FIELD OF INVENTION The present invention relates to the treatment or prevention of acute respiratory distress syndrome (ARDS) in a subject in need thereof. In particular, the present invention relates to the treatment or prevention of virus-related ARDS and more particularly, SARS-CoV-2-related ARDS. BACKGROUND OF INVENTION

ARDS (acute respiratory distress syndrome) is a manifestation of acute lung injury (ALI), commonly resulting from sepsis, trauma, and severe pulmonary infections. The evolution of ARDS is characterized by several phases: an exudative phase and a proliferative phase, which may be followed by a fibrotic phase in some patients. Although mechanical ventilation is the most important supportive therapy for patients with ARDS, it can paradoxically induce or aggravate lung injury, and thus may contribute to the development of lung fibrosis. Thus, there is a need to develop new therapies for treating ALI and ARDS, and in particular for preventing aggravation thereof toward lung fibrosis.

ARDS may in particular be induced by two types of virus: respiratory viruses that cause community-acquired viral pneumonia and Herpesviridae that cause nosocomial viral pneumonia. Among the respiratory viruses, the influenza H5N1 virus has been identified in 1997 in Hong Kong. Studies have demonstrated that the H5N1 virus was able to replicate in the respiratory tract of humans, leading to severe lesions with histopathologic features of ARDS. Accordingly, mice infected with the H5N1 virus exhibited clinical signs of respiratory disease, including visually prominent signs of respiratory distress. In January 2020, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was identified as the pathogen of the pandemic coronavirus disease 2019 (COVID-19), an infectious respiratory disease that emerged in December 2019 in the city of Wuhan, in the Chinese province of Hubei. The severity of COVID-19 symptoms can range from mild to severe. Early symptoms include headache, muscle pain, and fatigue. Then, fever, cough and respiratory discomfort occur secondarily. Most severe insults on the lung tissue can result in ARDS, which is one of the leading causes of mortality in COVID-19 disease. Emerging evidence suggest that a subset of COVID-19 patients develops a cytokine storm syndrome (corresponding to an excessive immune response) resulting in ARDS and multiple-organ failure. Indeed, severe patients were identified, exhibiting elevated levels of infection-related biomarkers, such as serum ferritin and C-reactive protein, and inflammatory cytokines including tumor necrosis factor (TNF)-a, interleukin (IL)-2, IL-10 and IL-6. In addition, recent evidences suggest that the levels of the pro-inflammatory cytokines IL-2 and IL-6 in the serum is positively correlated with the severity of the disease.

Recent observations have demonstrated that as well as their hemostatic function, platelets contribute to tissue injury. In particular, studies during the past decade have implicated platelets in experimental lung injury and clinical Acute Respiratory Distress Syndrome. Antiplatelet therapy may therefore offer a useful adjunctive strategy to fight virus-induced ARDS. Up to now, most studies on ARDS have focused on aspirin (ASA) with, so far, no definitive results. However, it should be noted that due to the aggravating effect of non-steroid anti-inflammatory drugs in Covid-19 infections, ASA at doses superior to 100 mg per day may not be a viable option in this case. Therefore, efficient therapies to treat virus-related ARDS, and especially SARS-CoV-2-related ARDS, are urgently needed.

The present invention describes a novel therapeutic strategy for treating or preventing ARDS. More specifically, the present invention relates to an isolated humanized protein binding to human Glycoprotein VI (hGPVI), in particular an anti-GPVI antibody for use in the treatment or prevention of ARDS, in particular virus-related ARDS and more particularly SARS-CoV-2-related ARDS. SUMMARY

The present invention relates to an isolated protein binding to human Glycoprotein VI (hGPVI) for treating or preventing the onset or aggravation of an acute respiratory distress syndrome (ARDS). In one embodiment, said isolated protein is an antibody molecule or an antigen-binding fragment thereof, preferably selected from the group consisting of a whole antibody, a single chain antibody, a Fv, a Fab, a unibody, a nanobody and a domain antibody; or a monomeric antibody mimetic selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer and a versabody.

In one embodiment, said isolated protein is a monovalent antibody molecule.

In one embodiment, said isolated protein is an antibody molecule or an antigen-binding fragment thereof wherein the variable region of the heavy chain comprises at least one of the following CDRs: VH-CDR1: GYTFTSYNMH (SEQ ID NO: 1);

VH-CDR2: GIYPGN GDTS YN QKFQG (SEQ ID NO: 2); and VH-CDR3: GTVVGDWYFDV (SEQ ID NO: 3), or any CDR having an amino acid sequence that shares at least 60% of identity with SEQ ID NO: 1-3, and/or the variable region of the light chain comprises at least one of the following CDRs:

VL-CDR1: RS S QS LENS N GNT YLN (SEQ ID NO: 4);

VL-CDR2: RVSNRFS (SEQ ID NO: 5); and VL-CDR3: LQLTHVPWT (SEQ ID NO: 6), or any CDR having an amino acid sequence that shares at least 60% of identity with SEQ ID NO: 4-6.

In one embodiment, said isolated protein is an antibody molecule or an antigen-binding fragment thereof wherein the variable region of the heavy chain comprises the following CDRs: GYTFTSYNMH (SEQ ID NO: 1), GIYPGN GDTS YN QKFQG (SEQ ID NO: 2) and GTVVGDWYFDV (SEQ ID NO: 3) and the variable region of the light chain comprises the following CDRs: RSSQSLENSN GNT YLN (SEQ ID NO: 4), RVSNRFS (SEQ ID NO: 5) and LQLTHVPWT (SEQ ID NO: 6) or any CDR having an amino acid sequence that shares at least 60% of identity with said SEQ ID NO: 1-6. In one embodiment, said isolated protein is an antibody molecule or an antigen-binding fragment thereof wherein the amino acid sequence encoding the heavy chain variable region is SEQ ID NO: 7 and the amino acid sequence encoding the light variable region is SEQ ID NO: 8, or any sequence having an amino acid sequence that shares at least 60% of identity with said SEQ ID NO: 7 or 8. In one embodiment, said isolated protein is an antibody molecule or an antigen-binding fragment thereof wherein the amino acid sequence encoding the heavy chain variable region is SEQ ID NO: 7 and the amino acid sequence encoding the light variable region is SEQ ID NO: 9, or any sequence having an amino acid sequence that shares at least 60% of identity with said SEQ ID NO: 7 or 9. In one embodiment, the ARDS is a virus-related ARDS, preferably a S ARS -Co V -2-related ARDS.

In one embodiment, the subject is suffering from mild to severe COVID-19.

In one embodiment, the subject presents with signs of a moderate progressive pulmonary disease, and/or exhibits respiratory distress symptoms. In one embodiment, the subject presents an elevation of D-dimers, an elevation of troponin T and/or signs of micro-angiopathy on a vascular enhanced chest CT scan.

In one embodiment, the protein is to be administered once a day during at least 3 consecutive days.

In one embodiment, a dose of protein ranging from about 100 mg to about 2000 mg is to be administered per administration to the subject. In one embodiment, said isolated protein is to be administered during at least 2 hours to the subject, preferably during at least 4 to 6 hours.

In one embodiment, said isolated protein is for administration with at least one further pharmaceutically active agent.

DEFINITIONS

In the present invention, the following terms have the following meanings:

“About” (preceding a figure): refers to plus or less 10% of the value of said figure.

“Adnectin” also known as monobody, is well known in the art and refers to proteins designed to bind with high affinity and specificity to antigens. They belong to the class of molecules collectively called “antibody mimetics”.

“Affibody” refers to an affinity protein based on a 58 amino acid residue protein domain, derived from one of the IgG binding domain of staphylococcal protein A.

“Affilin” is well known in the art and refers to an antibody mimetic technology, wherein the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual targeting protein, called Duocalins.

“Affitin” refers to an artificial protein structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaeal domain, that has the ability to selectively bind antigens. - “Anticalin” refers to an antibody mimetic technology, wherein the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual targeting protein, called Duocalins.

“Antibody” or “Immunoglobulin” refers to a protein having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity. “Antibodies” refers to such assemblies which have significant known specific immunoreactive activity to an antigen of interest (e.g. human GPVI). The term “anti-GPVI antibodies” is used herein to refer to antibodies which exhibit immunological specificity for human GPVI protein. As explained elsewhere herein, “specificity” for human GPVI does not exclude cross- reaction with species homologues of GPVI. Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood. The generic term “immunoglobulin” comprises five distinct classes of antibody that can be distinguished biochemically. All five classes of antibodies are within the scope of the present invention; the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, immunoglobulins comprise two identical light polypeptide chains of molecular weight of about 23,000 Daltons, and two identical heavy chains of molecular weight of about 53,000-70,000 Daltons.

The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region. The light chains of an antibody are classified as either kappa or lambda ([k], [l]). Each heavy chain class may be bonded with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” regions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (g, m, a, d,e) with some subclasses among them (e.g., gΐ - g4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgD, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc. are well characterized and are known to confer functional specialization.

Modified versions of each of these classes and isotypes are readily discemable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention. As indicated above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the light chain variable domain (VL domain) and heavy chain variable domain

(VH domain) of an antibody combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site presents at the end of each arm of the “Y”. More specifically, the antigen binding site is defined by three complementarity determining regions (CDRs) on each of the VH and VL chains. - “Antibody Mimetic” refers to molecules that can bind to antigens similar to antibodies; however, they are not generated by the immune system and have no structural relation to the antibodies. They are mostly unrelated protein scaffolds consisting of a-helices, b-sheets, or random coils that can bind to specific targets and could be designed to incorporate novel binding sites through common protein engineering strategies.

“Atrimer” refers to a molecule derived from a trimeric plasma protein known as tetranectin, a family of C-type lectins consisting of three identical units. The structure of the C-type lectin domain (CTLD) within the tetranectin has five flexible loops that mediate interaction with targeting molecules. - “Avimer” refers a molecule based on the A-domain of extracellular receptors.

Avimers are developed by the sequential selection of individual binding domains that enable generation of multidomain scaffolds to recognize different epitopes in the same ligand, single ligands, or multiple target ligands.

“Binding site” refers to a region of a protein which is responsible for selectively binding to a target antigen of interest (e.g. human GPVI). Binding domains or binding regions comprise at least one binding site. Exemplary binding domains include an antibody variable domain. The protein of the invention may comprise a single antigen binding site or multiple (e.g., two, three or four) antigen binding sites. Preferably, however, the protein of the invention comprises a single antigen binding site. - “Chimeric” refers to a protein comprising a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences may normally exist in separate proteins that are brought together in the fusion protein or they may normally exist in the same protein but are placed in a new arrangement in the fusion protein. A chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

“Conservative amino acid substitution” refers to substitutions in which the amino acid residue is replaced with an amino acid residue that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Other families of amino acid residues having similar side chains have been defined in the art, including basic side chains ( e.g ., lysine, arginine, histidine), acidic side chains ( e.g ., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains

(e.g., alanine, valine, leucine, isoleucine, pro line, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide may be replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. A variant may also, or alternatively, contain non-conservative changes. In one embodiment, variant proteins differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the protein.

“CDR” or “Complementarity Determining Region” refers to the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. CDRs were identified according to the following rules as deduced from Kabat (Kabat et al, 1991. J. Immunol. 147(5): 1709-19) and Chothia & Lesk (Chothia C. and A.M. Lesk, 1987. J. Mol. Biol. 196(4):901-17):

- CDR-L1:

Start - Approx residue 24;

Residue before is always a Cys;

Residue after is always a Trp. Typically TRP-TYR-GLN, but also,

TRP-LEU-GLN, TRP-PHE-GLN, TRP-TYR-LEU;

Length 10 to 17 residues;

- CDR-L2:

Start - always 16 residues after the end of LI;

Residues before generally ILE-TYR, but also, VAL-TYR, ILE-LYS, ILE-PHE;

Length always 7 residues;

- CDR-L3:

Start - always 33 residues after end of L2;

Residue before is always Cys;

Residues after always PHE-GLY -XXX-GLY (SEQ ID NO: 21);

Length 7 to 11 residues;

- CDR-H1:

Start - Approx residue 26 (always 4 after a CYS) [Chothia / AbM definition]

Kabat definition starts 5 residues later; Residues before always CYS-XXX-XXX-XXX (SEQ ID NO: 22);

Residues after always a TRP. Typically TRP-VAL, but also, TRP-ILE, TRP-ALA

Length 10 to 12 residues (AbM definition) Chothia definition excludes the last 4 residues;

- CDR-H2:

Start - always 15 residues after the end of Rabat / AbM definition) of CDR-H1 Residues before typically LEU-GLU-TRP-ILE-GLY (SEQ ID NO: 23), but a number of variations; Residues after LY S/ARG-LEU/ILE/V AL/PHE/THR/ALA-

THR/SER/ILE/ALA Length Rabat definition 16 to 19 residues (AbM definition ends 7 residues earlier);

- CDR-H3:

Start - always 33 residues after end of CDR-H2 (always 2 after a CYS); Residues before always CYS-XXX-XXX (typically CYS-ALA-ARG);

Residues after always TRP-GLY -XXX-GLY (SEQ ID NO: 24);

Length 3 to 25 residues.

“CH2 domain” refers to the region of a heavy chain molecule that usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain (Burton, Molec. Immunol. 22 (1985) 161-206). - “DARPin” (Designed Ankyrin Repeat Proteins) refers to an antibody mimetic DRP

(designed repeat protein) technology developed to exploit the binding abilities of non-antibody proteins. DARPins are artificial scaffolds based on human ankyrin repeat domain proteins, which are abundant intracellular adaptor molecules that mediate a broad range of protein interactions in variety of biological events. “Derived from” refers to the origin of the protein. In one embodiment, a protein or amino acid sequence which is derived from a particular starting protein or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a region thereof wherein the region consists of at least 3-5 amino acids, at least 5-10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.

“Diabodies” refers to small antibody fragments prepared by constructing sFv fragments with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.

“Domain antibody” refers to the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies.

“Engineered” refers to manipulation of nucleic acid or polypeptide molecules by synthetic means ( e.g ., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques). In one embodiment, the antibodies of the invention are engineered, including for example, humanized and/or chimeric antibodies, and antibodies which have been engineered to improve one or more properties, such as antigen binding, stability/half-life and/or effector function.

“Epitope” refers to a specific arrangement of amino acids located on a protein or proteins to which an antibody binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear or conformational, i.e., involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.

“Evasin” refers to a protein found in the saliva of Rhipicephalus sanguineus which binds to chemokines and inhibits their activity. “Fab” refers to an antigen-binding fragment of an antibody that contains one light chain in its entirety and the variable region and the constant CHI portion of one heavy chain.

