WO2025242732A1

WO2025242732A1 - Pan antibodies against sars-cov-2 spike protein and uses thereof for therapeutical purposes

Info

Publication number: WO2025242732A1
Application number: PCT/EP2025/063986
Authority: WO
Inventors: Pierre Martineau; Maria del Mar NARANJO GOMEZ; Hugo CALDERON; Manel OTERO JUAN; Mireia Pelegrin; Daouda ABBA MOUSSA
Original assignee: Institut De Investigaciones Biomedicas August Pi I Sunyer Idibaps; Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Montpellier; Institut Regional du Cancer de Montpellier
Current assignee: Institut De Investigaciones Biomedicas August Pi I Sunyer Idibaps; Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Montpellier; Institut Regional du Cancer de Montpellier
Priority date: 2024-05-21
Filing date: 2025-05-21
Publication date: 2025-11-27
Anticipated expiration: 2026-11-21

Abstract

The present invention relates to the treatment of the COVID-19, here, the inventors generated potent non-neutralizing pan-SARS-CoV-2 mAb, notably the antibody C10, targeting a conserved region of the virus. Noteworthy, C10 demonstrated remarkable efficacy in recognizing nearly all known variants of the virus and effectively binding infected cells. Leveraging this pan-SARS-CoV-2 mAb, they have engineered CAR-T cells capable of efficiently killing lung epithelial cells infected with the virus. Overall, their work identifies a pan-SARS-Cov-2 able to target bona fide infected cells and provides a proof-of-concept for the potential use of CAR-T cell therapy in combating SARS-CoV-2 infections. Their findings also highlight the potential of non-neutralizing mAbs in mediating immune protection against emerging infectious diseases. Thus, the present invention relates to anti-spike antibodies, particularly in a purified form or in an isolated form and their use to treat SARS-CoV-2. Particularly, the present invention is defined by the claims.

Description

PAN ANTIBODIES AGAINST SARS-COV-2 SPIKE PROTEIN AND USES THEREOF FOR THERAPEUTICAL PURPOSES

FIELD OF THE INVENTION:

The present invention relates to anti-spike antibodies and their uses.

BACKGROUND OF THE INVENTION:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped RNA virus that belongs to the Coronaviridae group. This virus is known for causing the COVID-19 disease (1,2). Since its’ outbreak in 2019, COVID-19 pandemic has been reported with millions of cases and deaths. Nowadays, thanks to the efforts from the global healthcare community, and worldwide vaccination programs, the pandemic has been controlled. Even though global vaccination has induced immunity in the global population, there are different high-risk subgroups such as solid organ transplantation, hematologic, and immunosuppressed patients, that are not capable of acquiring this immunity and are still at risk of suffering COVID-19 disease with a severe symptomatology (3-5).

An unprecedented effort by researchers around the world has resulted in the development of a spectrum of preventive and therapeutic approaches. Among them, several neutralizing antibodies directed against Spike protein have been developed and used to treat SARS-CoV-2 infection (6-9). However, sustaining their effectiveness in the new variants of concern (VOC) has been challenging because of the antibody escape mutations. It is therefore necessary to advance the development of immunotherapies that can effectively counter viral evolution. This could include the development of antibodies directed against highly conserved viral epitopes and able to target infected cells. Such antibodies and their derivatives hold promise for both antibody-based and cell-based therapeutic approaches. Most efforts for the development of protective anti-SARS-CoV-2 mAb have been focused thus far in targeting viral particles (6-9), while much less efforts have been concentrated in targeting infected cells. This is important to take into consideration as antiviral antibodies display multiple effector functions (i.e. antibody-dependent cellular cytotoxicity, ADCC; antibody-dependent cellular phagocytosis, ADCP, ...) than allow the killing/elimination of infected cells (10). In addition, neutralization does not always align with ADCC and some neutralizing antibodies such as Sotrovimab that display lower potency (high IC50) can still be efficient in reducing viral propagation (11). Worthy of note, the assessment of the effector functions of anti-SARS-CoV- 2 mAbs used in the clinic has mostly been done using Spike-expressing cells as a surrogate of infected cells (11,12). However, the critical question of whether such antibodies effectively bind and eliminate infected cells has been considerably understudied. Therefore, there is a pressing need to develop antibodies targeting highly conserved viral epitopes (known as "coldspots") (13) and directly targeting infected cells. This approach could emerge as a potent strategy against SARS-CoV-2 infection, poised to combat not only current variants but also future evolutions of the virus.

The discovery of anti-SARS-CoV-2 mAb able to target infected cells holds therapeutic promise, with applications spanning diverse treatment modalities. They can be used as full- length molecules exploiting their Fc-mediated polyfunctionality. In addition, antibody fragments offer a pathway to develop new cell-based immunotherapies, notably Chimeric Antigen Receptor T-cell therapy (CAR-T cell therapy), which harnesses the combined strengths of antibodies and T-cells. In the recent years, CAR-T cell therapy has demonstrated remarkable success in the haematological cancers area. Encouraged by these achievements, there is an increasing interest on developing CAR-T cell therapies for different diseases, including infectious diseases such as SARS-CoV-2 infection (14-17). This enthusiasm is fuelled by recent insights suggesting that SARS-CoV-2-specific T-cell therapy may confer clinical benefits in severe cases of COVID-19 (18). Given the absence of therapeutic options for high- risk populations vulnerable to SARS-CoV-2 infection, the aim of the inventors was to pioneer novel therapeutic strategies aimed at eliminating infected cells, thereby aiding in the resolution of viral infection. These approaches could harness the diverse therapeutic potential of mAb and their derivative to create both antibody-based and cell-based interventions.

SUMMARY OF THE INVENTION:

In the present work, the inventors generated potent non-neutralizing pan-SARS-CoV-2 mAb, notably the antibody CIO, targeting a conserved region of the virus. Noteworthy, CIO demonstrated remarkable efficacy in recognizing nearly all known variants of the virus and effectively binding infected cells. Leveraging this pan-SARS-CoV-2 mAb, they have engineered CAR-T cells capable of efficiently killing lung epithelial cells infected with the virus. Overall, their work identifies a pan-SARS-Cov-2 able to target bona fide infected cells and provides a proof-of-concept for the potential use of CAR-T cell therapy in combating SARS-CoV-2 infections. Their findings also highlight the potential of non-neutralizing mAbs in mediating immune protection against emerging infectious diseases. Thus, the present invention relates to anti-spike antibodies, particularly in a purified form or in an isolated form and their use to treat SARS-CoV-2. Particularly, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Antibodies of the invention:

A first aspect invention relates to an anti-spike antibody comprising or consisting:

(a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 1 or 9; a H-CDR2 having a sequence set forth as SEQ ID NO: 2 or 10; a H-CDR3 having a sequence set forth as SEQ ID NO: 3 or 11;

(b) a light chain wherein the variable domain comprises : a L-CDR1 having a sequence set forth as SEQ ID NO: 4 or 12; a L-CDR2 having a sequence set forth as SEQ ID NO: 5 or 13; a L-CDR3 having a sequence set forth as SEQ ID NO: 6 or 14.

The present invention thus provides antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies of the invention. A functional variant of a VL, VH, or CDR used in the context of a monoclonal antibody of the present invention still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity/avidity and/or the specificity/selectivity of the parent antibody and in some cases such a monoclonal antibody of the present invention may be associated with greater affinity, selectivity and/or specificity than the parent Ab. Such variants can be obtained by a number of affinity maturation protocols including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (supra) discusses these methods of affinity maturation. Such functional variants typically retain significant sequence identity to the parent Ab. The sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative substitutions; for instance, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, (e.g., about 65-95%, such as about 92%, 93% or 94%) of the substitutions in the variant are conservative amino acid residue replacements. The sequences of CDR variants may differ from the sequence of the CDRs of the parent antibody sequences through mostly conservative substitutions; for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected as follows:

Aliphatic residues I, L, V, and M

Cycloalkenyl-associated residues F, H, W, and Y

Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y

Negatively charged residues D and E

Polar residues C, D, E, H, K, N, Q, R, S, and T

Positively charged residues H, K, and R

Small residues A, C, D, G, N, P, S, T, and V

Very small residues A, G, and S

Residues involved in turn A, C, D, E, G, H, K, N, Q, R, S, P, and formation T

Flexible residues Q, T, K, S, G, P, D, E, and R

More conservative substitutions groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation in terms of hydropathic/hydrophilic properties and residue weight/size also is substantially retained in a variant CDR as compared to a CDR of the antibodies of the invention. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The retention of similar residues may also or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62, Open Gap= 1 1 and Extended Gap= 1). Suitable variants typically exhibit at least about 70% of identity to the parent peptide. According to the present invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence. According to the present invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence.

As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i. e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (NEEDLEMAN, and Wunsch). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification. According to the invention a first amino acid sequence having at least 80% of identity with a second amino acid sequence means that the first sequence has 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

In some embodiments, the antibody of the present invention is an antibody comprising a heavy chain having at least

70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98; or 99% of identity with SEQ ID NO: 7 or 15 and a light chain having at least 70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98; or 99% of identity with SEQ ID NO: 8 or 16.

In some embodiments, the antibody of the present invention is an antibody consisting a heavy chain having at least

70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98; or 99% of identity with SEQ ID NO: 7 or 15 and a light chain having at least 70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98; or 99% of identity with SEQ ID NO: 8 or 16 and which still comprises 100% of identity with the H-CDR1 having a sequence set forth as SEQ ID NO: 1 or 9; 100% of identity with the H- CDR2 having a sequence set forth as SEQ ID NO: 2 or 10; 100% of identity with the H-CDR3 having a sequence set forth as SEQ ID NO: 3 or 11; and 100% of identity with the L-CDR1 having a sequence set forth as SEQ ID NO: 4 or 12; 100% of identity with the L-CDR2 having a sequence set forth as SEQ ID NO: 5 or 13; 100% of identity with the L-CDR3 having a sequence set forth as SEQ ID NO: 6 or 14.

In some embodiments, the antibody of the present invention is an antibody consisting in a heavy chain having at least

In another embodiment, the invention relates to an anti-spike antibody comprising: a heavy chain wherein the variable domain has a sequence set forth as SEQ ID NO: 7 or 15; a light chain wherein the variable domain has a sequence set forth as SEQ ID NO: 8 or 16.

In another embodiment, the invention relates to the anti-spike antibody CIO which comprises or consists in:

(a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 1; a H-CDR2 having a sequence set forth as SEQ ID NO: 2; a H-CDR3 having a sequence set forth as SEQ ID NO: 3;

(b) a light chain wherein the variable domain comprises: a L-CDR1 having a sequence set forth as SEQ ID NO: 4; a L-CDR2 having a sequence set forth as SEQ ID NO: 5; a L-CDR3 having a sequence set forth as SEQ ID NO: 6.

In another embodiment, the invention relates to the anti-spike antibody C8 which comprises or consists in:

(a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 9; a H-CDR2 having a sequence set forth as SEQ ID NO: 10; a H-CDR3 having a sequence set forth as SEQ ID NO: 11;

(b) a light chain wherein the variable domain comprises: a L-CDR1 having a sequence set forth as SEQ ID NO: 12; a L-CDR2 having a sequence set forth as SEQ ID NO: 13; a L-CDR3 having a sequence set forth as SEQ ID NO: 14.

In some embodiments, the antibody of the present invention is the antibody CIO having a heavy chain identical to SEQ ID NO: 7 and a light chain identical to SEQ ID NO: 8.

In some embodiments, the antibody of the present invention is the antibody C8 having a heavy chain identical to SEQ ID NO: 15 and a light chain identical to SEQ ID NO: 16.

