any essential significance. Thus, a composition consisting essentially of the recited components would not exclude trace contaminants and pharmaceutically acceptable carriers and a polypeptide consisting essentially of the recited amino acid residues refers to the presence of such an amino acid sequence with optionally only a few additional and non-essential amino acid residues (the same for a nucleotide sequence). A polypeptide "consisting of” an amino acid sequence does not contain any amino acids but the recited amino acid sequence. In the present description, the term“comprising” (especially when referring to a specific sequence), gathers the three above-detailed cases and may be replaced with "consisting essentially of” or“consisting of”, if required.

Within the context of the present invention, the terms“nucleic acid”,“nucleic acid molecule”,“polynucleotide” and“nucleotide sequence” are used interchangeably and define a polymer of any length of either polydeoxyribonucleotides (DNA) (e.g. cDNA, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes, primers and any mixture thereof) or polyribonucleotides (RNA) (e.g. mRNA, antisense RNA, SiRNA) or mixed polyribo- polydeoxyribonucleotides. They encompass single or double-stranded, linear or circular, natural or synthetic, modified or unmodified polynucleotides.

The terms “polypeptide”, “peptide” and “protein” is to be understood to be any translational product of a nucleotide sequence comprising at least nine amino acid residues bonded via peptide bonds regardless of its size, the presence of post-translational components (e.g. glycosylation). No limitation is placed on the maximum number of amino acids comprised in a polypeptide. As a general indication, the term refers to both short polymers (typically designated in the art as peptide) and to longer polymers (typically designated in the art as polypeptide or protein). This term encompasses native polypeptides, modified polypeptides (also designated derivatives, analogs, variants or mutants), polypeptide fragments, polypeptide multimers (e.g. dimers), fusion polypeptides among others. The term also refers to a recombinant polypeptide expressed from a polynucleotide sequence which encodes said polypeptide. Typically, this involves translation of the encoding nucleic acid into a mRNA sequence and translation thereof by the ribosomal machinery of the cell to which the polynucleotide sequence is delivered.

The term“identity” refers to an amino acid to amino acid or nucleotide to nucleotide correspondence between two polypeptide or nucleic acid sequences. The percentage of identity between two sequences (e.g. a candidate sequence and a“prototype” sequence such as the vaccinia M2 protein disclosed in SEQ ID NO: 1) is a function of the number of identical positions shared by the sequences, taking into account the number of gaps which need to be introduced for optimal alignment and the length of each gap. Various computer programs and mathematical algorithms are available in the art to determine the percentage of identity between amino acid sequences, such as for example the Blast program available at NCBI or ALIGN in Atlas of Protein Sequence and Structure (Dayhoffed, 1981 , Suppl., 3: 482-9), or the algorithm of Needleman and Wunsh (J.Mol. Biol. 48,443-453, 1970). Programs for determining identity between nucleotide sequences are also available in specialized data base (e.g. Genbank, the Wisconsin Sequence Analysis Package, BESTFIT, FASTA and GAP programs). Those skilled in the art can determine appropriate parameters for measuring alignment including any algorithms needed to achieve maximum alignment over the sequences to be compared. For illustrative purposes,“at least 70%” means 70% or above (including 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. whereas“at least 80% identity” means 80% or above (including 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%and“at least 90%” 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%), etc

As used herein, the term“isolated” refers to a component (e.g., protein, polypeptide, peptide, nucleic acid molecule, vector, virus, etc.), that is removed from its natural environment (i.e. separated from at least one other component(s) with which it is naturally associated or found in nature). For example, a nucleotide sequence is isolated when it is separated of sequences normally associated with it in nature (e.g. dissociated from a genome) but it can be associated with heterologous sequences.

The term "obtained from",“originating” or“originate” and any equivalent thereof is used to identify the original source of a component (e.g. protein, polypeptide, peptide, nucleic acid molecule, vector, virus, etc.) but is not meant to limit the method by which the component is made which can be, for example, by chemical synthesis or recombinant means.

As used herein, the term “host cell” should be understood broadly without any limitation concerning particular organization in tissue, organ, or isolated cells. Such cells may be of a unique type of cells or a group of different types of cells such as cultured cell lines, primary cells and dividing cells. In the context of the invention, the term“host cells” preferably refers to cells capable of producing any of the components described herein (i.e.“producer cell”) whether prokaryotic or eukaryotic nature including mammalian (e.g. human or non- human) cells. This term also includes progeny of such cells.

The term“treatment” (and any form of treatment such as“treating”,“treat”) as used herein encompasses prophylaxis and/or therapy, possibly in association with one or more additional therapeutic modality(ies). Prophylaxis treatment concerns subjects at risk of having a disease or disorder associated with non-adequate immune responses (such as an autoimmune disease and at risk of rejecting a transplant) and aims at delaying development of such a disease or disorder. Therapy treatment concerns subjects diagnosed as having said disease or disorder and aims at improving the subject’s clinical status. An improvement of the clinical status can be easily assessed by any relevant clinical measurement typically used by physicians or other skilled healthcare staff and may translate for example into amelioration or control of the progression or severity of the targeted pathological condition, alleviation of a symptom thereof, diminishment of any direct or indirect pathological consequence(s) of the disease, decrease of the rate of disease progression, amelioration of the survival, amelioration of the disease’s surrogate markers, and/or prevention of the disease’s recurrence. .

The term“administering” (or any form of administration such as“administered”) as used herein refers to the delivery to a patient of a component described herein.

The term“subject” generally refers to a host organism for whom any product and method of the invention is needed or may be beneficial. Typically, the organism is a mammal, particularly a mammal selected from the group consisting of domestic animals, farm animals, sport animals, and primates. Preferably, the subject is a human who has been diagnosed as having a disease or disorder associated with non-adequate immune responses (e.g. an autoimmune disease or a subject that has been transplanted or is in the process of being transplanted). The terms“subject” and“patients” may be used interchangeably especially when referring to human and encompasses male and female. The subject to be treated may be a new-born, an infant, a young adult, an adult or an elderly.

As used herein the term "oligomer" refers to the association of at least two polypeptides (which may have the same or different amino acid sequences) such as at least two M2 polypeptides described herein. The polypeptides may interact with each other through covalent and/or non- covalent association(s). Oligomers of M2 polypeptides are preferably formed by one or more intermolecular disulphide bonding involving one or more cysteine (Cys) residues on each polypeptide forming the oligomer such that disulphide bond(s) can form between the oligomerized polypeptides Preferably, the oligomer is formed between at least two M2 polypeptides (dimer), three M2 polypeptides (trimer), four M2 polypeptides (tetramer), five M2 polypeptides (pentamer), six M2 polypeptides (hexamer), seven M2 polypeptides (heptamer), eight M2 polypeptides (octamer) to about ten M2 polypeptides (dodecamer), with a preference for hexa, hepta or octamer.

As used herein, the term“autoimmune disease” is a condition arising from a non- adequate immune response to a normal body part in the sense that it is directed against a subject's own tissue, organ, cell component, or soluble factor, etc.). Typically, various clinical and laboratory markers of autoimmune diseases exist including, but not limited to, hypergammaglobulinemia, high levels of autoantibodies, antigen-antibody complex deposits, and lymphoid cell aggregates in affected body part(s). The term "transplant rejection" refers to a partial or complete destruction of a transplanted cell, tissue, organ, or the like on or in the transplanted recipient subject.

The term“combination” or“association” as used herein refers to any arrangement possible of various components (e.g. a compound as described herein and one or more substance effective in the treatment of the targeted disease or disorder (e.g. autoimmune disease, transplant rejection). Such an arrangement includes mixture of said components as well as separate combinations for concomitant or sequential administrations. The present invention encompasses combinations comprising equal molar concentrations of each component as well as combinations with very different concentrations. It is appreciated that optimal concentration of each component of the combination can be determined by the artisan skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a composition comprising a poxviral M2 polypeptide or an expression vector thereof. Such a composition is for use as a medicament, and more specifically as an immunosuppressive drug.

The term "immunosuppressive” or “immunosuppression” to qualify a product, composition, drug or use as used herein refers to a product, composition or drug comprising a compound that acts to suppress or mask the immune system of the subject being treated. According to the present disclosure, there is provided a compound (e.g. a poxviral M2 polypeptide or an expression vector thereof) that interferes with the ligation of at least one of the B7 antigens (i.e. CD80 or CD86 or both) to their cognate receptors present at the surface of some immune effector cells (e.g. T or B lymphocytes) with the effect of inducing an immunosuppressive signal either directly on APC (reverse signalling) and/or by inhibiting the interaction of CD80/CD86 with CD28 and/or by potentializing the interaction of CD80 with PD- L1 and/or interfering with the binding of CD80 to CTLA-4 T cell receptor. The binding of a M2 polypeptide to CD80 and/or CD86 and the resulting immunosuppressive signal can be evidenced by any method and routine technique known in the art including those described hereinafter.

Poxyiral M2 protein

Poxyirus

Poxviruses are a broad family of viruses which contain a double-stranded genome of approximately 200 kb having the potential of encoding nearly 200 proteins with different functions. Like most viruses, poxviruses have developed self-defence mechanisms through a repertoire of proteins involved in immune evasion and immune modulation aimed at blocking many of the strategies employed by the host to combat viral infections (Smith and Kotwal, 2002, Crit. Rev. Microbiol. 28(3): 149-85). Typically, the poxvirus genome encodes more than 20 host response modifiers that allow the virus to manipulate host immune responses and, thus, facilitate virus replication, spread, and transmission. These include growth factors, anti-apoptotic proteins, inhibitors of the NFkB pathway, interferon signalling, complement activation and down-regulators of the major histocompatibility complex (MHC).

As used herein, the term “poxvirus” or“poxviral” refers to any Poxviridae virus identified at present time or being identified afterwards which genome in the native context contains a M2L locus encoding a M2 protein. The term“virus” as used herein encompasses the viral genome as well as the viral particle (encapsided and/or enveloped genome).

For general guidance, the wild type vaccinia virus (VV) comprises a M2L gene which coding sequence encodes a protein called M2 produced during the early stage of the virus life cycle and involved in the host’s antiviral response during poxviral infection. It is either secreted or localized in the reticulum endoplasmic (RE) and likely glycosylated (Hinthong et al., 2008, Virology 373: 248-262). Although its function is still under investigation, it is involved in core uncoating and viral DNA replication (Liu et al., 2018 J. Virol., doi/10.1128/JVI.02152- 17) but it is dispensable for in vitro viral replication (Smith, 1993, Vaccine 11 : 43-53). In addition, its function of downregulating the cellular NF-KB transcription factor via Erk1 phosphorylation inhibition is now well established (Gedey et al., 2006, J. Virol. 80: 8676-85). The VV“M2L” gene is present in the 5’ third part of the wild-type VV genome; specifically, the coding sequence is located between position 27324 and position 27986 of the Copenhagen VV genome. The VV M2L-encoded gene product is a protein of 220 amino acids composed of a mature polypeptide long of 203 amino acid residues including 8 Cys residues and comprising at its N-terminus a 17 amino acid residue long signal peptide also having a Cys residue.

The inventors have identified M2 proteins orthologs in a vast variety of poxviruses; more specifically in seven strains of vaccinia virus, in seven strains of myxoma virus, in 4 strains of Monkeypox, in multiple strains of cowpox virus, in eight strains of variola virus as well as in a variety of other poxviruses including, but not limited to, Horsepox, Taterapox, Camelpox, Raccoonpox, Shunkpox, Yokapox, Rabbit fibroma virus, Murmansk pox, Eptesipox, Deerpox, Tanapox, Cotia virus and Volepox. For illustrative purposes, M2 protein orthologs of Horsepox, Variola virus, Monkeypox, Camelpox, cowpox display more than 90% identity with the reference VV M2 protein (as represented by SEQ ID NO: 1) and those of myxoma, Skunk, Cotia and Volepox viruses show respectively 50%, 74%, 70% and 72% sequence identity with the VV M2 protein.

For sake of clarity, the gene nomenclature used herein to designate the poxviral M2L locus and the encoded M2 gene product is that of vaccinia virus (and more specifically that of Copenhagen strain). It is also used herein for other poxviruses containing functionally equivalent M2L genes and M2 proteins to those referred herein unless otherwise indicated. Indeed, gene and respective gene product nomenclature may be different according to the poxvirus families, genus and strains but correspondences between vaccinia virus and other poxviruses are generally available in the literature. For illustrative purposes, equivalents of the VV M2L gene is designated M154L in myxoma’s genome, CPXV040 or P2L in cowpox genome, 02L in monkeypox genome, RPXV023 in rabbitpox genome and 02L or Q2L in variola virus genome.

However, the genome of the attenuated vaccinia virus strain MVA lacks many of the VV immunomodulatory genes including M2L (Antoine et al., 1998, Virology 244(2) 365-96) due to large genomic deletions during the attenuation process. In the context of the invention, the term“poxvirus” does not include poxviruses which in the native context have genomic deletion(s) or mutation(s) encompassing M2L locus (or equivalent) which thus do not encode any M2 polypeptide, such as Pseudocowpox (PCPV), MVA and NYVAC viruses.

M2 polypeptide As used herein, the term“M2 polypeptide” refers to a polypeptide comprising all or a part (at least 60 amino acid residues) of a protein originally encoded by a so-called M2L locus present in the genomic sequences of a poxvirus.

This term“M2 polypeptide” encompasses native M2 polypeptides encoded by wild- type poxviral genomic sequences as well as any variant, fragment or fusion thereof including but not limited to those described herein. This term also refers to mature M2 polypeptides (i.e. without its signal peptide) and to M2 polypeptides containing a N-terminal peptide signal which can be native (present in the natural context at the N-terminus of the selected M2 polypeptide to allow its secretion) or heterologous (i.e. a peptide signal which is not naturally present at the N-terminus of the selected M2 polypeptide).

Identification of a M2 polypeptide is within the reach of the skilled artisan using the information given herein and the general knowledge in the art. To determine if a candidate protein belongs to the M2 polypeptide family or not, one may use various techniques which are routine in the art. First, the candidate protein must be obtained from or encoded by coding sequences present in a poxviral genome as described herein. In one embodiment, the M2 polypeptide comprised in or expressed by the composition described herein can be identified by its ability to bind CD80 or CD86 or both CD80 and CD86 co-stimulatory antigen(s).

The ability of a M2 polypeptide to bind CD80 and/or CD86 can be evaluated by routine techniques. For example, appropriate cells can be transfected with a vector encoding a candidate M2 polypeptide and the cell supernatant used to probe CD80 or CD86 either immobilized on plate (ELISA) or displayed on cell surface (FACS). Sandwich competition ELISA assays (see the Example section) are particularly appropriate due to the fact that there is no need to generate a tagged recombinant protein to get a result. For example, ELISA plates may be coated with a ligand of interest (e.g. CD86-Fc) before adding the sample to be tested (e.g. a supernatant of cells infected with a poxvirus). If the sample comprises a M2 polypeptide, it will bind to the coated ligand. Then, a detection ligand is added which is usually labelled to be detected, e.g. by the action of an enzyme that converts the labelling substance into a coloured product which can be measured using a plate reader (e.g. CTLA4-Fc with a Histag recognized by anti-Histag antibodies coupled to HRP (for horseradish peroxidase). A reduction of chromogenic detection in the presence of a candidate sample as compared to no sample or a negative control sample is indicative that the sample contains a M2 polypeptide competing with the detection ligand for binding to the coated ligand. One may also proceed vice versa, e.g. by using CTLA-4-Fc as coated ligand and CD80-Fc-Histag as detection ligand. Other conventional methods are also suitable such as Biacore™, calorimetry, fluorometry, Bio-Layer Interferometry and Immunoblot.

In an additional or alternative embodiment, the M2 polypeptide comprised in or expressed by the composition described herein can be identified by its ability to induce immunosuppressive signalling. In particular, said M2 polypeptide is able (i) to block the CD80/CD86 interaction with CD28; and/or ii) to promote the interaction of CD80 with PD-L1 ; and/or iii) to trigger a reverse signalling to the CD80/CD86 positive cells. Such measurements can be carried out by any technique known in the art.