“Fragment” refers to a part or region of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. The term “antigen-binding fragment” or “antibody fragment” refers to a protein fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding to human GPVI). As used herein, the term “fragment” of an antibody molecule includes antigen-binding fragments of antibodies, for example, an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody (scFv), a F(ab') ₂ fragment, a Fab fragment, an Fd fragment, an Fv fragment, a single domain antibody fragment (DAb), a one-armed (monovalent) antibody, diabodies or any antigen-binding molecule formed by combination, assembly or conjugation of such antigen binding fragments. Fragments can be obtained, e.g., via chemical or enzymatic treatment of an intact or complete antibody or antibody chain or by recombinant means. The "Fc" fragment of an antibody comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

“Framework region” or “FR region” refers to the amino acid residues that are part of the variable region, but are not part of the CDRs. Therefore, a variable region framework is between about 100-120 amino acids in length but includes only those amino acids outside of the CDRs. For the specific example of a heavy chain variable region and for the CDRs as defined by Kabat/Chothia, framework region 1 may correspond to the domain of the variable region encompassing amino acids 1-25; framework region 2 may correspond to the domain of the variable region encompassing amino acids 36-49; framework region 3 may correspond to the domain of the variable region encompassing amino acids 67-98, and framework region 4 may correspond to the domain of the variable region from amino acids 110 to the end of the variable region. The framework regions for the light chain are similarly separated by each of the light chain variable region CDRs. In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding site as the antibody assumes its three dimensional configuration in an aqueous environment.

The remainders of the heavy and light variable domains show less inter- molecular variability in amino acid sequence and are termed the framework regions. The framework regions largely adopt a [beta] -sheet conformation and the CDRs form loops which connect, and in some cases form part of, the [beta] -sheet structure. Thus, these framework regions act to form a scaffold that provides for positioning the six

CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The position of CDRs can be readily identified by one of ordinary skill in the art.

“Fv” refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute to antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

“Fynomer” refers to small globular proteins (7 kDa) that evolved from amino acids 83-145 of the Src homology domain 3 (SH3) of the human Fyn tyrosine kinase. Their binding scaffolds contain two antiparallel b-sheets and two flexible loops. Fynomers are attractive binding molecules due to their high thermal stability, cysteine-free scaffold, and human origin, which reduce any potential immunogenicity.

“Glycoprotein VI” or “GPVI” refers to a platelet membrane glycoprotein that is involved in platelet-collagen interactions. GPVI is a transmembrane collagen receptor encoded by the GP6 gene and expressed on the surface of platelets. The extracellular domain of GPVI is composed of two Ig-like C2-type domains, namely D1 and D2, linked by a hinge interdomain.

“Heavy chain region”: refers to amino acid sequences derived from the constant domains of an immunoglobulin heavy chain. A protein comprising a heavy chain region comprises at least one of: a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In an embodiment, a binding molecule of the invention may comprise the Fc region of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, a binding molecule of the invention lacks at least a region of a constant domain (e.g., all or part of a CH2 domain). In certain embodiments, at least one, and preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one preferred embodiment, the heavy chain region comprises a fully human hinge domain. In other preferred embodiments, the heavy chain region comprising a fully human Fc region

(e.g., hinge, CH2 and CH3 domain sequences from a human immunoglobulin). In certain embodiments, the constituent constant domains of the heavy chain region are from different immunoglobulin molecules. For example, a heavy chain region of a protein may comprise a CH2 domain derived from an IgGl molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domains are chimeric domains comprising regions of different immunoglobulin molecules. For example, a hinge may comprise a first region from an IgGl molecule and a second region from an IgG3 or IgG4 molecule. As set forth above, it will be understood by one of ordinary skill in the art that the constant domains of the heavy chain region may be modified such that they vary in amino acid sequence from the naturally occurring (wild-type) immunoglobulin molecule. That is, the proteins of the invention disclosed herein may comprise alterations or modifications to one or more of the heavy chain constant domains (CHI, hinge, CH2 or CH3) and/or to the light chain constant domain (CL). Exemplary modifications include additions, deletions or substitutions of one or more amino acids in one or more domains. “High human homology” refers to an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) will be considered as having high human homology if the VH domains and the VL domains, taken together, exhibit at least 70, 75, 80, 85, 90, 95% or more amino acid sequence identity to the closest matching human germline VH and VL sequences. Antibodies having high human homology may include antibodies comprising VH and VL domains of native non-human antibodies which exhibit sufficiently high % sequence identity human germline sequences, as well as engineered, especially humanized, variants of such antibodies and also “fully human” antibodies. In an embodiment the VH domain of the antibody with high human homology may exhibit an amino acid sequence identity or sequence homology of 75%, 80% or greater with one or more human VH domains across the framework regions FR1, FR2, FR3 and FR4. In other embodiments the amino acid sequence identity or sequence homology between the VH domain of the protein of the invention and the closest matching human germline VH domain sequence may be 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100%. In an embodiment the VH domain of the antibody with high human homology may contain one or more ( e.g ., 1 to 20) amino acid sequence mismatches across the framework regions FR1, FR2, FR3 and FR4, in comparison to the closest matched human VH sequence. In another embodiment the VL domain of the antibody with high human homology may exhibit a sequence identity or sequence homology of 80% or greater with one or more human VL domains across the framework regions FR1, FR2, FR3 and FR4. In other embodiments the amino acid sequence identity or sequence homology between the VL domain of the protein of the invention and the closest matching human germline VL domain sequence may be 85% or greater 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100%.

In an embodiment the VL domain of the antibody with high human homology may contain one or more (e.g., 1 to 20, preferably 1 to 10 and more preferably 1 to 5) amino acid sequence mismatches across the framework regions FR1, FR2, FR3 and FR4, in comparison to the closest matched human VL sequence. Before analyzing the percentage sequence identity between the antibody with high human homology and human germline VH and VL, the canonical folds may be determined, which allow the identification of the family of human germline segments with the identical combination of canonical folds for HI and H2 or LI and L2 (and L3). Subsequently the human germline family member that has the highest degree of sequence homology with the variable region of the antibody of interest is chosen for scoring the sequence homology. The determination of Chothia canonical classes of hypervariable loops LI, L2, L3, HI and H2 can be performed with the bioinformatics tools publicly available on webpage www.bioinf.org.uk/abs/chothia.html.page. The output of the program shows the key residue requirements in a data file. In these data files, the key residue positions are shown with the allowed amino acids at each position. The sequence of the variable region of the antibody of interest is given as input and is first aligned with a consensus antibody sequence to assign the Kabat/Chothia numbering scheme. The analysis of the canonical folds uses a set of key residue templates derived by an automated method developed by Martin and Thornton (Martin et al, 1996. J. Mol. Biol. 263(5):800-815). With the particular human germline V segment known, which uses the same combination of canonical folds for HI and H2 or LI and L2 (and L3), the best matching family member in terms of sequence homology can be determined. With bioinformatics tools the percentage sequence identity between the VH and VL domain framework amino acid sequences of the antibody of interest and corresponding sequences encoded by the human germline can be determined, but actually manual alignment of the sequences can be applied as well. Human immunoglobulin sequences can be identified from several protein data bases, such as VBase (http://vbase.mrc-cpe.cam.ac.uk/) or the Pluckthun/Honegger database (http://www.bioc.unizh.ch/antibody/Sequences/ Germlines). To compare the human sequences to the V regions of VH or VL domains in an antibody of interest a sequence alignment algorithm such as available via websites like www.expasy.ch/tools/#align can be used, but also manual alignment with the limited set of sequences can be performed. Human germline light and heavy chain sequences of the families with the same combinations of canonical folds and with the highest degree of homology with the framework regions 1, 2, and 3 of each chain are selected and compared with the variable region of interest; also the FR4 is checked against the human germline JH and JK or JL regions. Note that in the calculation of overall percent sequence homology the residues of FR1, FR2 and FR3 are evaluated using the closest match sequence from the human germline family with the identical combination of canonical folds. Only residues different from the closest match or other members of the same family with the same combination of canonical folds are scored (NB - excluding any primer-encoded differences). However, for the purposes of humanization, residues in framework regions identical to members of other human germline families, which do not have the same combination of canonical folds, can be considered “human”, despite the fact that these are scored “negative” according to the stringent conditions described above. This assumption is based on the “mix and match” approach for humanization, in which each of FR1, FR2, FR3 and FR4 is separately compared to its closest matching human germline sequence and the humanized molecule therefore contains a combination of different FRs as was done by Qu and colleagues (Qu et al, Clin. Cancer Res. 5:3095-3100 (1999)) and Ono and colleagues (Ono et al, Mol. Immunol. 36:387-395 (1999)). The boundaries of the individual framework regions may be assigned using the IMGT numbering scheme, which is an adaptation of the numbering scheme of Chothia (Lefranc et al, Nucleic acid res 27: 209-212 (1999); http://im.gt.cines.fr). Antibodies with high human homology may comprise hypervariable loops or CDRs having human or human-like canonical folds, as discussed in detail below. In an embodiment at least one hypervariable loop or CDR in either the VH domain or the VL domain of the antibody with high human homology may be obtained or derived from a VH or VL domain of a non-human antibody, yet exhibit a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies. It is well established in the art that although the primary amino acid sequences of hypervariable loops present in both VH domains and VL domains encoded by the human germline are, by definition, highly variable, all hypervariable loops, except CDR H3 of the VH domain, adopt only a few distinct structural conformations, termed canonical folds (Chothia et al, J. Mol. Biol. 196:901-917 (1987); Tramontano et al, Proteins 6:382- 94 (1989)), which depend on both the length of the hypervariable loop and presence of the so-called canonical amino acid residues (Chothia et al, J. Mol. Biol. 196:901-917 (1987)). Actual canonical structures of the hypervariable loops in intact VH or VL domains can be determined by structural analysis ( e.g X-ray crystallography), but it is also possible to predict canonical structure on the basis of key amino acid residues which are characteristic of a particular structure (discussed further below). In essence, the specific pattern of residues that determines each canonical structure forms a

“signature” which enables the canonical structure to be recognized in hypervariable loops of a VH or VL domain of unknown structure; canonical structures can therefore be predicted on the basis of primary amino acid sequence alone. The predicted canonical fold structures for the hypervariable loops of any given VH or VL sequence in an antibody with high human homology can be analyzed using algorithms which are publicly available from www.bioinf.org.uk/abs/chothia.html, www.biochem.ucl.ac.uk/~martin/antibodies.html and www.bioc.unizh.ch/antibody/Sequences/Germlines/Vbase_hVk.htm l. These tools permit query VH or VL sequences to be aligned against human VH or VL domain sequences of known canonical structure, and a prediction of canonical structure made for the hypervariable loops of the query sequence. In the case of the VH domain, HI and H2 loops may be scored as having a canonical fold structure “substantially identical” to a canonical fold structure known to occur in human antibodies if at least the first, and preferable both, of the following criteria are fulfilled: 1. An identical length, determined by the number of residues, to the closest matching human canonical structural class.

2. At least 33% identity, preferably at least 50% identity with the key amino acid residues described for the corresponding human HI and H2 canonical structural classes (note for the purposes of the foregoing analysis the HI and H2 loops are treated separately and each compared against its closest matching human canonical structural class). The foregoing analysis relies on prediction of the canonical structure of the HI and H2 loops of the antibody of interest. If the actual structures of the HI and H2 loops in the antibody of interest are known, for example based on X-ray crystallography, then the HI and H2 loops in the antibody of interest may also be scored as having a canonical fold structure “substantially identical” to a canonical fold structure known to occur in human antibodies if the length of the loop differs from that of the closest matching human canonical structural class (typically by +1 or +2 amino acids) but the actual structure of the HI and H2 loops in the antibody of interest matches the structure of a human canonical fold. Key amino acid residues found in the human canonical structural classes for the first and second hypervariable loops of human VH domains (HI and H2) are described by Chothia et ah, J. Mol.

Biol. 227:799-817 (1992), the contents of which are incorporated herein in their entirety by reference. In particular, Table 3 on page 802 of Chothia et ah, which is specifically incorporated herein by reference, lists preferred amino acid residues at key sites for HI canonical structures found in the human germline, whereas Table 4 on page 803, also specifically incorporated by reference, lists preferred amino acid residues at key sites for CDR H2 canonical structures found in the human germline. In an embodiment, both HI and H2 in the VH domain of the antibody with high human homology exhibit a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies. Antibodies with high human homology may comprise a VH domain in which the hypervariable loops HI and H2 form a combination of canonical fold structures which is identical to a combination of canonical structures known to occur in at least one human germline VH domain. It has been observed that only certain combinations of canonical fold structures at HI and H2 actually occur in VH domains encoded by the human germline. In an embodiment HI and H2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, yet form a combination of predicted or actual canonical fold structures which is identical to a combination of canonical fold structures known to occur in a human germline or somatically mutated VH domain. In non-limiting embodiments HI and H2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, and form one of the following canonical fold combinations: 1-1, 1-2, 1-3, 1-6, 1-4, 2- 1, 3-1 and 3-5. An antibody with high human homology may contain a VH domain which exhibits both high sequence identity/sequence homology with human VH, and which contains hypervariable loops exhibiting structural homology with human VH. It may be advantageous for the canonical folds present at HI and H2 in the VH domain of the antibody with high human homology, and the combination thereof, to be “correct” for the human VH germline sequence which represents the closest match with the VH domain of the antibody with high human homology in terms of overall primary amino acid sequence identity. By way of example, if the closest sequence match is with a human germline VH3 domain, then it may be advantageous for HI and H2 to form a combination of canonical folds which also occurs naturally in a human VH3 domain. This may be particularly important in the case of antibodies with high human homology which are derived from non-human species, e.g., antibodies containing VH and VL domains which are derived from camelid conventional antibodies, especially antibodies containing humanized camelid VH and VL domains. Thus, in an embodiment the VH domain of the anti- GP VI antibody with high human homology may exhibit a sequence identity or sequence homology of 70% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with a human VH domain across the framework regions FR1, FR2, FR3 and FR4, and in addition HI and H2 in the same antibody are obtained from a non-human VH domain, but form a combination of predicted or actual canonical fold structures which is the same as a canonical fold combination known to occur naturally in the same human VH domain. In other embodiments, LI and L2 in the VL domain of the antibody with high human homology are each obtained from a VL domain of a non-human species, and each exhibits a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies. As with the VH domains, the hypervariable loops of VL domains of both VLambda and VKappa types can adopt a limited number of conformations or canonical structures, determined in part by length and also by the presence of key amino acid residues at certain canonical positions. Within an antibody of interest having high human homology, LI, L2 and L3 loops obtained from a VL domain of a non-human species, e.g., a Camelidae species, may be scored as having a canonical fold structure “substantially identical” to a canonical fold structure known to occur in human antibodies if at least the first, and preferable both, of the following criteria are fulfilled: 1. An identical length, determined by the number of residues, to the closest matching human structural class.