As used herein the term "antibody" or "immunoglobulin" have the same meaning, and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L- CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.

The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NTH, USA (hereafter “Kabat et al ”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. (http://www.bioinf.org.Uk/abs/#cdrdef)

As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as spike, while having relatively little detectable reactivity with non-spike proteins or structures. Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g., an about 10: 1, about 20: 1, about 50: 1, about 100: 1, 10.000: 1 or greater ratio of affinity/avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is spike).

The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1/Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments.

The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

In some embodiment, the antibody of the invention is a monoclonal antibody.

The antibodies of the present invention are produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Typically, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques well-known in the art. For example, antibodies can be obtained as DNA expression products after incorporation of DNA sequences encoding the antibodies into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired antibodies, from which they can be later isolated using well-known techniques.

In one embodiment, the antibody of the invention is a chimeric antibody, particularly a chimeric mouse/human antibody.

According to the invention, the term "chimeric antibody" refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.

In some embodiments, the human chimeric antibody of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. As the CH domain of a human chimeric antibody, it may be any region which belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used. Also, as the CL of a human chimeric antibody, it may be any region which belongs to Ig, and those of kappa class or lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244). In another embodiment, the monoclonal antibody of the invention is a humanized antibody. In particular, in said humanized antibody, the variable domain comprises human acceptor frameworks regions, and optionally human constant domain where present, and nonhuman donor CDRs, such as mouse CDRs.

In one embodiment, the humanized antibody can be derived from a chimeric antibody (obtained from the antibody of the invention).

According to the invention, the term "humanized antibody" refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody.

The humanized antibody of the present invention may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell. The humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred. Examples of tandem type humanized antibody expression vector include pKANTEX93 (WO 97/10354), pEE18 and the like. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (See, e. g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5, 565, 332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

In one embodiment, the antibody of the invention is an antigen biding fragment selected from the group consisting of a Fab, a F(ab)’2, a single domain antibody, a ScFv, a Sc(Fv)2, a diabody, a triabody, a tetrabody, an unibody, a minibody, a maxibody, a small modular immunopharmaceutical (SMIP), minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody as an isolated complementary determining region (CDR), and fragments which comprise or consist of the VL or VH chains as well as amino acid sequence having at least 70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100% of identity with an heavy chain wherein the variable domain has a sequence set forth as SEQ ID NO: 7 or 15; and with a light chain wherein the variable domain has a sequence set forth as SEQ ID NO: 8 or 16.

The term “antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically binds to a given antigen (e.g., the spike protein). Antigen biding functions of an antibody can be performed by fragments of an intact antibody. Examples of biding fragments encompassed within the term antigen biding fragment of an antibody include a Fab fragment, a monovalent fragment consisting of the VL,VH,CL and CHI domains; a Fab’ fragment, a monovalent fragment consisting of the VL,VH,CL,CH1 domains and hinge region; a F(ab’)2 fragment, a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of VH domains of a single arm of an antibody; a single domain antibody (sdAb) fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain or a VL domain; and an isolated complementary determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (ScFv); see, e.g., Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883). "dsFv" is a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. Such single chain antibodies include one or more antigen biding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies. A unibody is another type of 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. Antigen binding fragments can be incorporated into single domain antibodies, SMIP, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The term "diabodies" “tribodies” or “tetrabodies” refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Antigen biding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) Which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).

The Fab of the present invention can be obtained by treating an antibody which specifically reacts with the spike protein with a protease, papaine. Also, the Fab can be produced by inserting DNA encoding Fab of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote (as appropriate) to express the Fab.

The F(ab')2 of the present invention can be obtained treating an antibody which specifically reacts with the spike protein with a protease, pepsin. Also, the F(ab')2 can be produced by binding Fab' described below via a thioether bond or a disulfide bond.

The Fab' of the present invention can be obtained treating F(ab')2 which specifically reacts with the spike protein with a reducing agent, dithiothreitol. Also, the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.

The scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well-known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) from a donor scFv fragment, and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, e. g., W098/45322; WO 87/02671; US5,859,205; US5,585,089; US4,816,567; EP0173494). Domain Antibodies (dAbs) are the smallest functional binding units of antibodies - molecular weight approximately 13 kDa - and correspond to the variable regions of either the heavy (VH) or light (VL) chains of antibodies. Further details on domain antibodies and methods of their production are found in US 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245; US 2004/0110941; EP 1433846, 0368684 and 0616640; WO 2005/035572, 2004/101790, 2004/081026, 2004/058821, 2004/003019 and 2003/002609, each of which is herein incorporated by reference in its entirety.

UniBodies are another antibody fragment technology, based upon the removal of the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a univalent binding region rather than a bivalent binding region. Furthermore, because UniBodies are about smaller, they may show better distribution over larger solid tumors with potentially advantageous efficacy. Further details on UniBodies may be obtained by reference to WO 2007/059782, which is incorporated by reference in its entirety.

In another aspect, the invention provides an antibody that competes for binding to the spike protein with the antibodies of the invention.

As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10'⁷ M or less, such as about 10'⁸ M or less, such as about 10'⁹ M or less, about 10'¹⁰ M or less, or about 10'¹¹ M or even less when determined by for instance surface plasmon resonance (SPR) technology in a Biacore 8K or Biacore 8K+ instrument using a soluble form of the antigen as the ligand and the antibody as the analyte. BIACORE® (Cytiva) is one of a variety of surface plasmon resonance assay format that are routinely used to epitope bin panels of monoclonal antibodies. Other variety of instrument useful can be the Bio-layer by Forte-Bio. Typically, an antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g., BSA, casein), which is not identical or closely related to the predetermined antigen. When the KD of the antibody is very low (that is, the antibody has a high affinity), then the KD with which it binds the antigen is typically at least 10,000-fold lower than its KD for a non-specific antigen. An antibody is said to essentially not bind an antigen or epitope if such binding is either not detectable (using, for example, plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100 fold, 500 fold, 1000 fold or more than 1000 fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.

Additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in standard spike protein binding assays. The ability of a test antibody to inhibit the binding of antibodies of the present invention to spike protein demonstrates that the test antibody can compete with that antibody for binding to spike protein; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on spike protein as the antibody with which it competes. Thus, another aspect of the invention provides antibodies that bind to the same antigen as, and compete with, the antibodies disclosed herein. As used herein, an antibody “competes” for binding when the competing antibody inhibits spike protein binding of an antibody or antigen binding fragment of the invention by more than 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79, 80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 or 99% in the presence of an equimolar concentration of competing antibody.

In other embodiments the antibodies or antigen binding fragments of the invention bind to one or more epitopes of the spike protein. In some embodiments, the epitopes to which the present antibodies or antigen binding fragments bind are linear epitopes. In other embodiments, the epitopes to which the present antibodies or antigen binding fragments bind are non-linear, conformational epitopes.

The antibodies of the invention may be assayed for specific binding by any method known in the art. Many different competitive binding assay format(s) can be used for epitope binding. The immunoassays which can be used include, but are not limited to, competitive assay systems using techniques such western blots, radioimmunoassays, ELISA, "sandwich" immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion precipitin assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York).

Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR- mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the invention. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell - epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

In some embodiments, the glycosylation of an antibody is modified. Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U. S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

Thus, according to the invention, the antibody of the invention is aglycosylated and particularly aglycosylated in the Fab fragment and not in the Fc region of the antibody.

In some embodiments, some mutations are made to the amino acids localized in aggregation “hotspots” within and near the first CDR (CDR1) to decrease the antibodies susceptibility to aggregation (see Joseph M. Perchiacca et al., Proteins 2011; 79:2637-2647).

The antibody of the present invention may be of any isotype. The choice of isotype typically will be guided by the desired effector functions. IgGl and IgG3 are isotypes that mediate such effectors functions as ADCC or CDC, when IgG2 and IgG4 don’t or in a lower manner. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of a monoclonal antibody of the present invention may be switched by known methods. Typical, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGl to IgG2. Thus, the effector function of the monoclonal antibodies of the present invention may be changed by isotype switching to, e.g., an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses.

In some embodiments, the antibody of the present invention is a full-length antibody. In some embodiments, the full-length antibody is an IgGl antibody. In some embodiments, the full-length antibody is an IgG3 antibody.

In some embodiments, the antibody of the present invention is a full-length antibody. In some embodiments, the full-length antibody is an IgG2 antibody. In some embodiments, the full-length antibody is an IgG4 antibody.

In some embodiments, the antibody of the present invention does not comprise an Fc domain capable of substantially binding to a FcyRIIIA (CD 16) polypeptide. In some embodiments, the antibody of the present invention lacks an Fc domain (e.g. lacks a CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype

In some embodiments, the antibody of the present invention is an antibody of a non- IgG2/4 type, e.g. IgGl or IgG3 which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated. Such mutations have e.g. been described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol. 75(24): 12161-12168 (2001) or Bruhns, P.; Jonsson, F. Mouse and human FcR effector functions. Immunological Reviews 2015, 268, 25-51, doi: 10.1111/imr.l2350.

In some embodiments, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In some embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al. In one embodiment, the antibody of the invention has a Fc region modified as followed: mutant S239D/H268F/S324T/I332E (see Moore, G.L.; Chen, H.; Karki, S.; A, G. Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions. MAbs 2010, 2, 181-189, doi: 10.4161/mabs.2.2.11158). In a particular embodiment, the Fc region is an afucosylated antibody or an antibody with at least one mutation described in Bruhns, P.; Jonsson, F. Mouse and human FcR effector functions. Immunological Reviews 2015, 268, 25-51, doi: 10.1111/imr.12350. For example, this mutation can be the N297A(NA), the L234A/L235A (LALA) or the D270A.

In some embodiments, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

In some embodiments, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.

In some embodiments, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fc receptor by modifying one or more amino acids. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgGI for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al., 2001 J. Biol. Chen. 276:6591- 6604, W02010106180).

The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well understood in the art, and refers to a cell-mediated reaction in which non- specific cytotoxic cells that express Fc receptors (FcRs) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Non-specific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

In a particular embodiment, the antibodies invention mediates cell killing through NK- mediated ADCC. Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or no fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the present invention to thereby produce an antibody with altered glycosylation. For example, EPl, 176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation or are devoid of fucosyl residues. Therefore, in some embodiments, the monoclonal antibodies of the present invention may be produced by recombinant expression in a cell line which exhibit hypofucosylation or non-fucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransferase. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al, 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N acetylglucosaminyltransf erase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al, 1999 Nat. Biotech. 17: 176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation pattern devoid of fucosyl residues (http://www.eurekainc.com/a&boutus/companyoverview.html). Alternatively, the monoclonal antibodies of the present invention can be produced in yeasts or filamentous fungi engineered for mammalian- like glycosylation pattern and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1).

In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 by Ward. Alternatively, to increase the biological half life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121 ,022 by Presta et al. Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the foetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitutions of Fc region residue 434 (US Patent No. 7,371,826).

Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl- CIO) alkoxy- or aryloxypolyethylene glycol or polyethylene glycol-mal eimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.

Another modification of the antibodies that is contemplated by the invention is a conjugate or a protein fusion of at least the antigen-binding region of the antibody of the invention to serum protein, such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule. Such approach is for example described in Ballance et al. EP0322094. Another possibility is a fusion of at least the antigen-binding region of the antibody of the invention to proteins capable of binding to serum proteins, such human serum albumin to increase half-life of the resulting molecule. Such approach is for example described in Nygren et al., EP 0 486 525.