The immunosuppressive ability provided by the M2 polypeptide for use herein can be evaluated in vitro or in vivo in suitable animal models (e.g. models of disease states) or in biological samples collected from a treated subject using techniques routinely used in laboratories. For example, a reduction of proinflammatory cytokines as compared to before treatment will be indicative of an immunosuppressive effect.

For example, T cell proliferation can be measured in vitro in the presence and in the absence of the M2 polypeptide by determining the amount of ³ H-labeled thymidine incorporated into the replicating DNA of cultured cells. The rate and amount of DNA synthesis and, in turn, the rate of cell division can thus be quantified. A lack or a reduction in the amount of cytokine production or T cell proliferation by stimulated T cells upon culture with said M2 polypeptide (recombinant or expressed through an expression vector) indicates the capacity of said M2 polypeptide of delivering an immunosuppressive signal to the T cells.

The ability of a M2 polypeptide to block the CD80/CD86 interaction with CD28 can be assessed as described above.

The ability of a M2 polypeptide to promote the interaction of CD80 with PD-L1 can be evaluated. Sandwich competition ELISA assays (see the Example section) are particularly appropriate due to the fact that there is no need to generate a tagged recombinant protein to get a result. For example, ELISA plates may be coated with ligand of interest (e.g. CD80-Fc) before adding the sample to be tested (e.g. a supernatant of cells infected with a poxvirus). If the sample comprises a M2 polypeptide, it will bind to the coated ligand. Then, PD-L1 is added at a concentration that give about 50% of the maximal signal in order to be able to detect both reduction and increase of signal. The added PD-L1 is usually labelled to be detected, e.g. by the action of an enzyme that converts the labelling substance into a coloured product which can be measured using a plate reader (e.g. PD-L1-Fc with a Histag recognized by anti-Histag antibodies coupled to HRP. An increase of chromogenic detection in the presence of a candidate sample as compared to no sample or a negative control sample is indicative that the sample contains a M2 polypeptide potentiating the interaction of the detection ligand with to the coated ligand. One may also proceed vice versa, e.g. by using PD-LI-Fc as coated ligand and CD80-Fc-Histag as detection ligand.

The ability of a M2 polypeptide to trigger a reverse signalling activity on APC that express CD80 and/or CD86 can be monitored by expression of the immunosuppressive enzyme IDO (for Indoleamine 2,3-dioxygenase), STAT3 (for Signal transducer and activator of transcription 3) phosphorylation and/or by the down regulation of CD86 and CD80 themselves (Tan et al., 2005, Blood 106(9): 2936-43). Such molecules are involved in inflammation, apoptose and immunity. In one additional or alternative embodiment, the amino acid sequence of M2 polypeptide comprised in or expressed by the composition described herein can be aligned against available databases. The candidate polypeptide is considered as a member of the M2 polypeptide family if the outcome after search in domain databases (e.g, Gene3D, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY or TIGRFAMs) is the same as the outcome of the M2 VV protein (referenced in uniprot under accession number P21092; also disclosed herein as SEQ ID NO: 1). Therefore, a candidate polypeptide belongs to the poxviridae M2 family membership if, when submitted to a Blast analysis using the above-cited databases, it is assigned in Uniprot a PFAM motif n°PF04887 or an Interpro motif n° IPR006971 signature. In still an additional or alternative embodiment, the M2 polypeptide can be identified by reference to its amino acid sequence. Typically, the“mature” M2 polypeptide (i.e. without any signal peptide) comprises 150 to 250 amino acids, preferably 180 to 220 amino acid residues, more preferably 190 to 210 amino acid residues and even more preferably 195 to 208 amino acid residues and even more preferably 200 to 205 according to the poxviral species or genus from which it originates. It is also preferred that its amino acid sequence comprises at least four Cys residues, preferably at least six, or seven Cys residues and even more preferably about 8 Cys residues.

In a preferred embodiment, a M2 polypeptide appropriate in the context of this invention comprises an amino acid sequence displaying at least 40%, desirably at least 50%, advantageously at least 60%, preferably, at least 70%, more preferably at least 80% and as an absolute preference at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO 1 (corresponding to the M2 protein of Copenhagen VV strain as disclosed in Uniprot under P21092 accession number) or in SEQ ID NO: 16 (corresponding to the exemplified myxoma M2 protein) , preferably starting at residue 18 (mature M2 polypeptides) or a part thereof containing at least 60 contiguous amino acids. Particularly preferred are M2 polypeptides described hereinafter.

In a preferred embodiment, the M2 polypeptide comprised in or encoded by the composition described herein is generated from or encoded by a Chordopoxvirinae, preferably selected from the group of genus consisting of Avipoxvirus, Capripoxvirus, Leporipoxvirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, Cervidpoxvirus and Yatapoxvirus. Genomic sequences of these poxviruses are available in the art, notably in specialized databases such as Genbank (see e.g., Table 1). Table 1 provides an overview of the Genbank’s accession numbers for the genomic sequences of various poxviruses and an indication of the amino acid identity of their M2 protein with respect to VV M2 protein (Uniprot’s accession number P21092 and SEQ ID NO: 1)·

A particularly preferred poxviral M2 polypeptide is generated from or encoded by an Orthopoxvirus genome, with a specific preference for a poxvirus selected from the group of Orthopoxvirus consisting of vaccinia virus, cowpoxvirus (CPXV), raccoon poxvirus (RCN), Monkey poxvirus, Horsepox virus, Volepox virus, Skunkpox virus, variola virus (or smallpox), Tateropox and Camelpox. For sake of clarity, the present invention provides exemplary M2 polypeptides defined by reference to an amino acid sequence. However, it is intended that the present invention also encompasses any fragment of at least 60 contiguous amino acids as well variant having at least 70%, preferably at least 80% and more preferably at least 90% identity with any of the exemplified sequences. The present invention encompasses M2 polypeptides with the native signal peptide (starting at residue 1 of the recited sequence) and mature M2 polypeptides without native signal peptide starting at a further residue (usually located between position 17 to 24, and preferably at position 18 or 19). Desirably, such a M2 polypeptide comprises an initiator methionine at its N terminus to be properly translated (the skilled person is able to add to a recited sequence a N-term initiator Met residue if needed). In a preferred aspect of this embodiment, the poxviral M2 polypeptide is generated from or encoded by a vaccinia virus. Any vaccinia virus strain can be used in the context of the invention, including, without limitation, Elstree, Lister (Genbank accession AY678276), Wyeth, Copenhagen (M35Q27), Tian Tan (AF095689 1) and Western Reserve (WR) (NC_006998). A typical VV M2 polypeptide comprises the amino acid sequence disclosed in SEQ ID NO: 2 starting either at the M residue in position 1 (VV M2 polypeptide with peptide signal) or at the V residue in position 18 (mature VV M2 polypeptide) with one or more of the following amino acid variations: Xaa could be any amino acid residue but, more particularly, Xaa (X1) (position 36) being either F or L, X2 (position 39) being E or V, X3 (position 99) being D or N, X4 (position 143) being I or F, X5 (position 189) being G or S and X6 (position 211) being E or K. Exemplary M2 polypeptides comprise (i) the amino acid sequence shown in SEQ ID NO: 1 starting at the M residue in position 1 (with peptide signal) and (ii) the amino acid sequence shown in SEQ ID NO: 1 starting at the V residue in position 18 (mature VV M2 polypeptide without peptide signal).

In another aspect, a suitable poxviral M2 polypeptide can be generated from or encoded by a Variola virus and any strain can be used in this context (e.g. VARV_UNK44- harv_022; VARV_IND53-ndel_022; VARV_UNK46-hind_022; VARV_BOT72-143_022, VARV_BEN68-59_022 whose sequences are disclosed in Genbank under accession numbers ABF28404.1 ; ABF25191.1 ; ABF28606.1 ; ABF22985.1 and ABF22782.1 , respectively) as well as Variola Major Virus strains (such as Human/lndia/lnd3/1967 strain described in Genbank under accession number AAA60767.1). A typical variola M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3, starting either at the M residue in position 1 (Variola M2 polypeptide with peptide signal) or at the V residue in position 18 (mature Variola M2 polypeptide) with one or more of the following amino acid variations: Xaa could be any amino acid residue but, more particularly, X1 (position 11) being either I or V, X2 (position 129) being N or S and X3 (position 162) being N or D. A preferred variola M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4 starting either at the M residue in position 1 (with peptide signal) or at the V residue in position 18 (mature variola M2 polypeptide without peptide signal).

In still another aspect, a suitable poxviral M2 polypeptide can also be generated from or encoded by a Monkeypox virus and any strain can be used in this context. Including M PXV_SU D2005_01 _026 (AGF36581.1), Zaire-96-1-16 (NP_536453.1 or AAL40484.1), MPXV_DRC_Yandongi_026 (AGF36788.1), MPXV-lkubi-019 5AIE40112.1), MPXV- Congo_8-020 (AIE40290.1), MPXV-UTC-015 (AIE40463.1), MPXV-W_Nigeria-019 (AIE40639.1); MPXV-PCH-021 (AIE40817.1); MPXV-Nig_SEV71_2_82-019 (AIE40993.1); MPXV297957-015 (All W64108.1) and MPXV298464_009 (AUW64264.1) strains among others. For general guidance, the equivalent of the VV M2L locus is designated 02L in Monkeypox and the encoded gene product MPXVgp026. Exemplary Monkeypox-originating M2 polypeptides include without limitation polypeptides comprising (i) the amino acid sequence disclosed in SEQ ID NO: 1 having the substitution of the A residue in position 37 with a V residue, the substitution of the D residue in position 179 with a N residue, and the substitution of the G in position 189 with a S residue, (ii) the amino acid sequence shown in SEQ ID NO: 5) and (iii) a variant of SEQ ID NO: 5 having the substitution of the P residue in position 27 with a H residue and/or the substitution of the I residue in position 78 with a V residue; any of these sequences starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the V residue in position 18 (mature M2 polypeptide).

In a further aspect, the poxviral M2 polypeptide can also be generated from or encoded by a Horsepox virus and any strain can be used in this context (e.g. HSPV034 strain; Genbank accession number ABH08137.1). Preference is directed to a M2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 except a G to S substitution in position 189; starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the V residue in position 18 (mature M2 polypeptide).

In a further aspect, the poxviral M2 polypeptide can also be generated from or encoded by a Camelpox virus and any strain can be used in this context (e.g. CMP29L, CMLV029 which sequences are disclosed in Genbank under accession numbers AAG37485.1 and NP_570419.1 , respectively). An exemplary M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6; starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the V residue in position 18 (mature M2 polypeptide).

In a further aspect, the poxviral M2 polypeptide can also be generated from or encoded by a Volepox virus and any strain can be used in this context (e.g. sequences disclosed in Genbank under accession numbers YP_009281778.1 or AOP31720.1). An exemplary M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7; starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the E residue in position 18 (mature M2 polypeptide).

In a further aspect, the poxviral M2 polypeptide can also be generated from or encoded by a Skunkpox virus and any strain can be used in this context (e.g. the SKPV-USA- 1978-WA strain available at ATCC®VR-1830™ or those with sequences disclosed in Genbank, e.g. under accession number AOP31509.1). An exemplary M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 8; starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the E residue in position 18 (mature M2 polypeptide). In a further aspect, the poxviral M2 polypeptide can also be generated from or encoded by a cowpox virus and any strain can be used in this context (e.g. CPXV_FIN2000_

MAN 038 Genbank accession number ADZ29155.1 ; CPXV_GER1980_ EPA _ 038 Genbank accession number ADZ29583.1 ; CPXV_GER2002_ MKY 038 Genbank accession number ADZ30226.1 ; CPXV_GER1998_ 2_038 Genbank accession number ADZ30012.1 ; C PXV_ F R A2001 _ NANCY_038 Genbank accession number ADZ29368.1 ;

C PXV_G E R 1990_ 2_038 Genbank accession number ADZ29796.1 ; CPXV_NOR1994_

MAN _ 038 Genbank accession number ADZ30437.1 ; CPXV_UK2000_ K2984_038

Genbank accession number ADZ30649.1 as well as those described under accession numbers YP_009282724.1 and AOP31509.1 among many others). Typically, a cowpox virus M2 polypeptide comprises an amino acid sequence disclosed in SEQ ID NO: 9 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the V or A residue in position 18 (mature M2 polypeptide) with at least one of the following amino acid variations: Xaa could be any amino acid residue but, more particularly, X1 (position 9) being either F or L, X2 (position 11) being I or A, X3 (position 12) being A or V, X4 (position 18) being V or A, X5 (position 24) being I or V, X6 (position 29) being Q or E, X7 (position 40) being L or V, X8 (position 47) being D or N, X9 (position 49) being N or S, X10 (position 53) being I or M, X11 (position 59) being S or N, X12 (position 63) being I or F, X13 (position 84) being D or N, X14 (position 105) being L or M, X15 (position 122) being T or I X16 (position 133) being T or I, X17 (position 135) being Y, D or H, X18 (position 136) being D or E, X19 (position 137) being S, I or G, X20 (position 138) being D or E, X21 (position 145) being K or H, X22 (position 146) being S or K, X23 (position 175) being L or I, X24 (position 179) being D or N, X25 (position 184) being H or R, X26 (position 187) being E or K, X27 (position 189) being G or S, X28 (position 190) being M or T, X29 (position 191) being R or H, X30 (position 196) being V or M, X31 (position 199) being I or M, X32 (position 204) being Q or K, X33 (position 206) being L or F, X34 (position 212) being L or V and X35 (position 215) being D or N. Exemplary M2 polypeptides comprise the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the V or A residue in position 18 (mature M2 polypeptide).

In a further aspect, the poxviral M2 polypeptide can also be generated from or encoded by a raccoonpox virus and any strain can be used in this context (e.g. Genbank under accession number AKJ93661.1 or AOP31292.1 ; Fleischauer et al. 2015, J. General Virology 96(9) doi:10.1099/vir). An exemplary M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the E residue in position 18 (mature M2 polypeptide). In a further aspect, a suitable poxviral M2 polypeptide can also be generated from or encoded by a Murmansk poxvirus and any strain can be used in this context (e.g. Genbank under accession number AST09387.1). An exemplary M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 13, starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the A residue in position 17 (mature M2 polypeptide).

In a further aspect, a suitable poxviral M2 polypeptide can also be generated from or encoded by a Taterapox virus and any strain can be used in this context (e.g. Genbank under accession number ABD97599.1). An exemplary M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 14 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the V residue in position 18 (mature M2 polypeptide).

In another embodiment, a suitable poxviral M2 polypeptide comprised in or encoded by the composition according to the present invention may also originate from a poxvirus belonging to the Leporipoxvirus genus, with a preference for myxoma virus, Rabbit Fibroma Virus and Squirrel Fibroma Virus. For general guidance, the equivalent of the VV M2L locus is designated M154L locus for Leporipoxvirus viruses and the encoded gene product gp120- like protein. In a specific aspect, the poxviral M2 polypeptide can be generated from or encoded by a myxoma virus and any strain can be used in this context (e.g. Genbank accession numbers AGW26952.1 ; ADI75814.1 , AQT34643.1 , AQT35833.1 , AQT37703.1 ; AGU99690.1 and AFU77085.1 among many others). Typically, a myxoma virus M2 polypeptide comprises an amino acid sequence disclosed in SEQ ID NO: 15 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the Xaa (A/T) residue in position 19 (mature M2 polypeptide) with at least one of the following amino acid variations: Xaa could be any amino acid residue but, more particularly, X1 in position 2 being either A or T, X2 in position 8 being V or L, X3 in position 11 being C or Y, X4 in position 18 being S or T, X5 in position 19 being A or T, X6 in position 20 being T or K, X7 in position 21 being Q or Y, X8 in position 27 being H or Y, X9 in position 53 being Y or C, X10 in position 55 being H or N, X11 in position 56 being K or H, X12 in position 58 being S or P, X13 in position 71 being I or V, X14 in position 110 being Y or D, X15 in position 114 being T or M, X16 in position 118 being P or no amino acid, X17 in position 124 being T or N, X18 in position 126 being G or T, X19 in position 141 being I or L, X20 in position 153 being L or M, X21 in position 158 being I or L, X22 in position 165 being T or K, X23 in position 169 being S or G, X24 in position 172 being V or M, X25 in position 176 being K or T, and X26 in position 179 being V or T. An exemplary myxoma virus M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 16 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the A residue in position 19. In another aspect, the poxviral M2 polypeptide can also be generated from or encoded by a Rabbit Fibroma Virus and any strain can be used in this context (e.g. Genbank accession number AAF18030.1). An exemplary M2 polypeptide preferably comprises the amino acid sequence shown in SEQ ID NO: 17 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the T residue in position 19.