2. At least 33% identity, preferably at least 50% identity with the key amino acid residues described for the corresponding human LI or L2 canonical structural classes, from either the VLambda or the VKappa repertoire (note for the purposes of the foregoing analysis the LI and L2 loops are treated separately and each compared against its closest matching human canonical structural class). The foregoing analysis relies on prediction of the canonical structure of the LI, L2 and L3 loops in the VL domain of the antibody of interest. If the actual structure of the LI, L2 and L3 loops is known, for example based on X-ray crystallography, then LI, L2 or L3 loops derived from the antibody of interest may also be scored as having a canonical fold structure “substantially identical” to a canonical fold structure known to occur in human antibodies if the length of the loop differs from that of the closest matching human canonical structural class (typically by +1 or +2 amino acids) but the actual structure of the loops in the antibody of interest matches a human canonical fold. Key amino acid residues found in the human canonical structural classes for the CDRs of human VLambda and VKappa domains are described by Morea et al., Methods, 20: 267-279 (2000) and Martin et al., J. Mol. Biol., 263:800-815 (1996). The structural repertoire of the human VKappa domain is also described by Tomlinson et al, EMBO J. 14:4628-4638 (1995), and that of the VLambda domain by Williams et al, J. Mol. Biol., 264:220-232 (1996). The contents of all these documents are to be incorporated herein by reference. LI and L2 in the VL domain of an antibody with high human homology may form a combination of predicted or actual canonical fold structures which is identical to a combination of canonical fold structures known to occur in a human germline VL domain. In non-limiting embodiments LI and L2 in the VLambda domain of an antibody with high human homology may form one of the following canonical fold combinations: 11-7, 13-7(A,B,C), 14-7(A,B), 12-11, 14-11 and 12-12 (as defined in Williams et al., J. Mol. Biol. 264:220 -32 (1996) and as shown on http://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hV L.html). In non- limiting embodiments LI and L2 in the VKappa domain may form one of the following canonical fold combinations: 2- 1, 3-1, 4- 1 and 6- 1 (as defined in Tomlinson et ah, EMBO J. 14:4628-38 (1995) and as shown on http://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hV K.html).

In a further embodiment, all three of LI, L2 and L3 in the VL domain of an antibody with high human homology may exhibit a substantially human structure. It is preferred that the VL domain of the antibody with high human homology exhibit both high sequence identity/sequence homology with human VL, and also that the hypervariable loops in the VL domain exhibit structural homology with human VL.

In an embodiment, the VL domain of the anti- GP VI antibody with high human homology may exhibit a sequence identity of 70% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with a human VL domain across the framework regions LR1, LR2 , LR3 and LR4, and in addition hypervariable loop LI and hypervariable loop L2 may form a combination of predicted or actual canonical fold structures which is the same as a canonical fold combination known to occur naturally in the same human VL domain.

It is, of course, envisaged that VH domains exhibiting high sequence identity/sequence homology with human VH, and also structural homology with hypervariable loops of human VH will be combined with VL domains exhibiting high sequence identity/sequence homology with human VL, and also structural homology with hypervariable loops of human VL to provide antibodies with high human homology containing VH/VL pairings with maximal sequence and structural homology to human-encoded VH/VL pairings.

“Isolated antibody” refers to an antibody that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous components. In preferred embodiments, the antibody is purified: (1) to greater than 80, 85, 90, 91, 92, 93, 94, 95% or more by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity as shown by SDS-PAGE under reducing or non-reducing conditions and using Coomassie blue or, preferably, silver staining. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

“Hinge region” refers to the region of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et ah, 1998. J. Immunol. 161(8):4083-90).

“Hypervariable loop” and “complementarity determining region” are not strictly synonymous, since the hypervariable loops (HVs) are defined on the basis of structure, whereas complementarity determining regions (CDRs) are defined based on sequence variability (Rabat, Elvin A. (1983). Sequences of proteins of immunological interest (5 ^th edition). Besthesda, MD: Public Health Service, National Institutes of Health) and the limits of the HVs and the CDRs may be different in some VH and VL domains. The CDRs of the VL and VH domains can typically be defined by the Kabat/Chothia definition (see above). In one embodiment, the CDRs of the VL and VH domains may comprise the following amino acids: residues 24-39 (CDRL1), 55-61 (CDRL2) and 94-102 (CDRL3) in the light chain variable domain, and residues 26-35 (CDRH1), 50-66 (CDRH2) and 99-109 (CDRH3) in the heavy chain variable domain. Thus, the HVs may be comprised within the corresponding CDRs and references herein to the “hypervariable loops” of VH and VL domains should be interpreted as also encompassing the corresponding CDRs, and vice versa, unless otherwise indicated. The more highly conserved regions of variable domains are called the framework region (FR), as defined herein. The variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a [beta] -sheet configuration, connected by the three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs and, with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies. Structural analysis of antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et ah, 1992. J. Mol. Biol. 227(3):799-817; Tramontano et al., 1990. J. Mol. Biol. 215(1): 175-182). Despite their high sequence variability, five of the six loops adopt just a small repertoire of main-chain conformations, called “canonical structures”. These conformations are first of all determined by the length of the loops and secondly by the presence of key residues at certain positions in the loops and in the framework regions that determine the conformation through their packing, hydrogen bonding or the ability to assume unusual main-chain conformations.

“Humanized”: refers to chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from a murine immunoglobulin. For example, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of the original antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody.

“Identity” or “identical”, when used in a relationship between the sequences of two or more amino acid sequences, refers to the degree of sequence relatedness between amino acid sequences, as determined by the number of matches between strings of two or more amino acid residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e. , “algorithms”). Identity of related amino acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Arthur M. Lesk,

Computational Molecular Biology: Sources and Methods for Sequence Analysis (New-York: Oxford University Press, 1988); Douglas W. Smith, Biocomputing: Informatics and Genome Projects (New-York: Academic Press, 1993); Hugh G. Griffin and Annette M. Griffin, Computer Analysis of Sequence Data, Part 1 (New Jersey: Humana Press, 1994); Gunnar von Heinje, Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit (Academic Press, 1987); Michael Gribskov and John Devereux, Sequence Analysis Primer (New York: M. Stockton Press, 1991); and Carillo el al., 1988. SIAM J. Appl. Math. 48(5): 1073-1082. Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., 1984. Nucl. Acid. Res. 12(1 Pt l):387-395; Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI), BLASTP, BLASTN, TBLASTN and FASTA (Altschul et al, 1990. J. Mol. Biol. 215(3):403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., 1990. J. Mol. Biol. 215(3):403-410). The well-known Smith Waterman algorithm may also be used to determine identity. - “Mammal” refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human.

“Modified antibody” refers to synthetic forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain regions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules joined to scFv molecules and the like. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019. In addition, the term “modified antibody” includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen). In another embodiment, a modified antibody of the invention is a fusion protein comprising at least one heavy chain region lacking a CH2 domain and comprising a binding domain of a protein comprising the binding region of one member of a receptor ligand pair. “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et ah, Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et ah, Nature, 352:624-628 (1991) and Marks et ah, J. Mol. Biol., 222:581-597 (1991), for example.

“Native sequence” refers to a polynucleotide that has the same nucleotide sequence as a polynucleotide derived from nature. Accordingly, a “native sequence” protein is one that has the same amino acid sequence as a protein (e.g., antibody) derived from nature (e.g., from any species). Such native sequence polynucleotides and proteins can be isolated from nature or can be produced by recombinant or synthetic means.

“Nanobody” refers to an antibody-derived therapeutic protein that contains the unique structural and functional properties of naturally-occurring heavy chain antibodies. These heavy chain antibodies may contain a single variable domain

(VHH) and two constant domains (CH2 and CH3).

“NEWS2” is the latest version of the National Early Warning Score (NEWS), first produced in 2012 and updated in December 2017, which advocates a system to standardize the assessment of and response to acute illness. NEWS is based on a simple aggregate scoring system in which a score is allocated to physiological measurements, already recorded in routine practice, when patients present to, or are being monitored in hospital. Six simple physiological parameters form the basis of the scoring system: o 1. Respiration rate; o 2. Oxygen saturation; o 3. Systolic blood pressure; o 4. Pulse rate; o 5. Level of consciousness or new confusion. The patient has new-onset confusion, disorientation and/or agitation, where previously their mental state was normal - this may be subtle. The patient may respond to questions coherently, but there is some confusion, disorientation and/or agitation. This would score 3 or 4 on the Glasgow coma scale (GCS) (rather than the normal 5 for verbal response), and scores 3 on the NEWS system o 6. Temperature.

A score is allocated to each parameter as they are measured, with the magnitude of the score reflecting how extremely the parameter varies from the norm (Table 1). The score is then aggregated and uplifted by 2 points for people requiring supplemental oxygen to maintain their recommended oxygen saturation. Table 1: NEWS 2 With A for Alert, V for responsive to Voice, P for responsive to Pain and U for Unresponsive.

“Pharmaceutically acceptable excipient” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Said excipient does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EM A. - “Polypeptide” is used in its conventional meaning, i.e., as a sequence of less than

100 amino acids. A polypeptide usually refers to a monomeric entity. The term “protein” refers to a sequence of more than 100 amino acids and/or to a multimeric entity. The proteins of the invention are not limited to a specific length of the product. This term does not refer to or exclude post-expression modifications of the protein, for example, glycosylation, acetylation, phosphorylation and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A protein may be an entire protein, or a subsequence thereof. Particular proteins of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen. An “isolated protein” is one that has been identified and separated and/or recovered from a component of its natural environment. In preferred embodiments, the isolated protein will be purified (1) to greater than 80, 85, 90, 95% by weight of protein as determined by the Lowry method, and most preferably more than 96, 97, 98, or 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated protein includes the protein in situ within recombinant cells since at least one component of the protein’s natural environment will not be present. Ordinarily, however, isolated protein will be prepared by at least one purification step. “Prevent”, “preventing” and “prevention” refer to prophylactic and preventative measures, wherein the object is to reduce the chances that a subject will develop the pathologic condition or disorder over a given period of time. Such a reduction may be reflected, e.g., in a delayed onset of at least one symptom of the pathologic condition or disorder in the subject.

“Respiratory support” refers to any measure administered to a subject in order to compensate for a respiratory distress or failure experienced by the subject. Examples of such measures include non-invasive ventilation (NIV) such as supplemental oxygen (also called oxygen therapy) by mask or nasal prongs, positive pressure, high flow nasal oxygen; invasive mechanical ventilation (IVM) requiring tracheal intubation or tracheostomy; and extracorporeal membrane oxygenation (ECMO). As used herein, “respiratory support” or “oxygen therapy” thus encompass both non-invasive ventilation (NIV) and invasive mechanical ventilation (IVM).

“SARS-Cov-2” or “Severe acute respiratory syndrome-coronavirus 2” refers to a positive-sense single-stranded RNA virus belonging to the Coronaviridae family. It is contagious in humans and the cause of the coronavirus disease 2019 (COVID-19).

“Single chain antibody” or “single chain Fv” or “SFv” or “scFv” refers to antibody fragments that comprise the VH and VL antibody domains connected into a single amino acid chain. Preferably, the sFv amino acid sequence further comprises a peptidic linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. 1995, infra.

“Specificity” refers to the ability to specifically bind (e.g., immunoreact with) a given target, e.g., GPVI.

“Specific for an antigen” or “to specifically bind to an antigen” refers to an antibody that reacts at a detectable level with the antigen, preferably with an affinity constant, K _A , of greater than or equal to about 10 ⁶ M ¹ , greater than or equal to about 10 ⁷ M ¹ , or greater than or equal to 10 ⁸ M ¹ , or greater than or equal to 1.5 10 ⁸ M ^1, , or greater than or equal to 10 ⁹ M ¹ or greater than or equal to 5 10 ⁹ M ¹ . Affinity of an antibody for its cognate antigen is also commonly expressed as a dissociation constant KD, and in certain embodiments, an antibody specifically binds to antigen if it binds with a KD of less than or equal to 10 ⁶ M, less than or equal to 10 ⁷ M, or less than or equal to 1.5 10 ⁸ M, or less than or equal to 10 ⁸ M, or less than or equal to 5 10 ⁹ M or less than or equal to 10 ⁹ M. Affinities of antibodies can be readily determined using conventional techniques, for example, those described by Scatchard G el al. (The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51: 660- 672). Binding properties of an antibody to antigens, cells or tissues thereof may generally be determined and assessed using immunodetection methods including, for example, ELISA, immunofluorescence-based assays, such as immuno-histochemistry

(IHC) and/or fluorescence-activated cell sorting (FACS) or by surface plasmon resonance (SPR, BIAcore).

“Subject” refers to a mammal, preferably a human. In one embodiment, a subject may be a “patient”, i.e. a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease.

“Therapeutically effective amount” means level or amount of agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of ARDS; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of ARDS;

(3) bringing about ameliorations of the symptoms of ARDS; (4) reducing the severity or incidence of ARDS; or (5) curing ARDS. A therapeutically effective amount may be administered prior to the onset of ARDS, for a prophylactic or preventive action. Alternatively or additionally, the therapeutically effective amount may be administered after initiation of ARDS, for a therapeutic action.

“Treating” or “treatment” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject is successfully “treated” for the targeted pathologic condition or disorder if, after receiving a therapeutic amount of a protein according to the present invention, the subject or mammal shows observable and/or measurable improvement in one or more of the following: relief to some extent of one or more of the symptoms associated with the specific disease or condition; improvement of a respiratory distress, absence of requirement of respiratory support, e.g., oxygenation or mechanical ventilation (in particular invasive ventilation), improvement of respiratory failure (for example assessed by measuring the Time-to clinical improvement of the NEWS2 score), reduced morbidity and mortality, and/or improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

“Unibody” refers to an antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies.

“Variable region” or “variable domain” refers to the fact that certain regions of the variable domains VH and VL differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called “hypervariable loops” in each of the VL domain and the VH domain which form part of the antigen binding site. The first, second and third hypervariable loops of the VLambda light chain domain are referred to herein as LI (l), L2 (l) and L3 (l) and may be defined as comprising residues 24-33 (Ll(k), consisting of 9, 10 or 11 amino acid residues), 49-53 L2 (l), consisting of 3 residues) and 90-96 (L3(k), consisting of 6 residues) in the VL domain

(Morea et ah, Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the VKappa light chain domain are referred to herein as L1(K), L2(K) and L3(K) and may be defined as comprising residues 25-33 (L1(K), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(K), consisting of 3 residues) and 90-97 (L3(K), consisting of 6 residues) in the VL domain (Morea et ah, Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the VH domain are referred to herein as HI, H2 and H3 and may be defined as comprising residues 25-33 (HI, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3, highly variable in length) in the VH domain (Morea et ah, Methods 20:267-279 (2000)). Unless otherwise indicated, the terms LI, L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and encompass hypervariable loops obtained from both VKappa and VLambda isotypes. The terms HI, H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including [gamma], [epsilon], [delta], [alpha] or [mu]. The hypervariable loops LI, L2, L3, HI, H2 and H3 may each comprise part of a “complementarity determining region” or “CDR”, as defined hereinabove.