Polysialytion is another technology, which uses the natural polymer polysialic acid (PSA) to prolong the active life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment on conjugation. This increases the active life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system. The PSA polymer is naturally found in the human body. It was adopted by certain bacteria which evolved over millions of years to coat their walls with it. These naturally polysialylated bacteria were then able, by virtue of molecular mimicry, to foil the body's defense system. PSA, nature's ultimate stealth technology, can be easily produced from such bacteria in large quantities and with predetermined physical characteristics. Bacterial PSA is completely non-immunogenic, even when coupled to proteins, as it is chemically identical to PSA in the human body.

Another technology includes the use of hydroxy ethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer derived from waxy maize starch and can be metabolized by the body's enzymes. HES solutions are usually administered to substitute deficient blood volume and to improve the rheological properties of the blood. Hesylation of an antibody enables the prolongation of the circulation half-life by increasing the stability of the molecule, as well as by reducing renal clearance, resulting in an increased biological activity. By varying different parameters, such as the molecular weight of HES, a wide range of HES antibody conjugates can be customized.

In certain embodiments of the invention antibodies have been engineered to remove sites of deamidation. Deamidation is known to cause structural and functional changes in a peptide or protein. Deamidation can result in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. (Anal Chem. 2005 Mar 1 ;77(5): 1432-9).

In certain embodiments of the invention the antibodies have been engineered to increase pl and improve their drug-like properties. The pl of a protein is a key determinant of the overall biophysical properties of a molecule. Antibodies that have low pls have been known to be less soluble, less stable, and prone to aggregation. Further, the purification of antibodies with low pl is challenging and can be problematic especially during scale-up for clinical use. Increasing the pl of the anti-spike antibodies of the invention or fragments thereof improved their solubility, enabling the antibodies to be formulated at higher concentrations (>100 mg/ml). Formulation of the antibodies at high concentrations (e.g. >100mg/ml) offers the advantage of being able to administer higher doses of the antibodies into eyes of patients via intravitreal injections, which in turn may enable reduced dosing frequency, a significant advantage for treatment of chronic diseases including cardiovascular disorders. Higher pls may also increase the FcRn- mediated recycling of the IgG version of the antibody thus enabling the drug to persist in the body for a longer duration, requiring fewer injections. Finally, the overall stability of the antibodies is significantly improved due to the higher pi resulting in longer shelf-life and bioactivity in vivo. Preferably, the pl is greater than or equal to 8.2.

Glycosylation modifications can also induce enhanced anti-inflammatory properties of the antibodies by addition of sialylated glycans. The addition of terminal sialic acid to the Fc glycan reduces FcyR binding and converts IgG antibodies to anti-inflammatory mediators through the acquisition of novel binding activities (see Robert M. Anthony et al., J Clin Immunol (2010) 30 (Suppl 1): S9— S 14; Kai-Ting C et al., Antibodies 2013, 2, 392-414).

In some embodiments, the heavy and light chains, variable regions domains and CDRs that are disclosed can be used to prepare polypeptides that contain antigen binding region that can specifically bind to the spike protein. For example, one or more of the CDRs listed in table 1 can be incorporated into a molecule (e.g., a polypeptide) covalently or noncovalently to make an immunoadhesion. An immunoadhesion may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDR(s) enable the immunoadhesion to bind specifically to a particular antigen of interest (e.g., the spike protein or epitope thereof).

The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well to naturally occurring amino acids polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

In some embodiments, the antigen biding fragment of the invention is grafted into non-immunoglobulin based antibodies also called antibody mimetics 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.

The term “antibody mimetic” is intended to refer to molecules capables of mimicking an antibody’s ability to bind an antigen, but which are not limited to native antibody structures. Examples of such antibody mimetics include, but are not limited to, Adnectins, Affibodies, DARPins, Anticalins, Avimers, and versabodies, all of which employ binding structures that, while they mimic traditional antibody binding, are generated from and function via distinct mechanisms. Antigen biding fragments of antibodies can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). An affibody is well known in the art and refers to affinity proteins based on a 58 amino acid residue protein domain, derived from one of the IgG binding domain of staphylococcal protein A. DARPins (Designed Ankyrin Repeat Proteins) are well known in the art and refer to an antibody mimetic DRP (designed repeat protein) technology developed to exploit the binding abilities of non-antibody proteins. Anticalins are well known in the art and refer to another antibody mimetic technology, wherein the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual targeting protein, called Duocalins. Avimers are well known in the art and refer to another antibody mimetic technology, Avimers are derived from natural A-domain containing protein. Versabodies are well known in the art and refer to another 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. Such antibody mimetic can be comprised in a scaffold. The term “scaffold” refers to a polypeptide platform for the engineering of new products with tailored functions and characteristics.

In one aspect, the invention pertains to generating non-immunoglobulin-based antibodies also called antibody mimetics using non-immunoglobulins scaffolds onto which CDRs of the invention can be grafted. Known or future non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the target the spike protein.

The fibronectin scaffolds are based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (see US 6,818,418). These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprise the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non- immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomisation and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques. The Ankyrin technology is based on using proteins with Ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The Ankyrin repeat module is a 33 amino acid polypeptide consisting of two antiparallel a-helices and a P-tum. Binding of the variable regions is mostly optimized by using ribosome display.

Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on “A-domains” monomers (2-10) linked via amino acids linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, U.S. patent Application publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of protein A. protein A is a surface protein form the bacterium Staphylococcus aureus. This scaffold domain consist of 58 amino acids, 13 of which are randomized to generate affibody librairies with a large number of ligand variants (See e.g., US 5,831,012). Affibody molecules mimic antibodies, they have a molecular weight of 6kDa. In spite of its small size, the binding site of affibody molecules is similar to that of an antibody.

Anticalins are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acids residues, being just marginally bigger than a single immunoglobulin domain. The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity. One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris Brassicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing anticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules. New affilin molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are employed, one of which is gamma crystalline, a human structural eye lens protein and the other is “ubiquitin” superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of “ubiquitin-like” proteins are described in W02004106368.

Versabodies are highly soluble and can be formulated to high concentrations. Versabodies are exceptionally heat stable and offer extended shelf-life. Additional information regarding Versabodies can be found in US 2007/0191272, which is hereby incorporated by reference in its entirety.

The above descriptions of antibody fragment and mimetic technologies is not intended to be comprehensive. A variety of additional technologies including alternative polypeptide- based technologies, such as fusions of complementarity determining regions as outlined in Qui et al., Nature Biotechnology, 25(8) 921-929 (2007), as well as nucleic acid- based technologies, such as the RNA aptamer technologies described in US 5,789,157; 5,864,026; 5,712,375; 5,763,566; 6,013,443; 6,376,474; 6,613,526; 6,114,120; 6,261,774; and 6,387,620; all of which are hereby incorporated by reference, could be used in the context of the instant invention.

In some embodiments, the invention provides a multispecific antibody comprising a first antigen binding site from an antibody of the present invention molecule described herein above and at least one second antigen binding site.

In some embodiments, the second antigen-binding site is used for recruiting a killing mechanism such as, for example, by binding an antigen on a human effector cell as a BiTE (Bispecific T-Cell engager) antibody which is a bispecific scFv2 directed against target antigen and CD3 on T cells described in US7235641, or by binding a cytotoxic agent or a second therapeutic agent. As used herein, the term "effector cell" refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, mast cells and granulocytes, such as neutrophils, eosinophils and basophils. Some effector cells express specific Fc receptors (FcRs) and carry out specific immune functions. In some embodiments, an effector cell is capable of inducing ADCC, such as a natural killer cell. For example, monocytes, macrophages, which express FcRs, are involved in specific killing of target cells and presenting antigens to other components of the immune system. In some embodiments, an effector cell may phagocytose a target antigen or target cell. The expression of a particular FcR on an effector cell may be regulated by humoral factors such as cytokines. An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell. Suitable cytotoxic agents and second therapeutic agents are exemplified below, and include toxins (such as radiolabeled peptides), chemotherapeutic agents and prodrugs

In some embodiments, the second antigen-binding site binds to an antigen on a human B cell, such as, e.g., CD19, CD20, CD21, CD22, CD23, CD46, CD80, CD138 and HLA-DR.

In some embodiments, the second antigen-binding site binds a tissue- specific antigen, promoting localization of the bispecific antibody to a specific tissue.

Exemplary formats for the multispecific antibody molecules of the invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to spike and another with a specificity to a second antigen; (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single-chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically- linked bispecific (Fab')2 fragment; (vi) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vii) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (viii) a so called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (ix) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (x) a diabody. Another exemplary format for bispecific antibodies is IgG-like molecules with complementary CH3 domains to force heterodimerization. Such molecules can be prepared using known technologies, such as, e.g., those known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies. In some embodiments, the bispecific antibody is obtained or obtainable via a controlled Fab-arm exchange, typically using DuoBody technology. In vitro methods for producing bispecific antibodies by controlled Fab-arm exchange have been described in W02008119353 and WO 2011131746 (both by Genmab A/S). In one exemplary method, described in WO 2008119353, a bispecific antibody is formed by "Fab-arm" or "half- molecule" exchange (swapping of a heavy chain and attached light chain) between two monospecific antibodies, both comprising IgG4-like CH3 regions, upon incubation under reducing conditions. The resulting product is a bispecific antibody having two Fab arms which may comprise different sequences. In another exemplary method, described in WO 2011131746, bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is the antibody of the present invention : a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is the antibody of the present invention and the second antibody has a different binding specificity, or vice versa. The reducing conditions may, for example, be provided by adding a reducing agent, e.g. selected from 2-mercaptoethylamine, dithiothreitol and tris(2- carboxyethyl)phosphine. Step d) may further comprise restoring the conditions to become nonreducing or less reducing, for example by removal of a reducing agent, e.g. by desalting. Preferably, the sequences of the first and second CH3 regions are different, comprising only a few, fairly conservative, asymmetrical mutations, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO 2011131746, which is hereby incorporated by reference in its entirety. The following are exemplary embodiments of combinations of such assymetrical mutations, optionally wherein one or both Fc-regions are of the IgGl isotype.

In some embodiments, the first Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and the second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and wherein the first and second Fc regions are not substituted in the same positions.

In some embodiments, the first Fc region has an amino acid substitution at position 405, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 407 and 409, optionally 409.

In some embodiments, the first Fc region has an amino acid substitution at position 409, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, and 407, optionally 405 or 368.

In some embodiments, both the first and second Fc regions are of the IgGl isotype, with the first Fc region having a Leu at position 405, and the second Fc region having an Arg at position 409.

Table A: sequences of interest of the antibodies of the present application (Kabat definition)

As used herein, the term “spike protein” or “protein S” “or spike” refers to the SARS- Cov-2 spike glycoprotein that binds its cellular receptor (i.e. ACE2), and mediates membrane fusion and virus entry. Each monomer of trimeric S protein is about 180 kDa, and contains two subunits, SI and S2, mediating attachment and membrane fusion, respectively. In particular, Spike protein SI attaches the virion to the cell membrane by interacting with host receptor (i.e. human ACE2 receptor) via its “receptor-binding domain” also named “RBD.” Spike protein S2 mediates fusion of the virion and cellular membranes by acting as a class I viral fusion protein. Under the current model, the protein has at least three conformational states: pre-fusion native state, pre-hairpin intermediate state, and post-fusion hairpin state. During viral and target cell membrane fusion, the coiled coil regions (heptad repeats) assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes. Spike protein S2' acts as a viral fusion peptide which is unmasked following S2 cleavage occurring upon virus endocytosis. Typically, the spike protein has the amino acid sequence as set forth in SEQ ID NO: 17. In particular, the RBD consists of the amino acid sequence that ranges from the amino acid residue at position 319 to the amino acid residue at position 541 in SEQ ID NO: 17.