In another embodiment, Cervidpoxvirus are also suitable for generating the M2 polypeptide for use herein, with a preference for Deerpox viruses. Any strain may be used in this context such as Deerpox virus W-1170-84 (Genbank reference ABI99004.1), White-tailed deerpox species (Genbank reference AU 180579.1) and Deerpox W-848-83 species (Genbank reference ABI99174.1). Exemplary M2 polypeptides comprise an amino acid sequence as shown in any of SEQ ID NO: 18-20 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the F or L or S residues in position 20 (for ABI99004.1 , AUI80579.1 and ABI99174.1 respectively).

In another embodiment, the poxviral M2 polypeptide for use herein can also be generated from a Cotia virus (e.g. the SPAn232 disclosed under the accession number NC_016924; Alfonso et al., 2012, J. Virol. 86(9): 5039-54). An exemplary M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 21 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the S in position 18.

In another embodiment, the poxviral M2 polypeptide can also be generated from a virus belonging to the Centapox virus genus with a preference for Yokapox viruses (e.g. accession number YP_004821523.1 ; Alfonso et al., 2012, J. Virol. 86(9): 5039-54). An exemplary M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 22 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the V residue in position 19.

In another embodiment, the poxviral M2 polypeptide can also be generated from a virus belonging to the Yatapox virus genus with a preference for Tanapox viruses (e.g. accession numbers ABQ43480.1 and ABQ43636.1). An exemplary M2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 23 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the Y residue in position 23. The poxviral M2 polypeptide for use herein may also be generated from a Yaba-like disease virus (GenBank CAC21247.1 ; Hu et al., 2001 , J. Virol. 75(8): 10300-8). An exemplary M2 polypeptide comprises the amino acid sequences shown in SEQ ID NO: 24 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the Y residue in position 23.

In another embodiment, the poxviral M2 polypeptide may also be generated from a

NY-014 poxvirus (GenBank AST09586.1 ; Smithson et al., 2017, Virus Genes 53(6): 883-97). An exemplary M2 polypeptide comprises the amino acid sequences shown in SEQ ID NO: 25 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the T residue in position 18.

In another embodiment, the poxviral M2 polypeptide may also be generated from a Eptesipox virus (GenBank ASK51372.1). An exemplary M2 polypeptide comprises the amino acid sequences shown in SEQ ID NO: 26 starting either at the M residue in position 1 (M2 polypeptide with peptide signal) or at the E residue in position 21.

Certain embodiments contemplate the presence of a signal peptide at the N-terminus of the M2 polypeptide (or fragment or fusion thereof) to enhance the processing through ER (endoplasmic reticulum)-and/or secretion. Briefly, signal peptides usually comprise 15 to 35 essentially hydrophobic amino acids, are inserted at the N-terminus of the polypeptide downstream of the codon for initiation of translation and are then removed by a specific ER- located endopeptidase to give the mature polypeptide. In the native context, the M2 protein encoded by the poxviral genome is equipped with a signal peptide (about 16-23 residue long). A preferred embodiment contemplates the use of the native signal peptide which is naturally present at the N-terminus of the selected M2 polypeptide (i.e. in the native context) as mentioned above. For example, a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 27 (MVYKLVLLFCIASLGYS) is typically present at the N-terminus of the native CopVV M2 protein.

In another embodiment, the native signal peptide of a selected M2 polypeptide can be replaced by a signal peptide of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide. Appropriate signal peptides are known in the art. They may be obtained from cellular or viral polypeptides such as those of immunoglobulins, tissue plasminogen activator, insulin, rabies glycoprotein, the HIV virus envelope glycoprotein or the measles virus F protein or may be synthetic (see e.g. W02008/138649). For guidance, a suitable signal peptide for use in the context of the present invention comprises the amino acid sequence shown in SEQ ID NO: 28 (MGLGLQWVFFVALLKGVHC). Fusion protein

In one embodiment, the M2 polypeptide comprised in or encoded by the composition of the present invention is fused to at least one fusion partner to generate a fusion protein. Fusion proteins comprising all or part of the M2 polypeptide disclosed herein and at least one fusion partner are also provided. The term "fusion" as used herein refers to the fusion of the M2 polypeptide with at least one fusion partner in a single polypeptide chain. The fusion can be direct (i.e. without any additional amino acid residues in between) or through a linker. Typically, linkers are 1 to 30 amino acids long peptides (typically 3 to 10) composed of amino acid residues such as glycine, serine, threonine, asparagine, alanine and/or proline (e.g. GSG, GAS, GTS, etc.,). It is within the reach of the skilled person to assess the need to include a linker or not between the M2 polypeptide and its fusion partner(s).

Moreover, each fusion partner can independently be located either at the N terminus or at the C-terminus or even within the M2 polypeptide sequence (internal fusion).

Appropriate fusion partners include, without limitation polypeptides capable of increasing the solubility, biological half-life, tissue accessibility, affinity and/or binding properties to CD80/CD86, augmenting biophysical characteristics such as stability and/or bioavailability, improving efficiency of production of the M2 polypeptide, or decreasing immunogenicity of fusion proteins or fragments thereof.

In one embodiment the fusion partner is a polypeptide from a non-poxviral source.

In another embodiment, the fusion partner contains one or more domains of an immunoglobulin heavy chain constant region, preferably a Fc (fragment crystallizable) domain. Recombinant M2-lg fusion proteins can be prepared by fusing the M2-polypeptide or a fragment thereof to the Fc region of human or mouse lgG1 , lgG2, or lgG4 Fc region, as described previously (e.g. in Chapoval, et al., 2000, Methods Mol Med. 45: 247-55).

In another embodiment, the M2 polypeptide is fused to serum albumin or to a serum albumin binding protein. The fusion or interaction of a poypeptide to serum albumin is known to increase its in vivo half-life.

In another embodiment the fusion partner may have a conjugation domain through which additional molecules can be bound to the M2 fusion proteins. In one aspect, the conjugated molecule can target the fusion protein to a particular organ or tissue. In another aspect, the conjugated molecule is another immunomodulatory compound that can augment the immunosuppressive effect of the M2 fusion protein. In still another aspect, the conjugated compound is Polyethylene Glycol (PEG).

In still another embodiment, the fusion partner may be one or more tag peptide(s). A

“tag is typically a short peptide sequence able to be recognized by available antisera or compounds with the goal of facilitating for example, visualisation and/or purification of the tagged protein. Tag peptides can be detected by immunodetection assays using anti-tag antibodies. A vast variety of tag peptides can be used in the context of the invention including, without limitation, PK tag, FLAG octapeptide, MYC tag, HIS tag (usually a stretch of 4 to 10 histidine residues) and e-tag (US 6,686,152). The tag peptide(s) may be independently positioned at the N-terminus of the M2 polypeptide or alternatively at its C-terminus or alternatively internally or at any of these positions when several tags are employed. In a preferred embodiment, the M2 polypeptide is equipped with tag peptides introduced at the N- terminus of the mature M2 polypeptide. Such tags are preferably His and Flag tags optionally followed by a cleavage site (e.g by the thrombin). Exemplary Tags for tagging a mature M2 polypeptide or a signal peptide-comprising M2 polypeptide comprise the amino acid sequence disclosed in SEQ ID NO: 29 (HHHHHHDYKDDDDKLVPRGS) or SEQ ID NO: 30 (DYKDDDDK). Exemplary tagged M2 polypeptides comprise the amino acid sequence shown in SEQ ID NO: 31. (tagged VV M2) and the amino acid sequence shown SEQ ID NO:32 (tagged myxoma virus M2), both with a heterologous signal peptide at the N-terminus.

Many modifications useful in construction of disclosed fusion proteins and methods for making them are known in the art.

Fragment(s) of the M2 polypeptide

Whatever the type of fragments generated in the context of the present invention, it is required that fragments of M2 polypeptides are biologically « active » fragments meaning that they retain binding activity to CD80 or CD86 or both.

In one embodiment, the M2 polypeptide fragment is a soluble fragment. Soluble fragments of M2 polypeptides include some or the entire extracellular domain of the polypeptide, and lack some or all of the intracellular and/or transmembrane domains. Variants

The present invention also encompasses variants of a M2 polypeptide comprising one or more amino acid modification(s) with respect to the native poxviral M2 counterpart. Various modifications can be contemplated including, without limitation, one or more deletions, insertions, and/or substitutions of one or more amino acid(s). The modifications may encompass contiguous residues or not and each amino acid substitution can be conservative or non-conservative. Such modification(s) preferably involve(s) one or more amino acid residues of the M2 polypeptide which are not essential for M2 binding to CD80 and/or CD86 so that the resulting variant retains binding properties of the same range as the M2 polypeptide from which it originates.

Useful variants include those that provide at least one improved property that may translate as described above in connection with the fusions proteins in an increase of solubility, biological half-life, tissue accessibility, affinity and/or binding properties to CD80 and/or CD86, augment biophysical characteristics such as stability and/or bioavailability, decrease immunogenicity or improve efficiency of production of the M2 polypeptides, fusion proteins or fragments thereof compared to the native (or parental) poxviral M2 polypeptide.

In one embodiment, the M2 polypeptide described herein can be modified to provide variants having stronger binding properties to CD80 and/or CD86 so as to enhance the immunosuppressive signal transmitted into the T cell. The binding capacity of a M2 variant to CD80 and/or CD86 may be determined by one skilled in the art, including the methods disclosed herein, Increased binding refers to binding that is at least 10%, 20%, 30%, 40%, 50% or greater for the targeted CD80 and/or CD86 ligands than the native M2 polypeptide.

In another embodiment, the M2 polypeptide described herein can be engineered to have an increased half-life or stability relative to the native M2 polypeptide. These variants typically are modified to resist proteolytic degradation especially in vivo. Exemplary modifications include modified amino acid residues and modified peptide bonds that resist enzymatic degradation. Various modifications to achieve this are known in the art. For example, one or more motif which could potentially be recognized and cleaved by a protease for example can be removed or substituted.

In another embodiment, modification(s) of M2 polypeptide does not include the substitution of the cysteine residue(s) involved in oligomerization and maintenance of the 3D structure.

The M2 polypeptide can be further modified for other purposes, e.g., to increase solubility, or enhance therapeutic efficacy in specific indications or diseases. In addition, amino acid side chains of the M2 polypeptide, fusion or fragment thereof can be chemically modified.

As another example, the glycosylation of the M2 polypeptide can be altered, for example to increase its affinity for its target ligands. Such modifications can be accomplished, for example, by mutating one or more residues within the site(s) of glycosylation or by selecting the host cells according to their glycosylation machinery. For illustrative purposes, expression in E. coli produce non-glycosylated protein.

Numerous processes are known in the art for modifying one or more residue(s) in a polypeptide. For example, mutations can be introduced into the encoding nucleic acid molecule by any method well known in the art, such as PCR mutagenesis or standard site- directed mutagenesis (protocols and reagents can be obtained commercially e.g., from Amersham). M2-encoding nucleic acid molecule

The present invention also relates to a nucleic acid molecule encoding the M2 polypeptide comprised in or encoded by the composition of the invention.

The M2-encoding nucleic acid molecule or part thereof can be obtained using standard molecular biology techniques, sequence data accessible in the art and the information provided herein. For example, it can be isolated from a poxviral genome or any available source by conventional techniques (e.g. PCR amplification, DNA cloning, chemical synthesis).

In the context of the present invention, the nucleic acid molecule encoding the M2 polypeptide may be optimized for increasing expression levels in a particular host cell. It has been indeed observed that, the codon usage patterns of organisms are highly not-random, and the use of codons may be markedly different between different host cells. As the M2 polypeptide originates from a virus, it may have an inappropriate codon usage pattern for efficient expression in the selected host vector system. Typically, one or more "native" (viral) codon(s) may be substituted with alternative codon(s) encoding the same amino acid to account for differences in codon usage between the poxvirus from which the M2 polypeptide or encoding nucleic acid molecule originates and the expression host cell. It is not necessary to replace all native codons with host preferred codons since increased expression can be achieved even with partial replacement.

Further to optimization of the codon usage, expression can further be improved through additional sequence modifications with the goal of suppressing clustering of rare, non-optimal codons present in concentrated areas and/or suppressing or modifying "negative" sequence elements which are expected to negatively influence expression levels. Such negative sequence elements include without limitation the regions having very high (>80%) or very low (<30%) GC content; AT -rich or GC-rich sequence stretches; unstable direct or inverted repeat sequences; R A secondary structures; and/or internal cryptic regulatory elements such as internal TATA-boxes, chi-sites, ribosome entry sites, and/or splicing donor/acceptor sites.

In certain embodiments as described herein, a nucleic acid molecule encoding a signal sequence can be incorporated in-frame at the 5' end of the M2-encoding nucleic acid molecule by standard recombinant DNA techniques, such as by PCR or by ligation.

Methods of making the M2 polypeptide

The nucleic acid molecules encoding the M2 polypeptide described herein may be expressed in a variety of systems as set forth below. The techniques for cloning and expressing the M2-encoding nucleotide sequence are well-established in the art (e.g., chemical synthesis, PCR, vector construction, expression systems, and the like), and most practitioners are familiar with the standard resource materials for specific conditions and procedures. For example, the cDNA may be excised by suitable restriction enzymes and ligated into suitable prokaryotic or eukaryotic expression vectors for the expression of the M2 polypeptide in a suitable host cell or organism. However, the following paragraphs are provided for convenience and may serve as a guideline.

Expression vector

The present invention also provides a vector for expression of the poxviral M2 polypeptide described herein in a suitable host cell or subject and a composition comprising such a vector. Specifically, such a vector comprises the M2 polypeptide-encoding nucleic acid molecule placed under the control of suitable regulatory elements for its expression in a host cell or organism. Insertion of the M2-encoding nucleic acid molecule into an expression vector can be performed by routine molecular biology, e.g. as described in Sambrook et al. (2001 , Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory).

A variety of host-vector systems may be used or engineered to express the M2 polypeptide, including prokaryotic hosts such as bacteria and eukaryotic hosts such as yeasts, insect cell systems; plant cell systems and mammalian systems (e.g. cultured cells). Typically, such vectors and host cells are commercially available (e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depositary institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them. Preference is given to a vector selected from the group of vectors consisting of plasmids for expression in prokaryotic, yeast and mammalian hosts and viral vectors.

Suitable vectors for use in prokaryotic hosts include plasmids such as pBR322 (Gibco BRL), pUC (Gibco BRL), pbluescript (Stratagene), p Poly (Lathe et al., 1987, Gene 57: 193- 201), pTrc (Amann et al., 1988, Gene 69: 301-15); pET lid (Studier et al., 1990, Gene Expression Technology: Methods in Enzymology 185: 60-89); pIN (Inouye et al., 1985, Nucleic Acids Res. 13: 3101-9; Van Heeke et al., 1989, J. Biol. Chem. 264: 5503-9); and pGEX where the M2-encoding nucleic acid molecule can be expressed in fusion with glutathione S-transferase (GST) (Amersham Biosciences Product). Suitable vectors for expression in yeast (e.g. S. cerevisiae) include, but are not limited to pYepSed (Baldari et al., 1987, EMBO J. 6: 229-34), pMFa (Kujan et al., 1982, Cell 30: 933-43), pJRY88 (Schultz et al., 1987, Gene 54: 113-23), pYES2 (Invitrogen Corporation) and pTEF-MF (Dualsystems Biotech Product).

Suitable plasmid vectors for expression in eukaryotic hosts (e.g. mammalian host cells) include, without limitation, pREP4, pCEP4 (Invitrogene), pCI (Promega), pCDM8 (Seed, 1987, Nature 329: 840), pMT2PC (Kaufman et al., 1987, EMBO J. 6: 187-95), pVAX and pgWiz (Gene Therapy System Inc; Himoudi et al., 2002, J. Virol. 76: 12735-46).

