FR3128713A1 - Binding agent with enhanced affinity for the prevention and treatment of sarbecovirus-related diseases - Google Patents

Abstract

The present invention relates to a novel binding agent, in particular an antibody or a functional fragment of an antibody (such as a single domain antibody), comprising the amino acid sequence SEQ ID NO:1, comprising at least one substitution of an amino acid residue selected from the residue at position 57, the residue at position 58, the residue at position 60, the residue at position 61, the residue at position 62, the residue at position 98, the residue at position 101 , the residue at position 102, the residue at position 103, the residue at position 104, of SEQ ID NO:1, and any combination thereof. The present invention also relates to a nucleic acid molecule encoding one of these binding agents, a vector comprising at least one of these nucleic acid molecules and a host cell comprising at least one of these nucleic acid molecules or at least one of these vectors. The present invention further relates to the uses of these binding agents, nucleic acid molecules, vectors, and host cells. tricks

Description

LIAISON AGENT with enhanced affinity for the prevention and treatment of Sarbecovirus-related DISEASES

Field of invention

The present invention falls within the fields of binding agents, in particular antibodies, and diseases linked to sarbecoviruses, in particular infections by a sarbecovirus.

The present invention relates in particular to variants of VHH72 having an improved affinity, which can be used in the prevention and treatment of diseases linked to sarbecoviruses.

The invention relates in particular to a binding agent (such as an antibody, in particular a single-domain antibody) comprising the amino acid sequence SEQ ID NO: 1, comprising at least one substitution of an amino acid residue selected from the residue at position 57, the residue at position 58, the residue at position 60, the residue at position 61, the residue at position 62, the residue at position 98, the residue at position 101, the residue at position 102, the residue at position 103, the residue at position 104, of SEQ ID NO:1, and any combination thereof.

The present invention also relates to a nucleic acid molecule encoding one of these binding agents, a vector comprising at least one of these nucleic acid molecules and a host cell comprising at least one of these nucleic acid molecules or at least one of these vectors. The present invention further relates to the use of these binding agents, nucleic acid molecules, vectors, and host cells, for treating sarbecovirus-related disease. The present invention also relates to a composition or a kit comprising at least one of these binding agents and/or at least one of these nucleic acid molecules, as well as their uses. The present invention also relates to a method for diagnosing a disease linked to sarbecoviruses, as well as a method for monitoring, prognosis and/or evaluation of the efficacy of a treatment for a disease linked to sarbecoviruses, comprising the use of at least one binding agent and/or at least one nucleic acid molecule and/or an aforementioned composition or kit.

State of the art

Therapeutic antibodies have benefited from rapid development since the 1980s, they represent a major group among biological drugs. In 2019, more than 80 therapeutic antibodies received marketing authorization in Europe and/or the United States. Their indications cover a wide field of pathologies with a strong dominance in the field of oncology. Antibodies are also found for autoimmune diseases, infectious diseases, allergies, transplant rejection and cardiovascular diseases.

These diseases, in particular infectious diseases, continue to rage and decimate populations throughout the world, including emerging viruses capable of adaptation and rapid evolution, giving rise to new diseases, or even pathogenic strains with facilitating pathogenicity. Global epidemics caused by these viruses and other emerging pathogens can cause significant human harm and cost to affected communities and economies.

Coronavirus infections, including SARS-CoV2, are recent examples. The SARS-CoV2 virus (Severe acute respiratory syndrome Coronavirus 2) is at the root of the current COVID-19 pandemic, with devastating consequences in many countries. To date, the vaccination of populations seems to be the most effective approach for controlling the extent of the epidemic and effectively protecting the health of individuals. There are indeed few therapeutic means, the only molecules having demonstrated efficacy in infected patients being anti-inflammatories and monoclonal antibodies.

SARS-CoV2 anti-protein S antibodies

To complete the therapeutic arsenal against the SARS-CoV2 virus, passive immunotherapy, in this case the injection of monoclonal antibodies, constitutes an interesting and complementary approach to vaccination. Indeed, the acquisition by patients of sufficient titers of neutralizing antibodies seems to be inversely correlated with a progression of severe forms of COVID-19 (1). Several studies have also demonstrated that the early administration of monoclonal antibodies neutralizing the S protein of the virus makes it possible to prevent an evolution towards severe forms (2-4)

Since the appearance of the SARS-CoV2 virus, a large number of studies have made it possible to obtain monoclonal antibodies targeting the S protein and more particularly the RBD domain (5). Among the most advanced, nine antibodies have been studied in clinical phase (IMDB), alone or in combination. To date, the combination of indevimab and casivirimab developed by Regeneron (6) as well as the combination of bamlanivimab with etesevimab developed by the Lilly laboratory have received temporary marketing authorization from the FDA and the EMA. Even more recently, a monotherapy based on sotrovimab developed by Vir Biotechnology and GlaxoSmithKline received temporary authorization for use by the FDA.

However, since the emergence of the SARS-CoV2 virus at the end of 2019 in China, new strains have appeared in different countries. These include new mutations compared to the original virus (original SARS-CoV2 virus), in particular in the RBM (Receptor Binding Motif) region of the RBD (Receptor Binding Domain) responsible for the interaction with the ACE2 receptor which is the entry point for the virus into human cells. The strains of most concern are classified as variants of concern by the WHO or VoC (“variant of concern”). These include the B.1.1.7 strains identified in the United Kingdom, the B.1.351 strain identified in South Africa, the P.1 strain identified in Brazil, as well as the B.1.617 strains identified in India. Certain mutations in these strains may confer increased virus transmissibility but may also contribute to evasion of host immunity (7-10).

Several studies have shown that certain strains can be resistant to neutralization by monoclonal antibodies, as is the case in particular for bamlanivimab or etesevimab (11, 12). In particular, the E484K and L452R mutations are located within the bamlanivimab epitope and the K417N/T mutations within the etesevimab epitope and induce a reduction in neutralization by these monoclonal antibodies by a factor greater than to 1000 (12).

In addition, literature data show antigenic evolution in response to host immune pressure related to prior infection (13, 14) or vaccination. Several studies show an increase in the frequency of mutations on the E484 residue or in the loop comprising amino acids 443-450 (15). Unfortunately, many monoclonal antibodies target this immunodominant region frequently recognized by polyclonal antibodies (12).

The use of antibody cocktails targeting distinct epitopes seems to be a relevant strategy to maintain the efficacy of passive immunotherapies and reduce the susceptibility to mutations of emerging strains. Nevertheless, these data illustrate the need to develop broad-spectrum neutralizing antibodies, with activity against different sarbecoviruses related to SARS-CoV2 and not very sensitive to the immune escape of the different viral strains circulating in human populations. Also, it is essential to select monoclonal antibodies targeting epitopes conserved by their structural role or their function, but also distinct from the main immuno-dominant zones to be less susceptible to the evolution of the SARS-CoV2 virus.

Broad-spectrum neutralizing anti-SARS-CoV antibodies

Numerous examples in the literature describe SARS-CoV2 antibodies targeting the receptor binding motif (RBM), ie, the site of interaction of the RBD domain of protein S with ACE2. These monoclonal antibodies can demonstrate excellent affinities, very good efficacy in neutralizing the SARS-CoV2 virus, as well as a protective effect in vivo . On the other hand, several studies indicate that most of these antibodies have a relatively restricted recognition spectrum, with an absence of recognition of RBD domains from other members of the sarbecovirus subgenus, in particular SARS-CoV1 (16). Moreover, a certain number of these antibodies, such as bamlanivimab or etesevimab, are subject to significant losses of affinity for the emerging variants of SARS-CoV2, whereas these are often only point mutations.

The homology of the S protein of the SARS-CoV2 virus with the S proteins of other human coronaviruses is highly variable, with approximately 30% similarity with the other beta-coronavirus lineages (MERS-CoV, HCoVHKU1, and HCoV-OC43) and only 24% similarity with alpha-coronaviruses (HCoV-229E and HCoV-NL63) (17). The S proteins of other members of the sarbecovirus subgenus reveal a greater proximity, with nearly 77% sequence similarity with the S protein of SARS–CoV1. Sarbecoviruses or SARS-like beta-coronaviruses include coronaviruses linked to severe acute respiratory syndrome including SARS-CoV1 and SARS-CoV2. These viruses interact with the ACE2 receptor via the Spike S protein to enter target cells, as well as with host transmembrane serine proteases, such as TMPRSS2 or TMPRSS11D, for their activation (18, 19).

Rapidly after the start of the current pandemic, the proximity of the S protein sequences of the SARS-CoV1 and SARS-CoV2 strains led several teams to search for "cross-reactive" antibodies, in particular from samples obtained from patients. having contracted the SARS-CoV1 virus or by screening banks of monoclonal antibodies obtained following the 2003 epidemic (20-22).

Among the rare examples of “cross-reactive” and neutralizing antibodies described in the literature to date, we can find the S309 antibodies (21) (from which sotrovimab is derived), the ADG-2 antibody (23), the antibody S2H97 (16) as well as the camelid antibody VHH72 (22). Another antibody, CR3022 has SARS-CoV1/SARS-CoV2 “cross-reactivity” but is not able to neutralize SARS-CoV2 in vitro (24, 25).

The S309 antibody was obtained from memory B cells from PBMCs of a patient who was infected with the SARS-CoV1 virus in 2003 (21). This antibody has a very good affinity (0.2 nM) for RBD SARS-CoV2 and shows neutralization of the virus in vitro . This antibody has very recently demonstrated clinical efficacy in humans (under the name VIR-7831) and has just obtained emergency use authorization from the FDA.

The ADG-2 antibody is also derived from an antibody from memory B cells of a patient who was infected with the SARS-CoV1 virus in 2003. The engineered enhanced antibody has high affinity and a broad spectrum. This antibody showed a protective effect in mice, in a model of SARS-CoV1 and SARS-CoV2 infection (23).

Unlike the S309 and ADG-2 antibodies, the S2H97 antibody was obtained from memory B cells from PBMCs of a patient who was infected with the SARS-CoV2 virus in 2020 (16). S2H97 demonstrates excellent affinity for RBD SARS-CoV2 (30 pM) good affinity for RBD SARS-CoV1 (1.6 nM), but also recognizes all RBD domains of sarbecoviruses, including within the most divergent clade , grouping Asian strains that do not use ACE2 as a receptor. However, the neutralizing effect of the S2H97 antibody for SARS-CoV2 turns out to be less efficient than the antibodies showing the most effective neutralization of the virus (e.g., S2E12, S2D106) but having less conserved epitopes in the RBM (16) . These data seem to illustrate a necessary compromise between therapeutic efficacy and "cross-reactivity", i.e. the spectrum of strains that can be recognized.

In addition to conventional monoclonal antibodies, there are antibodies from camelids (VHH). These VHHs contain a single variable domain consisting of a single heavy chain (VH) unlike conventional antibodies which have a heavy chain and a light chain (VH VL). These molecules are very stable and can have excellent affinities for their antigen. Thanks to the reduced size of their variable region, these molecules can target epitopes that are difficult to access by conventional antibodies.

VHH-72: a broad-spectrum VHH neutralizing SARS-CoV2 despite modest affinity

VHH-72 was obtained by immunizing a llama with the S proteins of SARS-CoV-1 and MERS-CoV followed by screening for specific VHHs using a phage display approach. VHH-72 shows good affinity for the RBD protein of SARS-CoV-1 (1.15 nM), an absence of recognition of RBD MERS-CoV but a modest affinity for RBD SARS-CoV-2 (39-63 nM ), characterized by rapid dissociation (22). In addition, once expressed in fusion with an Fc fragment of a human antibody, VHH-72 has a very modest capacity to neutralize SARS-CoV2 pseudoviruses (IC50 of approximately 0.2 μg/ml).

It is therefore necessary to increase the efficiency and affinity of VHH-72 in order to be able to neutralize the SARS-CoV2 virus.

Work has allowed the identification of several point mutants of VHH72 (in particular the VHH72-S56A mutant) with a slightly improved affinity for RBD SARS-CoV-2 compared to the parental molecule (Kd ≈ 8.3nM) (26) . This enhancement also results in increased binding of VHH to the trimeric Spike protein of SARS-CoV2 and increased neutralization of SARS-CoV2 pseudoviruses. However, the affinity and partial cross-reactivity of this VHH72 mutant remain insufficient to effectively target and neutralize the largest possible number of SARS-CoV2 variants.

In view of the persistence of the COVID-19 pandemic and the high frequency of appearance of new variants, as well as the risk of the emergence of new sarbecoviruses, it is essential to develop new antibodies with high affinity for different variants of SARS-CoV2, combined with a strong neutralizing power.

The present invention makes it possible to meet this need.

In the context of the present invention, the inventors have developed innovative binding agents, in particular innovative single-domain antibodies, capable of specifically recognizing and neutralizing the different variants of SARS-CoV2.

These optimized binding agents were notably obtained by an innovative approach of antibody molecular engineering and affinity maturation from the single-domain antibody VHH72, combining Deep-Mutational Scanning and Yeast Surface techniques. Display. This approach allowed the identification of amino acid positions and mutations likely to confer an increase in affinity to the antibody. Assembly in combinatorial libraries combined with high-throughput sequencing identified a large number of enriched variants with stronger antigen binding than the parental antibody VHH72.

The inventors have in particular shown that, surprisingly, the binding agents thus developed have very good affinities for the various RBD domains of the variants of the SARS-CoV2 virus, unlike binding agents (in particular antibodies with a domain unique) described in the prior art. Remarkably, the data also show that these optimized binding agents are able to prevent the binding between the ACE2 receptor and the RBD domain, more effectively than the binding agents (including single domain antibodies) described in l prior art. These data thus reveal the therapeutic potential of these binding agents to treat diseases linked to sarbecoviruses, in particular coronaviruses.

The data also show that these binding agents are tools for the detection of sarbecoviruses.

The present invention therefore provides both powerful and broad-spectrum treatment methods for diseases linked to sarbecoviruses, as well as methods for the effective and reliable diagnosis of these diseases, methods for screening molecules but also tools for research in the field of diseases related to sarbecoviruses.

The present invention relates in particular to variants of VHH72 having an improved affinity, which can be used in the prevention and treatment of diseases linked to sarbecoviruses.

The present invention relates in particular to a binding agent (such as an antibody or a functional fragment of an antibody, in particular a single-domain antibody) comprising, or consisting essentially of, or consisting of, the amino acid sequence SEQ ID NO:1, comprising at least one substitution of an amino acid residue selected from the group consisting of:

1. The residue at position 57 of SEQ ID NO:1, substituted by an amino acid selected from G, N, and M (preferably by an amino acid G);
2. The residue at position 58 of SEQ ID NO:1, substituted with an amino acid selected from: S, W, R, and H (preferably with an S amino acid);
3. The residue at position 60 of SEQ ID NO: 1, substituted by an amino acid preferably selected from: V, L, I, T, M, H, Q, Y, and F (preferably by an amino acid selected from V, L, and I);
4. The residue at position 61 of SEQ ID NO:1, substituted with an amino acid selected from: S, R, P, Q, I, M, and F (preferably with an amino acid selected from S, and R);
5. The residue at position 62 of SEQ ID NO: 1, substituted by an amino acid selected from any amino acid except D, C, and Q (in particular substituted by an amino acid preferably selected from: F , L, G, P, I, M, W, Y, A, G, S, T, N, E, H, K, and R; more preferably by an amino acid selected from F, and L);
6. The residue at position 98 of SEQ ID NO: 1, substituted by an amino acid preferably selected from any amino acid except A, P, and D (in particular substituted by an amino acid preferably selected from : V, W, R, Q, I, E, L, F, V, M, Y, G, C, S, T, N, H, and K; more preferably by an amino acid selected from V, W , R, and Q; more preferably by an amino acid V);
7. The residue at position 101 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, F, Y, C, E, and W;
8. The residue at position 102 of SEQ ID NO:1, substituted by an amino acid preferably selected from: E, Y, and N (more preferably by an amino acid selected from E and Y);
9. The residue at position 103 of SEQ ID NO:1, preferably substituted with an amino acid V;
10. The residue at position 104 of SEQ ID NO:1, substituted by an amino acid selected from: W, and Y (preferably by an amino acid W); And
11. Any combination of these.

The present invention further relates to a binding agent (such as an antibody or a functional fragment of an antibody, in particular a single domain antibody) comprising, or consisting essentially of, or consisting of the amino acid sequence SEQ ID NO :1, comprising a combination of at least two amino acid residue substitutions, the substituted amino acid residue being selected from the group consisting of:

1. The residue at position 57 of SEQ ID NO:1, substituted by an amino acid preferably selected from: G, N and M (preferably by an amino acid G);
2. The residue at position 58 of SEQ ID NO:1, substituted by an amino acid preferably selected from: S, W, R, and H (preferably by an S amino acid);
3. The residue at position 60 of SEQ ID NO: 1, substituted by an amino acid preferably selected from: V, L, I, T, M, H, Q, Y, and F (preferably by an amino acid selected from V, L, and I);
4. The residue at position 61 of SEQ ID NO: 1, substituted by an amino acid preferably selected from: S, R, P, Q, I, M, W, and F (preferably by an amino acid selected from S, and R);
5. The residue at position 62 of SEQ ID NO: 1, substituted by an amino acid preferably selected from any amino acid except D, C, and Q (in particular substituted by an amino acid preferably selected from : F, L, V, P, I, M, W, Y, A, G, S, T, N, E, H, K, and R; more preferably by an amino acid selected from F, and L) ;
6. The residue at position 98 of SEQ ID NO: 1, substituted by an amino acid preferably selected from any amino acid except A, P, and D (in particular substituted by an amino acid preferably selected from : V, W, R, Q, I, E, L, F, V, M, Y, G, C, S, T, N, H, and K; more preferably by an amino acid selected from V, W , R, and Q; more preferably by an amino acid V);
7. The residue at position 101 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, F, Y, C, E, and W;
8. The residue at position 102 of SEQ ID NO:1, substituted by an amino acid preferably selected from: E, Y, and N (more preferably by an amino acid selected from E and Y);
9. The residue at position 103 of SEQ ID NO:1, preferably substituted with an amino acid V; And
10. The residue at position 104 of SEQ ID NO:1, substituted by an amino acid preferably selected from: W, and Y (preferably by an amino acid W); And
11. Any combination of these.

The present invention also relates to a binding agent as defined above, which has been humanized (that is to say whose sequence has been humanized).