SEQ ID NO: 17:

MF VFLVLLPL VS SQCVNLTTRTQLPP AYTNSFTRGVYYPDKVFRS S VLHSTQD LFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDS KTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFE YVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLV DLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTI TDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNA TRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFV IRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFE LLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTT DAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTP TWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVAS QSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC SNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPD PSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTD EMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIA NQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILS RLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRV DFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVS NGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKY FKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP WYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKL HYT

Nucleic acids, vectors and host cells of the present invention:

Accordingly, a further object of the invention relates to a nucleic acid molecule encoding an antibody according to the invention. More particularly the nucleic acid molecule encodes, the CDRs or the heavy chain and light chain of an antibody of the present invention.

Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. So, a further aspect of the invention relates to a vector comprising a nucleic acid of the invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSGl beta d2-4-(Miyaji H et al. 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.

As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase “nucleotide sequence that encodes a protein or an RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

A further aspect of the invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid and/or a vector according to the invention.

The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been "transformed".

As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.

The nucleic acids of the invention may be used to produce an antibody of the present invention in a suitable expression system. The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., HEK 293 cells, Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2/0-Agl4 cell (ATCC CRL1581), mouse P3X63- Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2/3HL.P2.G11.16Ag.2O cell (ATCC CRL1662, hereinafter referred to as "YB2/0 cell"), and the like. The present invention also relates to a method of producing a recombinant host cell expressing an antibody or antibody-format according to the invention, said method comprising the steps of: (i) introducing in vitro, ex vivo or in vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and/or secrete said antibody. Such recombinant host cells can be used for the production of antibodies of the present invention.

Antibodies of the present invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. CAR-T Cells of the invention

The inventors demonstrated that antibodies of the invention can be used to generate CAR-T cells. Notably, the antibody CIO demonstrated remarkable efficacy in recognizing nearly all known variants of the virus and effectively binding infected cells. The CAR-T cells engineered with the CIO antibody is capable of efficiently killing lung epithelial cells infected with the virus.

Thus, a further aspect of the invention refers to a chimeric antigen receptor (CAR) comprising an antigen binding domain of the antibodies of the present invention. Typically, said chimeric antigen receptor comprises at least one VH and/or VL sequence of the antibodies of the present invention. The chimeric antigen receptor of the present invention also comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

In particular embodiment, said chimeric antigen receptor comprises a VH sequence and a VL sequence of the CIO or C8 antibodies of the invention.

As used herein, the term “chimeric antigen receptor” or “CAR” has its general meaning in the art and refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cell infected by the virus SARS-CoV-2, and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below. In some aspects, the set of polypeptides are contiguous with each other. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27 and/or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane. In particular aspects, CARs comprise fusions of single chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. In some embodiments, CARs comprise domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD 137, DAP 10, and/or 0X40. In some embodiments, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

A further aspect of the invention refers to a T-cell comprising a chimeric antigen receptor of the invention (“CAR-T cell”).

As used herein the term “CAR-T” has its general meaning in the art and refers to a T lymphocyte that has been genetically engineered to express a CAR. The definition of CAR T- cells encompasses all classes and subclasses of T-lymphocytes including CD4+ T cells, CD8+ T cells, gamma delta T cells as well as effector T cells, memory T cells, regulatory T cells, and the like. The T lymphocytes that are genetically modified may be "derived" or "obtained" from the patient who will receive the treatment using the genetically modified T cells or they may be "derived" or "obtained" from a different patient. In a particular embodiment, the chimeric antigen receptor of the present invention also comprises an extracellular hinge domain, a transmembrane domain, and an intracellular NK cell signaling domain.

Thus, a further aspect of the invention refers to a NK-cell comprising a chimeric antigen receptor of the invention (“CAR-NK cell”).

As used herein the term “CAR-NK” refers to natural killer (NK) cells that has been genetically engineered to express a CAR. NK cells are defined as CD56+ and CD3- cells and are subdivided into cytotoxic and immunoregulatory. They are of great clinical interest because they contribute to the graft-vs-leukemia/graft-vs-tumor effect but are not responsible for graft- vs-host disease. NK cells can be generated from various sources such as umbilical cord blood, bone marrow, human embryonic stem cells, and induced pluripotent stem cells. However, tumors can escape the cytotoxicity of NK cells when they are directed against NKG2D ligands MICA and MICB (major histocompatibility complex class I chain-related protein A/B). Henceforth, preclinical research has been reported for CAR-modified primary human NK cells redirected against CD 19, CD20, CD244, and HER2, as well as CAR-expressing NK-92 cells targeted to a wider range of cancer antigens. Primary NK cells engineered to express CARs have potential benefits compared to CAR-T cells. NK cells have spontaneous cytotoxic activity and can generate target cell death independent of tumor antigen, while T lymphocytes only kill their targets by a CAR-specific mechanism. Therefore, in the setting of antigen downregulation by tumor cells attempting to escape immune detection, NK cells would still be effective against tumor cells. In addition, primary human NK cells produce cytokines, such as interferon gamma, interleukin 3, and granulocyte-macrophage colony-stimulating factor, that differ from the proinflammatory cytokines produced by T cells that are responsible for the onset of cytokine release syndrome. Individual NK cells can survive after contacting and killing multiple target cells, possibly reducing the number of cells that need to be adoptively transferred (i.e. the ex vivo stimulation and expansion of autologous or allogeneic lymphocytes, followed by reinfusion of the expanded lymphocyte population into the patient, in contrast to T cells).

In a particular embodiment, the invention refers to a NK-cell armed with an antibody of the invention. Particularly, the NK-cell can be a NK-cell with an Fc-engineered of the antibodies of the invention.

A further aspect of the invention refers to a macrophage-cell comprising a chimeric antigen receptor of the invention (“CAR-macrophage cell” or CAR-M cell). As used herein the term “CAR-M” refers to macrophage that has been genetically engineered to express a CAR. Based on their ability to penetrate solid tumors and traffic through the TME, Macrophages engineered with CAR constructs demonstrate sufficient potency. Similar to CAR-T, the core components of CAR-M contain an extracellular domain that provides specific recognition by a single-chain variable fragment (scFv) (eg, CD 19 and HER2), a hinge domain, a transmembrane domain (mostly CD8), and an intracellular domain that presents dedicated downstream signalling (eg, CD3^, FcyR).

Antigen binding domain

The extracellular domain of the CAR comprises an antigen binding domain that specifically binds or recognizes a target antigen. Here and according to the invention the target antigen will be the protein spike of the SARS-CoV-2 virus.

As used herein, "bind" or "binding" refer to peptides, polypeptides, proteins, fusion proteins and antibodies (including antibody fragments) that recognize and contact an antigen. Preferably, it refers to an antigen-antibody type interaction. By “specifically bind” it is meant that the antigen binding domain of the CAR recognizes a specific antigen but does not substantially recognize or bind other molecules in a given sample. The "specific binding" is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope). As used herein, the term “specific binding” means the contact between an antigen binding domain of the CAR and an antigen with a binding affinity of at least 10-6 M. In certain aspects, the antigen binding domain of the CAR binds with affinities of at least about 10-7 M, and preferably 10-8 M, 10-9 M, 10-10 M. The binding affinity can be measured by any method available to the person skilled in the art, in particular by surface plasmon resonance (SPR).

In one embodiment, such antigen binding domain is an antibody, preferably a single chain antibody. Preferably, the antibody is a humanized antibody. Particularly, such antigen binding domain is an antibody fragment selected from fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, single chain variable fragments (scFv), single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multi-specific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFv. Particularly, such antigen binding domain is selected from a Fab and a scFv. Particularly, the antigen binding domain is one of the antibodies of the invention and particularly the antibody CIO or C8.

In one embodiment, the antigen binding domain is a scFv, the scFv can be derived from the variable heavy chain (VH) and variable light chain (VL) regions of an antigen-specific mAb linked by a flexible linker. The scFv retains the same specificity and a similar affinity as the full antibody from which it is derived. The peptide linker connecting scFv VH and VL domains joins the carboxyl terminus of one variable region domain to the amino terminus of the other variable domain without compromising the fidelity of the VH-VL paring and antigen-binding sites. Peptide linkers can vary from 10 to 30 amino acids in length. In one embodiment, the scFv peptide linker is a Gly/Ser linker and comprises one or more repeats of these amino acids.

The extracellular domain of the CAR may comprise one or more antigen binding domain(s).

As used herein, the term “antigen” has its general meaning in the art and generally refers to a substance or fragment thereof that is recognized and selectively bound by an antibody or by a T cell antigen receptor, resulting in induction of an immune response. Antigens according to the invention are typically, although not exclusively, peptides and proteins. Antigens may be natural or synthetic and generally induce an immune response that is specific for that antigen.

All term related to the antibodies are already defined in the first part relating to the antibodies of the invention.

Spacer or hinge domain

The CAR optionally comprises a spacer or hinge domain linking the antigen binding domain to the transmembrane domain.

In some embodiments, the CAR comprises a hinge sequence between the antigen binding domain and the transmembrane domain and/or between the transmembrane domain and the cytoplasmic domain. One ordinarily skilled in the art will appreciate that a hinge sequence is a short sequence of amino acids that facilitates flexibility.

In particular, the spacer or hinge domain linking the antigen binding domain to the transmembrane domain is designed to be sufficiently flexible to allow the antigen binding domain to orient in a manner that allows antigen recognition.

The hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgGl Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain embodiments, the hinge domain includes at least a portion of an IgGl, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains.

Exemplary hinges include, but are not limited to, a CD8a hinge, a CD28 hinge, IgGl/IgG4 (hinge-Fc part) sequences, IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number WO2014031687, U.S. Pat. No. 8,822,647 or published app. No. US2014/0271635. As hinge domain, the invention relates to all or a part of residues 118 to 178 of CD8a (GenBank Accession No. NP_001759.3), residues 135 to 195 of CD8 (GenBank Accession No. AAA35664), residues 315 to 396 of CD4 (GenBank Accession No. NP_000607.1), or residues 137 to 152 of CD28 (GenBank Accession No. NP_006130.1) can be used. Also, as the spacer domain, a part of a constant region of an antibody H chain or L chain (CHI region or CL region) can be used. Further, the spacer domain may be an artificially synthesized sequence.

In some embodiments, for example, the hinge sequence is derived from a CD8 alpha molecule or a CD28 molecule.

Transmembrane domain

The transmembrane domain of the CAR functions to anchor the receptor on the cell surface. The choice of the transmembrane domain may depend on the neighboring spacer and intracellular sequences.

The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane -bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D, and DAP molecules. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. A transmembrane domain is thermodynamically stable in a membrane. It may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular signaling domain(s) of the CAR. A glycine-serine doublet may provide a suitable linker.

Intracellular domain

The terms “intracellular domain”, “cytoplasmic domain” and “intracellular signaling domain” are used interchangeably herein. The role of the intracellular domain of the CAR is to produce an activation signal to the T cell as soon as the extracellular domain has recognized the antigen.

Examples of intracellular domain sequences that are of particular use in the invention include those derived from an intracellular signaling domain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3(^, FcRy, FcRp, CD3y, CD35, CD3s, CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), FcsRI, DAP10, and DAP12. It is particularly preferred that the intracellular domain in the CAR comprises a cytoplasmic signaling sequence derived from CD3(^.