Viral vectors can also be utilized in the context of the invention derived from a variety of different viruses (e.g. baculovirus, retrovirus, adenovirus, AAV (for Adenovirus associated virus), poxvirus, measles virus, and the like). As used herein, the term "viral vector" encompasses vector DNA as well as viral particles generated thereof. Viral vectors are can be replication-competent but, also and preferably, replication-defective or replication- impaired. Insertion into an adenoviral vector or a poxviral vector can be performed through homologous recombination as described respectively in Chartier et al. (1996, J. Virol. 70: 4805-10) and Paul et al. (2002, Cancer gene Ther. 9: 470-7). Retroviral vectors are particularly suitable for providing long-term expression in the selected host cell. Baculovirus are also suitable for expression in insect cells whereas plant viral vectors such as cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV) and potato virus X (PVX) are useful for expression in plant cell systems.

Further to the M2 polypeptide expression cassette (where the nucleic acid molecule encoding said M2 polypeptide is placed under the control of suitable regulatory elements such as a promoter and a transcription termination sequence for expression in a given host cell or subject), the vector may include additional elements enabling maintenance, propagation or expression of said M2-encoding nucleic acid molecule in a desired host cell. Such elements include, but are not limited to, an origin of replication (e.g. 2pm origin of replication), and one or more marker genes.

Marker gene(s) permit(s) to facilitate identification and isolation of the recombinant host cells (e.g. by complementation of a cell auxotrophy or by antibiotic resistance). Suitable marker genes for expression in prokaryotic host cells include tetracycline and ampicillin- resistance genes. Also, resistance genes can be used for expression in mammalian host cells such as dihydrofolate reductase (dhfr) which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981 , Proc. Natl. Acad. Sci. USA 78: 1527); gpt which confers resistance to mycophenolic acid (Mulligan and Berg, 1981 , Proc. Natl. Acad. Sci. USA 78: 2072); neo which confers resistance to the aminoglycoside G- 418 (Colberre-Garapin et al., 1981 , J. Mol. Biol. 150: 1); zeo which confers resistance to zeomycin, kana which confers resistance to kanamycin and hygro which confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147). URA3 and LEU2 genes can be used for expression in yeast systems, which provide for complementation of ura3 or Ieu2 yeast mutants.

Importantly, the nucleic acid molecule encoding the poxviral M2 polypeptide is in a form suitable for its expression in a host cell. For this purpose, the nucleic acid molecule encoding the M2 polypeptide is placed under the control of a promoter. It will be appreciated by those skilled in the art that the choice of the promoter can depend on such factors as the nucleic acid molecule itself, the expression vector as well as the host cell and the level of expression desired, etc. The promoter can be constitutive directing expression of the M2 polypeptide in many types of host cells or specific to certain host cells (e.g. tissue-specific regulatory sequences) or regulated in response to specific events or exogenous factors (e.g. by temperature, nutrient additive, hormone, etc) or according to the phase of a viral cycle (e.g. late or early).

Commonly used prokaryotic control sequences usually include a promoter for transcription initiation, optionally with an operator, along with Shine-Dalgarno (SD) sequence. Such commonly used promoters include for example, the beta-lactamase (penicillinase), lactose (lac) (Chang et al., 1977, Nature 198:1056), and the tryptophan (trp) promoter systems (Goeddel et al., 1980, Nucleic Acids Res. 8: 4057) and the lambda derived pL promoter and N-gene ribosome binding site (Shimatake et al., 1981 , Nature 292:128). Additional promoters are also suitable in the context of the present invention such as the phoA promoter, the alkaline phosphatase promoter, as well as IPTG-inducible promoters and hybrid promoters such as pTAC promoter.

Promoters suitable for expression in yeast include the promoters for the synthesis of glycolytic enzymes (Hess et al., 1968, J. Adv. Enzyme Reg. 7:149; Holland et al., 1978, Biochemistry 17:4900), and MF alpha (Inokuchi et al., 1987, Mol. Cell. Biol. 7: 3185-93) promoters. Other promoters, which have the additional advantage of transcription controlled by growth conditions are promoters controlling transcription of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization (e.g. CYC-1 (Guarente et al, 1981 , Proc. Natl. Acad. Sci. USA 78: 2199), GAL-1 , GAL4, GAL10, PH05, glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH), and alcohol dehydrogenase (ADH) (Denis et al. , 1983, J. Biol. Chem. 25: 1165) promoters). Expression of the poxviral M2 polypeptide in eukaryotic organisms may be controlled by promoters obtained from the genomes of viruses or from mammalian genes and adapted to the expression system used. Exemplary viral promoters include, without limitation, promoters present in polyoma virus, fowlpox virus, adenovirus (such as the adenovirus major late or the E4 promoter), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (e.g. the CMV immediate early promoter disclosed in US 5,168,062), retrovirus (e.g. LTR), Simian Virus 40 (SV40), herpes simplex virus (HSV) (e.g. HSV-1 thymidine kinase (TK) promoter), T7 polymerase promoter (W098/10088) and Vaccinia virus (e.g. 7.5K, H5R, 11 K7.5 (Erbs et al. , 2008, Cancer Gene Ther. 15(1): 18-28), p28, p11 and K1 L promoters, as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques 23: 1094-7); Hammond et al. (1997, J. Virol Methods 66: 135-8); and Kumar and Boyle (1990, Virology 179: 151-8) as well as early/late chimeric promoters). Mammalian promoters include without limitation, the phosphoglycerate kinase (PGK) promoter (Adra et al., 1987, Gene 60: 65-74), the actin promoter, any of immunoglobulin promoters and heat-shock promoters. Inducible eukaryotic promoters regulated by exogenously supplied compounds are also suitable in the context of the invention such as the zinc-inducible metallothionein (MT) promoter (Me Ivor et al., 1987, Mol. Cell Biol. 7: 838-48), the eedysone insect promoter (No et al. , 1996, Proc. Natl. Acad. Sci. USA 93: 3346-51), the tetracycline-repressible promoter (Gossen et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5547-51), the tetracycline-inducible promoter (Kim et al., 1995, J. Virol. 69: 2565-73), the RU486-inducible promoter (Wang et al., 1997, Nat. Biotech. 15: 239-43) and the rapamycin-inducible promoter (Magari et al. , 1997, J. Clin. Invest. 100: 2865- 72).

Those skilled in the art will appreciate that additional regulatory sequences may be use for controlling the expression of the M2 polypeptide in the host cell, more specifically for proper initiation, regulation and/or termination of transcription (e.g. polyA transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals), stability (e.g. introns and non-coding 5' and 3' sequences), translation (e.g. an initiator Met, tripartite leader sequences, IRES ribosome binding sites, signal peptides, etc.).

Recombinant DNA technologies can also be used to improve expression of the M2- encoding nucleic acid molecule in the host cell, e.g. by using high-copy number vectors, substituting or modifying one or more transcriptional regulatory sequences (e.g. adding to the promoter an enhancer and the like), optimizing the codon usage to the host cell, and suppressing negative sequences that may destabilize the transcript as described above. In one embodiment, the poxviral M2 polypeptide is delivered as a protein produced by recombinant means (i.e. a proteinaceous composition), for instance in prokaryotic, yeast or mammalian cultured cells. Another aspect of this invention pertains to expression vectors for use as immunosuppressive drug capable of expressing the M2 polypeptide in situ upon administration to a subject. Specifically, the M2 polypeptide may be delivered to the subject in need thereof in the form of a vector expressing the M2 polypeptide (i.e. a vector composition). Any of the vectors described herein can be used in this context, especially viral vectors such as lentiviral vector or AAV or plasmid vectors adapted to eukaryotic hosts.

Production of the M2 protein The M2 polypeptide comprised in or encoded by the composition of the present invention can be produced by recombinant means using suitable expression vectors and host cells.

. Therefore, the present invention also provides a method for producing such a poxviral M2 polypeptide comprising (a) introducing a M2-encoding expression vector into a suitable producer host cell to produce a transfected or infected producer host cell, (b) culturing in-vitro said transfected or infected producer host cell under conditions suitable for its growth, (c) recovering the M2 polypeptide from the cell culture, and (d) optionally, purifying the recovered M2 polypeptide.

In step a), the M2-expressing vector can be introduced into the producer host cells via conventional infection or transfection techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation or viral infection. For stable transfection of mammalian cells, identification of the recombinant host cells having incorporated the M2-expression vector can be identified by drug selection corresponding to the selectable marker gene (e.g., resistance to antibiotics) present in the expression vector. Cells having incorporated the selectable marker gene will survive, while the other cells die. The surviving cells can then be screened for production of the M2 polypeptide. In addition, the M2 expression vector can, where appropriate, be combined with one or more substances which improve the transfection efficiency and/or stability of the vector into the producer cell. These substances are widely documented in the literature. Representative examples of transfection reagents able to facilitate introduction of the vector in the producer cell, include without limitation polycationic polymers (e.g. chitosan, polymethacrylate, PEI, etc), cationic lipids (e.g. DC-Chol/DOPE, transfectam lipofectin now available from Promega) and liposomes. In the context of the invention, producer host cells include prokaryotic cells, lower eukaryotic cells such as yeast, and other eukaryotic cells such as insect cells, plant and mammalian (e.g. human or non-human) cells.

Suitable prokaryotic producer cells encompass Gram-negative as well as Gram- positive bacteria, for example, Escherichia (e.g., E. coli), Enterobacter, Klebsiella, Proteus, Shigella, as well as Bacilli such as Bacillus subtilis, Pseudomonas such as P. aeruginosa, and Streptomyces. Exemplary E. coli host cells include without limitation E. coli 294 (ATCC 31 ,446), E. coli BL21(DE3 Amersham Biosciences), E. coli XI 776 (ATCC 31 ,537), and E. coli W3110 (ATCC 27,325). In addition to prokaryotes, yeasts are also suitable as expression host cells, in particular Saccharomyces cerevisiae, or common baker's yeast as well as Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045) and Pichia pastoris (EP 183,070). Plant and insect cells are also suitable as producer host cells. Suitable insect cells to be used for baculovirus- mediated expression include Sf 9 cells (ATCC CRL-1711) and Tabacco BY-2 cells are adequate as producer cells with plant viral vectors (Huang and McDonald, 2009, Biochemical Engineering Journal 45(3): 168-84).

In a preferred embodiment, the producer host cells are mammalian cells, with a specific preference for mammalian cell lines that are adapted to grow in suspension. Examples of useful mammalian cell lines are African monkey kidney cell line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 cells; Graham et al., 1977, J. Gen Virol. 36: 59-74), baby hamster kidney cells (e.g. BHK-21 , ATCC CCL 10), Chinese hamster ovary cells (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-51), monkey kidney cells (CV1 ; ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL- 1587), human cervical carcinoma cells (HeLa, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), MRC 5 cells (ATCC CCL-171), mouse NIH/3T3 cells (ATCC CRL-1658) and PERC.6 cells (Fallaux et al., 1998, Hum Gene Ther. 9: 1909-17).

In step b), the producer cells can be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Culturing can be carried out in fermentation bioreactors, flasks and petri plates at a temperature, pH and oxygen content appropriate for a given producer cell. No attempts to describe in detail the various methods known for the production of proteins in prokaryotic and eukaryotic cells will be made here. Production of the M2 polypeptide can be periplasmic, intracellular or preferably secreted outside the producer cell (e.g. in the culture medium).

In step c), preferred embodiment is directed to the recovery of the M2 polypeptide directly in the cell culture (i.e. culture medium). However, step c) may include a disruption step if needed, e.g. if the M2 polypeptide is not or not completely secreted outside the producer cell. The disruption step may be carried out by various physical or chemical means including freeze thaw, sonication, mechanical disruption, chemical lysis (for example with a detergent such as Tween®) among others.

Optional step d) contemplates that the M2 polypeptide can be at least partially purified to remove contaminants before being used according to the present invention. Preferably, the resulting M2 preparation is substantially free of at least 50% of contaminants with which the M2 polypeptide naturally occurs in the producer host cell. Various methods of protein purification may be employed and such methods are known in the art and described for example in Methods in Enzymology, 182 (1990); Protein Purification: Principles and Practice, Springer- Verlag, New York. Such methods include without limitation precipitation (ethanol; ammonium sulphate precipitation), acid extraction, gel electrophoresis, filtration and chromatographic methods (e.g. reverse phase HPLC, size exclusion, ion exchange, affinity, phosphocellulose, hydrophobic-interaction or hydroxylapatite chromatography, etc). The conditions and technology used to purify a particular protein will depend on factors such as net charge, molecular weight, hydrophobicity, hydrophilicity and will be apparent to those having skill in the art. Moreover, the level of purification will depend on the intended use. It is also understood that depending upon the producer cell, the M2 polypeptide can have various glycosylation patterns or may be non-glycosylated (e.g. when produced in bacteria).

In a preferred embodiment, the recombinantly produced M2 polypeptide prepared according to the method of the present invention is oligomeric, preferably pentameric, hexameric, heptameric or octameric M2 or a mixture thereof.

An exemplary method for producing the M2 polypeptide described herein comprises (a) introducing a M2-encoding expression vector (preferably a plasmid or a retrovirus vector) into a Chinese Hamster Ovary (CHO) cell line to produce a stably transfected or infected CHO cell, wherein said vector encodes the M2 polypeptide comprising a signal peptide and optionally a tag, (b) culturing in-vitro said transfected or infected CHO cell line under conditions suitable for its growth, (c) recovering the M2 polypeptide in the cell supernatant, and (d) optionally, purifying the recovered M2 polypeptide (e.g. by affinity chromatography). Composition and Formulation of the M2 polypeptide

The M2 polypeptide described herein or an expression vector thereof can be utilized in compositions suitable for pharmaceutical administration as described in detail herein. Therefore, the present invention also pertains to a composition comprising a therapeutically effective amount of the M2 polypeptide (or any variant, fragment or fusion thereof) or the M2 expression vector described herein and a pharmaceutically acceptable carrier. Also provided is a composition, preferably a composition, comprising a poxviral M2 polypeptide or an encoding vector thereof for use as a medicament, and more specifically as an immunosuppressive drug. Compositions of the present invention are formulated, dosed, and administered in a fashion consistent with good medical practice.

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic beneficial results described herein (e.g. delivering an immunosuppressive effect). Factors for consideration in this context include the particular disorder being treated, the particular subject (e.g. age and weight), the clinical condition of the individual subject and his ability to respond to the treatment, the cause of the disorder, the severity and course of the disease, the mode of administration (e.g. site of delivery, administration route, the scheduling of administration), kind of concurrent treatment and other factors known to medical staff. Typically, a therapeutically effective amount is also one in which any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects. For general guidance, an "amount therapeutically effective to inhibit transplant rejection" refers to an amount of M2 polypeptide or expression vector that when administered to a transplant recipient, inhibits, either partially or completely, rejection of the transplant relative to an untreated control. As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. For general guidance, appropriate carriers for use herein are well known the art (see for example the most current edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams&Wilkins). Desirably, the composition of the invention is formulated appropriately to ensure its stability under the conditions of manufacture and long-term storage (i.e. for at least 6 months, with a preference for at least two years) at freezing (e.g. -70°C, -20°C), refrigerated (e.g. 4°C) or ambient (e.g. 20-25°C) temperature and it must also be preserved against the contaminating action of microorganisms such as bacteria and fungi. Exemplary physiologically acceptable carriers that may be used in the context of the present invention include, but are not limited to, water, saline, buffers (such as phosphate, Tris-HCI, HEPES, citrate, and other organic acids), antioxidants (such as ascorbic acid and methionine), proteins (such as serum albumin or gelatin), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), polyol (such as, glycerol, propylene glycol, and liquid polyetheylene glycol), as well as other aqueous physiologically balanced salt solutions and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars (e.g. monosaccharides, disaccharides and other carbohydrates such as glucose, mannose, or dextrins), polyalcohols (such as manitol, sorbitol) and sodium chloride in the composition.