In addition to the substitutions or combinations of substitutions listed above, the humanized binding agent according to the invention may in particular comprise at least one additional substitution of an amino acid residue selected from the group consisting of:

• The residue at position 1 of SEQ ID NO:1, preferably substituted with an amino acid E;
• The residue at position 5 of SEQ ID NO:1, preferably substituted with an amino acid selected from V, and L;
• The residue at position 14 of SEQ ID NO:1, preferably substituted by an amino acid P;
• The residue at position 27 of SEQ ID NO:1, preferably substituted by an amino acid F;
• The residue at position 31 of SEQ ID NO:1, preferably substituted with an S amino acid;
• The residue at position 35 of SEQ ID NO:1, preferably substituted with an S amino acid;
• The residue at position 37 of SEQ ID NO:1, preferably substituted by an amino acid V;
• The residue at position 44 of SEQ ID NO:1, preferably substituted with an amino acid G;
• The residue at position 45 of SEQ ID NO:1, preferably substituted with an L-amino acid;
• The residue at position 47 of SEQ ID NO:1, preferably substituted with an amino acid W;
• The residue at position 49 of SEQ ID NO:1, preferably substituted with an S amino acid;
• The residue at position 50 of SEQ ID NO:1, preferably substituted with an amino acid A;
• The residue at position 53 of SEQ ID NO:1, preferably substituted with an amino acid G;
• The residue at position 61 of SEQ ID NO:1, preferably substituted with an amino acid A;
• The residue at position 75 of SEQ ID NO:1, preferably substituted with an S amino acid;
• The residue at position 79 of SEQ ID NO:1, preferably substituted with an L-amino acid;
• The residue at position 87 of SEQ ID NO:1, preferably substituted with an amino acid R;
• The residue at position 88 of SEQ ID NO:1, preferably substituted with an amino acid A;
• The residue at position 89 of SEQ ID NO:1, preferably substituted with an amino acid E;
• The residue at position 120 of SEQ ID NO:1, preferably substituted by an amino acid Q; And
• Any combination of these.

The present invention also relates to a nucleic acid molecule encoding one of these binding agents, a vector comprising at least one of these nucleic acid molecules and a host cell comprising at least one of these nucleic acid molecules and/or at least one of these vectors. The present invention further relates to the use of these binding agents, nucleic acid molecules, vectors, and host cells, for treating sarbecovirus-related disease. The present invention also relates to a composition or a kit comprising at least one of these binding agents and/or at least one of these nucleic acid molecules, as well as their uses. The present invention also relates to a method for diagnosing a disease linked to sarbecoviruses, as well as a method for monitoring, prognosis and/or evaluation of the efficacy of a treatment for a disease linked to sarbecoviruses, comprising the use of at least one binding agent and/or at least one nucleic acid molecule and/or a composition and/or a kit mentioned above.

The present invention relates to a binding agent comprising, or consisting essentially of, or consisting of the amino acid sequence SEQ ID NO:1, comprising at least one substitution of an amino acid residue selected from the group consisting of :

1. The residue at position 57 of SEQ ID NO:1, substituted with an amino acid selected from G, N, and M; preferably by an amino acid G;
2. The residue at position 58 of SEQ ID NO:1, substituted with an amino acid selected from: S, W, R, and H; preferably by an S-amino acid;
3. The residue at position 60 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, L, I, T, M, H, Q, Y, and F; preferably by an amino acid selected from V, L, and I;
4. The residue at position 61 of SEQ ID NO:1, substituted with an amino acid selected from: S, R, P, Q, I, M, and F; preferably by an amino acid selected from S, and R;
5. The residue at position 62 of SEQ ID NO:1, substituted with an amino acid selected from any amino acid except D, C, and Q; in particular substituted with an amino acid preferably selected from: F, L, V, P, I, M, W, Y, A, G, S, T, N, E, H, K, and R; more preferably by an amino acid selected from F, and L;
6. The residue at position 98 of SEQ ID NO:1, substituted with an amino acid preferably selected from any amino acid except A, P, and D; in particular substituted with an amino acid preferably selected from: V, W, R, Q, I, E, L, F, V, M, Y, G, C, S, T, N, H, and K; more preferably by an amino acid selected from V, W, R, and Q; more preferably by an amino acid V;
7. The residue at position 101 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, F, Y, C, E, and W;
8. The residue at position 102 of SEQ ID NO:1, substituted by an amino acid preferably selected from: E, Y, and N; more preferably by an amino acid selected from E and Y;
9. The residue at position 103 of SEQ ID NO:1, preferably substituted with an amino acid V;
10. The residue at position 104 of SEQ ID NO:1, substituted with an amino acid selected from: W, and Y; preferably by an amino acid W; And
11. Any combination of these.

According to one embodiment, the binding agent comprises at least one substitution of an amino acid residue chosen from: the substitution of the residue in position 57 of SEQ ID NO: 1 by an amino acid selected from G, N , and M (preferably by an amino acid G); the substitution of the residue at position 103 of SEQ ID NO:1, preferably by an amino acid V; the substitution of the residue at position 104 of SEQ ID NO:1 by an amino acid selected from: W, and Y (preferably by an amino acid W); and any combination thereof.

The present invention further relates to a linker comprising, or consisting essentially of, or consisting of the amino acid sequence SEQ ID NO:1, comprising a combination of at least two amino acid residue substitutions , the substituted amino acid residue being selected from the group consisting of:

1. The residue at position 57 of SEQ ID NO:1, substituted by an amino acid preferably selected from: G, N and M; preferably by an amino acid G;
2. The residue at position 58 of SEQ ID NO:1, substituted by an amino acid preferably selected from: S, W, R, and H; preferably by an S-amino acid;
3. The residue at position 60 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, L, I, T, M, H, Q, Y, and F; preferably by an amino acid selected from V, L, and I;
4. The residue at position 61 of SEQ ID NO:1, substituted with an amino acid preferably selected from: S, R, P, Q, I, M, W, and F; preferably by an amino acid selected from S, and R;
5. The residue at position 62 of SEQ ID NO:1, substituted with an amino acid preferably selected from any amino acid except D, C, and Q; in particular substituted with an amino acid preferably selected from: F, L, V, P, I, M, W, Y, A, G, S, T, N, E, H, K, and R; more preferably by an amino acid selected from F, and L;
6. The residue at position 98 of SEQ ID NO:1, substituted with an amino acid preferably selected from any amino acid except A, P, and D; in particular substituted with an amino acid preferably selected from: V, W, R, Q, I, E, L, F, V, M, Y, G, C, S, T, N, H, and K; more preferably by an amino acid selected from V, W, R, and Q; more preferably by an amino acid V;
7. The residue at position 101 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, F, Y, C, E, and W;
8. The residue at position 102 of SEQ ID NO:1, substituted by an amino acid preferably selected from: E, Y, and N; more preferably by an amino acid selected from E and Y;
9. The residue at position 103 of SEQ ID NO:1, preferably substituted with an amino acid V; And
10. The residue at position 104 of SEQ ID NO:1, substituted by an amino acid preferably selected from: W, and Y; preferably by an amino acid W; And
11. Any combination of these.

Advantageously, the linker comprises at least one combination of amino acid residue substitutions chosen from:

1. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1, preferably by an amino acid selected from: G, N and M (preferably by an amino acid G), and of the substitution of the residue at position 103 of SEQ ID NO: 1 preferably by an amino acid V;
2. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1 preferably by an amino acid selected from: G, N and M (preferably by an amino acid G), and of the substitution of the residue at position 104 of SEQ ID NO:1 preferably by an amino acid selected from: W, and Y (preferably by an amino acid W); And
3. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1 preferably by an amino acid selected from: G, N and M (preferably by an amino acid G), and of the substitution of the residue at position 103 of SEQ ID NO: 1 preferably with an amino acid V, and the substitution of the residue at position 104 of SEQ ID NO: 1 preferably with an amino acid selected from: W, and Y (preferably with an amine W).

The binding agent may in particular comprise, or consist essentially of, or consist of, an amino acid sequence chosen from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

According to one embodiment, the binding agent comprises, consists essentially of, or consists of, at least one binding agent chosen from: an antibody, a functional antibody derivative, and a functional antibody fragment; the antibody functional fragment preferably being selected from: a single domain antibody, a nanobody, and a VHH (single variable domain heavy chain), and any combination thereof.

Advantageously, the linker is humanized, the humanized linker preferably comprising an amino acid sequence having at least 85% identity with the sequence SEQ ID NO: 7, preferably having at least 86% d identity, preferably having at least 87% identity, preferably having at least 88% identity, preferably having at least 89% identity, preferably having at least 90% identity, preferably having at least 91% identity, preferably having at least 92% identity, preferably having at least 93% identity, preferably having at least 94% identity, preferably having at least 95% identity, preferably having at least 96% identity, preferably having at least 97% identity, preferably having at least 98% identity, preferably having at least 99% identity, with the sequence SEQ ID NO: 7.

According to one embodiment, the binding agent further comprises an antibody heavy chain fragment, preferably an antibody Fc fragment (crystallizable antibody fragment), the antibody heavy chain fragment and/or the Fc fragment preferably being human.

Advantageously, the liaison agent is able to recognize:

• an RBD domain (Receptor Binding Domain) of a Spike protein of sarbecovirus, the sarbecovirus being preferably chosen from the SARS-CoV1 virus, the variants of the SARS-CoV1 virus, the SARS-CoV2 virus, and the variants of the SARS- CoV2; the binding agent preferably being capable of recognizing an RBD domain of a SARS-CoV1 virus Spike protein and an RBD domain of a SARS-CoV2 virus Spike protein; and or
• an RBD domain of a Spike protein of the original SARS-CoV2 virus, and an RBD domain of a Spike protein of at least one variant of the original SARS-CoV2 virus, the variant preferably being chosen from a variant Alpha, Beta Variant, Gamma Variant, Delta Variant, Epsilon Variant, Zeta Variant, Eta Variant, Teta Variant, Iota Variant, Kappa Variant, Lambda Variant, and any combination thereof; preferably selected from an Alpha variant, a Beta variant, a Gamma variant, a Delta variant, and any combination thereof.

Preferably, the dissociation constant K _{d (substituted VHH72)} of the binding agent, measured for an RBD domain of a Spike protein of SARS-CoV2 virus is lower than the dissociation constant K _{d (unsubstituted VHH72)} of the original VHH72 single domain antibody, comprising the amino acid sequence SEQ ID NO:1, measured for an RBD domain of a Spike protein of SARS-CoV2 virus.

The present invention also relates to a nucleic acid molecule encoding a binding agent according to the invention, comprising, or consisting essentially of, or consisting of, preferably a nucleic acid sequence having at least 85% identity with a sequence chosen from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO 13, preferably having at least 86% identity, preferably having at least 87% identity, preferably having at least 88% identity, preferably having at least 89% identity, preferably having at least 90% identity, preferably having at least 91% identity, preferably having at least 92% identity, preferably having at least 93% identity, preferably having at least 94% identity, preferably having at least 95% identity, preferably having at least 96 % identity, preferably having at least 97% identity, preferably having at least 98% identity, preferably having at least 99% identity, with a sequence chosen from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, more preferably a sequence chosen from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO 12, and SEQ ID NO: 13.

The present invention also relates to a vector comprising the nucleic acid molecule according to the invention, as well as a host cell comprising the nucleic acid molecule according to the invention and/or the vector according to the invention.

The present invention further relates to a binding agent according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, or a host cell according to the invention, for its use in the prevention or the treatment of a disease, the disease being preferably chosen from diseases linked to sarbecoviruses, the disease linked to sarbecoviruses being preferably chosen from infections by a sarbecovirus, the sarbecovirus being preferably chosen from the SARS-CoV1 virus of origin, origin SARS-CoV1 virus variants, origin SARS-CoV2 virus, and origin SARS-CoV2 virus variants.

The present invention also relates to the in vitro use of at least one binding agent according to the invention, for:

1. the diagnosis of a sarbecovirus-related disease in a subject suspected of suffering from a sarbecovirus-related disease;
2. follow-up in a subject suffering from a disease linked to sarbecoviruses;
3. the stratification of a subject suffering from a disease linked to sarbecoviruses;
4. the evaluation of the effectiveness of a treatment (in particular curative) administered to a subject suffering from a disease linked to sarbecoviruses;
5. detect the presence or absence of at least one sarbecovirus in a sample;
6. determine the amount of at least one sarbecovirus in a sample;
7. screening compounds/molecules capable of detecting and/or recognizing at least one sarbecovirus; preferably screening compounds/molecules capable of neutralizing at least one sarbecovirus; Or
8. any combination of i. to vii. ;

the disease linked to sarbecoviruses being preferably chosen from infections by a sarbecovirus.

Definitions

In this document, the term " coronavirus " refers to a group of viruses belonging to the family Coronaviridae. Generally speaking, coronaviruses are enveloped viruses with a helically symmetrical capsid. They have a single-stranded positive RNA genome and are able to infect cells of birds and mammals. The morphology of the virions is typical, with a halo of proteinaceous protrusions ("Spike") which gave them their name. Among the four genera of coronaviruses (Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus), the Betacoronavirus genus (β-CoVs or Beta-CoVs) includes viruses infecting animals and/or humans. Coronaviruses generally comprise four structural proteins, including spike (S or S-protein), envelope (E), membrane (M), and nucleocapsid (N) proteins.

The term “ Betacoronavirus ” or “ Betacoronavirus ” refers to any virus belonging to the genus Betacoronavirus (β-CoVs or Beta-CoVs) within the family Coronaviridae, in particular any betacoronavirus belonging to one of the four lineages designated as A, B , C and D. It denotes a betacoronavirus infecting animals (preferably a mammal) and/or humans. In particular, this designation includes betacoronaviruses infecting human organisms chosen from the group consisting of OC43, HKU1, SARS-CoV-1, SARS-CoV-2 and MERS-CoV. The Betacoronavirus genus is subdivided into four lineages designated A, B, C, and D. The A lineage (also referred to as the Embecovirus subgenus) includes the HCoV-OC43 and HCoV-HKU1 viruses, which are capable of infecting various species. The B lineage (also referred to as the Sarbecovirus subgenus) includes the SARS-CoV-1, SARS-CoV-2, and Bat SL-CoV-WIV1 viruses. Lineage C (also called subgenus Merbecovirus) includes Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4), Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5) and MERS-CoV, capable of infect camels and humans in particular. Lineage D (also called subgenus Nobecovirus) includes the Rousettus bat coronavirus HKU9 (BtCoV-HKU9).

In humans, coronavirus infections can cause respiratory pathologies associated with symptoms similar to the common cold, bronchiolitis and more serious illnesses such as severe acute respiratory syndrome caused by SARS-CoV-1, which generated a outbreak in 2003, and Middle East respiratory syndrome caused by MERS-CoV, which generated an outbreak in 2012. SARS-CoV-2 (and its variants) is the beta-coronavirus that causes the 2019-2021 (ongoing) coronavirus outbreak, causing the form of pneumonia known as coronavirus disease 2019 or COVID-19. According to the World Health Organization (WHO), as of March 28, 2021, 126,372,442 cases of COVID-19 have been confirmed and 2,769,696 patients have died worldwide. This outbreak was declared a public health emergency of international concern by WHO on January 30, 2020.

By “ sarbecovirus ”, we mean a subgenus of Betacoronavirus grouping together the coronaviruses linked to severe acute respiratory syndrome, including SARS-CoV1 and SARS-CoV2. Viruses of the Sarbecovirus subgenus were previously known as group 2b5 coronaviruses. Sarbecoviruses use ACE2 as a receptor. The vast majority of sarbecoviruses infect horseshoe bats but some have adapted to other mammals such as civets, badgers or humans for SARS-CoV-1, pangolin and humans for the SARS cluster -CoV-2, but also different carnivores (cat, tiger, mink and dog). The Sarbecovirus subgenus is characterized by the presence of a single papain-like proteinase (PLpro) in the ORF18 open reading frame, instead of two in the Embecovirus subgenus and the Alphacoronavirus genus.

" SARS-CoV1 " or "SARS-CoV1" or "SARS-COV1" or "SARS-CoV-1" or "SARS-CoV1 virus" means the coronavirus responsible for the epidemic of severe acute respiratory syndrome (SARS) which raged from 2002 to 2004. It is a strain of the SARSr-CoV coronavirus species (for SARS-related coronavirus, or “coronavirus linked to SARS”). This infectious agent appeared in November 2002 in the province of Guangdong, China. Between November 1, 2002 and August 31, 2003, the virus is said to have infected 8,096 people in some thirty countries, causing 774 deaths, mainly in China, Hong Kong, Taiwan, and Southeast Asia. Coming from a species of coronavirus well established in bats (Rhinolophes), SARS-CoV-1 infects civet cats, badgers and humans.

" SARS-CoV2 " or "SARS-CoV2" or "SARS-COV2" or "SARS-CoV-2" or "SARS-CoV2 virus" means coronavirus 2 which causes severe acute respiratory syndrome. SARS-CoV2 belongs to the Coronavirus species, the Sarbecovirus subgenus, the Betacoronavirus genus and the Coronaviridae family. The term "SARS-CoV2" here refers to the SARS-CoV2 virus as it was originally identified, as well as all variants of SARS-CoV2. As used herein, the SARS-CoV2 virus as originally identified (referred to as the original SARS-CoV2 virus) refers to the SARS-CoV2 virus first identified in Wuhan, China, and sequenced in early 2020 by a team from Fudan University in Shanghai (Zhou, P. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature Mar;579(7798):270-273 .doi:10.1038/s41586-020-2012-7.(2020)). As used in this document, SARS-CoV2 virus variants refer to viruses related to this first identified viral strain, which appeared subsequently, and in particular the following SARS-CoV2 variants:

1. Alpha variants (including strains of lineage B.1.1.7);
2. Beta variants (including strains of lines B.1.351, B.1.351.2, B.1.351.3);
3. Delta variants (including strains of lines B.1.617.2, AY.1, AY.2, AY.3);
4. Gamma variants (including strains from lineages P.1, P.1.1, P.1.2);
5. Epsilon variants;
6. Eta variants (including strains of the B.1.525 lineage);
7. Iota variants (including strains of the B.1.526 lineage);
8. Kappa variants (including strains of the B.1.617.1 lineage);
9. Zeta variants;
10. Teta variants;
11. Iota variants;
12. Lambda variants;
13. Variants of the B.1.617.3 line;
14. "Wuhan-like" strains;
15. The hCoV-19/France/ARA-104350/2020 strain (GISAID ID: EPI_ISL_683350) of the B.1 line (this strain has at least the D614G mutation in its spike protein);
16. The British variant strain hCoV-19/France/ARA-SC2118/2020 (GISAID: EPI_ISL_900512) of lineage B.1.1.7;
17. The South African strain (501Y.V2. HV001) of the B.1.351 line;
18. Brazilian variant strains of lineages B.1.1.28 and P.1;
19. N501Y variants;
20. E484K variants;
21. The K417N variants;
22. The K417T variants;
23. L452R variants;
24. T478K variants
25. R346K variants;
26. The K417T variants;
27. Any other variant of SARS-CoV2 yet to be identified;
28. Any combination of i. to xxvii.