The intracellular domain of the CAR can be designed to comprise a signaling domain (such as the CD3(^ signaling domain) by itself or combined with costimulatory domain(s). A costimulatory molecule can be defined as a cell surface molecule that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4- 1BB (CD137), 0X40 (CD134), CD30, CD40, CD244 (2B4), ICOS, lymphocyte function- associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. The intracellular signaling portion of the above recited co-stimulatory domains can be used alone or in combination with other co-stimulatory domains. In particular, the CAR can comprise any combination of two or more co-stimulatory domains from the group consisting of CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, CD244 (2B4), ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.

Thus, for example, the CAR can be designed to comprise a signaling domain such as the CD3(^ signaling domain and two co-stimulatory signaling domains selected from CD28 and CD40, CD28 and 4-1BB (CD137), CD28 and 0X40 (CD134), and CD28 and LFA-1.

“First-generation CARs” contain a single signaling domain. CARs containing a signaling domain together with one additional costimulatory domain are termed “second generation” while those containing a signaling domain together with two additional costimulatory domains are listed as “third generation”. For example, first-generation CARs contain solely the CD3(^ chain as a single signaling domain. Second- and third-generation CARs consist of one or two additional costimulatory signaling domains, respectively, such as CD28, CD27, OX-40 (CD134) and 4-1BB (CD137). For example, second-generation CAR may contain CD3(^ and CD28 signaling domains, while third-generation CAR may contain CD3(^, CD28 and either 0X40 (CD 134) or 4- IBB (CD 137).

The CAR of the invention may be a first generation, a second generation, or a third generation CAR as described hereabove. Preferably, the CAR-T cells is a second or third generation CAR.

“TRUCKs” represent the recently developed ‘fourth-generation’ CARs. TRUCKs (T cells redirected for universal cytokine killing) are CAR-redirected T cells used as vehicles to produce and release a transgenic product that accumulates in the targeted tissue. The product, for example a pro-inflammatory cytokine, may be constitutively produced or induced once the T cell is activated by the CAR. Other substances such as enzymes or immunomodulatory molecules may be produced in the same way and deposited by CAR-redirected T cells in the targeted lesion. This strategy involves two separate transgenes expressing for example (i) the CAR-T cells and (ii) a cell activation responsive promoter linked to a cytokine such as IL-12. Consequently, immune stimulatory cytokine such as IL-12 is secreted upon CAR engagement.

In a particular embodiment, the CAR-T cells of the invention is a CAR-T cells of second generation as defined above.

In a particular embodiment, the CAR-T cells of the invention comprise an intracellular domain using a signaling domain such as CD28 or 4- IBB.

Methods to obtain a CAR-T cells

Methods and protocols to obtain CAR-T cells are well known in the art. To obtain CAR- T cells from T cells, transfection, transposon system like the sleeping beauty method or infection thanks to a lentivirus or retroviral vectors can be used (see for example Martinez Marina et al., 2019).

Methods using lentivirus able to transduce T cells to obtain CAR-T cells are well known. For example, and as shown in the present application, a lentivirus stock can be used. Protocols used to obtain CAR-T cells are well known in the art (see for example Okuma Atsushi, 2021. Generation of CAR-T Cells by Lentiviral Transduction). In this case and according to the invention, the inhibition of RINF and the transformation of the cells in CAR-T cells using a lentivirus can be done using the same lentivirus expressing a shRNA targeting RINF and the CAR construction.

Another method to obtain CAR-T cells from T cells is call sleeping beauty using DNA transposons to transfect the cells (see for example Izsvak et al. 2010).

According to the invention, a retrovirus can be used to generate CAR-T cells.

According to the invention, the CAR-T cells can be CAR-T cells from the first, the second, the third, the fourth or fifth generation.

In a particular embodiment, the antibodies of the invention can be used for any antibodybased engineered cell design.

Therapeutic uses and pharmaceutical compositions

Antibodies, fragments thereof and CAR-T cells of the invention are particularly suitable for inducing an immune response against SARS-Cov-2 and/or to block virus entry into cells and/or to induce mediates cell killing through ADCC.

The antibodies or CAR-T cells of the invention may be used alone or in combination with any suitable agent.

Therefore, a further object of the present invention relates to an antibody or a CAR or CAR-T cell of the present invention for use in the treatment of a SARS-CoV-2 in a subject in need thereof.

In other words, the invention also relates to a method for treating a subject in need thereof against SARS-CoV-2 comprising administering a therapeutically effective amount of an antibody (or antibody derivatives) or CAR-T cells of the present invention.

In a particular embodiment, the antibody of the invention is suitable for vaccine purposes against SARS-CoV-2 (see for example Naranjo-Gomez, Mar*; Pel egrin, Mireia. Vaccinal effect of HIV-1 antibody therapy. Current Opinion in HIV and AIDS: July 2019 - Volume 14 - Issue 4 - p 325-333).

In each of the embodiments of the treatment methods described herein, an anti-spike antibody or CAR-T cells according to the invention are delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of an antibody according to the invention is administered to a patient in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.

As used herein, the terms "treatment" and "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term "therapeutically effective amount" or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the antibody of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the antibody of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of the antibody of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, the ability of a compound to decrease viral load may, for example, be evaluated in an animal model system predictive of efficacy in human infection. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity of infected cells and decrease in viral load by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may decrease viral load, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, intranasal or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled antibody of the present invention, fragment or mini-antibody derived from the antibody of the present invention. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of an antibody of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of an antibody of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 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, 40, 45,

50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 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, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

As used herein, the term "subject" or "subject in need thereof', is intended for a human or non-human mammal. Typically, the patient is affected or likely to be infected with SARS- Cov-2.

In some embodiments, the subject can be human or any other animal (e.g., birds and mammals) susceptible to coronavirus infection (e.g. domestic animals such as cats and dogs; livestock and farm animals such as horses, cows, pigs, chickens, etc.). Typically said subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a farm animal or pet. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is an elderly human. In some embodiments, the subject is a premature human infant. In some embodiment, the subject is immunodeficient. Particularly, the subject is affected or likely to be infected with SARS-Cov-2 and is asymptomatic or symptomatic.

As used herein, the term “coronavirus” has its general meaning in the art and refers to any member of members of the Coronaviridae family. Coronavirus is a virus whose genome is plus-stranded RNA of about 27 kb to about 33 kb in length depending on the particular virus. The virion RNA has a cap at the 5’ end and a poly A tail at the 3’ end. The length of the RNA makes coronaviruses the largest of the RNA virus genomes. In particular, coronavirus RNAs encode: (1) an RNA-dependent RNA polymerase; (2) N-protein; (3) three envelope glycoproteins; plus (4) three non-structural proteins. These coronaviruses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mostly in infants >12 mo.), and possibly neurological syndromes. Coronaviruses are transmitted by aerosols of respiratory secretions.

As used herein, the term “Severe Acute Respiratory Syndrome coronavirus 2” or “SARS-Cov-2” has its general meaning in the art and refers to the strain of coronavirus that causes coronavirus disease 2019 (COVID-19), a respiratory syndrome that manifests a clinical pathology resembling mild upper respiratory tract disease (common cold-like symptoms) and occasionally severe lower respiratory tract illness and extra-pulmonary manifestations leading to multi-organ failure and death. In particular, the term refers to the severe acute respiratory syndrome coronavirus 2 isolate 2019-nCoV_HKU-SZ-005b_2020 (for which the complete genome is accessible under the NCBI access number MN975262) as well as several variants of concern (VOC) such as Omicron BA.2, Omicron BA.5, XBB.1.5, JN.l and EG.5.1.1, among others.

As used herein, the term “Covid- 19” refers to the respiratory disease induced by the Severe Acute Respiratory Syndrome coronavirus 2.

As used herein, the term "asymptomatic" refers to a subject who experiences no detectable symptoms for the coronavirus infection. As used herein, the term "symptomatic" refers to a subject who experiences detectable symptoms of coronavirus infection. Symptoms of coronavirus infection include: fatigue, anosmia, headache, cough, fever, difficulty to breathe, diarrhea.

Typically, the antibodies or CAR-T cells of the present invention are administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers 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, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a patient, the composition will be formulated for administration to the patient. The compositions of the present invention may be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intranasal and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention 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, for example as a solution in 1,3 -butanediol. 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 di glycerides. 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. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be affected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. For example, an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m² and 500 mg/m². However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g. 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of an antimyosin 18A antibody of the invention. In certain embodiments, the use of liposomes and/or nanoparticles is contemplated for the introduction of antibodies and antibody genes into host cells. The formation and use of liposomes and/or nanoparticles are known to those of skill in the art.

Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent useful to treat a viral infection or the symptoms induced by the viral infection. For example, further agent may be selected in the group consisting in bronchodilators like P2 agonists and anticholinergics, corticosteroids, beta2-adrenoceptor agonists like salbutamol, anticholinergic like ipratropium bromide or adrenergic agonists like epinephrine. Further agent may be also an antiviral compound like amantadine, rimantadine, pleconaril, azitromicine, ivementine or chloroquine or antibodies like Bebtelovimab, Cilgavimab, Tixacevimab.

Adoptive cell therapies, immunomodulators of immunosuppressors (like Anti-IL6R) may also added in the pharmaceutical composition of the invention, for example for immunodeficient subject.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Identification of broad reactivity of C8 and CIO mAbs. Binding of C8 and CIO mAb to SARS-CoV-2 mutated Spike proteins and multiple VOC. Figure 2. Targeting capacity of SARS-CoV2-infected cells by C8 and CIO mAbs. (A) Schematic representation of cytotoxicity assay timeline. (B) ADCC capacity of CIO mAb using lung-epithelial infected cells as targets (graphical representation of cell index data obtained through the RTCA Xcelligence over-time). (C) ADCC capacity of C8 (left) and CIO (right) mAbs using lung-epithelial infected cells as targets and primary NK cells as effector cells.

Figure 3. Evaluation of second-generation CAR-T cells activity against Spikeexpressing cells. (A). Schematic representation of cytotoxicity assay timeline. (B, C) Grouped analysis of CAR-T cells cytotoxicity against 293GS and A549GS cell lines at 48h. (D, E) Quantification of IL-2 and IFNy in cell-culture supernatants at 24h post co-culture.

Figure 4. Evaluation of in vitro CAR-T persistence. (A) Schematic representation of rechallenge experiment time course. (B) Graph shows grouped analysis of CAR-T cell cytotoxicity during 4^th challenge. (C) Proliferation of CAR-T cells during the rechallenge experiment evaluated by total count of CD3+ T cells by flow cytometry.

Figure 5. Evaluation of second-generation CAR-T cells activity against infected cells of CAR-T cells. (A) Graphical representation of cell index data obtained through the RTCA Xcelligence over-time in the absence (left) or in the presence of CAR-T cells (right) (B) Grouped analysis of CAR-T cells cytotoxicity against infected lung-epithelial cells (Calu-3) at 48h. (C) Viral titers in supernatants on infected cells in the presence of C8- and CIO-derived CAR T cells.

Figure 6. SPR analysis of S309 (sotrovimab) and CIO binding to trimeric Spike (S1+S2 ECD with a His tag). Ab binding to trimers of Spike variants captured using their poly- His tag. The S309 mAb was used as control.

EXAMPLE 1:

Material & Methods

Ethics.

All human blood samples were fully anonymized and given with previous written consent of the donor. Samples were given under ethical approval of appropriate institutions.

Antibody selection.