Prevention of contamination by microorganisms can be achieved by various antibacterial and antifungal agents, such as, parabens, chlorobutanol, phenol, ascorbic acid, octadecyldimethylbenzyl ammonium chloride, thimerosal, and the like.

The composition may also include a cryoprotectant so as to ensure its stability at low storage temperature. Suitable cryoprotectants include without limitation sucrose (or saccharose), trehalose, maltose, lactose, mannitol, sorbitol and glycerol, preferably in a concentration of 0.5 to 20% (weight in g/volume in L, referred to as w/v). For example, sucrose is preferably present in a concentration of 5 to 15% (w/v). High molecular weight polymers such as dextran or polyvinylpyrrolidone (PVP) are particularly suited for freeze-dried compositions, usually obtained by a process involving vacuum drying and freeze-drying and the presence of these polymers assists in the formation of the cake during freeze-drying (see e.g. WO03/053463; W02006/085082; W02007/056847; W02008/114021 and

WO2014/053571 ) .

The composition, and especially liquid composition, may further comprise a pharmaceutically acceptable chelating agent, and in particular an agent chelating di-cations for improving stability. The pharmaceutically acceptable chelating agent may notably be selected from ethylenediaminetetraacetic acid (EDTA), 1 ,2-bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA), ethylene glycol tetraacetic acid (EGTA), dimercaptosuccinic acid (DMSA), diethylene triamine pentaacetic acid (DTPA), and 2,3- Dimercapto-1-propanesulfonic acid (DMPS). The pharmaceutically acceptable chelating agent is typically present in a concentration of at least 50 mM with a specific preference for a concentration of 50 to 1000 pM. For example, a chelating agent such as EDTA may be present in a concentration close to 150 pM.

Additional compounds may further be present to increase stability of the composition. Such additional compounds include, without limitation, C2-C3 alcohol (desirably in a concentration of 0.05 to 5% (volume/volume or v/v)), sodium glutamate (desirably in a concentration lower than 10 mM), non-ionic surfactant (US7,456,009, US2007-0161085) such as Tween 80 (also known as polysorbate 80) at concentration below 0.1 %. Divalent salts such as MgCh or CaCh may also contribute to stabilization of liquid biological compositions (see Evans et al. 2004, J Pharm Sci. 93:2458-75 and US7,456,009).

The composition can be formulated to provide quick, sustained, or delayed release of the active agent(s) after administration. Suitable examples of sustained-release compositions include semipermeable matrices of solid hydrophobic polymers containing the drug, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

In one embodiment, the composition of the present invention is formulated for oral administration, e.g. under forms including solid, semi-solid and liquid systems such as tablets, soft or hard capsules containing multi- or nanoparticulates, liquids, powders, chews, gels, fast dispersing dosage forms, films, ovules, sprays and buccal/mucoadhesive patches. For example, the M2 polypeptide may be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose, gelatin or poly- (methylmethacylate) microcapsules, respectively.

In another and preferred embodiment, the composition is formulated for parenteral administration. Sterile injectable solutions can be prepared by incorporating the active agent (i.e. the M2 polypeptide or expression vector) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Each unit contains a predetermined quantity of the M2 active agent calculated to produce the desired therapeutic effect in association with the selected pharmaceutically acceptable carrier(s).

Depending on the route of administration, the composition may be coated in a material to protect the M2 active agent from the action of enzymes, acids and other natural conditions which may inactivate said active agent. In this regard, PEGylation can be also be envisaged using several PEG attachment moieties including, but not limited to N- hydroxylsuccinimide active ester, succinimidyl propionate, maleimide, vinyl sulfone, or thiol. A PEG polymer can be linked to the M2 polypeptide at either a predetermined position or can be randomly linked. PEGylation can also be mediated through a peptide linker attached to a domain of the polypeptide. Methods of PEGylating are well known in the art (see e.g. Chapman, et al. , 2002, Adv. Drug Deliv. Rev. 54(4): 531-45). Dosing of the M2 active agent

The amount of M2-based active agent in the composition of the present invention, the administration route and the periods of time necessary to achieve the desired result (immunosuppressive effect) can be routinely defined by medical staff considering the various factors mentioned above.

For illustrative purposes, a therapeutically effective amount of the poxviral M2 polypeptide to be included in the composition described herein (individual doses) would be in the range from about is 0.05 to about 100 mg/kg relative to the body weight of the patient (desirably from 1 to 50 mg/kg, preferably from 2 to 40mg/kg, more preferably from 5 to 30mg/kg, and even more preferably 7 to 20mg/kg). For example, 500mg of M2 polypeptide- based composition are suitable for injection to patients weighting less than 60kg, 750mg for injection to patients weighting between 60kg and 100kg and 1000mg for injection to patients weighting more than 100kg. Individual doses of 10mg/kg are also appropriate.

Alternatively, the skilled person in the art is capable of defining appropriate therapeutically effective amount of M2-expression vector to be included in the composition described herein, according to the type of vector used. Exemplary individual doses for M2- expressing viral vector contain from about 10 ³ to 10 ¹² vp (viral particles), vg (viral genome), iu (infectious unit) or pfu (plaque-forming units) of a specific viral vector. Various techniques are available to assess a virus titer, e.g. by counting the number of plaques following infection of permissive cells to obtain a plaque forming units (pfu) titer, by measuring the A260 absorbance (vp titers), by measuring the number of viral genome copy by qPCT (vg) or still by quantitative immunofluorescence, e.g. using anti-virus antibodies (iu titers). As a general guidance, individual doses which are suitable for a virus composition comprise from approximately 10 ³ to approximately 10 ¹² pfu, advantageously from approximately 10 ⁴ pfu to approximately 10 ¹¹ pfu, preferably from approximately 10 ⁵ pfu to approximately 10 ¹ ° pfu; and more preferably from approximately 10 ⁶ pfu to approximately 10 ⁹ pfu and notably individual doses of approximately 10 ⁷ , 5x10 ⁷ , 10 ⁸ or 5x10 ⁸ pfu are particularly preferred. Doses of 10 ¹⁰ to 10 ¹⁴ vg/kg are also appropriate. Further refinement of the calculations necessary to adapt the appropriate dosage for a subject or a group of subjects may be routinely made by a practitioner, in the light of the relevant circumstances.

Administration routes

The composition of the present invention is formulated for administration once or several times via the same or different routes. Any of the conventional administration routes is applicable in the context of the invention including parenteral, topical and mucosal routes. Parenteral routes are intended for administration as an injection or infusion and encompass systemic as well as local routes. Preferably, the composition is formulated for one or more parenteral administration(s), and preferably intravenous (into a vein), intravascular (into a blood vessel), intra-arterial (into an artery), intradermal (into the dermis), subcutaneous (under the skin), intramuscular (into muscle) or intraperitoneal (into the peritoneum) route. Administration can be in the form of a single bolus dose or may also be by a continuous perfusion pump. Mucosal administrations include without limitation oral/alimentary, intranasal, intratracheal, intrapulmonary, intravaginal or intra-rectal route. Topical administration can also be performed using transdermal means (e.g. patch and the like). Preferably, the M2-based composition is formulated for administration by intravenous infusion.

Administrations may use conventional syringes and needles (e.g. Quadrafuse injection needles) or any compound or device available in the art capable of facilitating or improving delivery of a virus in the subject (e.g. electroporation for facilitating intramuscular administration). An alternative is the use of a needleless injection device (e.g. Biojector TM device). Transdermal patches may also be envisaged.

Several doses within the indicated ranges may be administered to the patient. For repeated administrations over several days or longer, the treatment would generally be sustained until an observable clinical benefit occurs. Such doses may be administered intermittently, e.g. every day, every 2 or 3 days, every week, every 2 weeks, every three weeks or every month (e.g. such that the subject receives from about two to about twenty doses of the composition). Doses may also be adapted at each administration (e.g., one or more initial higher dose(s) followed by one or more lower dose(s)).

Methods for using the composition of the invention The composition and vector described herein are suitable for administration to subjects for use for delivering an immunosuppressive signal or for treating any of the foregoing disorders as described herein.

The compositions and methods of the invention can be used in many clinical situations, as described further below. Given the ability of the M2 polypeptide to bind CD80 and/or CD86, the effect of administration of the composition is inhibitory, leading to blockage by the M2 polypeptide of CD28 triggering resulting from T cell/APC contact, with the aim of downregulating an immune response. For example, the composition or vector described herein may be used to block T cell proliferation and, thus, antagonizing a T cell response. It may also be used to induce an immunosuppressive state of APC or B cells (reverse signalling).

Embodiments provide a method/use of the composition described herein in a subject in need thereof for downregulating an immune response, for inducing an immunosuppressive response as well as for inducing an immunotolerance response in an amount sufficient to downregulate said immune response, induce an immunosuppressive state or induce an immunotolerance response according to the modalities described herein.

The compositions and methods according to the present invention are particularly useful for providing at least one of the following properties:

for antagonizing a T cell response;

for inhibiting T cell activation;

for inhibiting the interaction of CD28-positive cells with its ligands CD80 and CD86; for inducing an immunosuppressive state of APC or B cells.

for inducing cell death or anergy of any CD80 and or CD86 positive cells like APC or B cells or T cells (reverse signalling) and/or

for inducing survival or proliferation of Treg cells.

Inhibiting as used herein refers to a decrease of at least 50% with respect to an appropriate control (e.g. after versus before treatment or treated versus non-treated subject). The composition described herein is particularly suited for use for treating diseases or disorders associates with non-adequate immune responses including autoimmune diseases and graft-related diseases. Another embodiment relates to a method of treating such diseases or disorders in a subject in need thereof comprising administering to the subject the composition described herein in an amount sufficient to improve the clinical status of said subject according to the modalities described herein.

Autoimmune diseases

Further provided is the composition described herein for use for treating a patient having or at risk of having an autoimmune disease and a method of treating an autoimmune disease comprising administering said composition with the goal of moderating the progression and/or severity of such an autoimmune disease.

Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self-tissue (i.e., reactive against autoantigens) and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases or destroying the“self” tissues (autoreactive cytolytic T cells). Preventing the activation of autoreactive T cells by providing an immunosuppressive signal thus may reduce or eliminate disease symptoms. Administration of the composition described herein to a subject suffering from an autoimmune disorder may at least partially inhibit autoantigen-specific T cell responses. Improvement of the clinical status may be evidenced by a decrease in production of autoantibodies or T cell-derived cytokines which are known to be involved in the disease process.

The composition and methods of the present invention can be used to treat a variety of autoimmune diseases and disorders having an autoimmune component encompassing both organ-specific autoimmune disease wherein the immune response is specifically directed against an organ system (e.g., the endocrine system (e.g., the thyroid), the hematopoietic system, the skin, the ear, the cardiopulmonary system, the gastrointestinal system, the renal system, the liver, the neuromuscular system, the central nervous system, etc.)) and systemic autoimmune disease which affects multiple organ systems. The present invention also encompasses chronic and acute autoimmune diseases.

Exemplary autoimmune diseases include autoimmune rheumatologic disorders (such as rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, gout, septic arthritis and Sjogren's syndrome), lupus (such as systemic lupus erythematosus (SLE) and lupus nephritis), polymyositis/dermatomyositis, cryoglobulinemia, anti phospholipid antibody syndrome), autoimmune gastrointestinal and liver disorders (such as inflammatory bowel diseases; ulcerative colitis, Crohn's disease, autoimmune gastritis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, aphthous ulcer and celiac disease), vasculitis (such as ANCA-negative vasculitis and ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and microscopic polyangiitis), autoimmune neurological disorders (such as multiple sclerosis (MS), opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as glomerulonephritis, Goodpasture's syndrome and Berger's disease), autoimmune dermatologic disorders (such as dermatitis including atopic dermatitis and eczematous dermatitis, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, autoimmune endocrine disorders (such as diabetic-related autoimmune diseases (e.g., insulin-dependent diabetes mellitus (IDDM)), Addison's disease, and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis), allergic condition and autoimmune inflammatory responses. Each of these diseases is characterized by T cell receptors that bind to endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases.

Specific aspect of this embodiment is directed to the M2 composition for use for treating diabetes and notably IDDM. An exemplary use or method for treating diabetes, may comprise transplanting insulin producing cells into a subject in need thereof and administering concomitantly or further transplantation an effective amount of the composition disclosed herein to inhibit or reduce the risk of rejection of the insulin-producing cells. Preferably the insulin producing cells are beta cells or islet cells but may also be recombinant cells engineered to produce insulin. The insulin producing cells may also be genetically modified to produce the M2 polypeptide by delivering a vector encoding the M2 polypeptide. In the context of the present invention, the insulin producing cells can be encapsulated within a matrix, such as a polymeric matrix, using suitable polymers, including but not limited to, alginate, agarose, hyaluronic acid, collagen, synthetic monomers, albumin, fibrinogen, fibronectin, vitronectin, laminin, dextran, dextran sulphate, chondroitin sulphate, dermatan sulphate, keratan sulphate, chitin, chitosan, heparan, heparan sulphate, or a combination thereof.

In still another specific aspect of this embodiment, the composition is for use for treating arthritis and notably, rheumatoid arthritis (RA). Typically, arthritis is characterized by a chronic systemic inflammation at a joint generated by a variety of causes with resultant injury to the articular cartilage and ultimately joint destruction. The main presenting symptoms of arthritis are pain, stiffness, swelling, and/or loss of function of one or more joints. Among various types of arthritis, rheumatoid arthritis is a systemic chronic inflammatory disease and an autoimmune disease. An exemplary method for treating rheumatoid arthritis comprises the administration of the M2 composition concomitantly or in sequential with conventional drugs such as nonsteroidal anti-inflammatory drugs (NSAID), disease-modifying anti-rheumatic drugs (DMARD), adrenocortical hormones, and tumor necrosis factor (TNF) antagonists.

In a further aspect, the composition is for use for treating allergic conditions and responses involving non-self-antigens. Exposure to the allergen may be environmental or may involve administering the allergen to the subject. Allergic reactions may be systemic or local in nature, depending on the route of entry of the allergen. Representative examples of such allergic immune diseases or conditions include, without limitation, allergic dermatitis (e.g. allergic contact dermatitis, non-specific dermatitis, primary irritant contact dermatitis), allergic intraocular inflammatory diseases, chronic allergic urticaria, food allergies, drug allergies, insect allergies, rare allergic disorders such as mastocytosis, allergic reaction, eczema (e.g. allergic or atopic eczema), asthma (such as bronchial asthma, and auto immune asthma), among others. The IgE antibody response in atopic allergy is highly T cell dependent and, thus, administering the M2 composition described herein may be useful therapeutically in the treatment of allergy and allergic reactions, to inhibit T cell mediated allergic responses in the subject. An exemplary method comprises administering the M2 composition described herein together with the allergen with the goal of inducing an immune tolerance during a process of allergen desensitization.

In an additional aspect, the composition described herein is for use for treating autoimmune inflammatory responses, particularly those resulting from infiltration of T cells. Representative examples include, but are not limited to, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, paediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE), such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes.

In an additional aspect, the composition of the invention is also for use for treating autoimmune reactions induced by drug treatment including treatment with immune checkpoint inhibitors (ICI).

The invention also provides a method of reducing the amount of proinflammatory cytokines or other molecules (e.g. IL- Ib, TNF-a, TGF-beta, IFN-g, IL-17, IL-6, IL-23, IL-22, IL- 21 , MMPs, etc) associated with or that promote inflammation and especially a method of treating chronic or acute inflammatory responses upon administration of the M2 composition disclosed herein. Preferably reduction of proinflammatory cytokines and other molecules is at least 10%, preferably at least 20% and more preferably at least 40%. Preferred conditions to be treated with the method or use disclosed herein are autoimmune inflammatory diseases such as inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease. The efficacy of the M2 composition described herein in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., 1989, Fundamental Immunology, Raven Press, New York, pp. 840-856). In addition, the composition described herein is particularly useful for treating patients that do not respond to conventional anti-inflammatory treatment, and particularly to TNF-a blockers such as Enbrel, Remicade, Cimzia and Humira.