By “ Spike ” or “S protein” or “S” is meant here a structural protein of a coronavirus. The spike protein is usually composed of two subunits, S1 and S2, which are derived from a single protein by proteolytic cleavage. The S1 subunit contains a receptor binding domain (RBD) that recognizes and binds to the host receptor, angiotensin converting enzyme 2 (ACE2), while the S2 subunit fuses the viral cell membrane by forming a six-helix bundle via the two-heptad repeat domain. The spike protein therefore plays a key role in receptor recognition, the process of cell membrane fusion and entry into the host cell. With a size of 180-200 kDa, the S protein comprises an extracellular N-terminus, a transmembrane (TM) domain anchored in the viral membrane, and a short intracellular C-terminal segment. The S-protein is preferably post-translationally modified, preferably by glycosylation (glycosylated S-protein). Protein S trimers visually form a characteristic bulb-and-crown-shaped halo surrounding the virus particle. The Spike protein of SARS-COV2 has been well characterized. The total length of the S protein of SARS-CoV2 is 1273 amino acids and consists of a signal peptide (amino acids 1-13) located at the N-terminus, of the S1 subunit (amino acids 14 -685) and the S2 subunit (amino acids 686-1273); the latter two regions are responsible for receptor binding and membrane fusion, respectively. Within the S1 subunit are an N-terminal domain (amino acids 14-305) and a receptor binding domain (RBD, amino acids 319-541); fusion peptide (FP) (amino acids 788-806), heptapeptide repeat 1 (HR1) (amino acids 912-984), HR2 (amino acids 1163-1213), TM domain (amino acids 1213-1237) , and the cytoplasmic domain (amino acids 1237-1273) constitute the S2 subunit.

By " Spike protein RBD domain " or "RBD domain" we refer herein to a receptor binding domain (RBD) (i.e. a domain that recognizes and binds to the receptor of the cell host) of the S protein of any virus, preferably any coronavirus, preferably any betacoronavirus, more preferably any SARS-CoV virus.

The terms " variant ", or " mutant ", " derivative " can be used interchangeably to generally designate a component or a species (protein, antibody, protein fragment, polypeptide, polynucleotide, oligonucleotide, nucleoside, nucleotide, vector, virus , etc.) exhibiting one or more modifications from a reference component (e.g. the wild type component as found in nature as originally identified, i.e. i.e. the "original" matching component called the original component). A nucleotide or nucleoside variant may have a modified base and/or a modified sugar and/or a modified bond. With respect to polypeptide, polynucleotide and antibody variants, any modification may be contemplated, including substitution, insertion, deletion, and any combination thereof, of one or more nucleotide residues /amino acids. With respect to viruses, any modification of the genome and/or proteome may be considered, including the substitution, insertion, deletion, and any combination thereof, of one or more nucleotide residues/d 'amino acids. The variant can be of natural or artificial origin (for example, mutated and/or manufactured). In the present document, the variants are preferably of natural origin. Examples of such naturally occurring variants in the context of viruses include naturally occurring coronavirus variants (including sarbecovirus variants, such as SARS-CoV virus variants, particularly SARS-CoV2 variants, such as SARS-CoV variants, CoV2 listed above including Alpha variants, Beta variants, Gamma variants, Delta variants, Epsilon variants, Zeta variants, Eta variants, Teta variants, Iota variant, Kappa variants, Lambda, the "Wuhan-like" strains, the hCoV-19/France/ARA-104350/2020 strain (GISAID ID: EPI_ISL_683350) of lineage B.1, the British variant strain hCoV-19/France/ARA-SC2118/ 2020 (GISAID: EPI_ISL_900512) of lineage B.1.1.7, the South African strain (501Y.V2.HV001) of lineage B.1.351, the Brazilian variant strains of lineage B.1 .1.28 and P.1, SARS-CoV2 N501Y, SARS-CoV2 E484K, SARS-CoV2 K417N, SARS-CoV2 K417T, any combination of these variants, and any other variants of SARS-CoV2 yet to be identified).

When several mutations are envisaged, they may concern consecutive residues and/or non-consecutive residues. Preferred are variants (e.g., respectively, protein variants, antibody variants, virus variants) which retain at least 80% degree of sequence identity with the reference component (e.g., respectively, the corresponding "original" protein, the corresponding "original" protein fragment, the corresponding "original" virus). For example, "at least 80% identity" means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, an identity of at least 80% also encompasses an identity of 100%.

By “ identity ” or “ sequence identity ”, is meant an exact correspondence of sequence between two polypeptides or amino acids, or between two molecules of nucleic acids or oligonucleotides. The percentages of identity to which reference is made in the context of the presentation of the present invention are determined after optimal global alignment of the sequences to be compared, which can therefore comprise one or more additions, deletions, truncations and/or substitutions. This percentage of identity can be calculated by any sequence analysis method well known to those skilled in the art. The percentage identity is determined after overall alignment of the sequences to be compared taken in their entirety, over their entire length. Besides manually, it is possible to determine the global alignment of sequences by means of the algorithm of Needleman and Wunsch (1970).

In particular, for the nucleotide sequences, the comparison of the sequences can be carried out using any software well known to those skilled in the art, such as for example the Needle software. The parameters used may in particular be the following: “ Gap Open ” equal to 10.0, “ Gap Extend ” equal to 0.5 and the EDNAFULL matrix (EMBOSS version of NCBI NUC4.4).

For the amino acid sequences, the comparison of the sequences can be carried out using any software well known to those skilled in the art, such as for example the Needle software. The parameters used may in particular be the following: “ Gap Open ” equal to 10.0, “ Gap Extend ” equal to 0.5 and the matrix BLOSUM62.

For illustrative purposes, "at least 80% sequence identity", as used herein, notably represents 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 % sequence identity .

The terms " binding agent " denote a molecule capable of binding to another molecule (known as the "target molecule"), said binding preferably being a specific binding. The binding agent recognizes a defined binding site, domain, region, pocket and/or epitope of the target molecule. The binding agent can be of any kind or of any type and does not depend on its origin. The binding agent can be chemically synthesized, naturally occurring, recombinantly produced (and optionally purified), or synthetically designed and produced. The binding agent can therefore be a small molecule, a chemical, a peptide, a polypeptide, an antibody, or any derivative thereof, such as a peptidomimetic, an antibody mimetic, a functionally active fragment, a derivative chemical, among others. Advantageously, the binding agent can be chosen from antibodies, derivatives thereof (preferably functional derivatives), and functional antibody fragments. The target molecule can in particular be a pathogenic agent (such as a virus) or a fragment thereof (for example a nucleic acid, a protein, a polypeptide, a peptide, an epitope, etc.). For example, when the target molecule is a sarbecovirus (such as SARS-CoV1 and SARS-CoV2), the target molecule may be the sarbecovirus or any part thereof (in particular the Spike protein, in particular the RBD domain).

By “ antibody ” is meant a protein or a glycoprotein belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are used interchangeably. In mammals, antibodies are mostly secreted by cells derived from B lymphocytes: plasma cells. In particular, they are used by the immune system to detect and neutralize foreign bodies (in particular pathogens, such as bacteria and viruses), in a specific way. Antibodies also include autoantibodies (produced for example in the case of an autoimmune disease). The antibody recognizes a unique part of the foreign target, its antigen.

Antibodies have a structure made up of 4 polypeptide chains (150,000 amu or dalton): two identical heavy chains (H for “heavy”, of 50,000 amu each) and two identical light chains (L for “light”, of 25,000 uma each) which are interconnected by a variable number of disulphide bridges ensuring the cohesion of the molecule. These chains form a Y structure (each light chain constitutes half of an arm of the Y) and consist of immunoglobulin domains which can comprise approximately 110 amino acids. Each light chain consists of a constant domain (named CL) and a variable domain (named VL); the heavy chains are composed of a variable domain (called VH) and, depending on the isotype, of three or four constant domains respectively called CH1, CH2, CH3, (CH4). For a given antibody, the two heavy chains are identical, likewise for the two light chains. The constant domains are characterized by an amino acid sequence very close from one antibody to another, characteristic of the species and of the isotype. The constant domains are generally not involved in the recognition of the antigen, but intervene in the activation of the complement system, as well as in the elimination of the immune complexes (antibodies bound to its antigen) by the immune cells possessing the receptors to constant fragments (RFc). An antibody has four variable domains located at the ends of the two "arms". The association between a variable domain carried by a heavy chain (VH) and the adjacent variable domain carried by a light chain (VL) constitutes the recognition site (or paratope) of the antigen. Thus, an immunoglobulin molecule has two antigen-binding sites, one at the end of each arm. These two sites are identical (but intended for different epitopes), hence the possibility of binding two molecules of antigen by antibody. The antigen recognition site (or paratope) comprises 6 regions called complementarity-determining regions (or CDRs). Each VH has 3 CDRs and each VL also has 3.

The specific enzymatic cleavage makes it possible to isolate different fragments:

• the Fc fragment (Crystallizable fragment). It supports the biological properties of immunoglobulin, in particular its ability to be recognized by immunity effectors or to activate complement. It is made up of constant fragments of the heavy chains (CH2) beyond the hinge region. It generally does not recognize the antigen;
• the Fv fragment (Fragment variable). It is the smallest fragment keeping the properties of the antibody possessed by the immunoglobulin. It consists solely of the VL and VH variable regions, so it binds the antigen with the same affinity as the complete antibody and is monovalent;
• the Fab fragment (antigen-binding fragment). This fragment has the same affinity for the antigen as the whole antibody. The Fab fragment is made up of the entire light chain (VL+CL) and part of the heavy chain (VH+CH1). It is monovalent;
• the F(ab')2 fragment. It corresponds to the association of two Fab fragments linked by a small part of the constant parts of the heavy chains, the hinge region. It has the same affinity as the antibody for the antigen and is divalent.

As used herein, the term antibody encompasses native antibodies and their functional derivatives, (e.g., mutated and/or modified antibodies as well as mimetic antibodies), provided that such derivative is capable of binding specifically to an antigen (we speak of “ functional antibody derivatives ”).

Antibodies can be produced by various systems known to those skilled in the art. Antibody production systems include, for example, animal systems (such as rodents, camelids, etc.), hybridoma systems, mammalian cell systems (including in particular CHO cell lines (hamster ovary cells Chinese; e.g. CHO-K1, CHO-DG44, etc.), mouse myeloma cell lines (e.g. NS0), baby hamster kidney cell lines (e.g. BHK), human embryonic kidney cell lines (for example HEK293), etc.), yeast systems (including in particular yeasts improved for glycolization), insect cell systems (including in particular insect cell lines improved for glycolization), plant cell systems (including in particular plant cell lines improved for glycolization), etc.

Preferably the antibody is an animal antibody, preferably a mammalian antibody, more preferably a human or humanized antibody. Advantageously, the antibody is humanized.

The different categories of antibodies and their production methods are well known to those skilled in the art, who may in particular refer to reference works in the field (such as Thomas D. Pollard, William C. Earnshaw, Jennifer Lippincott-Schwartz , Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, 1 Nov. 2016; Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10.1007/978-1-4419-0919-0, Springer-Verlag New York 2010; Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, 2014 Aug 22; Bayer V., An overview of monoclonal antibodies. Semin Oncol Nurs. 2019 Sep 2:150927; Wang W, Wang EQ, Balthasar JP. Pharmacokinetics and pharmacodynamics monoclonal antibodies Clin Pharmacol Ther. 2008 Nov;84(5):548-58).

The term " functional antibody fragment", as used herein, refers to one or more part(s) or fragment(s) of an antibody, retaining the ability to bind specifically to an antigen. Examples of binding fragments encompassed within the term "antibody functional fragment" include, but are not limited to, antigen-binding (Fab) fragment, Fab' fragment, F(ab')2 , a variable fragment (Fv), a single chain variable fragment (scFv for "single chain variable fragment"), corresponding to the VH and VL regions fused using a linker peptide), a dsFv fragment ("disulphide -bond stabilized Fv"), a ds-scFv fragment ("disulphide-bond stabilized scFv"), a VH domain, a VL domain, a di-scFv (divalent scFv, consisting of the association of two scFvs), a "diabody" (made up of the covalent or non-covalent association of two scFvs), a single chain "diabody", a triple body, a minibody ("minibody", made up of VL-VH-CH3 fragments), a nanobody ("nanobody "), single domain antibody (sdAb), single chain antibody fragment (scAb), heavy chain antibody (HcAb), VHH, VNAR (variable new antigen receptor), novel antigen receptor immunoglobulin (IgNAR), bispecific T cell engager (BITEs), dual affinity retargeting molecule (DART), and any combination thereof (eg. a fusion protein thereof).

The term " single domain antibody ", or "sdAb", as used herein, refers to antibody fragments consisting of a single monomeric variable domain of an antibody. These antibodies only comprise the monomeric variable regions of the heavy chain of the heavy chain antibodies produced in particular by camelids or cartilaginous fish. Due to their different origins, they are also referred to as VHH (camelid) or VNAR (variable new antigen receptor; cartilaginous fish) fragments. Single domain antibodies are also called nanobodies. Single domain antibodies can also be obtained by monomerizing the variable domains of conventional mouse or human antibodies through genetic engineering. They have a molecular mass of approximately 12-15 kDa and are therefore the smallest antibody fragments capable of recognizing an antigen.

By “ humanized binding agent ” or “ K _d ” or “ Kd ” is meant a binding agent whose sequence has been modified in order to optimize its function in a human. To do this, the sequence of the humanized linker can be modified to be closer to the sequences of linkers found in humans. The humanized binding agents therefore have a greater proportion (ie greater than that of the binding agent before humanization) of materials/substances of human origin (ie found naturally in humans), in particular a higher proportion large number of sequences, in particular nucleic acid and/or amino acid sequences, of human origin (ie, found naturally in humans). The humanized binding agents preferably have at least 80% of materials/substances (in particular sequences, in particular nucleic acid and/or amino acid sequences) of human origin. Preferably, the humanized binding agent comprises at least one sequence (in particular a sequence of nucleic acids and/or amino acids of the binding agent) comprising at least 80% of sequences (in particular of acid sequences nuclei and/or amino acids) of human origin, preferably at least 85% of sequences of human origin, preferably at least 86% of sequences of human origin, preferably at least 87% of sequences of of human origin, preferably at least 88% of sequences of human origin, preferably at least 89% of sequences of human origin, preferably at least 90% of sequences of human origin, preferably at least 91% of sequences of human origin, preferably at least 92% of sequences of human origin, preferably at least 93% of sequences of human origin, preferably at least 94% of sequences of human origin, preferably at least 95% of sequences of human origin, preferably having at least 96% of sequences of human origin, preferably at least 97% of sequences of human origin, preferably at least 98% of sequences of human origin, preferably at least at least 99% of sequences of human origin, more preferably 100% of sequences of human origin (ie, sequences found naturally in humans).

In the case where the binding agent is an antibody or a functional fragment of an antibody (such as a single domain antibody), the humanized antibody or the functional fragment of a humanized antibody comprises at least one acid sequence amino acids comprising at least 80% (preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequences of human origin (i.e., amino acid sequences found naturally in humans).

Various techniques are known to those skilled in the art for humanizing a bonding agent. For example, in the case where the binding agent is an antibody or a functional fragment of an antibody (such as a single domain antibody), the humanized antibody or the functional fragment of a humanized antibody can be obtained by grafting / inserting the CDRs of interest into the framework of a variable domain of a human antibody or of a functional fragment of a human antibody (this technique is called humanization by “CDR-grafting”). The humanized antibody or the functional fragment of a humanized antibody can also be obtained by making specific amino acid substitutions in their sequences in order to obtain a sequence closer to a sequence of human origin. Whatever the technique selected for humanizing the antibody or the functional fragment, it is possible advantageously to use the germline sequence of a human antibody or of a functional fragment of a human antibody (i.e. the sequence devoid of mutations linked to the maturation of antibody) encoded by the human gene closest to the sequence of the non-human antibody or the functional fragment of the non-human antibody to be humanized (i.e. from which the CDRs are derived). The closest germline sequence can in particular be identified by performing sequence alignments using sequence databases and tools known to those skilled in the art (this technique is called the “best-fit” technique).

By " equilibrium dissociation constant " or " K _d " or " Kd " is meant the constant which makes it possible to evaluate the affinity between two molecules (for example between an antibody and the RBD domain of the Spike protein or a sarbecovirus). This affinity is based on the nature, geometry and number of physicochemical interactions between the two molecules (electrostatic interaction, hydrogen bonds, van der Waals interaction and hydrophobic forces). The lower the Kd value, the higher the binding affinity between the two molecules. Kd is expressed in M (mol/l), often in nM or pM.

There are several techniques for determining/measuring dissociation constants, which are well known to those skilled in the art, such as ELISA, gel retardation assays, filtration, chromatography, thermophoresis, "pull -down", equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), spectroscopic assays, isothermal titration calorimetry (ITC), nitrocellulose membrane filtration, l interferometry (e.g. BioLayer interferometry), etc. The method to determine/measure the dissociation constants is preferably interferometry. The dissociation constant at equilibrium can in particular be determined, under standard conditions, using the representations of Scatchard and Lineweaver Burk, or even using Langmuir binding models (such as the Langmuir model global), well known to those skilled in the art.

By " polypeptide ", " protein " and " peptide ", we mean polymers of amino acid residues which comprise at least nine amino acids linked by peptide bonds. The polymer can be linear, branched or cyclic. The polymer may comprise natural amino acids and/or amino acid analogs and may be interrupted by non-amino acid residues. As a general indication and without however being bound by it in this document, if the polymer of amino acids contains more than 50 amino acid residues, it is preferably called a polypeptide or a protein, whereas if the polymer consists of 50 amino acids or less, it is preferably called a "peptide". The reading and writing directions of an amino acid sequence of a polypeptide, protein and peptide as used herein are the conventional reading and writing directions. The reading and writing convention for polypeptide, protein, and peptide amino acid sequences places the amine terminus to the left, with the sequence then being written and read from the amine terminus (N- terminus) to the carboxyl terminus (C-terminus), from left to right.

The amino acids constituting the polypeptides, proteins and peptides include in particular the so-called "standard" amino acids (also called "natural amino acids", of which Table 1 below provides a non-exhaustive list), as well as the non-standard amino acids (also called "rare amino acids", including for example pyrrolysine (symbolized by the letter O), selenocysteine (symbolized by the letter U), alloisoleucine, allothreonine, ornithine, etc.)