For the selection of antibodies, phage display technology was used and consecutive phage enrichment rounds (panning rounds) were performed. Typically, the antibody domains were superficially presented on the produced phages in junction to pill, a phage coat protein. For each round, 1 x 10¹¹ phages containing a Fab of a self-made antibody library in a pHen vector, were applied on pre-coated SARS -Co V-2- Spike- RBD-His (0.5pg/ml; Sino Biological) immobilized on 96-well plates via Nickel-Nitrilotriacetic acid-Biotin-Streptavidin linkage. Extensive blocking was performed by the alternate use of 2 % milk or bovine serum albumin (BSA) in PBS + 0.1 % Tween20 (PBS-T). In addition, washing stringency was intensified (more washing steps) with increasing panning rounds. Bound phages were eluted in phage elution buffer (50 mM Tris (pH 8), 125 pg/ ml Trypsin, 1 mM CaC12). The eluted phages were infected into Escherichia coli (E. coli) TGI. The next day, exponentially growing TGI were infected with 20x excess of M13KO7 helper phage (New England Biolabs) to facilitate new phage assembly. Produced phages were precipitated using 20 % PEG-6000/ 2.5 M NaCl. With this material, another round of panning was started.

Following the last selection rounds, eluted phages were infected into E. coli HB2151, plated and individual clones were used for monoclonal screening. The expression of the encoded antibody fragment was induced in exponentially growing E. coli HB2151 with 1 OOpM Isopropyl-P-D-thiogalactopyranosid (IPTG) and expression lasted overnight. The antibody fragment was shuttled out of the cell due to a pelB leader sequence and the supernatant was screened against the SARS-CoV-2-Spike-RBD in an Enzyme-Linked Immunosorbent Assay (ELISA). Binding was detected using an anti -human Fab antibody (A0293-1ML; Sigma).

Antibody production.

C8 and CIO mAbs were purchased at Evitria. They were produced in mammalian cells upon transfection with the light chain and heavy chain variable sequences of C8 and CIO mAbs in a IgGl backbone.

Surface Plasmon resonance (SPR).

Affinity of bNAbs for Fey Receptors was assessed by SPR experiments performed on a Biacore T200 (GE Healthcare). SPR experiments were performed on a T200 apparatus at 25 °C in PBS containing 0,05 % P20 surfactant (Cytiva). Anti-histidine antibody (R&D Systems) was covalently immobilized on a CM5-S sensor chip flowcell (Fc2) by amine coupling according to the manufacturer’s instructions (Cytiva). A control reference surface (flowcell Fcl) was prepared using the same chemical treatment but without anti-His antibody. All kinetic measurements in Fcl and Fc2 were performed by single-cycle titration at lOOpl/min. Each human FcyR (R&D Systems) was captured on immobilized anti-His antibodies at 100-200 RU level. Five increasing concentrations (3,6, 11, 33, 100, 300 nM) of antibody were injected (injection time = 60s) at lOOpl/min on captured receptors. After a dissociation step of 600s in running buffer, sensor surfaces were regenerated using lOpl of glycine-HCl pH1.5. All the sensorgrams were corrected by subtracting the low signal from the control reference surface and buffer blank injections. Kinetic parameters were evaluated from the sensorgrams using a two-states or a steady-state models from the T200 evaluation software.

Docking and Structural approaches

The docking model was built with Rosetta Dock and guided by experimental information on CIO binding to different RBDs mutants/variants and on CIO cross competition with other anti-RBD antibodies with known epitope.

Human blood samples and Primary cells purification.

Healthy donor blood buffy coats were obtained from the local reference blood bank: Banc de Sang i Teixits, (BST, Barcelona, Spain; #HCB/2022/0137) and Etablissement Frangais du Sang (EFS, Montpellier, France; #21PLER2018-0069). Human PBMCs were obtained from buffy-coats by density-gradient centrifugation (Lypmhoprep, StemCell Technologies), and were cultured in RPMI Medium 1640 (IX) (Gibco, 31870-025), 10% fetal bovine serum (Sigma, F9665-500ML), 0.1% penicillin-streptomycin.

Healthy donor T-cells were obtained from buffy-coats by density-gradient centrifugation (Lymphoprep, StemCell technologies) using the RosetteSep™ Human T-cell Enrichment Cocktail (Stemcell technologies, 15061) to isolate T-cells from whole blood by negative selection. The purified T-cells were cultured in in R10 Cell Medium, consisting in RPMI Medium 1640 (IX) (Gibco, 31870-025), 10% fetal bovine serum (Sigma, F9665- 500ML), 0.1% penicillin-streptomycin, and IL-2 (50 lU/mL; Miltenyi Biotec). Cells were then activated and expanded for 4 days using beads conjugated with CD3 and CD28 mAbs (Dynabeads, Gibco, 1113 ID), and they were transduced 24 hr later with the lentivirus. A period of cell expansion of 9-10 days was necessary before conducting experiments.

Healthy donor NK-cell were purified by from human PBMCs by negative selection using a NK magnetic isolation kit (Miltenyi Biotech #130-092-657). Isolated NK cells were cultured in RPMI Medium 1640 (IX) (Gibco, 31870-025), 10% fetal bovine serum (Sigma, F9665-500ML), 0.1% penicillin-streptomycin, and IL-2 (100 lU/mL; Miltenyi Biotec).

Cell lines culture.

Human embryonic kidney cells (293T HEK cells) (HEK 293T-ATCC-CRL-1575TM) and A549 cell (A549-ATCC-CCL185) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, cat #31966) supplemented with 10% fetal bovine serum, sodium pyruvate and antibiotics (penicillin-streptomycin). Cells were grown at 37°C in a 5% CO2 atmosphere and split twice per week keeping the cell density < 2.5 x 106 cells/ ml. SARS-CoV-2 Spikeexpressing cells (293GS and A549GS) were maintained in the same medium supplemented with 400 pg/ ml Hygromycin B (Gibco). Jurkat cell line was cultured in RPMI media supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin).

Vero E6 cells (African green monkey kidney cells) were obtained from ECACC (#Vero Cl 008, ECACC 85020206) and maintained in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin/ streptomycin (100 U/mL and 100 pg/mL, Gibco # 15140122) at 37 °C with 5% CO2 as described previously (REF). Human pulmonary Alveolar A549-hACE2 cells were obtained from original A549 (ECACC #86012804) transduced with a lentiviral vector expressing human ACE2 receptor (manufactured by FlashTherapeutics company, Toulouse, France) and sorted by cytometry for having more than 80% hACE2 on their surface. The sorted A549-hACE2 cells were maintained in RPMI supplemented with 10% heat inactivated fetal bovine serum (FBS), 1% sodium Pyruvate, 0.5% HEPES and antibiotics (penicillin/streptomycin) and cultivated at 37°C with 5% CO2. Calu-3 cell line (EP-CL-0054, Elabscience Biotechnology Inc) were cultivated in the presence of 10% DMEM (Sigma #D6429), 1% SVF (Gibco), 1% penicillin/streptomycin (#P4333), 25mM Hepes (Sigma #H0887). All cells were cultivated at 37°C with 5% CO2.

Virus production, purification and titration.

The strains BetaCoV/France/IDF0372/2020 (Lineage B); hCoV-19/France/IDF- IPP11324/2020 (Lineage B.1.1.7); and hCoV-19/France/PDL-IPP01065/2021 (Lineage B.1.351) were supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur and headed by Pr. Sylvie van der Werf. The human samples from which the lineage B, B.1.1.7 and B.1.351 strains were isolated were provided by Dr. X. Lescure and Pr. Y. Yazdanpanah from the Bichat Hospital, Paris, France; Dr. Besson J., Bioliance Laboratory, saint-Herblain France; Dr. Vincent Foissaud, HIA Percy, Clamart, France, respectively. These strains were supplied through the European Virus Archive goes Global (Evag) platform, a project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement #653316.

The virus strains were propagated in Vero E6 cells with DMEM containing 2.5% FBS at 37 °C with 5% CO2 and were harvested 72 h post-inoculation. Virus stocks were harvested and stored at - 80 °C as described (40).

The titers were determined by means of a plaque assay on a monolayer of Vero E6 cells as previously described (41). Briefly, 100 pL from infected cell culture supernatants were titrated using 12 serial dilutions. The plaque-forming unit (PFU) values were determined by scoring the wells displaying cytopathic effects from 6 replicates/condition. The virus titer was determined as the number of PFU/mL, and MOI was the PFU/cell ratio.

Microscopy immunolabeling and imaging.

Vero E6, A549-hACE2 and Calu-3 cells were cultured and infected with SARS-CoV-2 as described above. Cells were incubated with primary antibodies (C8 or CIO) at concentration indicated in the figures during Ih (4°C in PBS+2%SFV). We used as secondary antibody an Alexa Fluor® 647 anti-human IgG Fc (diluted 1 :200; clone M1310G05 from BioLegend #410713) for 45 min, respectively. After washing, nuclei were labeled with Hoechst dye (Molecular Probes). Images were acquired using a CellDiscoverer 7 LSM900 Airyscan2 microscope (ZEISS) equipped with a 5X (x2) objective. Image analysis was performed using ZEN lite.

Real-time Cytotoxicity assay.

The cytotoxicity assay was performed using an xCELLigence real-time cell analyzer (RTCA) System (ACEA Biosciences, San Diego). Impedance-based RTCA was used for label- free and real-time monitoring of cytolysis activity. The cell index (CI) based on the measured cell-electrode impedance was used to measure cell viability.

For cytotoxic activity against HEK- Spike expressing cells, basal readout was performed by addition of 50uL of DMEM supplemented with 10% FBS, afterwards 10.000 target cells were seed in E-plate 16 (Agilent, ref: 5469830001) and incubated for 16-24h at 37°C 5%CO2 in 50 uL DMEM supplemented with 10% FBS to allow their attachment. After the attachment period, CAR-T cells were added on top in lOOuL of RPMI-1640 supplemented with 10% FBS in an effector to target ratio 5: 1.

For cytotoxic activity against lung-epithelial infected cells, Calu-3 were used. Basal readout was performed by addition of 80uL of DMEM supplemented with 10% FBS. Afterwards, Calu-3 seeded at a density of 17 x 103 cells per well and grown for 72 hours at 37°C 5% CO2 in 80uL DMEM supplemented with 10% FBS to allow their attachment and growing. After the attachment period, Calu-3 were infected with SARS-CoV-2 (MOI 10-4) in the DMEM without FBS during two hours. Afterwards, the medium (supernatant) is replace by a fresh RPMI-1640 supplemented with 10% FBS. 7h later, UTD CAR-T or SARS-CoV-2- CAR-T cells were then added on top of the RTCA unit in 80uL of RPMI-1640 at 5: 1 ratio (effectortarget). For Antibody Dependent Cellular Cytotoxicity (ADCC), hCD16-NK92 cell line and primary NK cells were added at 5: 1 and 1 : 1 (effectortarget) ratios, respectively. NK cells were previously armed with CIO or C8 mAb (lOpg/ml) for Ih.

The impedance signals were recorded for 96-120h every 20min intervals.

Cytotoxicity was calculated via the following formula: ((CI (target cells only) - CI (target cells + T cells))/CI target cells only) x 100%.

Enzyme-linked immunosorbent assay (ELISA).

Co-culture supernatants were analysed for cytokine production (IFNy and IL-2) by Enzyme-linked immunosorbent assay (ELISA), following the manufacturer’s instructions (DuoSet Elisa, R&D Systems, Abington, UK). ELISA plates (NUNC -IMMUNO Maxisorp, Thermofisher, Waltham, USA) were coated (100 pl/well) with the capture antibody diluted at the working concentration in PBS at room temperature overnight. Then plates were washed and blocked by adding 300 pL PBS containing 1% bovine serum albumin (BSA) to each well and incubated at room temperature for a minimum of 1 hour. The ELISA assay procedures were performed, after sample dilution in Reagent Diluent. Briefly, 100 pL of diluted sample or standards in Reagent Diluent per well were added and incubated 2 hours at room temperature. After three wash steps with 400 pL Wash Buffer, 100 pL of the Detection Antibody, diluted in Reagent Diluent, to each well were added and incubated 2 hours at room temperature. After three wash steps, 100 pL of the working dilution of Streptavidin-HRP were added to each well and incubated for 20 minutes at room temperature. Again, after three wash steps, 100 pL of the working dilution of Substrate Solution to each well was added and incubated for 20 minutes at room temperature. Then, 50 pL of Stop Solution to each well was added and the plate was gently mixed. The optical density of each well immediately determined using Epoch microplate spectrophotometer (BioTek Instrumentals, Inc., Winooski, VT, USA).