Graft related diseases In another and also preferred embodiment, the composition described herein is suited for use for treating a transplanted patient or a patient in the process of being transplanted and at risk of having a graft-rejection or for reducing the risk of graft rejection in a transplanted patient or a patient in the process of being transplanted. Administration of said M2 composition may inhibit undesired T cell responses occurring during or after transplant rejection and favour immune tolerance; resulting in a prolonged (long-term) graft acceptance without the need for generalized immunosuppression or at a lesser extent. It has been shown that inhibiting the PDL1-CD80 interaction has a negative impact of the transplantation success. Given the fact that the M2 polypeptide comprised or expressed by said composition potentiates interaction of CD80 and PDL1 , it is particularly appropriate for treating transplanted subject with the goal of reducing the risk of transplant rejection.

In a specific embodiment, the composition for use according to the present invention is also appropriate for treating a graft versus host disease (GVHD). GVHD is a major complication associated with transplantation of allogeneic hematopoietic stem cells in which functional immune cells in the transplanted marrow recognize the recipient as "foreign" and mount a vigorous immunologic attack against the recipient’s organs. Inhibition of T cell proliferation and/or cytokine secretion by the composition may result in reduced destruction in tissue transplantation and induction of antigen-specific T cell unresponsiveness. Symptoms of GVHD include skin rash or change in skin colour or texture, diarrhoea, nausea, abnormal liver function, yellowing of the skin, increased susceptibility to infection, dry, irritated eyes, and sensitive or dry mouth.

Clinical improvement may be evidenced by, e.g., a reduction of transplant rejection events in the population of patients treated with the composition as compared to the non- treated, a prolongation of transplant survival (notably allograft or non-self-transplants) and/or the reduction of graft related complications including GVHD, a reduction in the amount of proinflammatory cytokines upon surgical transplantation and M2 treatment.

The composition described herein may be administered to the site of transplantation prior to, at the time of, or systemically following transplantation. In one aspect, the M2 composition is administered to the site of transplantation parenterally, such as by subcutaneously or intravenously. In another aspect, it may be administered ex vivo directly to the transplant. For example, the transplant may be incubated with the M2 composition prior to transplantation. Vectors encoding the M2 polypeptide as described herein can also be used to deliver the M2 polypeptide locally in vivo or ex vivo, for example to the transplant (e.g. islet cells).

An exemplary method or use for treating transplant rejection comprises at least 6 administrations of the M2 composition described herein at a dose of approximately 10mg/kg for about 4 months following transplantation (e.g., at day 1 , D5, D14 and then every two weeks up to the 16th week) followed by at least 4 administrations of the composition at a maintenance dose of approximately 5mg/kg once a month. The progress of this therapy is easily monitored by conventional techniques and assays.

In another aspect, the composition described herein is particularly suited for accompanying a gene therapy treatment. Typically, gene therapy is based on genes as therapeutic agents for preventing or treating inherited disorders. Typically, genetic material is introduced into cells to compensate for abnormal genes or to make a beneficial protein. In one aspect, the composition may be used so as to be expressed in genetically modified cells, so as to tolerize such cells prior to transplantation. Such a method comprises the step of transfecting the cells ex vivo with a vector enabling the cells to express the M2 polypeptide (together with the genetic material) for a period of time sufficient to inhibit or reduce transplant rejection in the transplanted subject. For example, such a method can include the steps of harvesting cells from a donor, culturing the cells, transducing them with an expression vector encoding the M2 polypeptide, and maintaining the cells under conditions suitable for expression of the encoded M2 polypeptide. The transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. A vast number of appropriate vectors for providing sustained expression of the M2 polypeptide are available in the art including those described above. AAV (for Adenovirus Associated Virus) vector are particularly preferred in this context. Cells that have been successfully transduced then can be selected, for example, for expression of the M2 polypeptide (e.g., by RT-qPCR for detecting mRNA or by ELISA, Western Blot, fluorescence means, etc for detecting M2) or by the mean of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject.

In certain embodiment, a viral vector is injected directly into patients with genetic disorder to restore an expression of the defective gene. One limitation of this approach is the duration of gene expression (a few months to a few years depending of dose, delivery vector, transgene, route of administration, etc.,) and immunogenicity itself of the viral vector that prohibit a second injection of the therapeutic viral vector. In this case, co-injection of M2 with the therapeutic viral vector or co-expression of M2, in addition of the transgene of interest, by the viral vector itself could reduce the immune response against the vector and allow a re- administration.

Additional agents that inhibit the generation of stimulatory signals in the T cells (e.g., an anti-IL-2R antibody etc.,) can be included in the culture. After transplantation, the recipient may be further treated by in vivo administration of the M2 composition and/or with conventional anti-inflammatory or other immunosuppressive drugs.

In certain embodiments, the methods or uses comprises a further step wherein the transplant can be treated with enzymes or other compounds that remove cell surface proteins, carbohydrates and/or lipids (that are known or suspected in being involved with immune responses leading to transplant rejection).

Transplant In the context of the invention, the transplant can be cells, tissues, organs (e.g. heart, spleen, etc), portions of the body (e.g. digits, limbs) or the like. The graft-related disease can further be an acute transplant rejection. Alternatively, the graft-related disease can be a chronic transplant rejection.

In one aspect, the transplant can be syngeneic, allogenic or xenograft with respect to the recipient subject. The term "syngeneic" refers to genetically identical or closely related organisms (organisms referring to the recipient on the one hand and the transplant donor on the other hand). The term "allogeneic" refers to organisms of the same species, but genetically different one from another. The term "xenogenic" applies when the donor and recipient are of different species. The transplants are preferably allogenic.

Populations of any types of cells can be transplanted into a subject. The cells to be transplanted can be homogenous or heterogenous (i.e. containing more than one type of cell) and can be progenitor cells or pluripotent cells. Exemplary cells to be transplanted include without limitation, islet cells, hematopoietic cells, muscle cells, cardiac cells, neural cells, embryonic stem cells, adult stem cells, T cells, lymphocytes, dermal cells, mesoderm, endoderm, and ectoderm cells which can be harvested from a donor and transplanted into a recipient subject. The cells are optionally treated prior to transplantation as mentioned above.

Any tissue can be used for being transplantation. Exemplary tissues include skin, adipose tissue, cardiovascular tissue such as veins, arteries, capillaries, valves, neural tissue, bone marrow, pulmonary tissue, ocular tissue such as corneas and lens, cartilage, bone, and mucosal tissue. The tissue can be modified as discussed above. Exemplary organs that can be used for transplantation include, but are not limited to kidney, liver, heart, spleen, bladder, lung, stomach, eye, tongue, pancreas, intestine, etc. The organ to be transplanted can also be modified prior to transplantation as discussed above.

Selection of the subject to be transplanted Neutrophils are thought to contribute to early allograph rejection (Healy, et al. , 2006,

Eur J Cardiothorac Surg, 29: 760-6). Therefore, in certain subjects, elevated levels of neutrophils may be predictive of transplantation rejection, particularly acute rejection. A biological sample from a subject to be transplanted can be assayed for neutrophil levels and compared to the « normal » levels of neutrophils in a healthy individual (ranges from about 15,000 to 20,000 cells/mI). Thus, a subject determined as having neutrophil levels above the « normal » level can be correlated to a higher risk of transplant rejection than a subject having neutrophil levels within the normal range. Methods for measuring a level of neutrophils in a biological sample are routinely carried out in medical laboratories. In one embodiment, the composition described herein is for use for treating a transplanted subject or a subject in the process of being transplanted, wherein said subject has neutrophil levels above a normal level as determined in one or more healthy individual(s)

The therapeutic efficacy of the compositions and the methods disclosed herein and its ability to induce immunosuppressive responses can be assessed by a variety of assays known to those skilled in the art, using various animal transplantation models and autoimmune disease models (e.g. lupus, multiple sclerosis, diabetes, and arthritis models). For example, the efficacy of the composition and method of the invention in inhibiting organ transplant rejection or GVHD can be assessed in various models of organ transplantation that may be predictive of efficacy in humans. For example, murine models of GVHD (see Paul ed., 1989, Fundamental Immunology, Raven Press, New York, pp. 846-7) can be used to determine the effect of M2 composition on the prevention of GVHD complications.

Models of allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, are available to ascertain the immunosuppressive ability of the composition in vivo (Lenschow et al., 1992, Science, 257: 789-92 and Turka et al., 1992, Proc. Natl. Acad. Sci. USA. 89: 11102-5). For example, recipient Lewis rats receiving a Brown-Norway rat strain cardiac allograft which is anastomosed to vessels in the neck are described in Bolling et al. (1992, Transplant, 453: 283-6). Preferentially, the model animal could be transgenic animal expressing human CD86 and/or CD80 to mimic the human situation. Grafts are monitored for mechanical function by palpation and for electrophysiologic function by electrocardiogram. Graft rejection is said to occur on the last day of palpable contractile function. As an initial test, animals are treated with injections of the M2 polypeptide of interest for about 7 days and the rejection of allografts by the M2-treated animals is assessed in comparison to untreated controls. Untreated animals typically reject allografts in about 7 days. Histological examination can be carried out on the grafted tissue in sacrificed untreated animal and M2-treated animals... For example, a prominent interstitial mononuclear cell infiltrate with oedema formation, myocyte destruction, and infiltration of arteriolar walls may be indicative of severe acute cellular rejection. The lymphocytes from the treated and untreated animals can also be tested for their functional responses (e.g. T cell proliferative response to ConA, etc). Additionally, the thymus and spleen from the untreated and treated animals can be compared in size, cell number and cell type (e.g. by flow cytometic analyses of thymus, lymph nodes and spleen cells) as well as the ability of the T cells to respond to ConA. Prolonged acceptance of grafts and lack of an acute cellular rejection in the tissue following M2 treatment in such model systems may be predictive of the therapeutic efficacy of M2 composition in human transplant situation.

As another application, the composition described herein can be used in screening assays to identify yet undefined compounds which inhibit an interaction between a T cell receptor such as PD-L1 , CD28 and CTLA4 and CD80 or CD86. For example, the screening method of the invention involves contacting the composition with CD80 or CD86 and a compound to be tested. Either the M2 polypeptide or the CD80 or CD86 ligand can be labelled with a detectable substance, such as a radiolabel or biotin, to allow detection and quantitation of the amount of binding the M2 polypeptide to CD80 or CD86. After allowing the M2 polypeptide and the CD80/CD86 ligand to interact in the presence of the compound to be tested, unbound labelled M2 polypeptide or unbound labelled CD80/CD86 ligand is removed and the amount of M2 polypeptide bound to the CD80/CD86 ligand is determined. A reduced amount of binding of M2 polypeptide to CD80/CD86 ligand in the presence of the compound tested relative to the amount of binding in the absence of the compound is indicative of an ability of the compound to bind CD80 and/or CD86 at the same site as M2. Since the compound shares the same binding site of CD80 or CD86, it could have some properties close to the one of M2. Suitably, one of the M2 polypeptide or CD80/CD86 ligand can be immobilized on a solid phase support, such as a polystyrene plate or bead, to facilitate removal of the unbound labelled protein from the bound labelled protein.

In another embodiment, the composition described herein may also be useful if destruction of the cell is needed. For instance, it is possible to generate an M2 polypeptide- based immunotoxin by linking a to a toxin such as ricin or diptheria toxin (chemical conjugation or recombinant means) with the goal of killing activated T cell and/or B cells and/or APC. Infusion of immunotoxin into a patient would result in the death of immune cells, particularly of activated B cells and APC that express high amounts of CD80 and/or CD86.

Combination therapies

The composition of the invention can be used alone or in combination therapy with one or more other therapeutic agent(s). The additional therapeutic agents may be used to improve the efficacy or safety of the M2-based composition and may be administered in a single composition with the M2 polypeptide (mixture) or as a separate composition administered at the same time (simultaneously) or separately (sequentially).

The term "in combination with" is not limited to the administration of said additional therapeutic agent(s) at exactly the same time. Instead, it is meant that the M2 composition disclosed herein and the other agent(s) may be administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment with only either the composition disclosed herein or the other therapeutic agent(s). The skilled medical practitioner can determine empirically, or by considering the pharmacokinetics and modes of action of the agents, the appropriate dose or doses of each therapeutic agent, as well as the appropriate timings and methods of administration on a patient-to-patient basis.

Useful additional therapeutic agents in the context of the present invention are therapeutic agents that influence immune responses through the same or different pathways. In particular, another immunomodulating agent can be used to downregulate further T or B cell mediated immune responses. Examples of such immunomodulating agents for use in association with the M2 composition include, without limitation, blocking antibodies, cytokines, anti-inflammatory agents and immunosuppressive agents.

Exemplary immunomodulating agents for use herein may be e.g., fusion proteins (e.g. CTLA4-Fc Nulojix® Orencia®) blocking antibodies (e.g., against CD28, CD80, CD86 ) or other lymphocyte surface markers (e.g., CD40, alpha-4 integrin) or against cytokines, (e.g., TNF-a blockers such as Enbrel®, Remicade, Cimzia and Humira, cyclophosphamide (CTX) (e.G. Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), methotrexate (MTX) (e.g. Rheumatrex®, Trexall®), belimumab (i.e. Benlysta®), or other immunosuppressive agents (e.g., cyclosporin A, FK506-like compounds, rapamycin compounds, or steroids) or any other agents that may assist in immunosuppression.

More specifically, the additional immunomodulating agent can also function in concert with the M2 composition on the B7-mediated co-stimulatory pathway. For example, Abatacept® or Belatacept® may be used in this context, especially for the treatment of RA and graft rejections. More precisely, the M2 composition could be used in the beginning of the treatment or at treatment stage where the immune response is exacerbated; and Abatacept® or Belatacept® as maintenance drug i.e., once the immunosuppression is well established or re-established by M2 treatment. An advantage of complementing M2 action with less potent CTLA4-Fc fusions is that they may be more tolerated by the subject and therefore allow a long-term treatment such as the one considered for organ transplantation. Alternatively, the additional immunomodulating agent may act through other pathways than B7-mediated one. For example, blocking CD40L activation of APCs with anti-CD40L antibodies may be contemplated as well as TNF-a blockers and Treg enhancing agents (such as glucocorticoid fluticasone, salmeteroal, antibodies to IL- 12, IFN-g, IL-4, vitamin D3, and dexamethasone).

Anti-inflammatory agents can also be used in combination or alternation with the disclosed M2 composition. The anti-inflammatory agent can be non-steroidal, steroidal. Representative examples of non-steroidal anti-inflammatory agents include, without limitation, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, such as aspirin, disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulmdac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fenfiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, and tolfenamic acids; propionic acid derivatives, such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone and trimethazone. Representative examples of steroidal anti-inflammatory drugs include, without limitation, corticosteroids such as hydrocortisone, hydroxyl- triamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fiuadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, fiucortine butylesters, fluocortolone, fluprednidene (fiuprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetates hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflurosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, difiurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate and triamcinolone. Antibodies to proinflammatory molecules such as IL-2, IL-23, IL-22 or IL-21 may also be employed in the context of the present invention.

In another embodiment, the M2 composition may also be useful in combination with a immunogenic drug which may induce unwanted immunogenic responses, thus reducing its efficacy. Combination of such an immunogenic drug with the immunosuppressive M2 composition may prevent such unwanted immunogenic responses.

Article of manufacture

The invention also pertains to an article of manufacture containing materials useful for the treatment of the disorders described above. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the targeted condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

All of the above cited disclosures of patents, publications and database entries are specifically incorporated herein by reference in their entirety. Other features, objects, and advantages of the invention will be apparent from the description and drawings and from the claims. The following examples are incorporated to demonstrate preferred embodiments of the invention. However, in light of the present disclosure, those skilled in the art should appreciate that changes can be made in the specific embodiments that are disclosed without departing from the spirit and scope of the invention. EXAMPLES

Material and Methods

Proteins and viruses

Recombinant Fc fusion proteins (human and murine) with or without a His-tag at their C-terminus were ordered at R&D Systems. Human CD80-Fc and CD86-Fc were biotinylated in house using Biotinamidohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester (Sigma).