“Versabody” refers to an antibody mimetic technology. They are small proteins of 3-5 kDa with >15% cysteines, which form a high disulfide density scaffold, replacing the hydrophobic core the typical proteins have.

DETAILED DESCRIPTION

The present invention relates to an isolated protein binding to Glycoprotein VI (GPVI), in particular human GPVI, for treating or preventing an acute respiratory distress syndrome (ARDS) in a subject. In one embodiment, the isolated protein is for preventing the onset of ARDS, or for preventing the aggravation of ARDS.

In one embodiment, the isolated protein is an isolated humanized protein.

In one embodiment, the isolated protein binds to human GPVI (hGPVI).

In one embodiment, the amino acid sequence of human GPVI is SEQ ID NO: 13 (accession number: BAA89353.1) or any amino acid sequence presenting at least about 90% identity with SEQ ID NO: 13, preferably at least about 91, 92, 93, 94, 95, 96, 97,

98, 99% identity or more with SEQ ID NO: 13.

(SEQ ID NO: 13). MSPSPTALFCLGLCLGRVPA (Signal peptide).

QS GPLPKPS LQ ALPS S LVPLEKP VTLRC QGPPG VDLYRLEKLS S S R Y QDQ A VLFI PAMKRS LAGR YRC S Y QN GS LW S LPS DQLELV ATG VF AKPS LS AQPGP A V S S GG DVTLQCQTRYGFDQFALYKEGDPAPYKNPERWYRASFPIITVTAAHSGTYRCY SFS S RDP YLW S APS DPLELV VTGT S VTPS RLPTEPPS S V AEFS E AT AELT V S FTNK VFTTET S RS ITT S PKES DS P AGP ARQ Y YTKGN (Extracellular domain).

LVRICLGAVILIILAGFLAEDWHSRRKRLRHRGRAVQRPLPPLPPLPQTRKSHGG QDGGRQDVHSRGLCS (Transmembrane and cytoplasmic domains).

The extracellular domain of GPVI is composed of two Ig-like C2-type domains, namely D1 and D2, linked by a hinge interdomain. In one embodiment, D1 comprises amino acid residues 21 to 109 of SEQ ID NO: 13. In one embodiment, the hinge interdomain between D1 and D2 comprises amino acid residues 110 to 113 of SEQ ID NO: 13. In one embodiment, D2 comprises amino acid residues 114 to 203 of SEQ ID NO: 13.

In one embodiment, the isolated protein binds to the extracellular domain of GPVI.

In one embodiment, the isolated protein binds at least the Ig-like C2-type domain 2 (D2) of human GPVI.

In one embodiment, the protein for use in the present invention is an antibody or an antigen-binding fragment thereof.

In one embodiment, the protein for use in the present invention is a blocking or antagonist antibody or antigen-binding fragment thereof.

In one embodiment, the protein for use in the present invention is a monovalent antibody or antigen-binding fragment thereof.

In one embodiment, the protein for use in the present invention is a monoclonal antibody or antigen-binding fragment thereof.

In one embodiment, the protein for use in the present invention is a whole antibody. In one embodiment, the protein for use in the present invention is an antigen-binding fragment selected from the group comprising or consisting of a single chain antibody, a Fv, a Fab, and a unibody.

In one embodiment, the protein for use in the present invention is an antigen-binding fragment selected from the group comprising or consisting of a nanobody and a domain antibody.

In one embodiment, the protein for use in the present invention is an antigen-binding fragment selected from the group comprising or consisting of a single chain antibody, a Fv, a Fab, a unibody, a nanobody and a domain antibody. In one embodiment, the protein for use in the present invention is a monomeric antibody mimetic selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer and a versabody.

In one embodiment, the protein for use in the present invention is monovalent. In one embodiment, the isolated protein for use in the present invention is purified.

In one embodiment, the antibody or antigen-binding fragment thereof for use in the present invention comprises in the variable region of the heavy chain at least one of the following CDRs:

VH-CDR1: GYTFTSYNMH (SEQ ID NO: 1); VH-CDR2: GIYPGN GDTS YN QKFQG (SEQ ID NO: 2); and

VH-CDR3: GTVVGDWYFDV (SEQ ID NO: 3). or any CDR having an amino acid sequence that shares at least 60% of identity with SEQ ID NO: 1-3.

CDR numbering and definition are according to the Kabat/Chothia definition. In one embodiment, the antibody or antigen-binding fragment thereof for use in the present invention comprises in the variable region of the light chain at least one of the following CDRs: VL-CDR1: RS S QS LENS N GNT YLN (SEQ ID NO: 4);

VL-CDR2: RVSNRFS (SEQ ID NO: 5); and VL-CDR3: LQLTHVPWT (SEQ ID NO: 6). or any CDR having an amino acid sequence that shares at least 60% of identity with SEQ ID NO: 4-6.

CDR numbering and definition are according to the Kabat/Chothia definition.

In one embodiment, the antibody or antigen-binding fragment thereof for use in the present invention comprises in the variable region of the heavy chain at least one of the following CDRs:

VH-CDR1: GYTFTSYNMH (SEQ ID NO: 1);

VH-CDR2: GIYPGN GDTS YN QKFQG (SEQ ID NO: 2); and VH-CDR3: GTVVGDWYFDV (SEQ ID NO: 3), and or any CDR having an amino acid sequence that shares at least 60% of identity with SEQ ID NO: 1-3, and/or in the variable region of the light chain at least one of the following CDRs:

VL-CDR1: RSSQS LENS NGNT YLN (SEQ ID NO: 4);

VL-CDR2: RVSNRFS (SEQ ID NO: 5); and VL-CDR3: LQLTHVPWT (SEQ ID NO: 6), or any CDR having an amino acid sequence that shares at least 60% of identity with SEQ ID NO: 4-6.

In one embodiment, the antibody or antigen-binding fragment thereof for use in the present invention comprises in the variable region of the heavy chain the following CDRs: GYTFTSYNMH (SEQ ID NO: 1), GIYPGN GDTS YN QKFQG (SEQ ID NO: 2) and GTVVGDWYFDV (SEQ ID NO: 3) and in the variable region of the light chain the following CDRs: RSSQS LENS NGNT YLN (SEQ ID NO: 4), RVSNRFS (SEQ ID NO: 5) and LQLTHVPWT (SEQ ID NO: 6) or any CDR having an amino acid sequence that shares at least 60% of identity with SEQ ID NO: 1-6.

In another embodiment of the invention, the antibody or antigen-binding fragment thereof for use in the present invention comprises in its heavy chain the 3 CDRs SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and in its light chain the 3 CDRs SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, wherein one, two, three or more of the amino acids in any of said sequences may be substituted by a different amino acid.

According to the invention, any of the CDRs 1, 2 and 3 of the heavy and light chains may be characterized as having an amino acid sequence that shares at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity with the particular CDR or sets of CDRs listed in the corresponding SEQ ID NO.

In one embodiment, the antibody or antigen-binding fragment for use in the present invention comprises a heavy chain variable region comprising or consisting of the sequence SEQ ID NO: 7.

(SEQ ID NO: 7).

Q V QLV QS G AE VKKPG AS VKV S C KAS G YTFTS YNMHW VRQ APGQGLE WMGGI YPGN GDT S YN QKF QGR VTMTRDT STS T V YMELS S LRS EDT A V Y Y C ARGT V V G D W YFD VW GQGTLVT V S S . In one embodiment, the antibody or antigen-binding fragment thereof for use in the present invention comprises a light chain variable region comprising or consisting of the sequence SEQ ID NO: 8.

(SEQ ID NO: 8).

DIQMTQS PS S LS AS V GDRVTITCRS S QS LENS N GNT YLNW Y QQKPGKAPKLLIY RV S NRF S G VPS RFS GS GS GTDFTFTIS S LQPEDIAT Y YCLQLTH VPWTF GQGTKV

EITR.

In one embodiment, the antibody or antigen-binding fragment thereof for use in the present invention comprises a light chain variable region comprising or consisting of the sequence SEQ ID NO: 9. (SEQ ID NO: 9). DIQMTQS PS S LS AS V GDRVTITCS AS QS LENS N GNT YLNW Y QQKPGKAPKLLIY RV S NRF S G VPS RFS GS GS GTDFTLTIS S LQPEDFAT Y YCLQLTH VPWTF GQGTKV EIKR.

In one embodiment of the invention, the antibody or antigen-binding fragment thereof for use in the present invention is the anti-GPVI antibody ACT017 which comprises a heavy chain variable region comprising or consisting of the sequence SEQ ID NO: 7 and a light chain variable region comprising or consisting of the sequence SEQ ID NO: 8.

In another embodiment of the invention, the antibody or antigen-binding fragment thereof for use in the present invention is the anti-GPVI antibody ACT006 which comprises a heavy chain variable region comprising or consisting of the sequence SEQ ID NO: 7 and a light chain variable region comprising or consisting of the sequence SEQ ID NO: 9.

According to the invention, one, two, three or more of the amino acids of the heavy chain or light chain variable regions as described hereinabove may be substituted by a different amino acid.

In one embodiment, the heavy chain variable region encompasses sequences that have 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 7.

In one embodiment, the light chain variable region encompasses sequences that have 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 8 or SEQ ID NO: 9.

In one embodiment, the antibody or antigen-binding fragment thereof for use in the present invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the ACT017 antibody described herein, wherein the antibody or antigen-binding fragment thereof retains the desired functional properties of the protein described in the present invention.

In another embodiment, the antibody or antigen-binding fragment thereof for use in the present invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the ACT006 antibody described herein, wherein the antibody or antigen-binding fragment thereof retains the desired functional properties of the protein described in the present invention.

In any of the antibodies or antigen-binding fragments thereof for use in the present invention (e.g., ACT017 or ACT006), the specified variable region and CDR sequences may comprise conservative sequence modifications. Conservative sequence modifications refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or fragment thereof containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antigen-binding fragment thereof for use in the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR- mediated mutagenesis. Conservative amino acid substitutions are typically those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Specified variable region and CDR sequences may comprise one, two, three, four or more amino acid insertions, deletions or substitutions. Where substitutions are made, preferred substitutions will be conservative modifications. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody or antigen-binding fragment thereof for use in the present invention can be replaced with other amino acid residues from the same side chain family and the altered antibody or antigen-binding fragment thereof can be tested for retained function, in particular affinity for GPVI.

In one embodiment, the antibody or antigen-binding fragment thereof for use in the present invention binds essentially the same epitope as ACT017 or ACT006 antibodies. In the present invention, an antibody that binds essentially the same epitope as ACT017 or ACT006 antibodies will be referred as an ACT017-like or ACT006-like antibody, respectively.

In one embodiment, the antibody or antigen-binding fragment thereof for use in the present invention competes for binding to hGPVI with an antibody as described hereinabove, in particular with an antibody selected from ACT017 and ACT006.

In some embodiments, anti-hGPVI antibodies or antigen-binding fragments thereof comprising VH and VL domains, or CDRs thereof may comprise CHI domains and/or CL domains, the amino acid sequence of which is fully or substantially human. Where the GPVI binding protein is an antibody or an antigen binding fragment thereof intended for human therapeutic use, it is typical for the entire constant region of the antibody, or at least a part thereof, to have a fully or substantially human amino acid sequence. Therefore, one or more or any combination of the CHI domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain if present) may be fully or substantially human with respect to its amino acid sequence. Advantageously, the CHI domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain if present) may all have a fully or substantially human amino acid sequence. In the context of the constant region of a humanized or chimeric antibody, or an antigen-binding fragment, the term “substantially human” refers to an amino acid sequence identity of at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% with a human constant region. The term “human amino acid sequence” in this context refers to an amino acid sequence which is encoded by a human immunoglobulin gene, which includes germline, rearranged and somatically mutated genes. The invention also contemplates the use of proteins comprising constant domains of “human” sequence which have been altered, by one or more amino acid additions, deletions or substitutions with respect to the human sequence, excepting those embodiments where the presence of a “fully human” hinge region is expressly required. The presence of a “fully human” hinge region in the anti-hGPVI antibodies for use in the present invention may be beneficial both to minimize immunogenicity and to optimize stability of the antibody. It is considered that one or more amino acid substitutions, insertions or deletions may be made within the constant region of the heavy and/or the light chain, particularly within the Fc region. Amino acid substitutions may result in replacement of the substituted amino acid with a different naturally occurring amino acid, or with a non-natural or modified amino acid. Other structural modifications are also permitted, such as for example changes in glycosylation pattern (e.g., by addition or deletion of N- or O-linked glycosylation sites). Depending on the intended use of the antibody or antigen-binding fragment thereof, it may be desirable to modify the antibody or antigen-binding fragment thereof with respect to its binding properties to Fc receptors, for example to modulate effector function. For example, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved effector function. See Caron et al., J. Exp. Med. 176: 1191 - 1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).

Thus, in one embodiment, the protein for use in the present invention is a chimeric, a humanized or a fully human antibody or antigen-binding fragment thereof. In one embodiment, the heavy chain variable region of sequence SEQ ID NO: 7 is encoded by SEQ ID NO: 10:

(SEQ ID NO: 10).

CAGGTTCAGCTGGTTCAGTCAGGGGCTGAGGTGAAGAAGCCTGGAGCCTCA GTGAAGGTGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGC ACTGGGTAAGACAGGCTCCTGGACAGGGCCTGGAATGGATGGGAGGTATTT ATCCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCCAGGGCCGAGTTA CTATGACTCGGGACACTTCCACCTCTACAGTGTACATGGAGCTCAGCAGCCT GAGATCTGAGGACACCGCGGTCTATTACTGTGCAAGAGGCACCGTGGTCGG CGACTGGTACTTCGATGTGTGGGGCCAAGGCACCCTGGTCACCGTGAGCAG T.

In one embodiment, the light chain variable region of sequence SEQ ID NO: 8 is encoded by SEQ ID NO: 11:

(SEQ ID NO: 11). GACATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGAC AGAGTGACCATCACCTGTAGAAGTAGTCAGAGCCTTGAGAACAGCAACGGA AACACCTACCTGAATTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCTG CTGATCTACAGAGTTTCCAACCGATTCTCTGGTGTGCCAAGCAGATTCAGCG GTAGCGGTAGCGGTACCGACTTCACCTTCACCATCAGCAGCCTCCAGCCAG AGGACATCGCCACCTACTACTGCCTCCAGCTGACTCATGTCCCATGGACCTT CGGTCAGGGCACCAAGGTGGAGATCACCCGG.