Table 1 : Standard amino acids

Name 3 letters 1 letter alanine To the AT arginine Arg R asparagine Asn NOT aspartate or aspartic acid Asp D cysteine Cys VS glutamate or glutamic acid Glue E glutamine Gln Q wisteria Gly G histidine His H isoleucine Island I leucine Leu I lysine lily K methionine met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tryptophan trp W tyrosin Tire Y valine Val V

The terms " polynucleotide ", " nucleic acid molecule " and " nucleic acid " are used interchangeably herein and refer to a polymeric or oligomeric macromolecule consisting of nucleotide monomers (preferably at least 5 nucleotide monomers, also called nucleotide residues). Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed by phosphodiester bonds between individual nucleotide monomers. Nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids (mixed polyribo-polydeoxyribonucleotides). These terms encompass single or double-stranded, linear or circular, natural or synthetic, unmodified or modified versions thereof (e.g., genetically modified polynucleotides; optimized polynucleotides), sense or antisense polynucleotides, chimeric mixture (e.g., hybrids RNA-DNA). Additionally, a polynucleotide may include nucleotides of non-natural origin and may be interrupted by non-nucleotide components. Examples of DNA nucleic acids include, without limitation, complementary DNA (cDNA), genomic DNA, plasmid DNA, vector DNA, viral DNA (e.g., viral genomes, viral vectors), oligonucleotides, probes, primers, satellite DNA, microsatellite DNA, coding DNA, non-coding DNA, antisense DNA, and any mixture thereof. Exemplary RNA nucleic acids include, but are not limited to, messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), small interfering RNA (siRNA), short hairpin RNA (cRNA), microRNA (miRNA), vector RNA, viral RNA, guide RNA (gRNA), antisense RNA, coding RNA, non-coding RNA, antisense RNA, satellite RNA, cytoplasmic small RNA, nuclear small RNA, etc. The polynucleotides described herein can be synthesized by standard methods known in the art, for example using an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.) or obtained from from a natural source (eg, genome, cDNA, etc.) or from an artificial source (such as a commercially available library, plasmid, etc.) using molecular biology techniques well known in the art (e.g. cloning, PCR, etc.). The nucleic acids can for example be chemically synthesized, for example according to the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584).

By " isolated molecule " is meant a molecule, in particular a protein, a polypeptide, a peptide, a molecule of nucleic acids, a vector (for example a plasmid vector or a viral vector) or a host cell, which is extracted from its natural environment (that is to say separated from at least one other component with which it is naturally associated).

By " vector " is meant a vehicle, preferably a nucleic acid molecule or a viral particle, which contains the elements necessary to allow the administration, propagation and/or expression of one or more molecule(s) ) of nucleic acids in a host cell or organism.

From a functional point of view, this term encompasses vectors for maintenance (cloning vectors), vectors for expression in various host cells or organisms (expression vectors), extrachromosomal vectors (e.g. multicopy plasmids ) or integrating vectors (e.g. designed to integrate into a host cell's genome and produce additional copies of the nucleic acid molecule it contains when the host cell replicates). This term also encompasses shuttle vectors (for example, functioning in both prokaryotic and/or eukaryotic hosts) and transfer vectors (for example for the transfer of nucleic acid molecule(s) into the genome of a host cell).

From a structural point of view, vectors can be natural, synthetic or artificial genetic sources, or a combination of natural and artificial genetic elements.

Thus, in the context of the invention, the term "vector" should be understood broadly to include plasmid (or plasmids) and viral vectors.

A " plasmid " as used herein means a replicable DNA construct. Typically, plasmid vectors contain selectable marker genes which allow host cells carrying the plasmid to be identified and/or positively or negatively selected in the presence of the compound corresponding to the selectable marker. A variety of positive or negative selection marker genes are known in the art. By way of illustration, an antibiotic resistance gene can be used as a positive selection marker gene making it possible to select a host cell in the presence of the corresponding antibiotic.

The term " viral vector " as used herein refers to a nucleic acid vector which comprises at least one element of a virus genome and may be packaged into a virus particle or virus particle. Viral vectors can be replication-competent or selective (e.g., designed to replicate better or selectively in specific host cells), or can be genetically disabled so as to be defective or replication-defective.

By “ host cell ”, is meant a cell containing at least one vector according to the invention, or at least one nucleic acid molecule according to the invention, or any mixture thereof. Also advantageously, the host cell is capable of expressing the genes encoded by the nucleic acid molecule according to the invention and/or the vector according to the invention and/or of producing the vector of the invention.

The host cell can consist of a single cell type or a group of different cell types. The host cell can also be a hybrid cell, that is to say resulting from the fusion of at least two cells of different types.

The host cell can belong to cultured cell lines, primary cells, stem cells or proliferating cells. The term "host cells" includes prokaryotic cells, lower eukaryotic cells such as yeast cells, and other eukaryotic cells such as fungus cells, insect cells, plant cells, algae, microalgae cells, parasite cells, animal cells and mammalian cells (eg human or non-human, preferably non-human). The host cell can be a differentiated cell, a pluripotent cell, a totipotent cell, a stem cell, an induced pluripotent stem cell (iPSC or iPSC), an induced totipotent stem cell, or even an embryonic cell or an embryonic stem cell . In the case of an embryonic cell or an embryonic stem cell, the cell is preferably a non-human cell.

The term “host cell” more broadly includes cells which contain or have contained the nucleic acid molecule according to the invention, as well as the progeny of such cells.

The host cell can for example be isolated or organized into a tissue, an organ or even be within a complete organism. In the case where the host cell is within a complete organism, said organism is not human.

By " disease " or "condition" or "disorder" or "pathology" is meant an alteration of the functions or health of a living organism. It is both a disease, referring to all health alterations, and a disease, which then designates a particular entity characterized by its own causes, symptoms, evolution and therapeutic possibilities.

“ Infection ” or “infectious disease” designates here a disease caused by the transmission of a micro-organism or an infectious agent: virus, bacterium, parasite, fungus, protozoan, etc.

By “ sarbecovirus-related disease ” or “sarbecovirus disease” is meant any disease which develops in a subject infected or who has been infected with a sarbecovirus. Sarbecovirus-related diseases include, but are not limited to, infections with a sarbecovirus (e.g. COVID disease, in particular COVID-19)), as well as any non-infectious disease which is caused, caused, amplified and/or maintained by at least one infection by a sarbecovirus and/or by occasional, occasional, regular, prolonged or repeated exposure to at least one sarbecovirus.

" Infection by a sarbecovirus " or "infection with sarbecovirus" denotes the fact that cells of an organism have been infected by at least one sarbecovirus, the whole of the organism being said to be affected by said infection by a sarbecovirus.

The terms " COVID disease " or " COVID " or "betacoronavirus disease" refer to the disease related (associated) with infection by at least one betacoronavirus, as listed above. In particular, the terms "COVID-19 disease" or " COVID-19 " or "coronavirus 19 disease" refer to the disease related to (or associated with) infection with (at least) SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2).

By " prevention " or "prevention of a disease" or "prevention of the appearance of a disease" is meant the reduction of the risk of appearance, development or amplification of a disease, the causes of a disease, symptoms of a disease, effects (or consequences, preferably adverse, deleterious effects/consequences) of a disease, or any combination thereof; and/or delay the onset, development or aggravation of a disease, the causes of a disease, the symptoms of a disease, the effects (or consequences, preferably the deleterious effects/consequences) of a disease, or any combination thereof. Prevention includes preventive treatments.

By " treatment " or "treatment of a disease" is meant the reduction, inhibition and/or disappearance of a disease, the causes of a disease, the symptoms of a disease, the effects (or consequences, preferably the harmful, deleterious effects/consequences) of a disease, or any combination thereof. The treatment is preferably a curative treatment.

A "treatment" or "therapy" includes, but is not limited to, one or more molecules and/or drugs (including any type of molecule or drug, such as chemical or biological compounds, antibodies, antigens, gene therapy, cell therapy, immunotherapy, chemotherapy, any combination thereof, etc.), and/or other treatments (such as radiotherapy, immunotherapy, chemotherapy, surgery, l endoscopy, interventional radiology, physical oncology, phototherapy, light therapy, ultrasound therapy, thermotherapy, cryotherapy, electrotherapy, electroconvulsive therapy, oxygen therapy, assisted ventilation, hydrotherapeutic massage, transplantation organs/tissues/fluids, implantation, and any combination thereof, etc.) Therapy can be delivered by various modes of administration. A person skilled in the art knows how to select the most appropriate mode or modes of administration depending on the therapy, the disease and the subject to be treated. For example, modes of administration include, but are not limited to, oral administration, administration by injection into a vein (intravenous, IV), into a muscle (intramuscular, IM), into the space around the spinal cord (intrathecal), under the skin (subcutaneous, sc); sublingual administration; buccal administration; rectal administration; vaginal administration; ocular pathway; otic pathway; nasal administration; by inhalation; by nebulization; cutaneous, topical or systemic administration; transdermal administration.

" Medicine " or "drug" means any substance or composition presented as having curative or preventive properties with regard to human or animal diseases. A drug therefore includes any substance or composition that can be used in humans or animals or administered to them for the purpose of establishing a medical diagnosis or restoring, correcting or modifying their physiological functions by exerting a pharmacological action, immunological or metabolic.

The term " vaccine " or "vaccine composition" refers to a pathogenic or tumor-producing substance which, when inoculated into an individual in a preferably harmless form, confers immunity against disease (or protection against disease). In general, a vaccine stimulates the body's immune response. A vaccine can be preventive, allowing the prevention of a disease. A vaccine can also be therapeutic, helping the patient fight an ongoing disease. Therefore, the term "vaccine" means any component or group of components that is believed to elicit a biological response when appropriately administered to a subject through the presence or expression of one or more biological substances (e.g. , a polypeptide such as an antigen or an antibody, a nucleic acid molecule, an enzyme, a cytokine, a SiRNA, etc.).

The terms " therapeutic " or "therapeutic uses" in the context of the present invention cover the uses "prevention", "treatment" and "vaccine".

A " therapeutically effective amount " is that amount of each active entity that is sufficient to produce a beneficial health outcome. An "immunologically effective amount" is that amount of each active entity which is sufficient to produce a detectable immune response. The active entity is for example

As used herein, a " pharmaceutically acceptable vehicle " or "pharmaceutically acceptable carrier" is intended to include all carriers, solvents, diluents, excipients, adjuvants, dispersing media, coatings, antibacterial and antifungal agents, and absorption, and the like, compatible with administration in a subject especially in an animal, and in particular in a human Suitable carriers for use herein are well known in the art (see for example the edition the most recent from Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins).

" Screening " means a process to test and select active compounds/agents for a specific effect/activity on a molecule, virus, parasite, bacterium, cell, tissue, organ, disease (preferably an infectious disease and/or cancer), an organism (excluding human beings, human embryos and human embryonic stem cells). For example, the compounds/active agents can be tested for antiviral activity/effect (such as anti-coronavirus, especially anti-sarbecovirus activity/effect), for antibacterial activity/effect, for anti-coronavirus activity/ an antitumor and/or anticancer effect, and any combination thereof.

By " diagnosis " is meant the identification/determination of a disease, or the absence of a disease, in a subject. Diagnosis includes, for example, the search for the causes (etiology) and effects (symptoms) of the disease, in particular on the basis of observations and/or measurements, carried out using various tools. In the case of sarbecovirus-related illnesses, diagnostic tools include observation of specific clinical signs such as sudden onset of unexplained asthenia (feeling tired); unexplained myalgia (muscle pain); headaches (headaches); ageusia (absence of the sense of taste) and for severe forms of the onset of respiratory disorders but also the detection/quantification of genetic and/or biochemical biomarkers. Genetic biomarkers can be alleles or mutations in the genome that have been identified as predisposing to sarbecovirus-related diseases. This type of marker thus makes it possible to identify a population “at risk”, which has a higher probability of developing diseases linked to sarbecoviruses. Biochemical markers are biomolecules whose presence and/or quantity is correlated with the evolution of the pathology (for example the accumulation of viral proteins or the detection of anti-S or anti-N antibodies for infections by a sarbecovirus).

By " stratification " is meant the separation/classification of subjects into subgroups by severity/severity of the disease. The various subgroups include in particular the subgroup of healthy subjects as well as various subgroups of subjects suffering from a disease, classified according to the stage of evolution/advancement of the disease. It is also possible to stratify the subjects according to the type of symptoms present. The stage of evolution and the symptoms can be determined on the basis of observations and/or measurements, carried out using different tools. In the case of sarbecovirus-related diseases, stratification tools include tools that are also used for their diagnosis.

By " prognosis " is meant the prediction/determination/assessment of the risks of progression of a disease in a subject. The prognosis includes in particular the evaluation of the future development of the subject's condition and the possible chances of improvement or even cure. The prognosis can be determined on the basis of observations and/or measurements, carried out using different tools. In the case of sarbecovirus-related diseases, prognostic tools include tools that are also used for their diagnosis or subject stratification.

By " monitoring " is meant determining/assessing the progress of a disease in a subject. Monitoring can be carried out on the basis of observations and/or measurements, carried out using different tools, at different time intervals. Intervals can be regular or irregular. Their frequency depends on the disease but also on the stage of evolution of the disease. It can be of the order of a few days (for example in the event of severe/advanced/serious disease and/or in the event of rapidly progressive disease and/or in the event of an exacerbation phase) to a few years ( e.g. early, mild or moderate disease, and/or slowly progressing disease). In the case of sarbecovirus-related diseases, monitoring tools include tools that are also used for disease diagnosis or prognosis, or subject stratification.

By “ evaluation of the efficacy of a treatment ”, is meant the determination of the clinical state of a subject subjected to a treatment. The treatment can be preventive, for example in the case of a predisposition to a disease, or it can be curative, for example in the case of a diagnosed disease. The effectiveness of the treatment can for example be evaluated by determining the state of the subject at different time intervals. The state of the subject can in particular be evaluated before the first dose of the treatment then at regular (or irregular) time intervals after this first dose (for example after each new dose of the treatment). A comparison of the state of the subject evaluated at these different intervals can then be carried out in order to identify any change. In the case of a curative treatment, an improvement, no worsening, or a worsening of the patient's condition less than that expected in the absence of treatment indicates that the treatment is effective, while a worsening of the patient's condition at least equal to that expected in the absence of treatment indicates that the treatment is not effective. In the case of preventive treatment, an absence of onset of the disease or an onset later and/or less severe than expected in the absence of treatment indicates that the treatment is effective, while such an early onset and severe than expected in the absence of treatment indicates that the treatment is not effective. The patient's condition can be assessed on the basis of observations and/or measurements, carried out using different tools. In the case of diseases related to sarbecoviruses, the tools for evaluating the effectiveness of a treatment include tools that are also used for the diagnosis, prognosis or monitoring of the disease, or the stratification of subjects.

By “ aggravation ” or “ aggravation phase ”, is meant a period during which the clinical signs of a disease linked to sarbecoviruses increase in a subject suffering from said disease.

By “ subject ” or “ patient ” is meant a human individual or an animal other than a human. The subject is for example a human or an animal susceptible to contracting a disease linked to sarbecoviruses, susceptible to suffering from a disease linked to sarbecoviruses, or suffering from such a disease. The subject is preferably a human being. The subject may be a child (human subject 16 years of age or younger) or an adult (human subject over 16 years of age). By “ healthy subject ” is meant a subject who does not suffer from the disease in question. In the context of the present invention, a healthy subject is preferably a subject who does not suffer from any disease linked to sarbecoviruses, in particular a subject who does not suffer from any infection by a sarbecovirus, more preferably a subject who does not suffer of no disease. By “ reference subject ” is meant a subject who suffers from a disease linked to known sarbecoviruses (in particular an infection by a sarbecovirus, and in particular by a SARS-CoV1 or a SARS-CoV2, at a known stage).

" Biological sample " or " sample " from a subject means an entire organ or tissue or part of such an organ or tissue, a fluid or a fraction of such a fluid, cells or cellular components , obtained from this subject, as well as a homogenate, a lysate or an extract prepared therefrom. In particular, a "biological sample" or "sample" is preferably any tissue (preferably portions or fractions thereof) that may contain sarbecovirus, including but not limited to plasma, blood , lymph, serum, urine, mucus, saliva, central nervous system (CNS; such as brain sample or spinal cord sample), respiratory tract sample ( such as lung sample, etc.), salivary gland sample, nasopharyngeal sample, oropharyngeal sample, digestive system sample (e.g. colon), etc.

The biological sample may have been obtained beforehand by any technique known in the profession. These techniques include, for example, collection using a swab, needle or syringe, surgery (such as stereotaxic surgery), puncture, explant, excision, biopsy. By "excision" is meant a surgical procedure consisting in cutting (excising) a more or less wide or deep part of the tissue, preferably an abnormality or growth of the tissue. An excision may be performed to remove and/or analyze a cancerous or suspicious tumor. The term “biopsy” here designates a sample of cells or tissues taken for analysis. Several types of biopsy procedures are known and practiced in the field. The most common types include (1) incisional biopsy, in which only a sample of the tissue is taken; (2) excisional biopsy (or surgical biopsy), which consists of completely removing a tumor mass, thus carrying out a therapeutic and diagnostic procedure; and (3) needle biopsy, in which a tissue sample is removed using a needle, which can be coarse or fine. Other types of biopsy exist, such as smear or curettage, and can also be used to obtain the sample. Therefore, the sample can be, for example, an explant, an excision, a biopsy, etc. The sample is preferably obtained by a minimally invasive procedure, such as stereotactic surgery.

In the detailed description which follows, the embodiments can be taken alone or in any appropriate combination by those skilled in the art, and the definitions above apply to all the embodiments described below as well as to their combinations.

Liaison agent

In the context of the present invention, the inventors have developed binding agents, in particular single-domain antibodies, which are innovative and capable of specifically recognizing and neutralizing the different variants of SARS-CoV2.

These binding agents were notably obtained by an innovative approach of antibody molecular engineering and affinity maturation from the single-domain antibody VHH72, combining Deep-Mutational Scanning and Yeast Surface Display techniques. . This approach allowed the identification of amino acid positions and mutations likely to confer an increase in affinity to the antibody. Assembly in combinatorial libraries combined with high-throughput sequencing identified a large number of enriched variants with stronger antigen binding than the parental single-domain antibody VHH72.

The inventors have in particular shown that, surprisingly, the binding agents thus developed have very good affinities for the RBD domains of different sarbecoviruses, including variants of the SARS-CoV2 virus, unlike binding agents ( in particular the single domain antibodies) described in the prior art. Remarkably, the data also show that these optimized binding agents are able to prevent the binding between the host cell ACE2 receptor and the RBD domain, more effectively than binding agents (especially single domain antibodies ) described in the prior art. These data thus reveal the therapeutic potential of these binding agents to treat diseases linked to sarbecoviruses, in particular infections by a sarbecovirus.

The data also show that these binding agents are tools for the detection of sarbecoviruses.

The present invention therefore provides both powerful and broad-spectrum treatment methods for diseases linked to sarbecoviruses, as well as methods for the effective and reliable diagnosis of these diseases, methods for screening molecules but also tools for research in the field of diseases related to sarbecoviruses.

The binding agents thus developed have several advantages over the binding agents (in particular single-domain antibodies) of the prior art: 1) they recognize the RBD domain of the Spike protein of sarbecovirus with high specificity; 2) they have a higher affinity for the RBD domain of the sarbecovirus Spike protein; 3) they present a broader spectrum of recognition of sarbecoviruses, in particular of variants of SARS-CoV2; 4) they are able to more effectively inhibit the interaction between the Spike protein of sarbecovirus and the ACE2 receptor of the host cell.