Flow Cytometry.

During the T-cell expansion, we employed the following mAbs panel: CD4-PerCP (BD Biosciences, 555348), CD8-AlexaFluor700 (BD Biosciences, 345773) and the CARs were detected using Biotin-SP AffiniPure Goat Anti-Mouse IgG, F(ab')2 fragment (Jackson ImmunoResearch, 115-065-072) and Streptavidin-PE (eBioscience, 12-4317-87). Concerning the T-cell subsets panel the following mAbs were used: CCR7-PerCP Cy5.5 (BD Biosciences, 561144), CD62L-FITC (BD Biosciences, 555543), CD8-APC H7 (BD Biosciences, 560179), CD45RA-PeCy7 (BD Biosciences, 560675). In the case of the T-cell exhaustion panel, we employed: PD1-APC (BD Biosciences, 558694), CTLA4-PE (BD Biosciences, 557301), TIM3-FITC (eBioscience, 11-3109-42), LAG3-PeCy7 (eBioscience, 25-2239-42), CD8-APC H7 (BD Biosciences, 560179).

For the detection of Spike expression, we employed a rabbit mAb against the spike protein, followed by a staining with a mouse-anti rabbit IgG-FITC monoclonal secondary antibody. Samples were run through the fluorescence-activated cell sorting flow cytometer Attune, and data were analyzed using the FlowJo Software.

Luminiscence assay.

For luminescence determination we employed the Pierce™ Gaussia Luciferase Glow Assay Kit (ThermoFischer Scientific), we plated 10.000 293GS or A549GS cells in a white 96 flat well plate (ThermoFischer Scientific). Cells were lysed and, coelenterazine was add to the cell lysate. After 10-minute incubation, luminescence was read using Synergy HT spectrophotometer (Biotek, Vermont, USA).

Transcriptomic studies.

RNA was extracted from neutrophils obtained from 4 healthy blood donors. Total RNA was then purified from 500,000 cells using the miRNeasy Micro (Qiagen GmbH) following the manufacturer's instructions. The quantification of all RNA samples was performed using the Nanodrop One (Thermo Fisher Scientific), and RNA integrity was evaluated using the Agilent 2100 Bioanalyzer system. RNA expression profiling was performed using CAR-T Characterization Panel (770 genes covering the core pathways and cellular phenotypes that are known to predict CAR-T activity). Gene expression analysis was conducted with the NanoString technology. Briefly, 5 pL/sample containing of 50 ng of total RNA was combined with the nCounter® reporter CodeSet (3 pL) and nCounter® capture ProbeSet (2 pL) along with hybridization buffer (5 pL) for an overnight hybridization reaction at 65 °C. The reaction was then cooled to 4 °C, and the samples were purified, immobilized on a cartridge, and the data were assessed using the nCounter SPRINT. All expression data was reviewed using NanoString® nSolverTM Analysis software 4.0. The up-regulated genes were classified into the main 5 categories proposed by NanoString: Activation, Phenotype, Metabolism, Persistence, and Exhaustion.

Statistics.

Statistical analyses were performed using Prism software version 9.5.1 (GraphPad). Simple group comparisons were performed using the Wilcoxon signed rank test for paired data or the Mann-Whitney test for unpaired data. Multiple group comparisons were performed using Friedmann test for paired data or using Kruskal-Wallis test for unpaired data with additional multiple comparisons Dunn test. Significance was assigned as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

Identification and characterization of anti-SARS-CoV-2 spike antibodies by phage display.

To select antibodies against SARS -Co V-2- Spike, a large naive synthetic scFv library was used for three consecutive rounds of biopanning on recombinant Wuhan Spike Receptor Binding Domain (RBD) captured by its poly-Histidine tag in microtiter plates (data not shown). After 2 and 3 rounds of panning the library was strongly enriched in RBD-positive clones, as shown by the signal of phage pools by ELISA (data not shown). Ninety-five clones were tested for binding and the best 7 clones with different VH sequences were conserved. These 7 VH were shuffled with the whole VL library of a diversity of 4.105, and the resulting library in a Fab format on phages was sorted in solution by capture in decreasing concentrations of RBD (data not shown). To evaluate efficiency of the process, polyclonal phages in presence or absence of 10 nM competing RBD were tested for binding by ELISA. Whereas the library before shuffling was only weakly inhibited (R3, 22%), after shuffling and selection all the libraries were strongly inhibited (R4 to R6.2, 67-88%) suggesting that most of the phage stocks contained Fab with high affinities (data not shown). In particular, the library after 2 rounds at 10 nM (R6.1) was inhibited by 88% but the final round at 1 nM (R6.2) was presumably too stringent and resulted in a decrease in the average affinity (R6.2, 59%). Individual clones from R5, R6.1 and R6.2 were tested by ELISA as soluble Fab, quantified and ranked by doseresponse (data not shown). Four clones showed a strong binding with an EC50 around 20-50 nM (2B3, 2B2, G12, 2B6), all isolated from R5, and 1 clone a high affinity with an EC50 around 2 nM (CIO) isolated from R6.1. No interesting clone was isolated from R6.2 as suggested by the decrease in the competition experiment. These 5 clones were derived from 4 different parental VH. We then cloned, produced and purified as human IgGl these 5 clones and the 7 clones isolated in R3. After reformatting, R3 clones B12 and D3 were negative in an ELISA and were not further characterized. The remaining clones were tested by cytometry for binding to HEK293 cells stably expressing full-length Wuhan Spike at their surface (data not shown). All the IgG recognized the RBD domain presented in its native conformation at the cell surface but with different efficiencies. In particular, C8 clone was very efficient and strongly stained the cell line, despite being a non-matured antibody with a moderate affinity. We also measured the affinity of the 10 IgG by SPR by capturing the IgG to the chip and applying the monomeric RBD in the flow to avoid avidity effects. In these conditions all the affinities are comparable in the nM range (1.1 - 2.6 nM) but with different profiles. The non-matured clones showed an association rate faster than 5.10-5 M-ls-1 and a moderate dissociation rate around 10-3 s-1, whereas the matured IgG showed slower association around 1.5.10-5 M-ls-1 and slow dissociation rates in the 10-4 s-1 range (data not shown). Based on their sequences, cytometry and SPR binding properties, we retained 2 clones for a conversion to CAR format, C8 because of its high binding to cells, and CIO for its high affinity by SPR. We first verified their binding in the scFv format used in CAR by ELISA and showed activities comparable to the Fab format, CIO showing a much lower EC50 than C8 clones (data not shown).

Finally, we evaluated the binding affinity of C8 and CIO mAbs to the Spike protein in its trimeric conformation. Our results demonstrate that both mAbs effectively bind to trimeric Spike forms of the Wuhan strain and the Delta variant. However, CIO exhibited superior performance compared to C8, as it also recognized the trimeric Omicron Spike, in contrast to C8, which did not (data not shown).

Identification of a broadly reactive coronavirus antibody.

To comprehensively characterize C8 and CIO mAbs, we next assessed their binding capacity to multiple SARS-CoV-2 mutated Spike proteins and VOC spanning from the original Wuhan strains to more recent Omicron VOC. Both mAbs demonstrated binding capabilities to most SARS-CoV-2 mutated Spike proteins, with the exception of C8's binding to the W436R mutation (data not shown). Notably, the CIO mAh exhibited the ability to bind to all tested VOC, including the Omicron XBB.1.5 variant, while C8 failed to bind to all Omicron (BA, BQ, and XBB) VOC (data not shown). Figure 1 shows heatmaps indicating the binding strength of C8 and CIO to RBDs variants/mutants (based on ELISA results). Computational analysis of CIO were also obtained. These findings suggest that CIO recognizes a highly conserved epitope, positioning it as a broadly reactive anti-SARS-CoV-2 mAb.

The CIO mAb binds a conserved region of the SARS-CoV-2 RBD,

In an attempt to identify the epitope recognized by broadly reactive CIO mAb, we built a docking model with Rosetta Dock and guided by experimental information on CIO binding to different RBDs mutants/variants and on CIO cross competition with other anti -RBD antibodies with known epitope.

Obtained results showed that the putative epitope recognized by CIO encompasses amino acid residues dispersed across various regions within the RBD, predominantly situated between residues 457 to 491 (data not shown). Interestingly, cross-competition analyses showed that CIO binds to different epitopes than clinically used mAbs such as Bebtelovimab, Cilgavimab, Tixacevimab (data not shown). Interestingly, it cross-competed with the neutralizing mAb rbd.042 that recognizes a coldspot of the subdomain 1 (SD1) of the SARS- CoV2 Spike glycoprotein (13), adjacent to the RBD (data not shown).

We analyzed in parallel CIO binding to the Spike protein in the context of SARS-CoV- 2 full S trimer. We observed that CIO mAb makes clashes with adjacent monomers in the context of full S trimer with all the 3 RBDs in “down” conformation. However, no clashes were observed when the RBDs are in “Up” conformation. Thus, our docking model revealed that CIO binds when RBD is in the “Up” conformation (data not shown). Overall, the docking model is in accordance with the cross-competition data on tested anti-RBD mAbs with known epitopes. It fits in a RBD region with no mutations (as CIO binds all the RBD mutants/variants tested). Finally, according to this model, CIO is able to bind Spike trimer with RBDs in the “Up” conformation.

CIO mAb binds SARS-CoV-2 infected cells and mediates cell killing through NK- mediated ADCC.

We assessed in parallel the functional properties of C8 and CIO mAbs. We studied their neutralization potential as well as their ability to target infected cells, both properties being important for countering viral spread. Neither C8 nor CIO displayed neutralizing capacity (data not shown). Nevertheless, both mAbs demonstrated binding to Vero cells infected with SARS- CoV-2, albeit with different efficiencies, with CIO showing superior performance over C8, as determined by dose-response experiments (data not shown). Both mAbs were able to target Vero cells as well as lung-epithelial cells (A549-hACE2 and Calu-3) infected with different VOC (data not shown). Based on these observations, we next assessed the cytotoxic capacity of CIO mAb. To this end, we performed a cytotoxic assay based on the measure of electrical current using RTCA XCelligence, which allows the determination of a cell index based on the electric impedance produced by adherent target cells (Figure 2A-B). We set a co-culture of effector cells (primary NK cells) and target SARS-CoV-2 infected lung-epithelial cells at a 1 : 1 ratio armed or not with C8 (left panel) or CIO mAb (right panel), respectively. We showed that CIO mediates the killing of SARS-CoV2 lung-epithelial infected cells through NK-mediated ADCC (Figure 2C). This highlights the capacity of CIO mAb to eliminate infected cells and contribute to the decrease of viral propagation.

Development of second-generation CAR-T cells targeting SARS-CoV-2 Spike protein.