In one embodiment, the light chain variable region of sequence SEQ ID NO: 9 is encoded by SEQ ID NO: 12. (SEQ ID NO: 12).

GACATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGAC AGAGTGACCATCACCTGTAGTGCCAGTCAGAGCCTTGAGAACAGCAACGGA AACACCTACCTGAATTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCTG CTGATCTACAGAGTTTCCAACCGATTCTCTGGTGTGCCAAGCAGATTCAGCG GTAGCGGTAGCGGTACCGACTTC ACCCTCACC ATC AGCAGCCTCCAGCCAG AGGACTTCGCCACCTACTACTGCCTCCAGCTGACTCATGTCCCATGGACCTT CGGTCAGGGCACCAAGGTGGAGATCAAACGC.

In one embodiment, the nucleic sequences encoding the antibody or antigen-binding fragment thereof for use in the present invention are comprised in an expression vector. In one embodiment, the expression vector comprises at least one of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 or any sequence having a nucleic acid sequence that shares at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity with said SEQ ID NO: 10-12.

In one embodiment, the vector comprises the sequence SEQ ID NO: 10 and a sequence encoding a constant region of a heavy chain. A non-limiting example of a sequence encoding a constant region of a heavy chain is SEQ ID NO: 14.

(SEQ ID NO: 14). GCCTCCACCAAGGGTCCCTCAGTCTTCCCACTGGCACCCTCCTCCAAGAGCA CCTCTGGTGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGA ACCAGTGACTGTGTCATGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC CTTCCCTGCTGTCTTGCAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT CACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTCGAGCCTAAGTCATG CGAC AAGACTC AC .

In one embodiment, the vector comprises the sequence SEQ ID NO: 11 or SEQ ID NO: 12 and a sequence encoding a constant region of a light chain. A non-limiting example of a sequence encoding a constant region of a light chain is SEQ ID NO: 15.

(SEQ ID NO: 15).

ACTGTGGCTGCACCAAGTGTGTTCATCTTCCCACCTAGCGATGAGCAGTTGA AATCTGGAACTGCCTCTGTCGTGTGCCTCCTGAACAACTTCTACCCACGGGA GGCCAAGGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCA GGAGAGTGTC AC AGAGC AAGATAGC AAGGAC AGC ACCTAC AGCCTC AGC A GCACCCTGACTCTGAGCAAAGCAGACTACGAGAAGCACAAGGTCTACGCCT GCGAAGTCACCCATCAGGGCCTGAGTTCCCCTGTCACAAAGAGCTTCAACC GGGGAGAGTGT.

In one embodiment, the vector comprises a sequence encoding a signal peptide. Non-limiting examples of signal peptides sequences include, but are not limited to, SEQ ID NO: 16 and SEQ ID NO: 17.

(SEQ ID NO: 16).

ATGGATATGCGTGTACCAGCTCAACTACTTGGACTTCTATTGCTTTGGCTTC

GTGGTGCTAGATGT.

(SEQ ID NO: 17). ATGGACTGGACTTGGAGAATCCTATTCTTGGTTGCTGCAGCTACAGGTGCTC ATTCA.

In one embodiment, the vector comprises SEQ ID NO: 10, and a sequence encoding a constant region of a heavy chain (such as, for example, SEQ ID NO: 14), and a signal peptide sequence. An example of such a vector is a vector comprising SEQ ID NO: 18. SEQ ID NO: 18 further comprises cloning sites.

(SEQ ID NO: 18).

GCGGCCGCCACCATGGACTGGACTTGGAGAATCCTATTCTTGGTTGCTGCAG CTACAGGTGCTCATTCACAGGTTCAGCTGGTTCAGTCAGGGGCTGAGGTGA AGAAGCCTGGAGCCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACAT TTACC AGTTAC AATATGC ACTGGGTAAG AC AGGCTCCTGGAC AGGGCCTGG AATGGATGGGAGGTATTTATCCAGGAAATGGTGATACTTCCTACAATCAGA AGTTCCAGGGCCGAGTTACTATGACTCGGGACACTTCCACCTCTACAGTGTA CATGGAGCTCAGCAGCCTGAGATCTGAGGACACCGCGGTCTATTACTGTGC AAGAGGCACCGTGGTCGGCGACTGGTACTTCGATGTGTGGGGCCAAGGCAC CCTGGTCACCGTGAGCAGTGCCTCCACCAAGGGTCCCTCAGTCTTCCCACTG GCACCCTCCTCCAAGAGCACCTCTGGTGGCACAGCTGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCAGAACCAGTGACTGTGTCATGGAACTCAGGCGCCC TGACCAGCGGCGTGCACACCTTCCCTGCTGTCTTGCAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGAC CTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAA AGTCGAGCCTAAGTCATGCGACAAGACTCACTGATGAGGATCC. In one embodiment, the vector comprises SEQ ID NO: 11, and a sequence encoding a constant region of a light chain (such as, for example, SEQ ID NO: 15), and a signal peptide sequence. An example of such a vector is a vector comprising SEQ ID NO: 19. SEQ ID NO: 19 further comprises cloning sites. (SEQ ID NO: 19).

GACGTCACCATGGATATGCGTGTACCAGCTCAACTACTTGGACTTCTATTGC TTTGGCTTCGTGGTGCTAGATGTGACATCCAGATGACCCAGAGCCCAAGCA GCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGAAGTAGTC AGAGCCTTGAGA AC AGC AACGGAAAC ACCT ACCTGAATTGGT ACC AGC AGA AGCCAGGTAAGGCTCCAAAGCTGCTGATCTACAGAGTTTCCAACCGATTCTC TGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTTCACCTT CACCATCAGCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCTCCA GCTGACTCATGTCCCATGGACCTTCGGTCAGGGCACCAAGGTGGAGATCAC CCGGACTGTGGCTGCACCAAGTGTGTTCATCTTCCCACCTAGCGATGAGCAG TTGAAATCTGGAACTGCCTCTGTCGTGTGCCTCCTGAAC AACTTCTACCC AC GGGAGGCCAAGGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACT CCCAGGAGAGTGTCACAGAGCAAGATAGCAAGGACAGCACCTACAGCCTC AGCAGCACCCTGACTCTGAGCAAAGCAGACTACGAGAAGCACAAGGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGTTCCCCTGTCACAAAGAGCTTC AACCGGGGAGAGTGTTGATGATATC.

In one embodiment, the vector comprises SEQ ID NO: 12, and a sequence encoding a constant region of a light chain (such as, for example, SEQ ID NO: 15), and a signal peptide sequence. An example of such a vector is a vector comprising SEQ ID NO: 20. SEQ ID NO: 20 further comprises cloning sites. (SEQ ID NO: 20).

GACGTCACCATGGATATGCGTGTACCAGCTCAACTACTTGGACTTCTATTGC

TTTGGCTTCGTGGTGCTAGATGTGACATCCAGATGACCCAGAGCCCAAGCA

GCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGTGCCAGTC AGAGCCTTGAGA AC AGC AACGGAAAC ACCT ACCTGAATTGGT ACC AGC AGA

AGCCAGGTAAGGCTCCAAAGCTGCTGATCTACAGAGTTTCCAACCGATTCTC

TGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTTCACCCT

CACCATCAGCAGCCTCCAGCCAGAGGACTTCGCCACCTACTACTGCCTCCAG

CTGACTCATGTCCCATGGACCTTCGGTCAGGGCACCAAGGTGGAGATCAAA

CGCACTGTGGCTGCACCAAGTGTGTTCATCTTCCCACCTAGCGATGAGCAGT

TGAAATCTGGAACTGCCTCTGTCGTGTGCCTCCTGAACAACTTCTACCCACG

GGAGGCCAAGGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTC

CCAGGAGAGTGTCACAGAGCAAGATAGCAAGGACAGCACCTACAGCCTCA

GCAGCACCCTGACTCTGAGCAAAGCAGACTACGAGAAGCACAAGGTCTACG

CCTGCGAAGTCACCCATCAGGGCCTGAGTTCCCCTGTCACAAAGAGCTTCA

ACCGGGGAGAGTGTTGATGAT ATC .

In one embodiment, the vector as described hereinabove is comprised in an isolated host cell, that may be used for the recombinant production of the antibodies or antigen-binding fragments thereof for use in the present invention. In an embodiment, host cells may be prokaryote, yeast, or eukaryote cells preferably mammalian cells, such as, for example: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et ah, J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et ah, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCC CRL 1581 ; ATCC CRL 8287) or NSO (HPA culture collections no. 85110503); 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); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et ah, Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), as well as DSM's PERC-6 cell line. Expression vectors suitable for use in each of these host cells are also generally known in the art. It should be noted that the term “host cell” generally refers to a cultured cell line. Whole human beings into which an expression vector encoding an antigen-binding protein for use according to the invention has been introduced are explicitly excluded from the definition of a “host cell”. Methods for producing an antibody or antigen-binding fragment thereof in the present invention are well known in the art. Examples of suitable methods comprise culturing host cells containing the isolated polynucleotide sequence encoding the antibody or antigen binding fragment thereof for use in the present invention under conditions suitable for expression of the anti-hGPVI antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof. This recombinant process can be used for large scale production of antibodies or antigen-binding fragments thereof for use in the present invention, including monoclonal antibodies and antigen binding fragments thereof intended for in vivo therapeutic uses. These processes are available in the art and will be known by the skilled person. In one embodiment, the protein for use in the present invention may be purified by chromatography, preferably by affinity chromatography, more preferably by affinity chromatography on protein L agarose.

Therefore, in one embodiment, the protein for use in the present invention comprises a domain for binding protein L (PpL). Methods for transferring PpL-binding activity onto proteins of the invention are described in Muzard et ah, Analytical Biochemistry 388, 331-338, 2009 and in Lakhrif et ah, MAbs. 2016;8(2):379-88, which are incorporated herein by reference.

Fragments and derivatives of antibodies for use in the present invention (which are encompassed by the term “antibody” or “antibodies” as used in this application, unless otherwise stated or clearly contradicted by context), preferably a ACT017-like or ACT006-like antibody, can be produced by techniques that are known in the art. “Fragments” comprise a portion of the intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a protein having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or “single chain protein”), including without limitation (1) single-chain Fv molecules (2) single chain proteins containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain proteins containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments. Fragments of the present antibodies can be obtained using standard methods. For instance, Fab or F(ab') ₂ fragments may be produced by protease digestion of the isolated antibodies, according to conventional techniques. It will be appreciated that immunoreactive fragments can be modified using known methods, for example to slow clearance in vivo and obtain a more desirable pharmacokinetic profile, the fragment may be modified with polyethylene glycol (PEG). Methods for coupling and site-specifically conjugating PEG to a Fab' fragment are described in, for example, Leong et ah, Cytokines 16 (3): 106-119 (2001) and Delgado et ah, Br. J. Cancer 73 (2): 175- 182 (1996), the disclosures of which are incorporated herein by reference.

Alternatively, the DNA encoding an antibody for use in the present invention, preferably an ACT017-like or ACT006-like antibody, may be modified so as to encode a fragment. The modified DNA is then inserted into an expression vector and used to transform or transfect an appropriate cell, which then expresses the desired fragment.

Another object of the invention is a composition, pharmaceutical composition or medicament for treating ARDS or for preventing the onset or the aggravation of an ARDS, in particular a virus-related ARDS, and more particularly a SARS-CoV-2-related ARDS, wherein said composition, pharmaceutical composition or medicament comprises or consists essentially of a protein as described hereinabove.

As used herein, the term “consisting essentially of’, with reference to a composition, pharmaceutical composition or medicament, means that the protein binding to GPVI as described herein is the only one therapeutic agent or agent with a biologic activity within said composition, pharmaceutical composition or medicament.

In one embodiment, the pharmaceutical composition for use in the present invention comprises a protein as described hereinabove and a pharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat. In one embodiment, the protein for use in the present invention present in a composition, pharmaceutical composition or medicament can be supplied in a liquid composition at a concentration ranging from about 1 to about 100 mg/mL, such as, for example, at a concentration of 1 mg/mL, 5 mg/mL, 10 mg/mL, 50 mg/mL or 100 mg/mL.

In one embodiment, the protein is supplied at a concentration of about 10 mg/mL in either 100 mg (10 mL), 500 mg (50 mL) or 1000 mg (100 mL) single-use vials.

In one embodiment, the composition, pharmaceutical composition or medicament may comprise a protein of the invention in PBS pH 7.2-7.7.

In one embodiment, the composition, pharmaceutical composition or medicament may comprise a protein of the invention in sodium citrate buffer 20 mM, NaCl 130 mM, pH 5.0.

The present invention further relates to the use of an isolated protein binding to GPVI as described hereinabove for the manufacture of a medicament for treating ARDS, or for preventing the onset or the aggravation of an ARDS, preferably a virus-related ARDS, and more preferably a SARS-CoV-2-related ARDS.

In the meaning of the invention, ARDS (acute respiratory distress syndrome) is defined as a form of acute lung injury (ALI) and occurs as a result of a severe pulmonary injury that causes alveolar damage heterogeneously throughout the lung. ARDS is associated with at least one of the following symptoms: shortness of the breath, rapid breathing. ARDS is a rapidly progressive disorder that initially manifests as dyspnea, tachypnea, and hypoxemia, then quickly evolves into respiratory failure.

In one embodiment, ARDS is defined according to the American-European Consensus Conference (AECC), i.e., ARDS is defined as a respiratory failure of acute onset with aPaO ₂ /FiO ₂ ratio < 200 mm Hg (regardless of the level of positive end-expiratory pressure, PEEP), bilateral infiltrates on frontal chest radiograph, and a pulmonary capillary wedge pressure <18 mmHg (if measured) or no evidence of left atrial hypertension.

In one embodiment, ARDS is evaluated according to the Berlin definition for ARDS. According to this definition, patients are considered as having ARDS if they have: (1) acute respiratory failure not fully explained by cardiac failure or fluid overload, as judged by the treating physician; (2) bilateral opacities consistent with pulmonary edema on the chest radiograph or the computed tomography scan; (3) onset within 1 week after a known clinical insult or new/worsening respiratory symptoms. The Berlin definition categorizes ARDS according to the ratio of arterial oxygen partial pressure (PaO ₂ ) to fractional inspired oxygen (FiO ₂ ) (PaO ₂ /FiO ₂ ): mild (PaO ₂ /FiO ₂ : 201-300 mmHg), moderate (PaO ₂ /FiO ₂ : 101-200 mmHg), and severe (Pa02/Fi02 < 100 mm Hg), with a PEEP> 5 cm H2O.