The invention therefore relates in particular to a binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody), comprising, or consisting essentially of, or consisting of, the amino acid sequence SEQ ID NO:1 comprising at least one substitution of an amino acid residue selected from the residue at position 57, the residue at position 58, the residue at position 60, the residue at position 61, the residue at position 62, the residue at position 98, residue at position 101, residue at position 102, residue at position 103, residue at position 104, of SEQ ID NO:1, and any combination thereof. Indeed, the data show that such binding agents have an improved affinity and neutralization capacity vis-à-vis the SARS-Cov2 virus and its various variants. The binding agent can therefore be used effectively in the prevention and treatment of diseases related to sarbecoviruses.

The sequence SEQ ID NO: 1 is as follows:

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSS (SEQ ID NO:1).

The present invention also relates to variants of VHH72 having an improved affinity, which can be used in the prevention and treatment of diseases related to sarbecoviruses.

The present invention relates in particular to a binding agent (such as an antibody or a functional fragment of an antibody, in particular a single-domain antibody) comprising, or consisting essentially of, or consisting of, the amino acid sequence SEQ ID NO:1, comprising at least one substitution of an amino acid residue selected from the group consisting of:

1. The residue at position 57 of SEQ ID NO:1, substituted by an amino acid selected from G, N, and M (preferably by an amino acid G);
2. The residue at position 58 of SEQ ID NO:1, substituted with an amino acid selected from: S, W, R, and H (preferably with an S amino acid);
3. The residue at position 60 of SEQ ID NO: 1, substituted by an amino acid preferably selected from: V, L, I, T, M, H, Q, Y, and F (preferably by an amino acid selected from V, L, and I);
4. The residue at position 61 of SEQ ID NO:1, substituted with an amino acid selected from: S, R, P, Q, I, M, and F (preferably with an amino acid selected from S, and R);
5. The residue at position 62 of SEQ ID NO: 1, substituted by an amino acid selected from any amino acid except D, C, and Q (in particular substituted by an amino acid preferably selected from: F , L, G, P, I, M, W, Y, A, G, S, T, N, E, H, K, and R; more preferably by an amino acid selected from F, and L);
6. The residue at position 98 of SEQ ID NO: 1, substituted by an amino acid preferably selected from any amino acid except A, P, and D (in particular substituted by an amino acid preferably selected from : V, W, R, Q, I, E, L, F, V, M, Y, G, C, S, T, N, H, and K; more preferably by an amino acid selected from V, W , R, and Q; more preferably by an amino acid V);
7. The residue at position 101 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, F, Y, C, E, and W;
8. The residue at position 102 of SEQ ID NO:1, substituted by an amino acid preferably selected from: E, Y, and N (more preferably by an amino acid selected from E and Y);
9. The residue at position 103 of SEQ ID NO:1, preferably substituted with an amino acid V;
10. The residue at position 104 of SEQ ID NO:1, substituted by an amino acid selected from: W, and Y (preferably by an amino acid W); And
11. Any combination of these.

Advantageously, the binding agent is chosen from antibodies and functional antibody fragments. According to a particularly preferred embodiment, the binding agent is a single domain antibody.

According to a preferred embodiment, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) comprises at least one substitution of an amino acid residue chosen from: substitution of the residue at position 57 of SEQ ID NO: 1 by an amino acid by an amino acid selected from G, N, and M (preferably by an amino acid G); the substitution of the residue at position 103 of SEQ ID NO:1, preferably by an amino acid V; the substitution of the residue at position 104 of SEQ ID NO: 1 by an amino acid selected from: W, and Y (preferably by an amino acid W); and any combination thereof.

According to a particularly preferred embodiment, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) comprises at least the substitution of the residue at position 57 of SEQ ID NO: 1 with an amino acid selected from G, N, and M; preferably by an amino acid G.

The inventors have shown in particular that, surprisingly, the affinity for the RBD domain of the Spike protein of sarbecovirus and the neutralizing effect of the optimized binding agents developed in the context of the present invention, increase when the binding agent combines two or more substitutions at positions distinct from each other.

The present invention therefore relates to a binding agent (such as an antibody or a functional fragment of an antibody, in particular a single-domain antibody) comprising, or consisting essentially of, or consisting of, the amino acid sequence SEQ ID NO :1, comprising a combination of at least two amino acid residue substitutions, the substituted amino acid residue being selected from the group consisting of:

1. The residue at position 57 of SEQ ID NO:1, substituted by an amino acid preferably selected from: G, N and M (preferably by an amino acid G);
2. The residue at position 58 of SEQ ID NO:1, substituted by an amino acid preferably selected from: S, W, R, and H (preferably by an S amino acid);
3. The residue at position 60 of SEQ ID NO: 1, substituted by an amino acid preferably selected from: V, L, I, T, M, H, Q, Y, and F (preferably by an amino acid selected from V, L, and I);
4. The residue at position 61 of SEQ ID NO: 1, substituted by an amino acid preferably selected from: S, R, P, Q, I, M, W, and F (preferably by an amino acid selected from S, and R);
5. The residue at position 62 of SEQ ID NO: 1, substituted by an amino acid preferably selected from any amino acid except D, C, and Q (in particular substituted by an amino acid preferably selected from : F, L, V, P, I, M, W, Y, A, G, S, T, N, E, H, K, and R; more preferably by an amino acid selected from F, and L) ;
6. The residue at position 98 of SEQ ID NO: 1, substituted by an amino acid preferably selected from any amino acid except A, P, and D (in particular substituted by an amino acid preferably selected from : V, W, R, Q, I, E, L, F, V, M, Y, G, C, S, T, N, H, and K; more preferably by an amino acid selected from V, W , R, and Q; more preferably by an amino acid V);
7. The residue at position 101 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, F, Y, C, E, and W;
8. The residue at position 102 of SEQ ID NO:1, substituted by an amino acid preferably selected from: E, Y, and N (more preferably by an amino acid selected from E and Y);
9. The residue at position 103 of SEQ ID NO:1, preferably substituted with an amino acid V; And
10. The residue at position 104 of SEQ ID NO:1, substituted by an amino acid preferably selected from: W, and Y (preferably by an amino acid W); And
11. Any combination of these.

According to a particularly preferred embodiment, the substitution combination comprises at least the substitution of the residue at position 57 of SEQ ID NO: 1 by an amino acid preferably selected from: G, N and M, preferably by an amino acid G.

According to a preferred embodiment, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) comprises at least one combination of amino acid residue substitutions chosen from:

1. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1, preferably by an amino acid selected from: G, N and M (preferably by an amino acid G), and of the substitution of the residue at position 103 of SEQ ID NO: 1 preferably by an amino acid V;
2. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1 preferably by an amino acid selected from: G, N and M (preferably by an amino acid G), and of the substitution of the residue at position 104 of SEQ ID NO:1 preferably by an amino acid selected from: W, and Y (preferably by an amino acid W);
3. the combination of the substitution of the residue at position 103 of SEQ ID NO: 1 preferably by an amino acid V, and the substitution of the residue at position 104 of SEQ ID NO: 1 preferably by an amino acid selected from: W, and Y (preferably by an amino acid W); And
4. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1 preferably by an amino acid selected from: G, N and M (preferably by an amino acid G), and of the substitution of the residue at position 103 of SEQ ID NO: 1 preferably with an amino acid V, and the substitution of the residue at position 104 of SEQ ID NO: 1 preferably with an amino acid selected from: W, and Y (preferably with an amine W).

The data shows that binding agents (such as an antibody or a functional fragment of an antibody, especially a single domain antibody) combining a substitution at position 57 and a substitution at position 103, or combining a substitution at position 57 and a substitution at position 104, or combining a substitution at position 57 and a substitution at position 103 and a substitution at position 104, exhibit a particularly high affinity for the RBDs of different sarbecoviruses and a neutralizing effect.

Thus, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) advantageously comprises at least one combination of amino acid residue substitutions chosen from:

1. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1, preferably by an amino acid selected from: G, N and M (preferably by an amino acid G), and of the substitution of the residue at position 103 of SEQ ID NO: 1 preferably by an amino acid V;
2. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1 preferably by an amino acid selected from: G, N and M (preferably by an amino acid G), and of the substitution of the residue at position 104 of SEQ ID NO:1 preferably by an amino acid selected from: W, and Y (preferably by an amino acid W); And
3. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1 preferably by an amino acid selected from: G, N and M (preferably by an amino acid G), and of the substitution of the residue at position 103 of SEQ ID NO: 1 preferably with an amino acid V, and the substitution of the residue at position 104 of SEQ ID NO: 1 preferably with an amino acid selected from: W, and Y (preferably with an amine W).

According to these embodiments, the binding agent (such as an antibody or a functional fragment of an antibody, in particular a single-domain antibody) may further comprise at least one additional substitution of an amino acid residue selected in the group consisting of:

1. The residue at position 58 of SEQ ID NO:1, substituted by an amino acid preferably selected from: S, W, and R, and H, (preferably by an S amino acid);
2. The residue at position 60 of SEQ ID NO: 1, substituted by an amino acid preferably selected from: V, L, I, T, M, H, Q, Y, and F (preferably by an amino acid selected from V, L, and I);
3. The residue at position 61 of SEQ ID NO: 1, substituted by an amino acid preferably selected from: S, R, P, Q, I, M, W, and F (preferably by an amino acid selected from S, and R);
4. The residue at position 62 of SEQ ID NO: 1, substituted by an amino acid preferably selected from any amino acid except D, C, and Q (in particular substituted by an amino acid preferably selected from : F, L, V, P, I, M, W, Y, A, G, S, T, N, E, H, K, and R; more preferably by an amino acid selected from F, and L) ;
5. The residue at position 98 of SEQ ID NO: 1, substituted by an amino acid preferably selected from any amino acid except A, P, and D (in particular substituted by an amino acid preferably selected from V, W, R, Q, I, E, L, F, V, M, Y, G, C, S, T, N, H, and K; more preferably by an amino acid selected from: V, W, R, and Q; more preferably by an amino acid V);
6. The residue at position 101 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, F, Y, C, E, and W;
7. The residue at position 102 of SEQ ID NO:1, substituted by an amino acid preferably selected from: E, Y, and N; more preferably by an amino acid selected from: E and Y; And
8. Any combination of these.

According to a particularly preferred embodiment, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) comprises an amino acid sequence chosen from SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

The sequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 are shown in Table 2 below.

Table 2 : Sequences Included in Preferred Linkers

SEQ-ID Description Sequence SEQ ID NO:2 VHH6: Antibody comprising substitutions S57G; T58S; Y60L; T61S; D62L A98V; T103V; V104W. QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGGSYLSLSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAVAGLGVWVSEWDYDYDYWGQGTQVTVSS SEQ ID NO: 3 VHH65: Antibodies comprising the substitutions S57G; Y60V; T61R; A98V; T103V; V104W. QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGGTYVRDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAVAGLGVWVSEWDYDYDYWGQGTQVTVSS SEQ ID NO:4 VHH66: Antibodies comprising substitutions S57G; Y60V; T61S; D62L; A98V; T103V; V104W. QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGGTYVSLSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAVAGLGVWVSEWDYDYDYWGQGTQVTVSS SEQ ID NO:5 VHH71: Antibody comprising substitutions S57G; Y60V; T61S; D62F; A98V; T103V; V104W. QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGGTYVSFSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAVAGLGVWVSEWDYDYDYWGQGTQVTVSS SEQ ID NO:6 VHH76: Antibodies comprising the substitutions S57G; Y60I; A98V; T103V; V104W. QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGGTYITDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAVAGLGVWVSEWDYDYDYWGQGTQVTVSS

According to one embodiment of the invention, the binding agent comprises, consists essentially of, or consists of, at least one binding agent chosen from: an antibody, a functional derivative of an antibody, and a functional fragment of antibodies; the antibody functional fragment preferably being selected from: a single domain antibody, a nanobody, and a VHH (single variable domain heavy chain), and any combination thereof.

Preferably, the binding agent is chosen from antibodies, functional antibody derivatives, and functional antibody fragments (in particular single-domain antibodies).

Advantageously, the binding agent (such as an antibody or a functional fragment of an antibody) comprises, consists essentially of, or consists of a single domain antibody, a nanobody (nanobody in English), or a VHH (domain single heavy chain variable). Advantageously, the binding agent (such as an antibody or a functional fragment of an antibody) is a single domain antibody, a nanobody, or a VHH. Single domain antibodies, nanobodies, and VHHs have the advantage of being very stable molecules with excellent affinities for their antigen. Thanks to the reduced size of their variable region, these molecules can target epitopes that are difficult to access by conventional antibodies.

The inventors have in particular developed antibodies (in particular single-domain antibodies) as defined above, fused to a constant fragment of human immunoglobulin. The data show that an antibody thus humanized retains both its specificity for recognizing sarbecovirus RBDs and its broad-spectrum affinity for the RBDs of different sarbecoviruses, as well as its neutralizing capacity towards sarbecoviruses.

Thus, according to an advantageous embodiment, the present invention also relates to a binding agent as defined above, which has been humanized (that is to say whose sequence has been humanized).

In addition to the substitutions or combinations of substitutions listed above, the humanized binding agent according to the invention may in particular comprise at least one additional substitution of an amino acid residue selected from the group consisting of:

• The residue at position 1 of SEQ ID NO:1, preferably substituted with an amino acid E;
• The residue at position 5 of SEQ ID NO:1, preferably substituted with an amino acid selected from V, and L;
• The residue at position 14 of SEQ ID NO:1, preferably substituted by an amino acid P;
• The residue at position 27 of SEQ ID NO:1, preferably substituted by an amino acid F;
• The residue at position 31 of SEQ ID NO:1, preferably substituted with an S amino acid;
• The residue at position 35 of SEQ ID NO:1, preferably substituted with an S amino acid;
• The residue at position 37 of SEQ ID NO:1, preferably substituted by an amino acid V;
• The residue at position 44 of SEQ ID NO:1, preferably substituted with an amino acid G;
• The residue at position 45 of SEQ ID NO:1, preferably substituted with an L-amino acid;
• The residue at position 47 of SEQ ID NO:1, preferably substituted with an amino acid W;
• The residue at position 49 of SEQ ID NO:1, preferably substituted with an S amino acid;
• The residue at position 50 of SEQ ID NO:1, preferably substituted with an amino acid A;
• The residue at position 53 of SEQ ID NO:1, preferably substituted with an amino acid G;
• The residue at position 61 of SEQ ID NO:1, preferably substituted with an amino acid A;
• The residue at position 75 of SEQ ID NO:1, preferably substituted with an S amino acid;
• The residue at position 79 of SEQ ID NO:1, preferably substituted with an L-amino acid;
• The residue at position 87 of SEQ ID NO:1, preferably substituted with an amino acid R;
• The residue at position 88 of SEQ ID NO:1, preferably substituted with an amino acid A;
• The residue at position 89 of SEQ ID NO:1, preferably substituted with an amino acid E;
• The residue at position 120 of SEQ ID NO:1, preferably substituted by an amino acid Q; And
• Any combination of these.

According to a particularly advantageous embodiment, the humanized binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) comprises an amino acid sequence having at least 85% identity with the sequence SEQ ID NO:7, preferably having at least 86% identity, preferably having at least 87% identity, preferably having at least 88% identity, preferably having at least 89% d identity, preferably having at least 90% identity, preferably having at least 91% identity, preferably having at least 92% identity, preferably having at least 93% identity, preferably having at least 94% identity, preferably having at least 95% identity, preferably having at least 96% identity, preferably having at least 97% identity, preferably having at least 98% identity, preferably having at least 99% identity, with the sequence SEQ ID NO: 7.

The sequence SEQ ID NO: 7 is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGGTYITDSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAVAGLGVWVSEWDYDYDYDYWGQGTLVTVSS (SEQ ID NO: 7).

Advantageously, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) further comprises at least one antibody heavy chain fragment, preferably an antibody Fc fragment (crystallizable antibody fragment), the antibody heavy chain fragment (in particular the antibody Fc fragment) preferably being human.

When the binding agent according to the invention (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) further comprises at least one antibody heavy chain fragment, said heavy chain fragment of antibody preferably comprises, or preferably consists essentially of, or preferably consists of, an amino acid sequence having at least 85% identity with the sequence SEQ ID NO: 8, preferably having at least 86% identity, preferably having at least 87% identity, preferably having at least 88% identity, preferably having at least 89% identity, preferably having at least 90% identity, preferably having at least 91% identity, preferably having at least 92% identity, preferably having at least 93% identity, preferably having at least 94% identity, preferably having at least 95% identity identity, preferably having at least 96% identity, preferably having at least 97% identity, preferably having at least 98% identity, preferably having at least 99% identity, with the sequence SEQ ID NO: 8.

The sequence SEQ ID NO: 8 is as follows:

SEQ ID NO: 8: KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The invention further relates to an antibody, a functional antibody derivative, or a functional antibody fragment, comprising, consisting essentially of, or consisting of, a binding agent according to the invention (in particular a VHH) fused to antibody constant regions, preferably human antibody constant regions.

According to one embodiment, the antibody formats, the antibody functional derivative formats, or the antibody functional fragment formats, are of the Fab type and are characterized by the association of two VHH domains or of two domains identical or different human VHs, or two human VH domains onto which are grafted the CDRs ("complementarity determining regions") of the VHHs, one of the domains being fused to the Cκ or Cλ constant region of a human immunoglobulin, the another to the CH1 constant region of a human immunoglobulin. The antibody formats, the antibody functional derivative formats, or the antibody functional fragment formats, can also be of the Fab' type and are then characterized by the association of two VHH domains or of two human VH domains , identical or different, or two human VH domains on which the CDRs of the VHH are grafted, one of the domains being fused to the constant region Cκ or Cλ of a human immunoglobulin, the other to the constant region CH1 followed by a hinge region H of a human immunoglobulin. The antibody formats, the functional antibody derivative formats, or the functional antibody fragment formats, can also be of the F(ab') ₂ type and are characterized by the association of two Fab' type formats as defined above. Also included are the antibody formats are of the F(ab′) ₂ type, characterized by the association of two Fab′ obtained by reduction of the F(ab′) ₂ type formats above. The antibody formats, the antibody functional derivative formats, or the antibody functional fragment formats, can also be of type (HCH2CH3) ₂ (H representing the "Hinge" hinge region of a human immunoglobulin; CH2 and CH3 representing the second and third constant domains of a heavy chain of a human Ig), and are characterized by the association of two identical human VHHs or two human VHs or of two human VHs on which the hypervariable regions are grafted VHHs, each being fused to the H-CH2-CH3 region of a human Ig. Also included are antibody formats, functional antibody derivative formats, or functional antibody fragment formats, of the mAb* type (this type designates the original variable domains replaced by all or part of the domains of VHH or of humanized VHH or of human VH, fused to constant regions of human antibodies), which are characterized by the association of two VHH or of two human VH, identical or different, or of two human VH on which are grafted the hypervariable regions of the VHHs, one being fused to the human Cκ or Cλ region, the other to the CH1-H-CH2-CH3 region of a human Ig.