Various vaccinia viruses were used:

Wild type vaccinia (Copenhagen, Wyeth and Western Reserve strains)

Double deleted vaccinia viruses (Copenhagen strain) defective for both thymidine kinase and ribonucleotide reductase activities (tk-; rr-; described in W02009/065546). Triple deleted vaccinia viruses (Copenhagen strain) defective for tk, rr- and m2 activities. The triple deleted virus was generated from the double deleted tk- rr- by specific homologous recombination into the open reading frame of the M2L Locus. More specifically, the M2L gene deletion, which encompasses 64 nucleotides upstream open reading frame (ORF) and the 169 first codons of the M2L ORF, was performed by homologous recombination using a transfer plasmid. This plasmid was transfected by electroporation into CEF infected by vaccinia virus encoding luciferase (/4L-; T -/luciferase) using the Amaxa Nucleofactor. A primary research stock was produced on CEFs. The deletion of the M2L gene was verified by PCR and sequencing.

Beside vaccinia virus, all poxviruses tested, unless otherwise specified, were wild type strains.

For in vivo studies, double (tk- rr-) and triple (tk- rr- m2-) deleted vaccinia viruses were engineered to encode the firefly luciferase at the J2R locus under the p11 K7.5 promoter. ELISA assay for B7 binding

Ninety-six well plates (Nunc immune plate Medisorp) were coated, overnight at 4°C, with 100 pL of 0.5 pg/mL of either B7, CTLA4 or CD28 proteins in coating buffer (50 mM Na carbonate pH 9.6). Microplates were washed by PBS/0.05% Tween20 and saturated by 200 pL of blocking solution (PBS; 0.05% Tween20; 5% Non-Fat Dry Milk (Biorad)). All antibody preparations and dilutions were made in blocking solution. One hundred pL of samples were added to each well in triplicate and in two-fold serial dilutions for some experiments (binding curves). Microplates were then incubated with 100 pL of anti-Flag-HRP (Sigma) diluted 10000-fold. Microplates were then incubated with 100 pL/well of 3, 3’, 5,5’- tetramethylbenzidine (TMB, Sigma) and reaction was stopped with 100 pl_ 2M H ₂ SO ₄ . Absorbance was measured at 450 nm with a plate reader (TECAN Infinite M200PRO). The absorbance values were transferred into the software GraphPadPrism for analysis and graphic representation.

Competition ELISA

Experimental conditions and solution, not otherwise specified, were identical to the ones described above. For CTLA4/CD80, CD28/CD80 and PDL1/CD80 competition assays, 100 mI_ of CTLA4, CD28 and PDL1 were coated at 0.25 (CTLA4) or 1 mg/mL (CD28 and PD- L1). Samples were added and diluted (two-fold serial dilution) in blocking solution containing constant concentration of CD80 (either 50, 250 or 500 ng/mL for CTLA4, CD28 and PD-L1 respectively). For CD86/CTLA4 and CD28/CD86 competition essays, 100 mI_ of CD86 or CD28 were coated at 0.25 (CD86) or 2 pg/mL (CD28). Samples were added and diluted (two fold serial dilution) in blocking solution containing constant concentration of either CTLA4 (100 ng/mL) or CD86 (500 ng/mL) respectively. Either anti-His tag- HRP (Qiagen) at 1/2000 or streptavidin HRP (Southern Biotech) at 1/1000 were used as conjugated reagents. The plates were further treated, and results analysed, as described above.

Western blot

Twenty-five microliters of samples were prepared in Laemmli buffer containing 5% b- mercaptoethanol (BME) (reducing condition) or not (non-reducing condition). After electrophoresis on Criterion TGX 4-15% stain free gel (Biorad) the proteins were transferred to PVDF membrane (Transblot Turbo System). I Bind Flex Western system (Invitrogen) was used for the proteins/antibodies incubations and washes. Blots were probed with 2.5 pg/mL CD80-FC, CD86-FC, CTLA4-Fc or anti-Flag-HRP at 1/1000. For CD80-Fc, CD86-Fc and CTLA4-Fc, a HRP anti-Human Fc (Bethyl) at 1/3000 was used as conjugated antibody. The 1X iBind Flex Solution was used to block, dilute the antibodies, wash and wet the iBind Flex Card. Immune complexes were detected using the Amersham ECL Prime Western Blotting reagents. Chemiluminescence was recorded with a Molecular Imager ChemiDOC XRS (Biorad). Affinity chromatography

Supernatant of Chicken embryo fibroblasts (CEF) infected (MOI 0.05) by either MVA or vaccinia virus Copenhagen (tk- rr-) were collected 72 hours post-infection. The supernatants supplemented with 0.05% Tween 20 were concentrated ~20-fold using vivaspin20 30 000 MWCO cut-off concentrator (Sartorius). Streptavidin magnetic beads (GE healthcare) were coated with either an irrelevant monoclonal biotinylated antibody (chCXIIG6), CTLA4-Fc-Biot or CD86-Fc-Biot. Four ml_ of concentrated supernatants (MVA and Vaccinia virus Copenhagen) were incubated with 24 pl_ of chCXIIG6-Streptavindin beads to remove unspecific binding. The flow-throughs of this first incubation were split in 2 equal parts and incubated either with CTLA4-Fc-Biot-streptavidin beads or CD86-Fc-Biot- streptavidin beads to yield the four following arms: MVA supernatant + CTLA4 beads (MVA A4); MVA supernatant + CD86 beads (MVA CD); Vaccinia virus + CTLA4 beads supernatant (VV A4) and Vaccinia virus + CD86 beads (VV CD86). The beads were extensively washed with PBS, 0.05% Tween20 followed by PBS and bound proteins were eluted two times by 50 mI_ 0.1 M acetic acid neutralized immediately by addition of 4 mI_ 2M Tris Base. The two elutions were then pooled before MS analysis.

Protein preparation for digestion.

Ten or 20 mI of sample were evaporated and submitted to reduction by solubilization in 10 mI of 10 mM DTT in 25 mM NH4HC03 (1 h at 57°C). Reduced cysteine residues were alkylated 10 mI of 55 mM iodoacetamide in 25 mM NH4HC03 30 min at room temperature in the dark. The trypsin (12.5 ng/pL; Promega V5111) freshly diluted in 25 mM NH4HC03 was added to the sample in a 1 : 100 (enzyme/protein) ratio to a final volume of 30 mI and incubated 5 hours at 37°C. The activity of the trypsin is inhibited by acidification with 5 mI of H20/TFA 5%.

MS/MS analysis.

Samples were analysed on a nanoUPLC-system (nanoAcquity, Waters) coupled to a quadrupole-Orbitrap hybrid mass spectrometer (Q-Exactive plus, Thermo Scientific, San Jose, CA). The UPLC system was equipped with a Symmetry C18 precolumn (20 x 0.18 mm, 5 pm particle size, Waters, Milford, USA) and an ACQUITY UPLC® BEH130 C18 separation column (75 pm ^c 200 mm, 1.7 pm particle size, Waters). The solvent system consisted of 0.1 % formic acid in water (solvent A) and 0.1 % formic acid in acetonitrile (solvent B). Two pL of each sample were injected. Peptides were trapped during 3 min at 5 pL/min with 99 % A and 1 % B. Elution was performed at 60 °C at a flow rate of 400 nL/min, using a 79 minutes linear gradient from 1-35 % B. To minimize carry-over, a column wash (50% ACN during 20 min.) was included in between each sample in addition to a solvent blank injection, which was performed after each sample.

The Q-Exactive Plus was operated in positive ion mode with source temperature set to 250°C and spray voltage to 1.8 kV. Full scan MS spectra (300-1800 m/z) were acquired at a resolution of 140,000 at m/z 200, a maximum injection time of 50 ms and an AGC target value of 3 x 106 charges with the lock-mass option being enabled (445.12002 m/z). Up to 10 most intense precursors per full scan were isolated using a 2 m/z window and fragmented using higher energy collisional dissociation (HCD, normalised collision energy of 27eV) and dynamic exclusion of already fragmented precursors was set to 60 sec. MS/MS spectra were acquired with a resolution of 17,500 at m/z 200, a maximum injection time of 100 ms and an AGC target value of 1 x 105. The system was fully controlled by theXCalibur software (v3.0.63; Thermo Fisher Scientific). MS/MS data interpretation

MS/MS data were searched against a Gallus gallus and Vaccinia virus Uniprot database derived combined target-decoy database (01-04-2018, containing 33939 target sequences plus the same number of reversed decoy sequences) using Mascot (version 2.5.1 , Matrix science, London, England). The targets proteins hCTLA4, hCD86 and hCXIIG6 and target-decoy were manually added to the database. The database including common contaminants (human keratins and porcine trypsin) and was created using an in-house database generation toolbox (http://msda.u-strasbg.fr). The following parameters were applied: one missed cleavage by trypsin and variable modifications (oxidation of Methionine (+16 Da), carbamidomethylation of Cysteine (+57 Da), were considered. The search window was set to 25 ppm for precursor ions and 0.07 Da for fragment ions. Mascot result files (.dat) were imported into Proline software (http://proline.profiproteomics.fr/) and proteins were validated on pretty rank equal to 1 , 1% FDR on peptide spectrum matches based on adjusted e-value, at least 1 specific peptide per protein, 1% FDR on protein sets and Mascot Modified Mudpit scoring. Mixed Lymphocyte Reaction (MLR)

The samples tested in MLR were either purified recombinant M2 protein or human CTLA4-Fc fusion protein from R&D Systems at final concentrations ranging from 0.01 to 10 pg/mL. Blood from different healthy donors were purchased at Etablissement Frangais du Sang (EFS Grand Est, 67065 Strasbourg). PBMC were purified by Ficoll-Paque method (Ficoll-Paque PLUS, GE Healthcare) and resuspended at about 10 ⁷ cells/mL in RPMI medium supplemented with 20% FBS (fetal bovine serum) and 10% DMSO and stored at - 150 °C until use. PBMC were thawed at 37 °C, resuspended in RMPI medium + 10% FBS and centrifugated 5 minutes at 300 g. Cells were resuspended in RMPI medium + 10% FBS. Cell concentration was adjusted to 3x10 ⁶ cells/mL. One hundred pL of PBMC from two different donors were mixed in a well of a 96-well microplate in triplicate. Twenty pL of M2 or CTLA4- Fc sample described above were added to each well and the microplates were incubated 72h at 37°C and 5% CO2. The MLR culture supernatants were then harvested and the quantity of human interleukin-2 (IL-2) was measured by ELISA using the human IL-2*-2ELISA MAX™ deluxe Set kit (BioLegend). The measures were normalized by dividing the mean of IL-2 concentration of the three replicates of a given sample by the mean of IL-2 concentration of the three replicates of PBMC incubated with buffer (mock treatment).

EXAMPLE 1 : Identification of the ability of the vaccinia virus M2 protein to interfere with B7-mediated costimulatory pathway and characterization of its binding properties

Supernatants of vaccinia virus infected cell inhibits the interaction of CTLA4 with CD80 or CD86. Two assays were set-up to monitor quantitatively the CD80/CTLA4 and CD86/CTLA4 blocking activities provided by the different virus candidates. In these assays, human CTLA4 (hCTLA4) was immobilized on ELISA plate and soluble tagged hCD80 or hCD86 were added. In this setting, any competitive molecule that binds to either the immobilized or the soluble partner will induce a decrease of signal (competition assay). The anti-hCTLA4 antibody Ipilimumab (Yervoy) and supernatant of uninfected DF1 (chicken cells line available; e.g. from ATCC® CRL-12208™) were used as positive and negative controls, respectively. Surprisingly, as Yervoy which interacts with the coated hCTLA-4, all supernatants of cells infected by vaccinia virus (Copenhagen, Wyeth and Western Reserve strains) were found to be competitive in dose-response manner with both CD80/CTLA4 and CD86/CTLA4 assays (Figures 1A and 1 B), whereas the supernatant of the uninfected DF1 cells did not have any effect. Interestingly, the supernatants of DF1 infected by modified vaccinia virus Ankara (MVA) were not producing any inhibition of the hCTLA4/hCD80 and hCTLA4/hCD86 interactions indicating that this interference ability is not conserved in this virus which has lost six genomic fragments (deletions I to VI) during its attenuation process (data not shown). These results suggest that something in VV supernatants interferes with the binding of CTLA- 4 with CD80 and CD86.

To rule out any artefact involving cell or medium components, different cells lines from different origins (avian primary and human tumoral cell lines) were tested and a method of FACS competition was also assayed.

Competition FACS analysis was carried out using a human cell line (i.e. KM-H2, Hodgkin lymphoma) displaying naturally hCD80 and hCD86 at its surface. Binding of soluble recombinant CTLA4-Fc to KM-H2 cells was shown using a fluorochrome-conjugated anti-Fc antibody. When co-incubated with CTLA4-Fc, supernatants of vaccinia virus-infected cells competed for CTLA4-Fc binding to KM-H2 cells in marked contrast to MVA-infected cells which behave as the negative control (data not shown).

Competition ELISA was carried out using supernatants of HeLa (instead of DF1) cells infected with different poxviruses to evaluate their capacity to interfere with the CTLA4 binding to CD80 or CD86. Various strains of vaccinia virus (Wyeth, WR and Copenhagen) were tested as well as other orthopox (e.g., raccoonpox, rabbitpox, cowpox, MVA) avipox (fowlpox) and parapox virus (pseudocowpox virus). Uninfected HeLa cells are used as negative control. In this screening experiment, HeLa cells were infected with different poxviruses at a high MOI (MOI 1) to guarantee an optimal infection and the resulting supernatants were collected and tested by evaluating their capacity to inhibit the CTLA4-Fc binding to CD80 (represented as OD450nm). As illustrated in Figure 2, all supernatants of cells infected with either the three strains of vaccinia virus or with raccoonpox (RCN), rabbitpox (RPX) and cowpox (CPX) were able to interfere with the binding of hCTLA4 to hCD80. These results indicate that a factor secreted during infection with these poxviruses is interfering with the CTLA4-B7 pathway. The new unknown factor involved in this inhibitory activity was called“interference factor” (IF). Again, supernatants of cells infected with MVA and some other poxviruses like the pseudocowpox virus (PCPV) and fowlpoxvirus (FPV) did not display any inhibition of the CTLA4/CD80-CD86 interactions as the uninfected HeLa cells (HeLa).

The“Interference factor” is present in vaccinia virus supernatants but not in MVA supernatants To figure out with which molecule present in VV-infected supernatants, the IF was interacting, a western blot of supernatants of chicken embryo fibroblast (CEF or CEP) uninfected or infected with either MVA or vaccinia virus was probed with the three components of the ELISA assay described above (namely hCD80, hCD86 and hCTLA4). CEF were chosen since they are permissive to both vaccinia virus and MVA that produce, or not, the IF, respectively. Each protein used to probe the western blot was a fusion with an Fc part that allows, among other things, their dimerization and their detection with the same anti-Fc conjugated antibody. Each supernatant was used either as such or concentrated 20 times (x20) The blots presented on Figure 3 demonstrated unambiguously that a large molecule of about 200 kDa was present only in the vaccinia virus infected supernatants and highlighted with both hCD80 and hCD86 but not with hCTLA4 (at least in these immunoblot conditions). This band was easily detected even in non-concentrated supernatants). Reactivity with both hCD80-Fc and hCD86-Fc was lost in reducing conditions (no detection of any band) indicating that intra and/or inter disulfide bonds are necessary to maintain the IF’s structure and interaction with CD80 and CD86 (data not shown). In marked contrast, no band was highlighted in MVA supernatants.