In one embodiment, ARDS is evaluated by the Murray score system. The Murray scoring system is based on 4 pulmonary variables: hypoxemia with PaO ₂ /FiO ₂ ratio, positive end-expiratory pressure (PEEP) in cmH ₂ O , respiratory system compliance in mL/ cmH ₂ O and chest X-rays (CXR) quadrants infiltrated. Each of the 4 criteria is awarded a value from 0 to 4 according to the severity of the condition. The final score can be interpreted as follows: 0 points: no lung injury; 1 to 2.5 points: mild to moderate lung injury; >2.5 points: ARDS.

In one embodiment, the subject is affected with ARDS. In one embodiment, the subject is diagnosed with ARDS. In one embodiment, the subject is diagnosed with an ARDS according to the AECC definition.

In one embodiment, the subject is diagnosed with a mild ARDS according to the Berlin definition. In one embodiment, the subject is diagnosed with a moderate ARDS according to the Berlin definition. In one embodiment, the subject is diagnosed with a severe ARDS according to the Berlin definition.

In one embodiment, the subject presents a Murray score ranging from 1 to 2.5 points. In one embodiment, the subject presents a Murray score superior to 2.5 points.

In one embodiment, the subject is at risk of developing an ARDS. Examples of risk factors for developing ARDS include, but are not limited to, diabetes (such as, for example, type 2 diabetes mellitus (T2DM)), high blood pressure, obesity, pneumonia, aspiration of gastric contents, inhalation injury, pulmonary contusion, pulmonary vasculitis, and drowning.

In one embodiment, the subject is affected with a viral infection. Thus, in one embodiment, the protein for use in the present invention is for treating ARDS or for preventing the onset or the aggravation of an ARDS, wherein said ARDS is caused by or related to infection by a vims. Examples of viruses that may cause ARDS include, but are not limited to, viruses of the coronaviridae family, viruses of the herpesviriadae family, respiratory syncytial vims and influenza viruses. Examples of viruses of the herpesviridae family that may cause ARDS include, without being limited to, herpes simplex vims (HSV) and cytomegalovims (CMV). Examples of influenza viruses that may cause ARDS include, without being limited to, H5N1 (avian influenza A) and H1N1 (influenza A). In one embodiment, the viral infection is caused by a coronavirus. Thus, in one embodiment, the protein for use in the present invention is for treating ARDS or for preventing the onset or the aggravation of ARDS, wherein said ARDS is caused by or related to infection by a coronavirus. In one embodiment, the coronavirus is a human coronavirus.

In one embodiment, the coronavirus is an alpha coronavirus or a beta coronavirus, preferably a beta coronavirus.

Examples of alpha coronavimses include, without being limited to, human coronavirus 229E (HCoV-229E) and human coronavirus NL63 (HCoV-NL63) also sometimes known as HCoV-NH or New Haven human coronavirus.

Examples of beta coronavimses include, without being limited to, human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKUl), Middle East respiratory syndrome -related coronavirus (MERS-CoV) previously known as novel coronavirus 2012 or HCoV-EMC, severe acute respiratory syndrome coronavirus (SARS-CoV) also known as SARS-CoV-1 or SARS-classic, and severe acute respiratory syndrome coronavirus (SARS-CoV-2) also known as 2019-nCoV or novel coronavirus 2019.

In one embodiment, the coronavirus is selected from the group comprising or consisting of HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKUl, MERS-CoV, SARS-CoV-1 and SARS-CoV-2.

In one embodiment, the viral infection is caused by SARS-CoV-2 (causing the disease COVID-19). Thus, in one embodiment, the protein for use in the present invention is for treating ARDS or for preventing the onset or aggravation of ARDS, wherein said ARDS is caused by or related to an infection by SARS-CoV-2. In one embodiment, the subject is suffering from COVID-19 and developed ARDS.

In one embodiment, the subject is suffering from COVID-19 and is at risk of developing ARDS. In one embodiment, the subject developed micro-thrombotic events (and optionally macro-thrombotic events).

In one embodiment, the subject presents at least one of the following symptoms: cough, shortness of breath or difficulty breathing. In one embodiment, the subject presents at least one, preferably at least two, of the following symptoms: fever (i.e., any body temperature over 38°C), chills, repeated shaking with chills, muscle pain, headache, sore throat and new loss of taste or smell.

In one embodiment, the severity of the disease is evaluated by the WHO COVID scoring scale. The WHO COVID scoring scale provides a score ranging from 0 to 8 depending on the patient’s state, as shown in Table 2 hereafter.

Table 2: WHO COVID scoring scale

In one embodiment, the subject is suffering from a mild form of COVID- 19, corresponding to a score of 3 or 4 according to the WHO COVID scoring scale. In another embodiment, the subject is suffering from a severe form of COVID- 19 corresponding to a score ranging from 5 to 7 according to the WHO COVID scoring scale. In one embodiment, the subject is suffering from mild to severe COVID-19. In one embodiment, the subject is diagnosed with a SARS-CoV-2 infection. Methods for diagnosing a SARS-CoV-2 infection include, but are not limited to, rRT-PCR (real-time reverse transcription polymerase chain reaction) test allowing to detect the presence of SARS-CoV-2 in a sample from a subject (such as a sample from a nasal swab, a sample from an oropharyngeal swab, a sputum sample, a lower respiratory tract aspirate, a bronchoalveolar lavage, a nasopharyngeal wash/aspirate or a nasal aspirate) or an antibody test (such as an enzyme-linked immunosorbent assay (ELISA)) allowing to detect the presence of antibodies against SARS-CoV-2 in a sample from a subject (such as a blood sample). Methods for diagnosing a SARS-CoV-2 infection also include a highly positive serology for SARS-CoV-2 and clear symptoms of COVID-19, such as the ones described hereinabove.

In one embodiment, the protein for use in the present invention prevents the onset of ARDS.

In one embodiment, the protein for use in the present invention prevents the aggravation of ARDS.

In one embodiment, the protein for use in the present invention prevents progressive status degradation of the subject.

In one embodiment, the protein for use in the present invention prevents the acute status degradation of the subject. In one embodiment, the protein for use in the present invention prevents further global and respiratory status degradation. In the meaning of the invention, global and respiratory status degradations include, but are not limited to, thrombotic complications, pulmonary embolism, cardiovascular failure, renal failure, liver failure, secondary infection or sepsis.

In one embodiment, the protein for use in the present invention prevents downstream complications due to thrombotic conditions.

In one embodiment, the protein for use in the present invention prevents the development of lung fibrosis. In one embodiment, the protein for use in the present invention prevents hyperinflammation and/or cytokine storm.

In one embodiment, the protein for use in the present invention prevents the clinical progression of the disease. In one embodiment, the protein for use in the present invention prevents the elevation of at least one of the following biological markers: C-reactive protein, Lactate dehydrogenase (LDH), Interleukin-6 (IL-6), N-terminal prohormone of brain natriuretic (NT proBNP) or Pro-calcitonin and/or the reduction of the lymphocyte count. In one embodiment, the protein for use in the present invention may prevent the elevation of the Ferritin. In one embodiment, the protein for use in the present invention has an anti-inflammatory effect. In one embodiment, the protein for use in the present invention has an anti-fibrotic effect. In one embodiment, the protein for use in the present invention has an anti- thrombolytic effect. In one embodiment, the protein for use in the present invention has 3 effects: an anti-inflammatory effect, an anti-fibrotic effect and an anti-thrombolytic effect.

In one embodiment, the subject is a mammal, preferably a human.

In one embodiment, the subject is hospitalized.

In one embodiment, the subject is hospitalized but does not require immediate admission to intensive care unit (ICU). In one embodiment, the subject is hospitalized in ICU.

In one embodiment, the subject does not require invasive mechanical ventilation such as, for example, intubation.

In one embodiment, the subject, preferably the COVID-19 patient, presents at least one of the following abnormalities in chest CT scan: ground-glass opacity (> 50%), bilateral patchy shadowing (>50%), local patchy shadowing (30-50%) and/or interstitial abnormalities (10-25%) compared to baseline. In one embodiment, the subject, preferably the COVID-19 patient, presents with signs of a moderate progressive pulmonary disease. Examples of signs of a moderate progressive pulmonary disease include, without being limited to, respiratory symptoms such as cough or dyspnea, uni- or bilateral ground-glass opacities or pulmonary infiltrates on chest radiograph and/or CT scan, and clinical evidence of progression over the last 48 hours.

In one embodiment, the subject, preferably the COVID-19 patient, presents with one or several signs associated with the onset of ARDS.

Examples of signs associated with the onset of ARDS include, but are not limited to, a respiratory rate (RR) equal or superior to 24 respirations per minute, an oxygen saturation (Sp0 ₂ ) equal or inferior to 93% in resting state and a ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaOi/FiO ₂ ) equal or inferior to 300 mmHg.

Examples of signs associated with the onset of ARDS include, but are not limited to, a respiratory rate (RR) comprised between 24 respirations per minute (included) and 30 respirations per minure (excluded), an oxygen saturation (SpOi) equal or inferior to 93% in ambient air and a ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO ₂ /FiO ₂ ) comprised between 100 (excluded) and 200 mmHg (included).

In one embodiment, the subject, preferably the COVID-19 patient, presents at least one of the following biological markers of progression of the disease: C-reactive protein (CRP) >10 mg/L, Lactate dehydrogenase (LDH) > 250 U/L, Interleukin-6 (IL-6) > 8 pg/mL, Lymphocyte count < 1X10 ⁹ /L, N-terminal prohormone of brain natriuretic peptide (NT proBNP) > 88 pg/mL or Pro-calcitonin > 0.5 ng/mL. In one embodiment, the subject, preferably the COVID-19 patient, may present the following biological marker of progression of the disease : Ferritin > 400 pg/L.

In one embodiment, the subject, preferably the COVID-19 patient, exhibits respiratory distress symptoms. Examples of respiratory distress symptoms are described above.

In one embodiment, the subject, preferably the COVID-19 patient, exhibits respiratory distress symptoms and requires respiratory support. In one embodiment, the subject is under non-invasive ventilation (such as supplemental oxygen (also called oxygen therapy) by mask or nasal prongs, positive pressure, high flow nasal oxygen). In one embodiment, the subject is under invasive ventilation (i.e. ventilation requiring tracheal intubation or tracheostomy). In one embodiment, the subject is under extracorporeal membrane oxygenation. In one embodiment, the subject presents with at least one sign of a prothrombotic status. In one embodiment, signs of a prothrombotic status include, but are not limited to, an elevation of D- dimers (in particular above 0,5 pg/mL), an elevation of troponin T (in particular above 2,5 pg/L), signs of micro-angiopathy on a vascular enhanced chest CT- scan, thrombocytopenia (in particular under 150,000/mm ³ ), increased fibrinogen (in particular above 2g/L) and prolonged Prothrombin Time (PT) (in particular above 12 seconds).

In one embodiment, the subject presents an elevation of D-Dimers, an elevation of troponin T and/or signs of micro-angiopathy on a vascular enhanced chest CT-scan.

In one embodiment, elevation of D-dimers corresponds to a concentration of D-dimers in the blood exceeding or equal to 0.5mg/L (or 0.5pg/mL). Methods to estimate the concentration of D-dimers are well known in the art and include, but are not limited to, Enzyme Linked ImmunoabSorbent Assay (ELISA).

In one embodiment, elevation of troponin T, a biological marker of cardiomyocyte necrosis corresponds to a concentration of troponin T in the blood exceeding 2,5 pg/L. A value of 10 ng/mL is very positive, and a value of 30 ng/mL is almost synonimous to a diagnosis of myocardial infarct. Methods to estimate the concentration of troponin T levels are well known in the art and include, without being limited to, 3 ^rd , 4 ^th , and 5 ^th generations of High-Sensitivity cTnT methods, blood tests initially developed for the rapid detection of myocardial infarct (hs-Trop T). 140 pg/mL roughly correlates with the reference Trop T O.lng/mL detection level. These methods are all based on sandwich electrochemiluminescence immunoassay (ECLIA) technique, and are run on Elecsys or COBAS e411 analyzers (Roche Diagnostics). In one embodiment, the subject presents with a high risk of thrombosis. A high risk of thrombosis may for example be associated to elevated blood level(s) of at least one of the following biomarkers: C-reactive protein, D-dimers, ferritin, IL-6.

In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is an adult human (> 18 years old).

In one embodiment, the subject is younger than 80, 75, 70, 65 or 60 years of age, preferably younger than 80 years of age. In one embodiment, the subject is older than 60, 65, 70 or 75 years of age. In one embodiment, the subject is 80 years of age or older.

In one embodiment, the subject is suffering from at least one comorbidity. As used herein, “comorbidity” refers to a disease or condition coexisting with a disease or condition, such as, for example, a viral infection, in particular a coronavirus infection, such as a SARS-CoV-2 infection causing COVID-19, in the subject.

Examples of comorbidities that may coexist with a viral infection, in particular a coronavirus infection, such as a SARS-CoV-2 infection causing COVID-19, in the subject to be treated according to the present invention include, without being limited to, diabetes, high blood pressure, obesity, asthma, autoimmune or auto-inflammatory diseases or conditions, cardiovascular diseases or conditions, chronic bronchitis, chronic kidney diseases, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, immunodeficiency, pulmonary hypertension, and severe respiratory conditions.

In one embodiment, the subject to be treated does not present with signs of disseminated intravascular coagulation (DIC).

In one embodiment, signs of DIC include, without being limited to, low platelet count (<100,000/mL), altered fibrinogen concentration (elevated >2g/L or low < 1 g/L) , prolonged prothrombin time (PT) (> 12 seconds), partial thromboplastin time (PTT) above 60 seconds, presence of fibrin degradation products in the plasma, with or without clinically visible hemorrhagic signs. )n one embodiment, signs of DIC include low platelet count, prolonged prothrombin time (PT), presence of fibrin degradation products in the plasma, with or without clinically visible hemorrhagic signs.

In one embodiment, the subject was not affected with an ischemic stroke or transient ischemic attack within one year before the administration of the protein of the present invention.

In one embodiment, the subject was not affected with a deep venous thrombosis or pulmonary embolism within one year before the administration of the protein of the present invention. In one embodiment, the subject does not present with a hereditary tendency to bleeding or coagulopathy.

In one embodiment, the subject is considered treated if said subject does not progress to severe respiratory distress following the administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, the subject is considered treated if said subject does not progress to severe respiratory distress after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, the subject is considered treated if said subject does not progress to severe respiratory distress after 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention.