The VHHs can be replaced by human VHs or humanized VHHs by grafting the CDRs of the VHHs onto human VHs.

The immunoglobulin used is preferably an IgG, such as a human IgG1, IgG2, IgG3 or IgG4 isoform, or a human IgA corresponding to an IgA1, IgA2 isoform, or any other human Ig.

Advantageously, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) according to the invention is capable of recognizing an RBD domain (Receptor Binding Domain) of a Spike protein of sarbecovirus, the sarbecovirus being preferably chosen from the SARS-CoV1 virus, the variants of the SARS-CoV1 virus, the SARS-CoV2 virus, and the variants of the SARS-CoV2 virus.

According to a preferred embodiment, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) is capable of recognizing an RBD domain of a Spike protein of SARS-CoV1 virus and an RBD domain of a Spike protein of the SARS-CoV2 virus, in particular as demonstrated by the data obtained by the inventors.

According to a particularly advantageous embodiment, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) is capable of recognizing an RBD domain of a Spike protein of the SARS- original CoV2, and an RBD domain of a Spike protein of at least one variant of the original SARS-CoV2 virus, the variant being preferably chosen from the variants listed in the definition section above, the variant being in particular chosen from an Alpha variant, a Beta variant, a Gamma variant, a Delta variant, an Epsilon variant, a Zeta variant, an Eta variant, a Teta variant, an Iota variant, a Kappa variant, a Lambda variant, and any combination of these; preferably selected from an Alpha variant, a Beta variant, a Gamma variant, a Delta variant, and any combination thereof.

The binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) according to the invention is in particular derived from the antibody VHH72. The inventors have shown that, surprisingly, the binding agents (in particular the single-domain antibodies) thus developed have an affinity for the various RBD domains of the variants of the SARS-CoV2 virus that is significantly higher than that of VHH72 for these domains. RBD.

Thus, according to a particularly advantageous embodiment, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) according to the invention has a dissociation constant K _{d (VHH72 substituted )} measured for an RBD domain of a Spike protein of SARS-CoV2 virus which is lower than the dissociation constant K _{d (unsubstituted VHH72)} of the single domain antibody VHH72 comprising the amino acid sequence SEQ ID NO: 1 without substitution (ie, the original VHH72) measured for an RBD domain of a Spike protein of SARS-CoV2 virus.

According to a preferred embodiment, the dissociation constant K _{d(substituted VHH72)} measured for an RBD domain of a Spike protein of SARS-CoV2 virus, is lower (preferably significantly lower) than the dissociation constant K _{d(VHH72 unsubstituted)} measured for an RBD domain of a Spike protein of SARS-CoV2 virus, preferably at least 10 times lower, more preferably at least 11 times lower, more preferably at least 12 times lower , more preferably at least 13 times lower, more preferably at least 14 times lower, more preferably at least 15 times lower, more preferably at least 20 times lower, more preferably d at least 25 times, more preferably at least 30 times less, more preferably at least 40 times less, more preferably at least 50 times less, more preferably at least 100 times less, more preferably at least 200 times lower, more preferably at least 300 times lower, more preferably at least 400 times lower, more preferably at least 500 times lower, more preferably at least 600 times, more preferably at least 700 times less, more preferably at least 800 times less, more preferably at least 900 times less, more preferably at least 1000 times less.

Nucleic acid molecule - Vectors - Host cells

The present invention also relates to a nucleic acid molecule encoding a binding agent (such as an antibody or a functional fragment of an antibody, in particular a single domain antibody) as described above.

Advantageously, the nucleic acid molecule comprises, or essentially consists of, or consists of, a nucleic acid sequence having at least 85% identity with a sequence chosen from SEQ ID NO: 9, SEQ ID NO: 10 , SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO 13, preferably having at least 86% identity, preferably having at least 87% identity, preferably having at least 88% identity, preferably having at least 89% identity, preferably having at least 90% identity, preferably having at least 91% identity, preferably having at least 92% identity, preferably having at least at least 93% identity, preferably having at least 94% identity, preferably having at least 95% identity, preferably having at least 96% identity, preferably having at least 97% identity , preferably having at least 98% identity, preferably having at least 99% identity, with a sequence chosen from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO : 12, and SEQ ID NO 13, more preferably a sequence chosen from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO 13.

The sequences SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO 13 are indicated in Table 3 below.

Table 3 : Sequences Included in Nucleic Acid Molecules Encoding Preferred Binding Agents

SEQ-ID Description Sequence SEQ ID NO:9 VHH6: Antibody comprising substitutions S57G; T58S; Y60L; T61S; D62L A98V; T103V; V104W. CAAGTCCAGTTGCAAGAATCTGGTGGTGTTGGTTCAAGCTGGTGGTTCTTTGAGATTGTCTTGTGCTGCTTCTGGTAGAACTTTTTCTGAATATGCTATGGGTTGGTTCAGACAAGCTCCAGGTAAAGAAAGAGAATTCGTTGCTACTATTTCTTGGTCAGGTGGTGGCTCGTACTTGTCGTTGTCTGTTAAGGGTAGATTCACCATTTCTAGAGATAACGCTAAGAACACCGTCTACTTGCAAATGAATTCTTT GAAGCCAGATGATACCGCTGTTTATTACTGTGCTGTAGCAGGTTTGGGAGTATGGGTTTCTGAATGGGATTATGATTACGACTATTGGGGTCAAGGTACTCAAGTTACTGTTTCCTCT SEQ ID NO: 10 VHH65: Antibodies comprising the substitutions S57G; Y60V; T61R; A98V; T103V; V104W. CAAGTCCAGTTGCAAGAATCTGGTGGTGTTGGTTCAAGCTGGTGGTTCTTTGAGATTGTCTTGTGCTGCTTCTGGTAGAACTTTTTCTGAATATGCTATGGGTTGGTTCAGACAAGCTCCAGGTAAAGAAAGAGAATTCGTTGCTACTATTTCTTGGTCAGGTGGTGGACGTACGTGAGGGACTCTGTTAAGGGTAGATTCACCATTTCTAGAGATAACGCTAAGAACACCGTCTACTTGCAAATGAATTCTTT GAAGCCAGATGATACCGCTGTTTATTACTGTGCTGTAGCAGGTTTGGGAGTATGGGTTTCTGAATGGGATTATGATTACGACTATTGGGGTCAAGGTACTCAAGTTACTGTTTCCTCT SEQ ID NO:11 VHH66: Antibodies comprising substitutions S57G; Y60V; T61S; D62L; A98V; T103V; V104W. CAAGTCCAGTTGCAAGAATCTGGTGGTGTTGGTTCAAGCTGGTGGTTCTTTGAGATTGTCTTGTGCTGCTTCTGGTAGAACTTTTTCTGAATATGCTATGGGTTGGTTCAGACAAGCTCCAGGTAAAGAAAGAGAATTCGTTGCTACTATTTCTTGGTCAGGTGGTGGGACGTACGTGTCGTTGTCTGTTAAGGGTAGATTCACCATTTCTAGAGATAACGCTAAGAACACCGTCTACTTGCAAATGAATTCTTT GAAGCCAGATGATACCGCTGTTTATTACTGTGCTGTAGCAGGTTTGGGAGTATGGGTTTCTGAATGGGATTATGATTACGACTATTGGGGTCAAGGTACTCAAGTTACTGTTTCCTCT SEQ ID NO:12 VHH71: Antibody comprising substitutions S57G; Y60V; T61S; D62F; A98V; T103V; V104W. CAAGTCCAGTTGCAAGAATCTGGTGGTGTTGGTTCAAGCTGGTGGTTCTTTGAGATTGTCTTGTGCTGCTTCTGGTAGAACTTTTTCTGAATATGCTATGGGTTGGTTCAGACAAGCTCCAGGTAAAGAAAGAGAATTCGTTGCTACTATTTCTTGGTCAGGTGGTGGCACGTACGTCTCGTTCTGTTAAGGGTAGATTCACCATTTCTAGAGATAACGCTAAGAACACCGTCTACTTGCAAATGAATTCTTT GAAGCCAGATGATACCGCTGTTTATTACTGTGCTGTAGCAGGTTTGGGAGTATGGGTTTCTGAATGGGATTATGATTACGACTATTGGGGTCAAGGTACTCAAGTTACTGTTTCCTCT SEQ ID NO:13 VHH76: Antibodies comprising the substitutions S57G; Y60I; A98V; T103V; V104W. CAAGTCCAGTTGCAAGAATCTGGTGGTGTTGGTTCAAGCTGGTGGTTCTTTGAGATTGTCTTGTGCTGCTTCTGGTAGAACTTTTTCTGAATATGCTATGGGTTGGTTCAGACAAGCTCCAGGTAAAGAAAGAGAATTCGTTGCTACTATTTCTTGGTCAGGTGGTGCACGTACATCACGGACTCTGTTAAGGGTAGATTCACCATTTCTAGAGATAACGCTAAGAACACCGTCTACTTGCAAATGAATTCTTTGA AGCCAGATGATACCGCTGTTTATTACTGTGCTGTAGCAGGTTTGGGAGTATGGGTTTCTGAATGGGATTATGATTACGACTATTGGGGTCAAGGTACTCAAGTTACTGTTTCCTCT

The invention also relates to a vector comprising said nucleic acid molecule. The vector is advantageously a viral vector.

The invention further relates to a host cell comprising said nucleic acid molecule and/or said vector.

Compositions and kits

The present invention further relates to a composition or a kit comprising, or consisting essentially of, at least one binding agent (such as an antibody or a functional fragment of an antibody, in particular a single-domain antibody) as defined below. above.

The present invention also relates to a composition or a kit comprising, or consisting essentially of, at least one nucleic acid molecule according to the invention (as defined above).

The present invention also relates to a composition or a kit comprising, or essentially consisting of, at least one vector according to the invention (as defined above).

The present invention also relates to a composition or a kit comprising, or essentially consisting of, at least one host cell according to the invention (as defined above).

The present invention also relates to a composition or a kit comprising, or consisting essentially of:

• at least one binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, or
• at least one nucleic acid molecule as defined above, or
• at least one vector as defined above, or
• at least one host cell as defined above, or
• any combination of these.

Advantageously, the composition or the kit comprises, or essentially consists of, a combination chosen from:

• a combination of at least two distinct binding agents (such as an antibody or a functional antibody fragment, in particular a single-domain antibody), as defined above;
• a combination of two or more distinct nucleic acid molecules, as defined above (i.e., each encoding a distinct single domain antibody);
• a combination of at least two distinct vectors, as defined above, or
• a combination of two or more distinct host cells, as defined above, or
• any combination of these.

The composition is preferably a pharmaceutical composition. It can therefore comprise a pharmaceutically acceptable vehicle.

According to one embodiment, the pharmaceutical composition is presented in a form specific to administration by injection, or in a form specific to administration using an adhesive transdermal therapeutic system (for example in the form of a patch), or in a form suitable for administration by spraying or vaporization (e.g. in the form of a spray, vaporizer, spray, etc.), or in a form suitable for topical administration (e.g. in a form of a cream, gel, milk, serum, etc.), etc.

Advantageously, the kit is a kit chosen from:

• a kit for treating at least one disease linked to at least one sarbecovirus in a subject, the disease being preferably chosen from infections by a sarbecovirus;
• a kit for detecting (preferably in vitro ) the presence or absence of at least one sarbecovirus in a sample;
• a kit for determining (preferably in vitro ) the amount of at least one sarbecovirus in a sample;
• a kit for screening (preferably in vitro ) compounds/molecules capable of detecting and/or recognizing at least one sarbecovirus, preferably for screening compounds/molecules capable of neutralizing at least one sarbecovirus;
• a kit for the diagnosis (preferably in vitro ) of a disease linked to sarbecoviruses in a subject likely to suffer from a disease linked to sarbecoviruses, the disease being preferably chosen from infections by a sarbecovirus;
• a kit for the prognosis and/or monitoring and/or stratification (preferably in vitro ) of a subject suffering from a disease linked to sarbecoviruses, the disease being preferably chosen from infections by a sarbecovirus;
• a kit for evaluating the efficacy of a treatment (in particular a curative and/or preventive treatment) administered to a subject suffering from a disease linked to sarbecoviruses or susceptible to contracting a disease linked to sarbecoviruses, the disease being of preferably chosen from infections by a sarbecovirus; And
• any combination of these.

Advantageously, the kit further comprises suitable means for detecting the presence or absence of at least one sarbecovirus in a sample.

The detection or quantification of sarbecoviruses can be carried out by any technique known to those skilled in the art. Such techniques include in particular any technique for the detection and/or quantification of proteins (for detecting the virus at the protein level, by detecting and/or quantifying viral proteins), such as flow cytometry (in particular cell sorting activated by fluorescence ( FACS)), western blot, enzyme immunoassay, LFA (lateral flow assay), ELISA, ELISPOT, antibody chips, immunoprecipitation, immunohistology, cell membrane staining by biotinylation or other equivalent techniques followed by immunoprecipitation with specific antibodies, dot blot, protein microarray, tissue microarray, antibody microarray, nucleic acid microarray, immunohistochemistry. Other suitable techniques include FRET or BRET, single cell microscopy or histochemistry methods using one or more excitation wavelengths and applying any of the suitable optical methods, electrochemical methods (technical voltammetry and amperometry), atomic force microscopy, radiofrequency methods (e.g. multipole resonance spectroscopy), confocal and non-confocal microscopy, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance and birefringence or refractive index, cell ELISA, radioisotope, magnetic resonance imaging, polyacrylamide gel electrophoresis analysis (SDS-PAGE), HPLC, mass spectroscopy , spectrometry, chromatography coupled to (eg, liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS)).

The amount of sarbecovirus can be measured, for example, at the nucleic level, by measuring the amount of sarbecovirus DNA and/or sarbecovirus transcripts (mRNA or regulatory RNA) and/or sarbecovirus cDNA. In this case, any technology usually used by those skilled in the art can be implemented. These technologies include well-known methods such as PCR (polymerase chain reaction, if starting from DNA), RT-PCR (reverse transcription-PCR, if starting from RNA), RT-PCR quantitative (RT-qPCR), nucleic acid-based sensors, nucleic acid microarrays (including DNA arrays and oligonucleotide arrays), transcriptome analysis, RNA-seq analysis (including including 3'RNASeq, 5'RNA-seq, 3'scRNA-Seq, 5'scRNA-Seq). It is also possible to use hybridization with a labeled nucleic acid probe, such as Northern blot (for mRNA) or Southern blot (for cDNA) or fluorescence in situ hybridization (FISH) , but also by techniques such as the Serial Gene Expression Analysis (SAGE) method and its derivatives, such as LongSAGE, SuperSAGE, DeepSAGE, etc. It is also possible to use tissue chips (also called TMA: “tissue microarrays”). Tests typically used with tissue chips are immunohistochemistry and fluorescent in situ hybridization. For mRNA analysis, tissue arrays can be coupled with fluorescent in situ hybridization. Finally, it is possible to use massive sequencing in parallel to determine the quantity of mRNA in the sample (RNA-Seq or "Whole Transcriptome Shotgun Sequencing").

At the viral particle level, the amount of sarbecovirus can be measured, for example, by measuring the amount of particles (infectious particles and/or non-infectious particles), using one of the techniques listed below to measure the particles infectious viruses. Any technique well known in the art for the detection/quantification of infectious viral particles can be used, for example techniques based on the use of anti-virus antibodies such as the antibody according to the invention, spectrophotometry, quantitative immunofluorescence, the quantification of sarbecovirus-positive cells after infection by quantifying viral protein, RNA or DNA in host cells (using any protein or nucleic acid quantification technique mentioned above; preferably after a cell purification step, for example after removing the cell culture and washing the cells using an appropriate medium), etc.

In addition, all the technologies mentioned above can be used to detect at least one sarbecovirus (at the level of nucleic acids, proteins and/or particles).

It is possible to use a combination of one or more of the techniques cited above.

Advantageously, the kit or the composition further comprises a molecule competing for the binding of the antibody to the RBD domain of sarbecovirus (that is to say molecules entering into competition with the antibody for binding to the RBD domain of sarbecovirus ). Such competitor molecules include in particular the cellular receptor ACE2 or fragments thereof (provided that these fragments are capable of binding to the RBD domain of sarbecovirus). Indeed, the use of such a molecule makes it possible to effectively and reliably detect the presence or absence of sarbecovirus in a sample, as shown by the data obtained by the inventors.

Alternatively or in combination, the antibody is linked to a molecule detectable by the methods listed above, so as to allow its detection in a sample.

According to one embodiment, the kit according to the invention is characterized in that, when the kit comprises several antibodies and/or several nucleic acid molecules and/or several vectors and/or several host cells and/or a combination of these, the different components of the kit are:

1. all in one composition, or
2. distributed in several separate compositions in separate containers, including the case where each of the aptamers is in a separate composition located in a separate container.

According to one embodiment, the kit further comprises instructions for use.

The composition or the kit according to the invention may also comprise an excipient (chosen for example from carriers, solvents, diluents, adjuvants, dispersion media, coatings, preservatives, antibacterial and antifungal agents, absorbents, and any combination thereof). In the case of a kit, the various excipients may each be in a separate composition located in a separate container, or may be mixed in pairs or more in separate compositions in separate containers, or all in the same composition. They can also be mixed with one or more antibodies from the kit.

Non-limiting examples of excipients include water, NaCl, saline solutions, saccharide solutions (e.g. glucose, trehalose, sucrose, dextrose, etc.), Ringer's milk, alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, etc. (see for example the most recent edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins).

Mention may in particular be made, as adjuvant, of swelling agents such as, for example, a sugar such as lactose, sucrose, trehalose, sorbitol, glucose, raffinose, mannitol, preferably lactose, sucrose, trehalose, glucose, or mannitol, an amino acid such as arginine, glycine, or histidine, preferably glycine, or polymers of dextran or polyethylene glycol type, or mixtures thereof. According to this embodiment, the pharmaceutical composition comprises 50 to 99% by weight of adjuvants, preferably 80 to 97% by weight, relative to the total weight of the composition.

The composition or the kit according to the invention can also comprise the compounds necessary for the amplification ( in vitro ) of the sarbecoviruses, in particular of the sarbecoviruses present in a sample (the sample being preferably chosen from nasopharyngeal samples, salivary samples , and oropharyngeal specimens), including specimens taken from patients with signs and symptoms suggestive of sarbecovirus-related disease (the disease preferably being selected from sarbecovirus infections, the disease more preferably being COVID- 19). The amplification of the sarbecoviruses can be carried out by any technique known to those skilled in the art. Such techniques include in particular methods for the amplification of viral nucleic acids, such as RT-PCR (reverse transcription-PCR), quantitative RT-PCR (RT-qPCR), gene amplification by TMA (Transcription Mediated Amplification ), loop-mediated isothermal amplification (LAMP), etc.