Based on the efficient binding and cell killing capacity of C8 and CIO mAbs, we next sought to repurpose these non-neutralizing antibodies for CAR-T cell therapy. To this aim we developed second generation CAR-T cells from their sequences. Four different CAR desings were generated, using C8 or CIO scFv sequences combined with either a CD28 or 4- IBB intracellular costimulation. These sequences were cloned in a lentiviral expression system (data not shown). Lentiviral vectors encoding for the different CAR designs were used to transduce healthy donor T cells to determine that they were properly expressed. Following T cell activation with CD3/CD28 stimulation, we next assessed CAR-T cell proliferation during a 10 day course as an indicator of T cell fitness. T cells expressing the different CAR molecules demonstrated a similar proliferation compared to control T cells (data not shown). Last, we aimed to detect CAR expression on T cell surface by flow cytometry staining after 10-day proliferation. For that, cells were stained using mAbs that specifically detect the scFv region of the CAR molecule. Sample acquisition revealed expression of all four CAR molecules on the surface of human T cells (data not shown). Altogether, these results allow us to conclude that the designed CAR constructs can be properly expressed on human T cells without affecting their fitness. Evaluation of CAR-T cell activity against SARS-CoV-2 Spike-expressing cells.

Subsequently, we endeavoured to establish a cell line model expressing the Spike protein on the cell surface. This model served as an initial platform to assess CAR-T cell activity, circumventing the stringent constraints associated with handling SARS-CoV-2 infected cells. To this end, we generated HEK293T and A549 cell lines expressing Gaussia Pierce Luciferase, and the Spike protein from the Wuhan strain, renamed as 293 GS and A549GS, respectively (data not shown). Both cell lines maintained the expression of Gaussia Luciferase (data not shown) and expressed the Spike protein on the cell surface for 40 days. Of note, 293GS cells displayed a higher expression of the Spike protein on cell surface.

We next sought to evaluate the functionality of the different CAR-T cells we developed. We performed a cytotoxic assay based on the measure of electrical current using RTCA XCelligence, as above mentioned (Figure 3A). We set a co-culture of CAR-T cells and target cells expressing the Spike protein at a 5: 1 ratio. The results show that there is a decrease in the cell index growth over time when target cells are co-cultured with CAR-T cells derived from the CIO scFv sequence, while C8-28z CAR-T does not have significant effect on target cell growth (data not shown). Repeated experiments using T cells derived from different donors demonstrate that C10-28z and ClO-BBz can recognize and eliminate the Spike-expressing cell lines 293GS and A549GS (Figure 3B-3C). The analysis of the fFNy and IL-2 in cell culture supernatants by ELISA revealed that untransduced cells (UTD) and C8-28z CAR-T cells do not produce detectable amounts of cytokines after 24h of co-culture. On the contrary, C10- derived CAR-T cells exhibited substantial cytokine production, with notably higher secretion levels observed in C10-28z CAR-T cells compared to ClO-BBz. This difference was particularly pronounced when co-cultured with 293GS cells (Figure 3D-3E), suggesting that the Spike-expression level is key for CAR-T cell activation. These results prove that C10- derived CAR-T cells can be activated and perform a cytotoxic activity after encountering target cells. They also show that C10-28z CAR-T cells have a more potent activation than ClO-BBz CAR-T cell.

CAR-T cells maintain their activity upon continuous stimulation by SARS-CoV-2 Spike

The next investigated C8- and CIO-derived CAR-T cell persistence to evaluate if they could maintain their activity when encountering a continuous stimulation by the targeted antigen. To address this issue, we designed a rechallenge experiment (Figure 4A). We evaluated whether the cytotoxic capacity of CAR-T cells was compromised after four consecutive challenges. Results show that even after facing a continuous stimulation, C10-28z and C10- BBz CAR-T cells can maintain their cytotoxicity towards target cells (Figure 4B). We also determined by flow cytometry the proliferation of CAR-T cells during consecutive challenges, measuring the total count of CD3+ cells. The results demonstrate that CIO-derived CAR-T cells, but not C8-derived, maintain their proliferation throughout the experiment. It is also important to note that C10-28z CAR-T cells exhibit a bigger expansion rate than the ClO-BBz construct (Figure 4C). Moreover, we evaluated CAR-T cells exhaustion profile after 4 challenges by assessing the expression of exhaustion markers such as LAG-3, CTLA-4 and PD- 1 by flow cytometry. Results show a lower expression of CTLA-4 and PD-1 exhaustion markers in C10-28z and ClO-BBz CAR-T cells as compared to UTD and C8-28z CAR-T cells (data not shown). Overall, the findings show a notably higher expansion rate and a reduced exhaustion phenotype in CIO-derived CAR-T cells when compared to those derived from the C8 mAb.

CAR-T cells has cytotoxic activity against SARS-CoV-2-infected lung epithelial cells.

We next examined the cytotoxic potential of various CAR-T cells against lung-epithelial cells infected with SARS-CoV-2 (Figure 5 A). This was important to asses as these cells secrete numerous cytokines and chemokines (data not shown) (19) that might affect CAR-T cell efficacy. As previously described (20,21), we observed a pronounced virus-induced cytopathic effect in lung-epithelial infected cells in the absence of CAR-T cells. However, despite this cytopathic effect reducing the window for assessing the cytotoxic potential of CAR-T cells, a significant decrease in the cell index growth over time was observed when infected cells were co-cultured with C10-28z CAR-T cells (Figure 5A-B). The cytotoxic capacity of CIO-derived CAR-T cells was also visualized using cutting-edge imaging approaches (data not shown). Simultaneously, we assessed the release of viral particles by infected target cells co-cultured with the different CAR-T cells generated, using UTD as control. Notably, a significant decrease in the viral titer was observed in supernatants recovered from infected cells co-cultured with both types CIO-derived CAR-T cells (C10-28z CAR-T and ClO-BBz), while C8-28z CAR-T cells showed no effect (Figure 5C). These findings corroborate the impact of C10-28z CAR-T cells on cell index growth and infected cell -killing capacity. Thus, our results demonstrate the cytotoxic activity of CIO-derived CAR-T cells against SARS-CoV-2-infected lung-epithelial cells. Furthermore, they underscore the superior cytotoxic efficacy of CIO-derived CAR-T cells against target cells, consistent with our observations in non-infected, Spike-expressing cells.

The observed disparity in cytotoxic efficacity between C8- and CIO-derived CAR-T cells prompted us to further characterize the different CAR-T cells, in an attempt to identify molecular mechanisms potentially involved in the enhancement of cytotoxic activity. To this end, we performed transcriptomic analysis using the NanoString nCounter technology. This approach allowed us to identify differentially expressed genes of the different CAR-T cells. Heatmap representation of upregulated and downregulated genes shows a completely distinct transcriptional profile between C8- and CIO-derived CAR-T cells co-cultured with infected cells. Notably, as compared to UTD co-cultured with infected cells, CIO-derived CAR-T cells upregulated multiple genes associated with T-cell activation and antiviral immune responses, distinguishing them from C8-derived CAR-T cells (data not shown). Moreover, differentially expressed gene (DEG) analysis revealed specific signatures for each intracellular costimulation molecule, in particular genes involved in T-cell phenotype and persistence (data not shown). These observations show that specific SARS-CoV-2-CAR-T cell gene signatures are associated with successful response.

EXAMPLE 2:

Material & Methods

SPR analysis of S309 (sotrovimab) and CIO binding to trimeric Spike (S1+S2 ECD with a His tag)

Ab binding to trimers of Spike variants captured using their poly-His tag (Omicron B.1.1.529 from R&D system (#11061-CV); JN.l (#40589-V08H59) and EG5.1 (#40589- V08H57) from Sinobiological). The S309 mAb was used as control. Antibodies in increasing concentration were applied in the flow. Data were fitted to a Langmuir 1 : 1 model.

Results

Results are depicted in Figure 6. Our evaluation using the trimeric Spike protein revealed that CIO exhibits binding capacity to EG5.1 and JN1 VOCs, at difference of S309 clone that does not show measurable binding to TN.1.

Conclusion

The inventors introduced a "pan" SARS-CoV-2 monoclonal antibody (mAb) with remarkable binding efficiency to RBD proteins harboring various mutations. Its discerning ability to identify cells infected by diverse VOCs underscores its specificity towards a highly conserved region of the virus. CAR-T-cells obtained with said antibody show a CAR-T-cell mediated killing of infected cells and reduce viral titers. Moreover CAR-T-cells retain their cytotoxic capacity in the strong pro-inflammatory environment associated with SARS-CoV-2 - infected lung-epithelial cells.

The antibodies of the invention and particularly the C8 and CIO antibodies offer numerous potential applications in both antibody-based and cell-based therapeutic approaches to target infected cells. This development significantly expands the therapeutic options available for combating SARS-CoV-2 infections.

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Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. An anti-spike antibody comprising: a. a heavy chain wherein the variable domain comprises: i. a H-CDR1 having a sequence set forth as SEQ ID NO: 1 or 9; ii. a H-CDR2 having a sequence set forth as SEQ ID NO: 2 or 10; iii. a H-CDR3 having a sequence set forth as SEQ ID NO: 3 or 11; b. a light chain wherein the variable domain comprises: i. a L-CDR1 having a sequence set forth as SEQ ID NO: 4 or 12; ii. a L-CDR2 having a sequence set forth as SEQ ID NO: 5 or 13; iii. a L-CDR3 having a sequence set forth as SEQ ID NO: 6 or 14.

2. An anti-spike antibody comprising a heavy chain having at least 70% of identity with SEQ ID NO: 7 or 15 and a light chain having at least 70% of identity with SEQ ID NO: 8 or 16.

3. An anti-spike antibody CIO according to the claim 1 which comprises or consists in: a. a heavy chain wherein the variable domain comprises: i. a H-CDR1 having a sequence set forth as SEQ ID NO: 1; ii. a H-CDR2 having a sequence set forth as SEQ ID NO: 2; iii. a H-CDR3 having a sequence set forth as SEQ ID NO: 3; b. a light chain wherein the variable domain comprises: i. a L-CDR1 having a sequence set forth as SEQ ID NO: 4; ii. a L-CDR2 having a sequence set forth as SEQ ID NO: 5; iii. a L-CDR3 having a sequence set forth as SEQ ID NO: 6.

4. An anti-spike antibody C8 according to the claim 1 which comprises or consists in: a. a heavy chain wherein the variable domain comprises: i. a H-CDR1 having a sequence set forth as SEQ ID NO: 9; ii. a H-CDR2 having a sequence set forth as SEQ ID NO: 10; iii. a H-CDR3 having a sequence set forth as SEQ ID NO: 11; b. a light chain wherein the variable domain comprises: i. a L-CDR1 having a sequence set forth as SEQ ID NO: 12; ii. a L-CDR2 having a sequence set forth as SEQ ID NO: 13; iii. a L-CDR3 having a sequence set forth as SEQ ID NO: 14.

5. An antibody that competes for binding to the spike protein with the antibodies according to the claims 1 to 4.

6. A chimeric antigen receptor (CAR) comprising an antigen binding domain of the antibodies of the claim 1 to 4.

7. A chimeric antigen receptor (CAR) according to the claim 6 wherein the CAR is a CAR- T cell.

8. A CAR-T cell according to the claim 7 wherein the antigen binding domain comes from the CIO or C 8 antibody.

9. An antibody according to the claims 1 to 5 or a CAR or CAR-T cell according to the claims 6 to 8 for use in the treatment of a SARS-CoV-2 in a subject in need thereof.

10. An antibody, a CAR or a CAR-T cell according to the claim 9 for use wherein the antibody, the CAR or the CAR-T cell are efficient against all variant of the SARS-CoV- - O - l l. A pharmaceutical composition which comprises an antibody according to the claims 1 to 5 or a Car or CAR-T cells o according to the claims 6 to 8 for use in the treatment of a SARS-CoV-2 in a subject in need thereof.