In one embodiment, progression to severe respiratory distress is assessed by a respiratory rate (RR) equal or superior to 30 respirations per minute, an oxygen saturation (SpO ₂ ) equal or inferior to 93% in resting sate, and/or a ratio of arterial oxygen partial pressure (PaO ₂ ) to fractional inspired oxygen (F1O2) (PaO ₂ /FiO ₂ ) equal or inferior to 200 mm Hg.

In one embodiment, progression to severe respiratory distress is assessed by a respiratory rate (RR) equal or superior to 30 respirations per minute, a decrease of the oxygen saturation (SpC ) superior to 5% in ambient air, a ratio of arterial oxygen partial pressure (PaO ₂ ) to fractional inspired oxygen (F1O2) (PaO ₂ /FiO ₂ ) equal or inferior to 100 mm Hg or death occurring during the administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, the subject is considered treated if the time to a clinical improvement of NEWS2 is 0 maintained for 24 hours over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after the administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, the subject is considered treated if the time to a clinical improvement of NEWS2 is 0 maintained for 24 hours over a period of at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days after the administration of the protein, composition, pharmaceutical composition or medicament of the present invention.

NEWS2 is a scoring system aiming to identify patients at risk of deterioration, in which a score is allocated to physiological measurements, already recorded in routine practice, when patients present to, or are being monitored in hospital. Six simple physiological parameters form the basis of the scoring system: respiration rate, oxygen saturation, systolic blood pressure, pulse rate, level of consciousness or new confusion and temperature. NEWS2 score is then associated with a frequency of monitoring: 0 with minimum 12 hourly, 1-4 with minimum 4-6 hourly, 5 or more or 3 in one parameter with 1 hourly and 7 or more with continuous monitoring of vital signs.

In one embodiment, the subject is considered treated if the NEWS2 score decreases from baseline (i.e., from the NEWS2 score measured before (preferably just before) the administration of the protein of the present invention) after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, the subject is considered treated if the NEWS2 score decreases from baseline (i.e., from the NEWS2 score measured before (preferably just before) the administration of the protein of the present invention) after 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention.

In one embodiment, the subject is considered treated if said subject presents a decreased score on the WHO COVID scoring scale after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, the subject is considered treated if said subject presents a decreased score on the WHO COVID scoring scale after 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention.

In one embodiment the subject is considered treated if said subject presents a less severe hypoxemia status after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment the subject is considered treated if said subject presents a less severe hypoxemia status after 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, hypoxemia status is classified as mild with 200<PaO ₂ /FiO ₂ £300 mmHg, moderate with 100<PaO ₂ /FiO ₂ £200 mmHg or severe with PaO ₂ /FiO ₂ £100 mmHg.

In one embodiment, the subject is considered treated if said subject does not progress to one of the following: admission to intensive care unit (ICU), invasive mechanical ventilation (with intubation) following the administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, the subject is considered treated if said subject does not progress to one of the following: admission to ICU, invasive mechanical ventilation (with intubation), after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, the subject is considered treated if said subject does not progress to one of the following: admission to ICU, invasive mechanical ventilation (with intubation), after 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention.

In one embodiment, the subject is considered treated if the surface area of total pulmonary lesions on chest CT-scan or radiograph is smaller in said subject after administration of the protein, composition, pharmaceutical composition or medicament of the present invention, compared to subjects treated with standard of care + placebo.

In one embodiment, the subject is considered treated if the respiratory failure is improved. In one embodiment, the subject is considered treated if the respiratory failure is improved after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention. In one embodiment, the subject is considered treated if the respiratory failure is improved after 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days (preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention.

In one embodiment, improvement of the respiratory failure corresponds to the possible removal of oxygenation or ventilation for the patient.

In one embodiment, the subject is considered treated if said subject present a better respiratory rate (RR) status after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days

(preferably consecutive days) of administration of the protein, composition, pharmaceutical composition or medicament of the present invention.. According to the present invention, the respiratory rate status are the following : normal: inferior to 20/min, mild: comprise between 20/min (included) and 24/min (excluded), moderate: comprised between 24/min (included) and 30/min (excluded), severe: superior or equal to 30/min, and the death.

In one embodiment, the subject is considered treated in case of a discharge from the intensive care unit. In one embodiment, the subject is considered treated in case of a discharge from hospital.

In one embodiment, the protein for use in the present invention is used in a monotherapy, i.e. the protein for use in the present invention is the only active agent to be administered.

In one embodiment, the protein for use in the present invention is for administration with at least one further pharmaceutically active agent. Examples of pharmaceutically active agents include, but are not limited to, anti-viral agents, anti-IL-6 agents and other agents such as chloroquine or hydroxychloroquine.

In one embodiment, the at least one further pharmaceutically active agent is an anti- viral agent. Examples of anti-viral agents that may be administered with the protein for use in the present invention include, but are not limited to, remdesivir and a combination of lopinavir and ritonavir (lopinavir/ritonavir).

In one embodiment, the at least one further pharmaceutically active agent is an anti-IL-6 agent selected from the group comprising or consisting of tocilizumab and sarilumab.

In one embodiment, such pharmaceutical active agents are used in standard of care COVID-19. In one embodiment, the at least one further pharmaceutically active agent is an anti inflammatory selected from the group comprising nonsteroidal anti-inflammatory drugs (NSAIDs) such as Aspirin, Naproxene, Celecoxib, Difhmisal, Etodolac, Ibuprofen, Indomethacin, Ketoprofen, Ketorolac, Nabumetone, Oxaprozin, Piroxicam, Salsalte, Sulindac, Tolmetin. In one embodiment, the at least one further pharmaceutically active agent is an anti inflammatory selected from the group comprising steroidal anti-inflammatory drugs such as Bethamethasone, Cortisone, Dexamethasone, Hydrocortisone, Triamcinolone acetonide, Methylprednisolone, or Prednisolone.

In one embodiment, the at least one further pharmaceutically active agent is corticosteroids. In one embodiment, the active agent is oxygen.

In one embodiment, the at least one further pharmaceutically active agent is an antibody binding to the spike protein of SARS-CoV-2, in particular the receptor-binding domain of the spike protein of SARS-CoV-2. In one embodiment, the at least one further pharmaceutically active agent is bamlanivimab, a combination of bamlanivimab and etesevimab, or a combination of casirivimab and imdevimab.

In one embodiment, the at least one further pharmaceutically active agent is not an anti platelet agent. Thus, in one embodiment, the protein for use in the present invention is the only anti-platelet agent that is to be administered.

In one embodiment, the at least one further pharmaceutically active agent is not an anti- coagulant agent. An example of an anti-coagulant agent includes, without limitation, fondaparinux, heparine and low-molecular- weight heparin. Thus, in one embodiment, the protein for use in the present invention is not administered with an anti-coagulant agent.

Another object of the invention is thus a kit of part comprising, in a first part, at least one isolated protein binding to GPVI as described hereinabove and, in a second part, another pharmaceutically active agent, as described hereinabove.

In one embodiment, the kit of part is for treating ARDS or for preventing the onset or the aggravation of an ARDS in a subject.

In one embodiment, the kit of part is for preventing further global and respiratory status degradation. In a preferred embodiment, the kit of part is for preventing the development of lung fibrosis.

In one embodiment, the kit of part is for preventing the clinical progression of the disease. In one embodiment, the subject receives a protein, composition, pharmaceutical composition or medicament as described hereinabove as part of a treatment protocol.

In one embodiment, said treatment protocol further comprises, before, concomitantly or after the administration of the protein, composition, pharmaceutical composition or medicament of the invention, the administration of another pharmaceutically active agent, as described herein. Therefore, the subject to be treated was previously treated or is to be treated with another pharmaceutically active agent.

In one embodiment, a dose of the protein as described herein ranging from about 30 mg to about 5000 mg is administered (or is to be administered) to the patient, preferably ranging from about 60 mg to about 4000 mg, more preferably of about 100 to about 2000 mg, and even more preferably of about 500 or about 1000 mg.

In one embodiment, a dose of the protein as described herein of about 30, 60, 62.5, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 2000, 2500 or of about 3000 mg is administered (or is to be administered) to the subject.

In one embodiment, a dose of the protein for use in the present invention ranges from about 100 mg to about 2000 mg, from about 125 mg to about 2000 mg, preferably from about 250 mg to about 1000 mg or from about 500 mg to about 1000 mg. In one embodiment, a dose of the protein as described herein ranging from about 0.5 mg/kg to about 50 mg/kg is administered (or is to be administered) to the patient, preferably ranging from about 1 mg/kg to about 32 mg/kg, more preferably of about 8 mg/kg or of about 16 mg/kg.

In one embodiment, a dose of the protein as described herein ranging from about 2.5 mg/kg to about 25 mg/kg, preferably from about 5 mg/kg to about 20 mg/kg, more preferably of about 8 mg/kg or of about 16 mg/kg is to be administered to the patient. In one embodiment, a dose of the protein for use as described herein of about 0.5 mg/kg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or of about 50 mg/kg is administered (or is to be administered) to the subject. The protein, composition, pharmaceutical composition or medicament of the present invention will be formulated for administration to the subject. The protein, composition, pharmaceutical composition or medicament may be administered parenterally, by inhalation spray, rectally, nasally, or via an implanted reservoir. The term administration used herein includes subcutaneous, intravenous (IV), intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

In one embodiment, the protein for use in the present invention is injected, preferably by intravenous infusion. In another embodiment, the protein for use in the present invention is injected intraperitoneally. In another embodiment, the protein for use in the present invention is injected intradermally.

Examples of forms adapted for injection include, but are not limited to, solutions, such as, for example, sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms and the like. Sterile injectable forms of the compositions may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

In one embodiment, the protein, composition, pharmaceutical composition or medicament for use in the present invention is to be administered as a single dose.

In another embodiment, the protein, composition, pharmaceutical composition or medicament for use in the present invention is to be administered as repeated doses, such as, for example, once an hour, once every 2 hours, once every 3 hours, 4 times a day, 3 times a day, 2 times a day, once every 24 hours (i.e., once a day), once every two days, 3 times a week, 2 times a week or once a week, preferably once every 24 hours.

In one embodiment, the protein composition, pharmaceutical composition or medicament for use in the present invention is to be administered during 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days (preferably consecutive days), or until complete healing of the subject. In one embodiment, the protein composition, pharmaceutical composition or medicament for use in the present invention is to be administered during 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days (preferably consecutive days), or until complete healing of the subject.In one embodiment, the protein, composition, pharmaceutical composition or medicament for use in the present invention is to be administered during at least 3 consecutive days, preferably once a day during at least 3 consecutive days.

In one embodiment, the protein, composition, pharmaceutical composition or medicament for use in the present invention is to be administered once every 24 hours during 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 consecutive days, preferably during at least 3 consecutive days. In one embodiment, the protein, composition, pharmaceutical composition or medicament for use in the present invention is to be administered once every 24 hours during 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive days, preferably during at least 3 consecutive days. In one embodiment, several doses of the protein are to be administered to the patient, with a total dose (corresponding to the dose administered during the complete duration of the treatment) of 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000,18000, 19000 or 20000 mg.

In one embodiment, the protein, composition, pharmaceutical composition or medicament for use in the present invention is to be administered with a dose escalation mode.

In one embodiment, the protein for use in the present invention is administered (or is to be administered) to the subject during about 2 hours, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or during about 12 hours. In one embodiment, the protein, composition, pharmaceutical composition or medicament for use in the present invention is to be administered during at least 2 hours to the subject, preferably during at least 4 to 6 hours.

In one embodiment, the protein for use in the present invention is continuously administered to the subject during at least 2 hours, preferably during at least 4 to 6 hours (e.g. during about 2 hours, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5,

11, 11.5 or during about 12 hours). As used herein, the terms “continuously administered” refers to the administration of a compound for a prolonged period of time with a substantially constant speed of administration.

In one embodiment, the protein, composition, pharmaceutical composition or medicament for use in the present invention is to be administered with a constant infusion rate. In one embodiment, the constant infusion rate is comprised between 100 and 200 mg/hour, preferably between 160 to 170 mg/hour, more preferably at about 167 mg/hour.

In one embodiment, the constant infusion rate is comprised between 100 and 200 mg/hour, preferably between 160 to 170 mg/hour, more preferably at about

167 mg/hour during at least 2, 4 or 6 hours, preferably during 6 hours.

Another object of the invention is a method for treating ARDS and/or preventing the onset or the aggravation of an ARDS in a subject, wherein said method comprises administering a protein binding to GPVI as described herein, or a composition, a pharmaceutical composition or a medicament as described hereinabove to the subject.

In one embodiment, a therapeutically effective amount of the protein described herein is administered to the subject.

In one embodiment, the method of the invention is for treating ARDS or for preventing the onset or the aggravation of ARDS, wherein said ARDS is a virus-related ARDS, preferably a SARS-CoV-2-related ARDS.

In one embodiment, the method of the invention comprises administering a dose of the protein as described in the present invention ranging from about 0.5 mg/kg to about 50 mg/kg, preferably ranging from about 1 mg/kg to about 32 mg/kg, more preferably of about 8 mg/kg or of about 16 mg/kg. In another embodiment, the method of the invention comprises administering a dose of the protein as described in the present invention ranging from about 2.5 mg/kg to about 25 mg/kg, preferably from about 5 mg/kg to about 20 mg/kg, more preferably of about 8 mg/kg or of about 16 mg/kg. In one embodiment, the method of the invention comprises administering a dose of the protein as described in the present invention of about 0.5 mg/kg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,

40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or of about 50 mg/kg.

In one embodiment, the method of the invention comprises administering a dose of the protein as described in the present invention ranging from about 30 mg to about 5000 mg, preferably ranging from about 60 mg to about 4000 mg, more preferably of about 100 to about 2000 mg, and even more preferably of about 500 mg or about 1000 mg. In one embodiment, the method of the invention comprises administering a dose of the protein as described in the present invention of about 30, 60, 62.5, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,

950, 1000, 1500, 2000, 2500 or of about 3000 mg. In another embodiment, the method of the invention comprises administering a dose of the protein as described in the present invention ranging from about 100 mg to about 2000 mg, from about 125 mg to about 2000 mg, preferably from about 250 mg to about 1000 mg or from about 500 mg to about 1000 mg.

In one embodiment, the protein as described in the present invention is injected, preferably by intravenous infusion.

In one embodiment, the method of the invention comprises administering a single dose of the protein. In one embodiment, the method of the invention comprises administering repeated doses of the protein, such as, for example, once an hour, once every 2 hours, once every 3 hours, 4 times a day, 3 times a day, 2 times a day, once every 24 hours (i.e. once a day), once every two days, 3 times a week, 2 times a week or once a week, preferably once every 24 hours. In one embodiment, the method of the invention comprises administering a dose of the protein during 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days (preferably consecutive days), or until complete healing of the subject. In one embodiment, the method of the invention comprises administering a dose of the protein during 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days (preferably consecutive days), or until complete healing of the subject. In one embodiment, the method of the invention comprises administering a dose of the protein during at least 3 consecutive days. In one embodiment, the method of the invention comprises administering a dose of the protein once every 24 hours during 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive days, preferably during at least 3 consecutive days. In one embodiment, the method of the invention comprises administering a dose of the protein once every 24 hours during 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive days, preferably during at least 3 consecutive days.