Uses and Therapeutic Methods

The inventors have in particular shown that, surprisingly, the binding agents (in particular the single-domain antibodies) thus developed have very good affinities for the RBD domains of different sarbecoviruses, including variants of the SARS-CoV2 virus. Remarkably, the data also show that these optimized antibodies are able to prevent the binding between the host cell ACE2 receptor and the RBD domain and to neutralize the virus, more effectively than binding agents (in particular antibodies single domain) described in the prior art. These data thus reveal the therapeutic potential of these binding agents to treat sarbecovirus-related diseases, including sarbecovirus infections (such as COVID-19).

The present invention therefore relates to a binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, a nucleic acid molecule as defined above, a vector as defined above, a host cell as defined above, a composition as defined above, or a kit as defined above, for its use as a medicament.

Advantageously, the medicament is a vaccine.

The present invention therefore relates to a binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, a nucleic acid molecule as defined above, a vector as defined above, a host cell as defined above, a composition as defined above, or a kit as defined above, for its use in the prevention and/or treatment of a disease, the disease preferably being chosen from the diseases linked to sarbecoviruses.

According to a preferred embodiment, the disease linked to sarbecoviruses is chosen from infections by a sarbecovirus, the sarbecovirus being preferably chosen from the original SARS-CoV1 virus, the variants of the original SARS-CoV1 virus, the virus Origin SARS-CoV2, and Origin SARS-CoV2 virus variants (including as listed in the “Definitions” section above). According to a particularly preferred embodiment, the sarbecovirus-related disease is a COVID disease, preferably COVID-19.

According to a preferred embodiment, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, the nucleic acid molecule as defined above above, the vector as defined above, the host cell as defined above, the composition as defined above, or the kit as defined above, is administered to a subject in need thereof, preferably in a therapeutically effective amount.

The subject in need thereof is preferably a subject susceptible to contracting a disease linked to sarbecoviruses, susceptible to suffering from a disease linked to sarbecoviruses, or suffering from such a disease (in particular a subject diagnosed as suffering from such disease).

The present invention also relates to the use of a binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, of a nucleic acid molecule as defined above, a vector as defined above, a host cell as defined above, a composition as defined above, or a kit as defined above above, for the manufacture of a medicine.

Advantageously, the medicament is a vaccine.

The present invention also relates to the use of a binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, of a nucleic acid molecule as defined above, a vector as defined above, a host cell as defined above, a composition as defined above, or a kit as defined above above, for the manufacture of a medicament for the prevention and/or treatment of a disease, the disease preferably being chosen from diseases linked to sarbecoviruses (the disease still preferably being COVID-19).

The present invention also relates to the use of a binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, of a nucleic acid molecule as defined above, a vector as defined above, a host cell as defined above, a composition as defined above, or a kit as defined above above, as medicine.

Advantageously, the medicament is a vaccine.

The present invention also relates to the use of a binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, of a nucleic acid molecule as defined above, a vector as defined above, a host cell as defined above, a composition as defined above, or a kit as defined above above, for the prevention and/or treatment of a disease, the disease preferably being chosen from diseases linked to sarbecoviruses.

The present invention also relates to a method for preventing and/or treating a disease, the disease preferably being chosen from the diseases linked to sarbecoviruses, comprising the administration to a subject in need thereof, of a binding agent ( such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, of a nucleic acid molecule as defined above, of a vector as defined above above, of a host cell as defined above, of a composition as defined above, or of a kit as defined above.

According to a preferred embodiment of the uses and methods described above, the disease linked to sarbecoviruses is chosen from infections by a sarbecovirus, the sarbecovirus being preferably chosen from the original SARS-CoV1 virus, the variants of the virus Origin SARS-CoV1, Origin SARS-CoV2 Virus, and Origin SARS-CoV2 Virus Variants (including as listed in the “Definitions” section above). According to a particularly preferred embodiment, the sarbecovirus-related disease is a COVID disease, preferably COVID-19.

According to a preferred embodiment of the uses and methods described above, the binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, the nucleic acid molecule as defined above, the vector as defined above, the host cell as defined above, the composition as defined above, or the kit as defined above, is administered to a subject in need thereof, preferably a therapeutically effective amount.

The subject in need thereof is preferably a subject susceptible to contracting a disease linked to sarbecoviruses, susceptible to suffering from a disease linked to sarbecoviruses, or suffering from such a disease (in particular a subject diagnosed as suffering from such disease).

In Vitro Uses and Methods

The data reveal that the binding agents (in particular single domain antibodies) according to the invention can be used as tools for diagnosis, prognosis, stratification or even monitoring of diseases linked to sarbecoviruses, in particular infections by a sarbecovirus , or to assess the effectiveness of a treatment.

The data show in particular that the antibodies according to the invention have a strong affinity for the RBD domain of a large number of sarbecoviruses. Remarkably, these antibodies are specific to sarbecoviruses.

The present invention therefore relates to the in vitro use of at least one binding agent according to the invention (as defined above), for:

1. the diagnosis of a sarbecovirus-related disease in a subject suspected of suffering from a sarbecovirus-related disease;
2. follow-up in a subject suffering from a disease linked to sarbecoviruses;
3. the stratification of a subject suffering from a disease linked to sarbecoviruses;
4. the evaluation of the effectiveness of a treatment (in particular curative) administered to a subject suffering from a disease linked to sarbecoviruses;
5. detect the presence or absence of at least one sarbecovirus in a sample;
6. determine the amount of at least one sarbecovirus in a sample;
7. screening compounds/molecules capable of detecting and/or recognizing at least one sarbecovirus; preferably screening compounds/molecules capable of neutralizing at least one sarbecovirus; Or
8. any combination of i. to vii. ;

the disease linked to sarbecoviruses being preferably chosen from infections by a sarbecovirus.

The present invention relates in particular to an in vitro method for diagnosing a disease linked to sarbecoviruses in a subject likely to suffer from a disease linked to sarbecoviruses, comprising:

1. bringing a biological sample from said subject into contact with at least one binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above;
2. detection of the presence or absence (and/or quantification) of at least one sarbecovirus in the biological sample from step a); And
3. diagnosing the presence or absence of the disease in said subject based on the result of step b);

the disease being preferably chosen from infections by a sarbecovirus, the sarbecovirus being preferably chosen from the original SARS-CoV1 virus, the variants of the original SARS-CoV1 virus, the original SARS-CoV2 virus, and variants of the original SARS-CoV2 virus (in particular as listed in the definition section above).

Advantageously, step b) of detection is carried out as described above in connection with the compositions and the kits according to the invention.

According to one embodiment, the subject is suffering from a disease linked to sarbecoviruses, if the presence of at least one sarbecovirus is detected in the sample. Conversely, the subject is not affected by a disease linked to sarbecoviruses, if the presence of at least one sarbecovirus is not detected in the sample.

According to one embodiment, the method further comprises a step a′) carried out before step a), of amplification ( in vitro ) of the sarbecoviruses possibly present in a biological sample of said subject (called sample (A)). The amplification of the sarbecoviruses can be carried out by any technique known to those skilled in the art. Such techniques are in particular described in the “Compositions and Kits” section above.

According to an alternative embodiment or in combination with the embodiments described above, the method further comprises a step b′) carried out between step b) and step c), the detection of the presence or the absence (and/or quantification) of at least one sarbecovirus, in one or more biological sample(s) from reference subject(s). The reference subject(s) preferably comprises at least one reference subject suffering from a sarbecovirus-related disease and/or at least one healthy reference subject. As a sample from a reference subject suffering from a disease linked to sarbecoviruses, it is possible in particular to use a sample from a subject suffering from an infection by a sarbecovirus, the sarbecovirus preferably being chosen from the SARS-CoV1 virus of origin, the variants of the original SARS-CoV1 virus, the original SARS-CoV2 virus, and the variants of the original SARS-CoV2 virus (in particular as listed in the definition section above).

Several samples from reference subjects suffering from different sarbecovirus infections can be used in step b’). According to this embodiment, the method may further comprise a step b'') carried out between step b') and step c), of comparing the detected sarbecoviruses (in particular the quantities of detected sarbecoviruses), for the steps b) and b'). In this case, step c) includes diagnosing the subject's sarbecovirus-related disease based on the comparison in step b''). The subject will be diagnosed as suffering from a disease linked to sarbecoviruses if the comparison of step b'') shows that the result of step b) is comparable to that obtained in step b') for a sample of reference of a reference subject suffering from a sarbecovirus-related disease (according to the different reference samples, a more precise diagnosis among the sarbecovirus-related diseases can potentially be made on the same principle), and as not suffering from a disease linked to sarbecoviruses if the result of step b) is comparable to that obtained in step b′) for a reference sample from a healthy reference subject.

Thus, the method according to the invention may comprise steps a′), a), b), and c) as described above, or steps a), b), b′), and c) such as described above, or steps a'), a), b), b'), and c) as described above, or steps a), b), b'), b''), and c) as described above, or else steps a'), a), b), b'), b''), and c) as described above.

The present invention also relates to an in vitro method for the prognosis, follow-up, stratification, or any combination thereof, of a subject suffering from a disease linked to sarbecoviruses, comprising:

1. bringing a biological sample from said subject into contact with at least one binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above;
2. detection of the presence or absence (and/or quantification) of at least one sarbecovirus in the biological sample from step a); And
3. the prognosis, follow-up, stratification, or any combination thereof, of the disease in said subject based on the result of step b);

the disease being preferably chosen from infections by a sarbecovirus, the sarbecovirus being preferably chosen from the original SARS-CoV1 virus, the variants of the original SARS-CoV1 virus, the original SARS-CoV2 virus, and variants of the original SARS-CoV2 virus (in particular as listed in the definition section above).

The present invention also relates to an in vitro method for evaluating the efficacy of a treatment (in particular curative) administered to a subject suffering from a disease linked to sarbecoviruses, comprising:

1. bringing a biological sample from said subject into contact with at least one binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above;
2. detection of the presence or absence (and/or quantification) of at least one sarbecovirus in the biological sample from step a); And
3. the evaluation of the effectiveness of the treatment in said subject according to the result of step b);

the disease being preferably chosen from infections by a sarbecovirus, the sarbecovirus being preferably chosen from the original SARS-CoV1 virus, the variants of the original SARS-CoV1 virus, the original SARS-CoV2 virus, and variants of the original SARS-CoV2 virus (in particular as listed in the definition section above).

The present invention further relates to an in vitro method for evaluating the efficacy of a preventive treatment for a sarbecovirus-related disease (such as a vaccine), administered to a subject susceptible to contracting the sarbecovirus-related disease, including:

1. bringing a biological sample from said subject into contact with at least one binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above;
2. detection of the presence or absence (and/or quantification) of at least one sarbecovirus in the biological sample from step a); And
3. the evaluation of the effectiveness of the preventive treatment in said subject according to the result of step b);

the disease being preferably chosen from infections by a sarbecovirus, the sarbecovirus being preferably chosen from the original SARS-CoV1 virus, the variants of the original SARS-CoV1 virus, the original SARS-CoV2 virus, and variants of the original SARS-CoV2 virus (in particular as listed in the definition section above).

Advantageously, step b) of detection (and/or quantification) of the different methods is carried out as described above in connection with the compositions and the kits according to the invention.

According to a preferred embodiment of methods for prognosis, monitoring, stratification, or any combination thereof, or evaluating the effectiveness of a treatment, the sarbecovirus-related disease is worsened, or the prognosis of the sarbecovirus-related disease is negative, or the sarbecovirus-related disease has progressed, or the treatment of the sarbecovirus-related disease is ineffective or not very effective, if the presence of at least one sarbecovirus is detected in the sample.

According to one embodiment, the method further comprises a step a′) carried out before step a), of amplification ( in vitro ) of any sarbecoviruses present in a biological sample of said subject (called sample (A)). The amplification of the sarbecoviruses can be carried out by any technique known to those skilled in the art. Such techniques are in particular described in the “Compositions and Kits” section above.

According to an alternative embodiment or in combination with the embodiments described above, the method further comprises a step b′) carried out between step b) and step c), of detecting the presence or absence (and/or quantification) of at least one sarbecovirus in one or more biological sample(s) from reference subject(s). The reference subject(s) preferably comprises at least one subject suffering from a sarbecovirus-related disease at a known prognosis/stage/level of stratification (preferably at least one reference subject suffering from a sarbecovirus-related disease at a known prognosis/stage/level of stratification, the sarbecovirus-related disease being the same for the reference subject and for the tested/prognosticated subject) and/or at least one healthy reference subject. As a sample from a reference subject suffering from a disease linked to sarbecoviruses, it is possible in particular to use a sample from a subject suffering from an infection by a sarbecovirus, the sarbecovirus preferably being chosen from the SARS-CoV1 virus of origin, the variants of the original SARS-CoV1 virus, the original SARS-CoV2 virus, and the variants of the original SARS-CoV2 virus (in particular as listed in the definition section above). Several samples from reference subjects suffering from different sarbecovirus infections can be used in step b’). Preferably, several samples from reference subjects suffering from the same sarbecovirus-related disease but with known different prognoses/stages/levels of stratification can be used in step b'). According to this embodiment, the method may further comprise a step b'') carried out between step b') and step c), of comparing the detected sarbecoviruses (in particular the quantities of detected sarbecoviruses), for the steps b) and b'). In this case, step c) includes the prognosis, monitoring, stratification, or any combination thereof, or the evaluation of the effectiveness of a treatment, of the sarbecovirus-related disease in the subject, depending on the comparison of step b''). The subject will have a comparable prognosis, and/or will be stratified as being at a comparable stage/level of stratification, to a reference subject with the same sarbecovirus-related illness at a known prognosis/stage/level of stratification, if the comparison of step b'') shows that the result of step b) is comparable to that obtained in step b') for a reference sample of the reference subject (according to the different reference samples, more precise prognosis, monitoring, stratification, and/or evaluation among the different stages can potentially be done on the same principle). The subject will have a more negative prognosis, and/or will be stratified as being at a more advanced (more severe) stage/level of stratification, than a reference subject with the same sarbecovirus-related illness at a prognosis/stage/level of known stratification or of a healthy reference subject, if the result of step b) shows a greater detection of sarbecovirus or a higher quantity of sarbecovirus, than that obtained in step b′) for a sample of reference of the reference subject or of the healthy reference subject. Conversely, the subject will have a better prognosis, and/or will be stratified as being at a less advanced stage/level of stratification (less severe), than a reference subject with the same sarbecovirus-related illness with a prognosis /stage/level of stratification known, if the result of step b) shows a lower detection of sarbecovirus, or a lower quantity of sarbecovirus, than that obtained in step b') for a reference sample of the subject reference.

Thus, the method according to the invention may comprise steps a′), a), b), and c) as described above, or steps a), b), b′), and c) such as described above, or steps a'), a), b), b'), and c) as described above, or steps a), b), b'), b''), and c) as described above, or else steps a'), a), b), b'), b''), and c) as described above.

According to an alternative embodiment or in combination with the embodiments described above, the in vitro method of stratifying a sarbecovirus-related disease, of prognosing a sarbecovirus-related disease, of monitoring a to sarbecoviruses, or for evaluating the effectiveness of a treatment for a disease linked to sarbecoviruses in a subject suffering from a disease linked to sarbecoviruses, further comprises a step b′) carried out between step b) and step c), of detecting the presence or the absence (and/or the quantification) of at least one sarbecovirus, in a second biological sample of the subject tested (called sample (B)). Said second sample (B) was preferably obtained/taken after sample (A), for example during a second visit (sample (A) then having been obtained during a first visit), preferably said sample (B) having been obtained at least 24 hours after sample (A), more preferably at least 48 hours after sample (A), more preferably at least 72 hours after sample (A ), more preferably at least 7 days after sample (A), more preferably at least 10 days after sample (A), more preferably at least 15 days after sample (A), more preferably at least 1 month after the sample (A), more preferably at least 2 months after the sample (A), more preferably at least 3 months after the sample (A), more preferably at least 4 months after the sample (A), more preferably at least 5 months after the sample (A), more preferably at least 6 months after the sample (A), more preferably at least 7 months after the sample ( A), more preferably at least 8 months after the sample (A), more preferably at least 9 months after the sample (A), more preferably at least 10 months after the sample (A), preferably still at least 11 months after sample (A), preferably still at least 12 months after sample (A). More preferably said sample (B) was obtained between 7 days and 6 months after sample (A), more preferably said sample (B) having been obtained between 10 days and 5 months after sample (A), more preferably between 15 days and 4 months, more preferably between 21 days and 3 months, more preferably between 30 and 60, more preferably between 40 and 50 days. According to this embodiment, the method may further comprise a step b'') carried out between step b') and step c), of comparing the detected sarbecoviruses (in particular the quantities of detected sarbecoviruses), for the steps b) (therefore for the sample (A) of the subject) and b′) (thus for the sample (B) of the subject). In this case, step c) includes stratification of the sarbecovirus-related disease, prognosis of the sarbecovirus-related disease, monitoring of the sarbecovirus-related disease, evaluation of the effectiveness of the treatment of the to sarbecoviruses, in the subject, according to the comparison of step b''). The subject will be stratified as being at an unchanged/comparable stage/level of stratification, or the subject will have an unchanged/comparable prognosis, or the subject will have little or no progression of sarbecovirus disease (will be stable), or the treatment of the disease linked to sarbecoviruses will be reasonably effective, if the comparison of step b′) shows that the result of step b) is comparable to that obtained in step b′).

The subject will be stratified as being at a more advanced (more severe) stage/level of stratification, or the subject will have a more negative prognosis, or the subject's sarbecovirus-related illness will have progressed (worsened, worsened) in the subject, or the treatment of the disease linked to sarbecoviruses will be ineffective or not very effective, if the result of step b) shows a greater detection of sarbecovirus, or a higher quantity of sarbecovirus, than that obtained in step b′). Conversely, the subject will be stratified as being at a less advanced stage/level of stratification (less severe), or the subject will have a better prognosis, or the sarbecovirus-related illness will have improved (will be less severe, will have receded) in the subject, or the treatment of the disease linked to sarbecoviruses will be effective, if the result of step b) shows a lower detection of sarbecovirus, or a lower quantity of sarbecovirus, than that obtained in step b' ).

The method according to the aforementioned embodiment may also further comprise a step a′) carried out before step a), of amplification ( in vitro ) of any sarbecoviruses present in a biological sample of said subject (called sample (A)) . The amplification of the sarbecoviruses can be carried out by any technique known to those skilled in the art. Such techniques are in particular described in the “Compositions and Kits” section above.

Thus, the method according to the invention may comprise steps a′), a), b), and c) as described above, or steps a), b), b′), and c) such as described above, or steps a'), a), b), b'), and c) as described above, or steps a), b), b'), b''), and c) as described above, or else steps a'), a), b), b'), b''), and c) as described above.