Characterization of the binding properties: the“Interference factor” present in vaccinia virus supernatant inhibits the binding of CD80 and CD86 to CTLA4 and CD28 but potentializes the binding of CD80 with PD-L1. As discussed above, CD80 and CD86 are important co-stimulation antigens involved in the regulation of the adaptive T cell response. Because CD80 and CD86 are involved in several molecular interactions with negative (CTLA4 for both, and PD-L1 for CD80 only) and positive (CD28) outcomes in term of immune response, different ELISAs were set up to decipher the effect of IF on each of these 5 specific interactions. The undiluted supernatants from CEF infected with non-recombinant vaccinia virus (VV) were tested in these different assays and compared to supernatant of MVA-infected CEF and the anti-hCTLA4 antibody Yervoy (10pg/ml). Supernatants of uninfected CEF cells are used as negative control. As illustrated in Figure 4, VV supernatant inhibited the interaction of CD80 and CD86 with CTLA4 (as evidenced by an impressive decrease of the OD450nm absorbance) similarly as Yervoy (as expected due to the binding of Yervoy to its CTLA-4 target that prevents access to CD80 and thus CTLA4/CD80 ligation). In marked contrast, MVA-infected cell supernatants had no effect (same absorbance as the negative CEF control). Moreover, VV supernatants were also able to abolish the positive interaction of CD80 or CD86 with CD28 (strong diminishment of the OD450nm absorbance with respect to absorbance measured with the supernatant of uninfected CEF cells). In contrast, MVA-infected cell supernatants and Yervoy (as expected for an antibody that target only CTLA4 receptor) had no effect (same absorbance as the negative control). These results confirm the presence of an“IF” in supernatants of VV infected cells whereas MVA genome does not produce such a factor. Surprisingly, the PD-L1/CD80 interaction was increased by the presence of vaccinia virus supernatants (strong increase of the OD450nm absorbance with respect to the negative control) reinforcing the PDL1-mediated immunosuppressive signalling. In contrast, Yervoy and MVA-infected CEF supernatants had no impact on PDL1/CD80 (same absorbance as uninfected control). As expected, recombinant hCD80, hCTLA4 and hPD1 abolished this interaction. This result indicates that the IF and CTLA4 binding sites on CD80 are not completely overlapping. It should be noted that the CD80/PD-L1 interaction has been recently involved to Treg survival.

These results highlight the improved immunosuppressive properties displayed by the poxviral M2 polypeptide. Indeed, M2 pushes toward immunosuppressive pathways by blocking CD80/CD28, CD86/CD28 and by potentializing PDL1-CD80 pathways whereas CTLA4-Fc inhibits these three pathways including the immunosuppressive PDL1-CD80 interaction.

Identification of the M2 poxyiral protein as being the interference factor Based on the apparent molecular weight of approximately 200 kDa and the fact that

IF was not present in MVA-infected supernatants, the 37 genes that are different between vaccinia Copenhagen strain and MVA were investigated for a potential candidate without finding any obvious one. No protein of about 200 kDa could be identified. The largest encoded protein, among these 37 gene candidates is the DNA-dependent RNA polymerase subunit rpo147 (J6R) with a theoretical mass of 147 kDa, thus lower than the 200 kDa observed. Based on primary structure, there was no obvious viral protein candidate that could be linked to IF.

Therefore, an experimental approach to identify IF was attempted using an affinity chromatography (see scheme Figure 5A) to capture IF. A 20-fold concentrated supernatant of vaccinia virus infected CEF (VV infected) was loaded on this affinity chromatography. A 20-fold concentrated supernatant of MVA infected cells (MVA infected) was processed in parallel. The VV and MVA supernatants were submitted to either immobilized CTLA4 (negative controls) or immobilized CD86-Fc fusion before being eluted by acid. The different elutions of the affinity chromatography arms were analyzed by MS/MS (mass spectrometry) after trypsic digestion. The obtained m/z data were used to probe the chicken (Gallus gallus) and vaccinia virus data banks. One hit was obtained only from the supernatant of vaccinia infected CEF incubated with CD86 coated beads which covers 75% (including the peptide signal) or 82% (without the peptide signal) of the vaccinia virus protein M2 protein encoded by the M2L locus (Figure 5B where the sequence covered by the detected peptides is indicated in bold). This result is in full agreement with the absence of M2L locus in MVA genome and with the fact that M2 has a predicted signal peptide making it a putative secreted protein.

However, M2 protein has a calculated molecular weight of only 25 kDa and has been reported to migrate on SDS-PAGE on reducing conditions as a 35 kDa protein (Hinthong et al. 2008) which is far from the 200 kDa mass of IF observed on SDS-PAGE. Nevertheless, to our knowledge, the behavior of M2 protein on SDS-PAGE in non-reducing conditions was not documented. Therefore, we hypothesize that IF could be a homo or hetero-multimeric complex involving the VV M2 protein with inter-subunit disulfide bonds resulting in an apparent mass on SDS-PAGE of approximately 200 kDa.

M2L deleted virus does not produce IF anymore

The involvement of M2 in the IF was further investigated by deleting the M2L gene in a vaccinia virus genome. Specifically, the M2L locus was disrupted in a double deleted (DD) vaccinia virus expressing the luciferase (i.e. ttc, rr- described in W02009/065546 and designated VVTG18277) resulting in a recombinant triple deleted (TD) virus (i.e. tk rr, m2 ) expressing the luciferase (COPTG19289). The M2L modification resulted in a suppressed expression of M2 protein (m2-) and did not have any significant impact on the virus replication on CEF compared to the parental one (data not shown). The supernatant obtained upon infection of human HeLa and avian DF1 cells with the DD and TD viruses were studied by competition ELISA as before. As shown in Figure 6, the supernatant collected upon infection with the M2L-deleted vaccinia virus COPTG19289 was not able anymore to inhibit the CTLA4/CD80 interactions (as evidenced by the same absorbance as the one measured in the uninfected HeLa or DF1 cells) unlike the parental DD virus (VVTG18277) which showed a strong decrease in the absorbance measurement compared to the negative controls.

Moreover, when submitted to western blotting as above, the large complex migrating at 200 kDa detected using CD80-Fc or CD86-Fc probes was no more detected with the supernatant of M2L-deleted virus (data not shown). These results confirmed that M2 is, at least, part of the IF. EXAMPLE 2: Production of recombinant W M2 and immunosuppressive activity

Recombinant M2 is oligomeric and inhibits the hCD80/hCTLA4 interaction.

In order to know if other viral or cellular factors are involved in the inhibition of the interaction of CD80 andCD86 with CTLA4 and CD28, recombinant tagged M2 was produced by transient transfection of HEK293 with a M2-expressing plasmid (pTG19262) and purified by affinity chromatography to near homogeneity (no other protein from human or vaccinia virus origin was detected by mass spectrometry analysis of the digested purified M2 protein). A gel electrophoresis analysis of M2 (7.5pg loaded) was performed either in reducing conditions (b-mercaptoethanol (BME) and heat) or in the presence of increasing amounts of DTT (0, 1 , 2, 4, 8 and 16mM in 200mM Tris pH 8,0 for 15 min before loading). As illustrated in Figure 7, in reducing condition tagged-M2 migrated slightly below the 37 kDa marker in agreement with the published 35 kDa size for a non-tagged M2 (Hinthong et al. 2008). Moreover, in non-reducing condition a band was observed at a size close to that of IF (i.e. 200 kDa area) indicating that M2 can auto-assemble into a large complex stabilized by inter- subunit disulfide bonds. The use of intermediary levels of reducing agent allowed to visualize the transitional products of assembly between the 35 kDa product with observation of dimers (x2), trimers (x3), tetramer (x4), pentamer (x5), hexamer (x6), heptamer (x7) and probably octamer (X8) below the approximately 200 kDa product, suggesting that this 200 kDa product results from auto-assembly of the 6 or 7 or maybe 8 M2 proteins or mixture thereof (formation of a homohexo or/and heptamer). Mass spectrometry analysis in non-reducing condition did not allow to determine the number of subunits involved in the oligomer mostly because of the large heterogeneity of glycosylation of the protomers.

M2 is necessary and sufficient to inhibit the CD80-CD86/CTLA4-CD28 interaction.

Moreover, purified M2 was submitted to competition ELISA for the 5 interactions involving CD80 and CD86 and the inhibitory effect provided by M2 was compared to recombinant CTLA4-Fc fusion, anti-hCD80 monoclonal antibody (MAB B7-1) and anti-hCD86 monoclonal antibody (MAB B7-2), Yervoy (as negative control) as well as the supernatant collected from CEF cells infected with Copenhagen VVTG18058 expressing M2 protein.

As illustrated in Figure 8B and C, purified M2 protein reproduced the inhibitory effect on hCD28/hCD80 and hCD28/hCD86 interactions observed with the VV-infected supernatants Interestingly, the competition provided by the purified M2 protein for hCD28/hCD80 (Figure 8B) interaction is as strong as the one obtained with the anti-CD80 monoclonal antibody (clone 37711 ref MAB140 R&D systems). Concerning hCD28/hCD86 interaction (Figure 8C), purified M2 competed stronger than the same amount of the anti- CD86 antibody (clone 37301 ref MAB141 R&D systems). Moreover, M2 polypeptide competed similarly to inhibit hCD80/hCTLA4 (Figure 8D) and hCD86/CTLA4 (Figure 8E) interactions. Inhibitory effect of serial dilution of purified M2 on hCTLA4/hCD80 interaction allowed to quantify the concentration of natural M2 in the VV supernatants to be around 0.5 pg/mL (see the Figure 8D which included a VV18058 supernatant). In particular, M2 was able to inhibit the hCTLA4/hCD80 interaction with an EC50 of 0.01 pg/ml. As observed with VV- infected cell supernatants, purified M2 polypeptide also potentialized hPD-L1/hCD80 interaction (Figure 8A).

These results demonstrate that recombinant M2 (in absence of any other cell or vaccinia virus protein) recapitulates the electrophoresis behavior and the interference activity towards CD80 and CD86 co-stimulatory antigens, suggesting that M2 is IF.

Recombinant VV M2 binds to murine and human CD80 and to human CD86 but slightly to murine CD86 Recombinant tagged M2 was used to investigate further the interactions with CD80 and CD86 co-stimulatory antigens. Several recombinant proteins from the human B7 family were immobilized on ELISA plate (0.5pg/ml), respectively human CD80 (hCD80), mouse CD80 (mCD80), human CD86 (hCD86), mouse CD86 (mCD86), human CTLA4 (hCTLA4), mouse CTLA4 (mCTLA4), human CD28 (hCD28), mouse CD28 (mCD28), human B7-H2 (hB7-H2), human B7-H3 (hB7-H3) and human PDL-1. Their interaction with the tagged M2 protein (undiluted supernatants of transfected cells with pTG19262) was monitored. Undiluted supernatants of transfected cells with GFP-expressing pTG15839 were used as negative control and as expected displayed no binding at all. Detection was carried out by adding an anti-Flag-HPR antibody. As illustrated in Figure 9A, recombinant tagged M2 interacted with both human and murine CD80 and CD86 but with no other protein of the B7 family tested (hCTLA4, mCTLA4, hCD28, mCD28, B7-H2, B7-H3 and hPDL1). The signal obtained with murine CD86 appeared lower than the one observed with human CD86.

T o document the binding of M2 to human and mouse CD80 and CD86, dose-response curves were generated for both species. The results presented in figure 9B demonstrated clearly that vaccinia virus M2 bound with the same apparent affinity murine CD80, human CD80 and human CD86. However, the apparent affinity for murine CD86 seemed to be lower than for its human counterpart or for the murine CD80. The binding of M2 to KM-H2 cells which naturally display hCD80 and hCD86 was also verified by staining the KMH2 cells with recombinant tagged M2 and it was found that M2 binds KM-H2 cells (data not shown). EXAMPLE 3: Production of myxoma virus M2 and immunosuppressive activity

M2 ortholog from myxoma virus interacts with CD80 and CD86

M2 protein from vaccinia virus has orthologs in others chordopoxviruses (see Table 1). To investigate if the CD80/CD86 binding activity is conserved along the poxvirus family, the Myxoma virus was chosen because of the low percentage of identity between the VV M2 protein and the M154L gene product designated Gp120-like protein (Gp120LP), reaching 50% at the protein level. For this purpose, M154L coding sequences (M2L equivalent) were cloned (giving pTG19348) and expressed in HEK293. The ability of supernatants of cells transfected with pTG19348 (producing a tagged myxoma Gp120-like protein)“pM154L” to interfere with the binding of human co-stimulatory B7 antigens to hCTLA-4 was tested by ELISA and compared to supernatants of pTG19262-transfected cells diluted 100 times (producing a tagged VV M2 protein and designated“pM2”) and purified recombinant VV M2 (1 pg/ml)). Non-transfected cells (Cells) and cells transfected with GFP-expressing plasmid (pGFP) were used as negative controls. CTLA4 binding to either CD80 or CD86 was monitored with an anti-Histag HRP conjugated antibody. As shown in Figure 10, no competition activity was detected for the negative controls “Cells” and “pGFP”, whereas pTG19262-transfected cells (pM2) strongly competed with human CTLA-4 for binding to CD80 and CD86 (translating in a very weak OD) as recombinant M2. pTG19348-transfected cells (pM154L) producing the M154L-encoded Gp120LP protein inhibited the binding of CTLA4 to human CD80 although at a lower extend than M2 counterpart as well as inhibited the binding of CTLA4 to human CD86 at the same range as its vaccinia M2 counterpart (produced in diluted cell-transfected supernatants). The lower inhibition capacity provided by the myxoma virus Gp120LP protein compared to VV M2 protein may be explained partly by the lower expression levels obtained in pTG19348-transfected cells. These results support the ability of Gp120LP to inhibit the interaction of hCD86 and hCD80 with hCTLA4. A plausible hypothesis is that binding of the VV and myxoma M2 proteins to the co-stimulatory antigens prevents their ligation with the T cell CTLA-4 receptor.

The supernatants of cells transfected with either pTG19262 (expressing VV M2) or pTG19348 (expressing myxoma Gp120-like protein or Gp120LP) were analysed by western blot performed in non-reducing (-BME) and reducing (+BME) conditions. Various supernatant volumes (1 OmI, 5mI, 2.5mI of non-diluted supernatants or 20mI diluted 100 times) were loaded on the gel. Proteins were blotted on PVDF membrane and the detection was performed by adding an anti-Flag-HPR antibody. As shown in Figure 11 , in reducing condition (+BME), the tagged-M2 migrated slightly below the 37 kDa marker in agreement with the data of Example 2 and the myxoma virus Gp120-like protein migrated just above 25kDa (this difference is due to the shorter amino acid sequence of myxoma protein than VV M2 and probably to the glycosylation profile). Interestingly, in non-reducing condition (-BME), the VV M2 band was observed at a size close to 200 kDa indicating the tendency of VV M2 protein to auto- assemble in oligomers of at least 6 or more proteins whereas myxoma virus Gp120-like protein was still detected just above 25kDa.

The results demonstrate that the CD80/CD86 binding capacity of the M2 protein is conserved among different poxvirus and notably showing no more than 50% identity with the “prototype” vaccinia M2. However, myxoma virus Gp120L protein is produced at least as non- crosslinked homo-oligomer contrary to VV M2 protein which is produced in oligomers of 6 to 8 proteins. It has to be confirmed that Gp120LP is a monomer or an oligomer without any intermolecular disulphide bonds.

EXAMPLE 4: Mixed Lymphocyte Reaction (MLR) assays

The purified recombinant M2 protein or human CTLA4-Fc fusion protein (R&D Systems) at final concentrations ranging from 0.01 to 10 pg/mL were evaluated in MLR for their ability to activate lymphocytes. PBMC were purified by Ficoll-Paque PLUS (GE healthcare) from blood collected from healthy donors. More specifically, 3x10 ⁵ PBMC from 2 different donors were mixed in 96-well microplate in the presence of 20pL of M2 protein or human CTLA4-Fc fusion samples (or buffer as a negative control) and cultured for 72h at 37°C in 5% CO2 atmosphere. IL-2 secretion was quantified in culture supernatants by ELISA (IL-2*-2ELISA MAX™ deluxe Set kit from BioLegend) as a marker of lymphocytes’ activation. The measures were normalized by dividing the mean of IL-2 concentration of the three replicates of a given sample by the mean of IL-2 concentration of the three replicates of PBMC incubated with buffer (mock treatment).

As illustrated in Figure 12, negative control represents a normalized lymphocyte activation status of 1 whereas PBMC incubated with M2 or human CTLA4-Fc fusion protein respectively secreted a reduced level of IL-2, confirming the immunosuppressive activity of these proteins. Interestingly, recombinant M2 protein seemed to be more immunosuppressive especially at low concentrations (at concentration of 0.01 pg/ml, normalized IL-2 secretion of 0.5 for M2 versus 0.75 for CTLA4-Fc fusion). REFERENCES

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