Thus, in one embodiment, the method of the invention comprises administering a total dose of 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000,18000, 19000 or 20000 mg of the protein. In one embodiment, the method of the invention comprises administering a dose of the protein with a dose escalation mode.

In one embodiment, the method of the invention comprises continuously administering the protein during at least 2 hours, preferably during at least 4 to 6 hours ( e.g during about 2 hours, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or during about 12 hours).

In one embodiment, the method of the invention comprises administering a dose of the protein with a constant infusion rate, such as between 100 and 200 mg/hour, preferably between 160 to 170 mg/hour, more preferably at 167 mg/hour.

In one embodiment, the constant infusion rate is comprised between 100 and 200 mg/hour, preferably between 160 to 170 mg/hour, more preferably at about

167 mg/hour during at least 2, 4 or 6 hours, preferably during 6 hours

In one embodiment, the method of the invention is for preventing global and respiratory status degradation. In one embodiment, the method of the invention is for preventing the development of lung fibrosis. In one embodiment, the method of the invention is for preventing the clinical progression of the disease. EXAMPLES

The present invention is further illustrated by the following examples.

Example 1: Clinical trial investigating the efficacy and safety of the anti-GPVI antibody ACT017 in SARS-CoV-2-related ARDS The clinical trial is a randomized, double bind, multicenter, placebo-controlled, parallel group, fixed dose, phase II study to evaluate the efficacy and safety of the anti-GPVI antibody ACT017 in patients presenting a SARS-CoV-2-related ARDS.

The primary objective is to evaluate the effect of the anti-GPVI antibody ACT017 in preventing clinical progression of disease in patients, when added to Standard-of-Care in Covid-19 patients presenting with acute respiratory distress syndrome.

The secondary objectives are:

For the efficacy aspect: to assess the impact of treatment on overall disease control and to assess the impact of treatment on symptomatology and biological parameters,

For the safety aspect: to assess the number of the following events: deaths, serious adverse events (SAEs), suspected unexpected serious adverse reactions (SUSARs), medically important events, bleeding-related events,

For the pharmacokinetics: to verify that actual PK profile in patients does not differ from that in healthy volunteers, and matches with PK-PD simulation,

For the exploratory aspect : evolution of pulmonary lesions on chest imaging, evolution of biological parameters related to hemostasis, coagulation and inflammation, determination of predictive factors for a response.

Study design Eligible patients (n=68) are randomized in a 1:1 ratio to ACT017 or placebo. Patient inclusions are fractioned into sequential (3-day apart) cohorts of growing size (2, 4 then 6 patients), each balanced between ACT017 and placebo in order to check safety in a gradual manner. A Data Safety Monitoring Board (DSMB) is met after 12 patients have been accrued, and again after the first 30 patients.

ACT017 is administered by IV infusion. The dosing regimen is 1000 mg over 6 hours, every 24 hours for 3 consecutive days. ACT017 is formulated for intravenous (IV) administration as a sterile product with 20 mM sodium citrate and 130 mM sodium chloride buffer at pH of 5.0 and is supplied for clinical trial use in vials containing 50 mL of the drug product at a concentration of 10 mg/mL. Each vial containing 500 mg of ACT017, two vials are administered concomitantly in eligible patients for a total daily dose of lg and a global dose of 3g during the three days of treatment.

The administered study treatment is ACT017 or placebo, as per central randomization allocation to the study group into which the patient is included.

In each study arm, patients receive either ACT017 or its matching placebo. ACT017 or the matching placebo is intended to be administered as an IV infusion over 6 hours. The objective is therefore to cover a period of time of 12 to 24 hours, for 3 consecutive days, a period of time assumed to be long enough to turn around the ongoing pro-thrombotic events and prevent later complications.

All patients receive in parallel the best medical care at the discretion of the investigating center, or per local guidelines. Unless patient’s condition worsens after the first infusion and/or an untoward adverse drug reaction precludes it, the treatment is readministered 24 hours after the initiation of the first infusion, and likewise after the second infusion, so that a total of 3 subsequent infusions, that represent the expected standard treatment is administered.

The study period is of a maximum of 40 days per patient. Patients are closely monitored during the first 7 days following randomization with complete evaluations being performed at 24 hours, 48 hours, 72 hours, then on Days 4 (96 hours), 5 (120 hours), 7 (+/- 1 day), 14 (+/- 2 days), 20 (+/- 2 days), 40 days (+/- 3 days). If a patient is discharged before Day 40, distant consultations by telemedicine are undertaken if it is not deemed desirable that the patient comes back to the institution.

Selection of patients

A total of 60 evaluable adult patients, hospitalized and presenting with an ARDS meeting the following study criteria are enrolled in the study.

Inclusion criteria:

1. Male or female hospitalized patients > 18 years (/. _<? ., at least 18 years old at the time of randomization) and < 80 years, having given their written consent; 2. Having a positive RT-PCR or antigenic test for COVID-19 or with a highly positive serology and clear symptoms of COVID-19;

3. Presenting with symptoms of COVID-19, including:

• Cough, or

• Shortness of breath or difficulty breathing, or at least 2 of the following:

• Fever, defined as any body temperature above 38°C

• Chills

• Repeated shaking with chills

• Muscle pain · Headache

• Sore throat

• New loss of taste or smell;

4. Presenting with signs of moderate but progressive pulmonary disease with:

• respiratory symptoms (cough, dyspnea, etc...) · uni- or bilateral ground-glass opacities, or pulmonary infiltrates on chest radiograph and/or CT scan performed within the past 96 hours,

• clinical and/or biological evidence of progression over the past 48 hours;

5. Presenting with one or several signs associated with the onset of ARDS such as:

• 24/min < Respiratory rate (RR) < 30/min, · Sp0 ₂ <93% in in ambient air, • 100 < Pa02/Fi02 £ 200mmHg;

6. Presenting with signs of a pro-thrombotic status characterized by: a. D-Dimers >0.5 pg/mL, b. and/or Troponin T > 2.5 pg/L (or by default Troponin I greater than local laboratory reference), c. and/or signs of micro-angiopathy on a vascular enhanced chest CT-scan (Thrombocytopenia <150,000/mm3 or prolonged Prothrombin Time (PT) >12s are additional signs of a pro-thrombotic status that are not necessary for eligibility); 7. With one or more of the following biological markers of progression:

• C-reactive protein (CRP) >10 mg/L,

• Lactate dehydrogenase (LDH) > 250 U/L,

• Interleukin-6 (IL6) > 8 pg/mL,

• Lymphocyte count < 1X10 ⁹ /L, · N-terminal prohormone of brain natriuretic peptide

(NT proBNP) > 88 pg/mL

• Pro-calcitonin > 0.5 ng/mL,

• Ferritin > 400 pg/L;

8. Effective birth control that should have been in place for at least 2 months in non-menopausal women, and 4 months for men after investigational medicinal product (IMP) administration. Birth control methods considered to be highly effective include:

• combined (estrogen-progestogen) hormonal contraception associated with the inhibition of ovulation: oral, intravaginal, transdermal;

• progesterone-only hormonal contraception associated with the inhibition of ovulation: oral, injectable, implantable;

• intrauterine device,

• intrauterine hormone -releasing system,

• bilateral tubal occlusion,

• vasectomized partner. 9. Women of child-bearing potential must have negative results of a urinary or plasma pregnancy test (serum HCG). Non-inclusion criteria:

1. Patients requiring invasive mechanical/assisted ventilation (intubation);

2. Obvious disseminated intravascular coagulation (DIC), (with e.g. a variable combination of the following: low platelet count (<100,000/mL), prolonged PT > 12sec and/or a PTT > 60sec, presence of fibrin degradation products in the plasma, with or without clinically visible hemorrhagic signs); An isolated change of one of these parameters does not qualify for DIC,

3. ARDS of another origin; 4. Concomitant pulmonary infection (pneumoniae) with another agent, notably bacterial or fungal;

5. Patients presenting with hemoglobin < 9g/dL,

6. Patients under immunosuppressive agents;

7. Patients receiving an anti-cancer treatment (radiotherapy, chemotherapy, immunotherapy);

8. Initiation of a treatment with aspirine (previous stable preventative aspirin regimen from 75 to 160 mg per day is allowed);

9. Patients under anticoagulant therapy (except heparin and low-molecular weight heparin (LMWH)), and anti-Xa drugs achieving effective anti-coagulation, as assessed by appropriate tests, or having received thrombolytics < 24 hours;

10. Patients receiving nonsteroidal anti-inflammatory drugs (NSAIDs) or anti-platelet agents with platelet suppression within the past 7 days;

11. Patients treated concomitantly with another monoclonal antibody (e.g. tocilizumab)

12. Ischemic stroke or transient ischemic attack within the past year; 13. Deep venous thrombosis or pulmonary embolism within the past year;

14. Severe renal insufficiency (Grades 4-5) with a glomerular filtration rate < 30mL/min/1.73m ² ;

15. One of the following severe organ failures: a. Hepatic with either Child Pugh score > C, or ASAT/ALAT > 5 U.N.L., b. Cardiac with NYHA> Class II, unstable angina pectoris, myocardial infarct < 1 year, supra- ventricular or ventricular arrythmia; 16. Hereditary tendency to bleeding or coagulopathy;

17. Severe vascular disease (aneurysms, arterial surgery < 6 months);

18. Unhealed wounds, gastrointestinal ulcers or perforation < 6 months;

19. Major surgery < 28 days, other surgery within the past 7 days; 20. Hemoptysis, gastrointestinal bleeding, central nervous system bleeding < 1 month;

21. Platelet count < 50,000/mm3 (50G/L);

22. Absolute Neutrophil Count < l,000/mm3 (1.0G/L);

23. Terminal illness, including cancer (life expectancy < 3 months);

24. Uncontrolled arterial hypertension (Systolic blood pressure > 185 mmHg and/or diastolic blood pressure > 110 mmHg despite appropriate antihypertensive therapy);

25. Childbirth within < 10 days;

26. Pregnancy or breastfeeding;

27. Prior cardiopulmonary resuscitation < 10 days;

28. Allergy or hypersensitivity to drugs of the same class; 29. Participation in another interventional clinical trial within 30 days prior to the inclusion.

Concomitant treatments:

Patients should not receive any other experimental therapies. In addition to the aforementioned non-inclusion criteria, patients should not receive any of the following: · Anticoagulants, except low-molecular-weight heparin or heparin whether oral or injectable, throughout the 3-day treatment period and during the 24 hours following the last infusion;

• Anti-platelet agents, whether oral or injectable, throughout the 3-day treatment period and during the 24 hours following the last infusion; · Any off-label treatment, except if officially recommended for COVID-19 treatment as standard of care (SOC).

All other symptomatic treatments used routinely for disease-related symptoms are allowed in all patients involved in this trial. Specific treatments for any adverse events are also permitted. The use of both antibiotics and antiviral agents are authorized. The use of treatments recently recommended for the treatment of SARS-CoV-2 infection is permitted. This includes corticosteroids. However, the use of other monoclonal antibodies, even recommended is not permitted.

Primary and secondary endpoints

Primary efficacy endpoints: Progression from moderate to severe respiratory distress assessed at Day 4.

The primary efficacy endpoint is a composite failure endpoint defined as the occurrence of at least one of the following failure events :

• RR > 30/min, or

• Sp0 ₂ decrease > 5% in ambient air,

• PaOi/FiOi < lOOmmHg,

• Death occurring prior to or on Day 4

Secondary efficacy endpoints:

• All cause Death at day 40 and Overall Survival

• WHO-CO VID- 19 Scale

• NEWS 2 Scale;

• Respiratory Rate status defined as::

- Normal: < 20/min,

- Mild: 20/min < RR < 24/min,

- Moderate: 24/min < RR < 30/min,

- Severe: > 30/min,

- Death;

• Hypoxemia status defined as:

- Normal: > 300 mmHg,

- Mild: 200 mmHg < Pa0 ₂ /FI0 ₂ £ 300 mmHg,

- Moderate: 100 mmHg < Pa0 ₂ /FI0 ₂ £ 200 mmHg,

- Severe: Pa02/FI02£ 100 mmHg,

- Death;

SpOi status defined as: - Normal: > 95%

- Mild: 93% < Sp0 ₂ < 95%,

- Moderate: 90% < Sp0 ₂ £ 93%,

- Severe: < 90%, - Death.

• Chest CT-Scan (or in exceptional cases, chest radiogram)

• Oxygen-free days (over the study period = 40 days),

• Need for mechanical ventilation,

• Mechanical ventilation-free days, · Hospital-free days (over study period = 40 days),

• Clinical recovery and Time to Clinical recovery (over study period = 40 days),

• Cure and Time-to-cure (over study period = 40 days). Safety Endpoints:

• Incidence, nature and severity of Adverse Events, SAEs, SUSARs and Treatment-Emergent Adverse Events (TEAEs);

• Incidence of bleeding-related events;

• Incidence of hypersensitivity reactions, · Changes to vital signs over the course of the study versus screening;

• Change to clinical laboratory assessments (hematology, coagulation, biochemistry, biological markers, urinalysis) over the course of the study versus screening;

• Electrocardiogram (ECG) over the course of the study versus screening. Change to soluble GPVI (sGPVI) levels :

Soluble GPVI levels are measured in aliquots of frozen citrated platelet poor plasma (PPP) using a sandwich immunoassay using the MesoScale Discovery (MSD) technology. The concentrations are extrapolated from standard curves generated by serial dilutions of recombinant GPVI ectodomain). Statistical methods

The nominal one-sided alpha level of significance considered for statistical tests is 0.025.

RESULTS Safety Data

Preliminary data were obtained from 50 patients that have been randomized and treated. Mean age is 56 years, 76% are males, and 82% are Caucasian. At inclusion, 82% had at least one comorbidity; most frequent (>5%) were hypertension, diabetes, obesity, and dyslipidemia. Dyspnea was present in 91%, cough in 86%, and fever in 47%. On data available in 31 patients, heparin and steroids were prescribed in >80% and one patient received tocilizumab. There have been no deaths or SUSARs, 22% of patients experienced a serious adverse event (SAE), similar to SAE rates reported in other clinical trials of hospitalized COVID-19 patients.

Thus, preliminary data indicate that ACT017 is safely tolerated in COVID-19 patients with ARDS.