According to one embodiment, if the subject is stratified as being at a more advanced (more severe) stage/level of stratification, or if the subject has a more negative prognosis, or if the sarbecovirus-related illness has progressed (worsened, has worsened) in the subject, or if the treatment of the illness linked to sarbecoviruses is ineffective or not very effective;

• treatment may be administered to the subject if the subject was not on treatment at the time the sample was collected; Or
• the treatment may be modified (for example by modifying the drug and/or the dose of drug administered and/or the frequency of drug administration), if the subject was already under treatment at the time the sample was taken .

According to one embodiment, if the subject is stratified as being at a less advanced stage/level of stratification (less severe), or if the subject has a better prognosis, or if the sarbecovirus-related illness has improved (is less severe, has receded) in the subject, or whether treatment for sarbecovirus-related illness is effective;

• the treatment may be modified (for example by modifying the drug and/or the dose of drug administered and/or the frequency of drug administration), if the subject was already under treatment at the time the sample was taken ; Or
• no treatment will be administered to the subject if the subject was not on treatment at the time the sample was collected; Or
• the treatment may be stopped in the event of complete recovery.

The present invention also relates to the in vitro use of at least one binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, for the diagnosis of sarbecovirus-related disease in a subject suspected of having sarbecovirus-related disease, or for follow-up in a subject with sarbecovirus-related disease, or for stratification of a subject with sarbecovirus-related disease, or for evaluating the efficacy of a treatment (in particular curative) administered to a subject suffering from a sarbecovirus-related disease, or any combination thereof.

According to a preferred embodiment, the disease linked to sarbecoviruses is chosen from infections by a sarbecovirus, the sarbecovirus being preferably chosen from the original SARS-CoV1 virus, the variants of the original SARS-CoV1 virus, the virus original SARS-CoV2, and variants of the original SARS-CoV2 virus (in particular as listed in the definition section above).

The use may include in particular:

1. detection of the presence or absence of at least one sarbecovirus in a biological sample; and or
2. determining the amount of at least one sarbecovirus in a biological sample.

Advantageously, steps i and ii of use are implemented as described above in connection with the compositions and kits according to the invention.

Advantageously, the uses are implemented as described above in connection with the methods of diagnosis, prognosis, monitoring, stratification, or evaluation of the efficacy of a treatment, as defined above.

The inventors have shown that, surprisingly, the binding agents (in particular the single-domain antibodies) thus developed have very good affinities for the RBD domains of different sarbecoviruses, including variants of the SARS-CoV2 virus. Remarkably, the data also show that these optimized antibodies are able to prevent the binding between the host cell ACE2 receptor and the RBD domain, more effectively than binding agents (including single domain antibodies) described. in the prior art.

The data also show that these antibodies are tools for detecting sarbecoviruses. The present invention therefore provides tools for research in the field of diseases linked to sarbecoviruses.

The present invention therefore relates to an in vitro method for detecting the presence or absence of at least one sarbecovirus in a sample, comprising:

a) bringing the sample into contact with at least one binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above; And

b) detecting the presence or absence of at least one sarbecovirus in the sample from step a).

The present invention also relates to an in vitro method for determining the amount of at least one sarbecovirus in a sample, comprising

a) bringing the sample into contact with at least one binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above;

b) quantification of sarbecoviruses in the sample from step a); And

The sample can be any sample taken from nature. Advantageously, the sample is a plant sample, a sample taken from a living being such as an animal, an air sample, a soil sample, a soil sample, a water sample, a waste water sample , a sample of circulating water in the pipes of a building or in the ground, or even a sample of natural watercourses or a sample of seawater.

The present invention also relates to an in vitro method for screening compounds/molecules capable of detecting and/or recognizing at least one sarbecovirus, preferably for screening compounds/molecules capable of neutralizing at least one sarbecovirus, the method comprising:

a) bringing at least one test compound/molecule into contact with at least one sarbecovirus to obtain a mixture;

b) adding a binding agent (such as an antibody or a functional fragment of an antibody, in particular a single-domain antibody) as defined above to the mixture of step a);

c) the detection of the presence or the absence of at least one sarbecovirus in the mixture of step b), and/or the detection of the presence or the absence of at least one compound/a molecule to test.

Advantageously, the detection (and/or quantification) step of the aforementioned methods is carried out as described above in connection with the compositions and the kits according to the invention, mutatis mutandis .

Advantageously, the methods are implemented as described above in connection with the methods of diagnosis, prognosis, monitoring, stratification, or evaluation of the effectiveness of a treatment, as defined above, mutatis mutandis .

According to one embodiment, the method further comprises a step a′) carried out before step a), of amplification ( in vitro ) of any sarbecoviruses present in the sample. The amplification of the sarbecoviruses can be carried out by any technique known to those skilled in the art. Such techniques are in particular described in the “Compositions and Kits” section above.

The present invention also relates to an in vitro use of at least one binding agent (such as an antibody or a functional antibody fragment, in particular a single-domain antibody) as defined above, for:

1. detect the presence or absence of at least one sarbecovirus in a sample;
2. determine the amount of at least one sarbecovirus in a sample;
3. screening compounds/molecules capable of detecting and/or recognizing at least one sarbecovirus; preferably screening compounds/molecules capable of neutralizing at least one sarbecovirus; Or
4. any combination of a) to c).

The sample can be any sample taken from nature. Advantageously, the sample is a plant sample, a sample taken from a living being such as an animal, an air sample, a soil sample, a soil sample, a water sample, a waste water sample , a sample of circulating water in the pipes of a building or in the ground, or even a sample of natural watercourses or a sample of seawater.

Advantageously, the step of detecting (and/or quantifying) the aforementioned uses is carried out as described above in connection with the compositions and the kits according to the invention, mutatis mutandis .

Advantageously, the uses are implemented as described above in connection with the methods of diagnosis, prognosis, monitoring, stratification, or evaluation of the effectiveness of a treatment, as defined above, mutatis mutandis .

According to one embodiment, the use also comprises a preliminary step of amplification ( in vitro ) of any sarbecoviruses present in the sample. The amplification of the sarbecoviruses can be carried out by any technique known to those skilled in the art. Such techniques are in particular described in the “Compositions and Kits” section above.

The following examples are intended to illustrate the present invention, and cannot be considered as limiting.

Description of figures

: Deep Mutational Scanning probing the binding of VHH-72 to the RBD domain of the Spike protein of SARS CoV2.

(A) Two DNA libraries of single mutants of VHH72 (corresponding to the N-terminal and C-terminal part of the nanobody) were transformed into yeast using gap repair recombination. (B) General principle of functional screening by surface display in yeast. Cells are incubated with antigen and clones carrying VHH72 mutations with increased SARS-CoV2 RBD binding relative to the parental antibody are sorted by FACS. (C) Bivariate flow cytometric analysis of L1 and L2 libraries of yeast cells expressing VHH72 variants on their surface. Cells were double-labeled with biotinylated antigen/Streptavidin-PE (RBD SARS-CoV2 binding) and anti-HA tag APC markers (VHH expression). Libraries were enriched with 10% clonal cells expressing parental VHH72 with egfp protein to distinguish cells with increased antigen binding. The selected cells (delimited by a “sorted cells” box) were sorted and sequenced with Illumina Deep Sequencing.

: Mapping of VHH72 nanobody paratopes and design of combinatorial libraries for the selection of improved clones.

NGS-based heat map depicting the enrichment values of each unique VHH72 mutant after functional sorting in FACS. The enrichment score is a base-2 logarithmic function of the enrichment between sorted and unsorted VHH72 yeast populations for a given amino acid substitution. The corresponding table is colored in light gray for enriched mutations and dark gray for depleted mutations. The substitutions in black were selected for the design of combinatorial libraries aimed at identifying variants of VHH72 with improved binding properties. Due to the high diversity generated, two separate libraries were designed (with respective theoretical diversities of 4.14e4 and 1.12e7 clones).

: Yeast Surface Display-Based Library Screening and Sorting of VHH72 Clones with Improved Binding for SARS-CoV2 RBD Antigen.

(A) Library A was incubated with 10nM SARS-CoV2 RBD and analyzed by FACS. Few clones with better antigen binding were detectable. Library B was double-sorted at 10 nM and 1 nM SARS-CoV2 RBD concentrations, respectively. In the second round, a selection based on K _off was undertaken. After equilibration with 1 nM antigen, an excess amount of 100 nM non-biotinylated antigen was introduced to drive dissociation and limit reassociation. (B) Clones selected from library B after two rounds of FACS selection. All selected clones show strong binding to RBD antigens of SARS-CoV-1 and SARS-CoV-2.

: (A) Sequence logo of clones contained in Library B before sorting (B) Sequence logo of selected clones after FACS-based affinity maturation selection.

: Affinity of the VHH-Fc Mut76 antibody for the RBD of SARS-CoV and SARS-CoV-2.

Measurement of the affinity of VHH-Fc Mut76 for the RBD of SARS-CoV-2 by biolayer interferometry. Antibodies were loaded onto anti-human Fc biosensors via protein A for measurement of SARS-CoV-2 RBD association and dissociation at varying concentrations.

: ELISA neutralization test of the link between the ACE2 receptor and the RBD domain of the SARS-CoV2 virus.

This test assesses the ability of selected VHH-Fc antibodies to block the interaction between ACE2 and different variants of the RBD domain. IC50s were determined using Prism 6.0 software. Values were generated from 3 independent experiments.

: Molecular modeling of the interaction between mutated VHH-72 (with S57G/T103V/V104W mutations) and SARS-CoV-2 RBD.

(A) Location of amino acid substitutions present in the different variants of concern (VOC) of SARS-CoV-2 in the homology pattern of the interaction between mutated VHH72 and the RBD domain of SARS-CoV- 2. (B) Interaction of mutated tryptophan residue W104 with a hydrophobic cavity bounded by RBD residues F377 and P384. The SARS-CoV2 Y369 RBD residues show a differential preferential conformation with respect to the SARS-CoV-1 structure, imposed by the presence of a proline at position 384 in SARS-CoV-2. Introduction of the S57G mutation could introduce more flexibility into CDR2 and could help accommodate the preferential orientation of Y369 to interface with VHH72.

Examples

EXAMPLE: Design and development of broad-spectrum VHH72 antibodies

1. Materials and methods

1.1. Design and generation of DMS (Deep Mutational Scanning) libraries

Two libraries of VHH72 variants (whose wild-type, unmutated sequence is SEQ ID NO:1) with single amino acid mutations were generated by splicing overlay extension PCR (SOE-PCR) using degenerate NNK primers. A library was generated for each of the two targeted regions (bank 1: amino acids 1 to 59 and bank 2: amino acids 61 to 125) corresponding respectively to a theoretical diversity of 1888 and 2112 DNA variants.

1.2. Generation of combinatorial libraries for affinity maturation

The design of mutagenic primers containing degenerate codons was performed using SwiftLib (http://rosettadesign.med.unc.edu/SwiftLib/). Two distinct libraries were designed, corresponding respectively to the CDRH1 and CDRH2+CDRH3 regions.

1.3. Display of VHH on the surface of yeasts

The preparation of EBY100 competent yeast cells (ATCC® MYA-4941) and the transformation of the libraries were carried out according to Benatuil et al [27]. The transformations using the gap-repair technique were carried out in the plasmid pNT VHH72 between the NheI and NotI restriction sites with 1 μg of digested vector and a molar ratio of 12:1 (linear library/digested vector; (AT)). After transformation, 250 mL of SD-CAA medium [6.7 g/L yeast nitrogenous base without amino acids, 20 g/L glucose, 5 g/L amino acids, 100 mM sodium phosphate, pH 6.0] were seeded with 400 µL of transformed cells and incubated overnight at 30°C, 200 rpm. The saturated culture (typically OD600 of 8-10) was passaged to obtain an initial culture OD600 of 0.25-0.50 in 50 mL. The culture was grown at 30°C until its OD600 reached 0.5-1.0. The cells were centrifuged and resuspended in 50 mL of SG-CAA galactose induction medium [6.7 g/L yeast nitrogenous base without amino acids, 20 g/L galactose, 5 g/L d amino acids, 100 mM sodium phosphate, pH 6.0] and induced for 16-36 h at 20°C, 200 rpm.

1.4. Flow cytometry

For sorting the banks, 10 ⁷ to 2.10 ⁸ induced cells from each bank were washed with 1 mL of PBSF buffer (PBS, 0.1% BSA). Cells were resuspended in PBSF with the appropriate concentration of SARS-CoV2 RBD antigen ( (B)), using adequate volume to avoid ligand depletion, as done in Hunter et al [28]. After incubation at 20°C with shaking for 3 hours, the cells were washed with ice cold PBSF to avoid dissociation. Cells were incubated on ice in the dark for 15 minutes with anti-HA antibody (Invitrogen HA Tag Mouse anti-Tag, DyLight® 650 conjugate, Clone: 2-2.2.14; 1:100 dilution) and Streptavidin- PE (Thermo Fisher scientific; catalog number S866; 1:100 dilution). Cells were then washed with ice-cold PBSF and sorted with a BD FACS Aria™ III cytometer using BD FACSdiva™ software. Diagonal gates were defined to sort cells with VHH expression and strong SARS-CoV-2 RBD binding ( (VS)).

1.5. Massive sequencing and analysis of NGS data

Plasmid DNA from each yeast population was extracted using Zymoprep Yeast Plasmid Miniprep II and prepared for sequencing as described in Medina-Cucurella and Whitehead [29]. A two-step PCR was performed to amplify the region of interest and add adapters and Illumina barcodes for multiplexing. Deep sequencing was performed with an Illumina ISeq 100 device (2x150 bp, 300 cycles) with at least 300,000 reads per population. Reads were demultiplexed and each sample was processed separately using the Galaxy platform (https://usegalaxy.org/) using functions described in Blankenberg et al [30]. First, the paired reads were joined (Fastq Joiner). A selection was then performed (Fastq Trimmer) on the reads to keep only the region of interest in the correct reading frame. A quality filter (Filter FASTQ) was applied to eliminate reads with a minimum quality score below 30. Then, DNA sequences were translated into protein sequences and identical sequences were grouped together. Sequences not repeated at least twice were filtered out. Using RStudio software, the calculation of enrichment ratios for each unique mutation was performed.

1.6. Production and purification of VHH72-Fc ligands

The VHH72-Fc and RBD SARS-CoV2 constructs were obtained by transient transfection of HEK293 Freestyle ^TM (Thermo). The synthetic genes corresponding to the selected VHH72 mutants were ordered from IDT in the form of "gblocks" and cloned into the mammalian expression vector pcDNA 3.4 VHH72-Fc. The genes encoding the different RBD domains were cloned into the pCAGGS RBD-SARS-CoV2 plasmid, which is a donation from Florian Krammer's laboratory. HEK293 Freestyle ^TM were transiently transfected at a density of 2.5 106 cells/mL in 100mL of Freestyle medium (Thermo-Fisher) by adding 150 µg of plasmid and 1.8 mL of linear polyethyleneimine ( PEI, 0.5 mg/ml) (Polysciences). After 24 h, 100 mL of fresh medium was added. After seven days at 37°C, 120 rpm, 8% CO2, the supernatant was purified using HiTrap Protein A for the VHH-Fc constructs or HisTrap Excel for the RBD constructs, following the manufacturer's instructions (GE Healthcare). Size exclusion chromatography was performed (HiPrep Sephacryl-S-200HR or S100HR) with PBS.

1.7. Affinity measurement by BioLayer interferometry

Binding kinetics were determined using an Octet RED96 instrument (ForteBio). Anti-hIgG Fc Capture (AHC) biosensors (Fortebio) were loaded with VHH-Fc molecules (50 nM) for 60 seconds. After a baseline using kinetic buffer (PBS, 0.5% (w/v) BSA and 0.05% (v/v) Tween 20), the combination of RBD SARS-CoV2 or RBD SARS -CoV1 (Sinobiological) was measured at different concentrations (50 nM to 0.78 nM) for 300 seconds before dissociation in kinetic buffer. Data from the antigen-free control were subtracted from all binding curves and binding kinetics were fitted using a 1:1 global Langmuir binding model.

1.8. Neutralization test

Attempts to neutralize the binding between the ACE2 receptor and the RBD domain of the SARS-CoV2 virus are carried out in ELISA. This test assesses the ability of selected VHH-Fc antibodies to block the interaction between ACE2 and different variants of the RBD domain. IC50s were determined using Prism 6.0 software. Values were generated from 3 independent experiments.

2. Results

To improve the affinity of VHH72 with respect to the RBD domain of the Spike protein of the SARS-CoV2 virus, the inventors undertook affinity maturation by molecular engineering.

In a first step, the influence of all possible substitutions for each amino acid of the VHH72 sequence was exhaustively mapped. This approach called Deep Mutational Scanning (DMS) is coupled with functional screening in Yeast Surface Display (YSD) to identify the variants displaying the best binding properties ( ). This makes it possible on the one hand to identify the residues involved in the recognition of the antigen, but also to identify the favorable substitutions likely to confer an increase in the affinity.

After high-throughput sequencing, the enrichment data of the different mutants can be represented in the form of a “heatmap”, with a color code making it possible to distinguish between favorable mutations in light gray and unfavorable mutations in dark gray ( ). The “heatmaps” obtained suggest three main areas of interaction with the antigens, at least partially overlapping with the 3 CDRs of HCV. Mutations within CDR2 and CDR3 are overwhelmingly unfavorable to interaction with the antigen. Some positions only seem to tolerate certain well-defined substitutions (eg A/G/I in position 57, W in 58, Y/W/P in position 101, as well as T103V or V104W. In addition, some positions seem permissive to the mutation , like the majority of the residues of CDR1, the amino acids downstream of CDR2 (positions 59 to 62) or position 98 in CDR3.

To potentiate the increase in affinity, the best substitutions were assembled within combinatorial libraries (Positions circled in black, ). These libraries were generated and then screened using the YSD technique to identify the clones showing increased binding to the antigen. The best variants were then selected by several successive rounds of selection. The cells expressing the best variants of VHH are sorted by flow cytometry, in the presence of decreasing concentrations of antigen. During a last step, a selection of the clones having the slowest antigen dissociation was undertaken, with a screen on the Koff kinetic parameter ( ). At the end of the screening steps, the analysis of the sorted population shows that all the clones selected have a very substantial gain in affinity for the RBD SARS-CoV-2 antigen with a notable binding to RBD in the presence of a concentration of 50 μM of antigen (Fig. 3B). Moreover, these same clones retained their ability to recognize the SARS-CoV1 antigen as evidenced by the strong signal observed in the presence of a concentration of 1 nM of RBD SARS-CoV1 antigen.

At the end of the screening steps, a new high-throughput sequencing step was undertaken. This sequencing made it possible to obtain the frequency of mutations within the mutated zones (represented in the form of a "Sequence Logo" graph, ). The data show that at the end of the FACS sorting steps, the selected clones overwhelmingly possess the S57G and V104W mutations, often associated with the T103V mutation. The other positions do not display such a marked consensus and remain more variable.

NGS sequencing also provided a list of the most frequently observed clones in the sorted population. To evaluate these antibodies from a functional point of view, some variants were chosen from among the most represented sequences (five variants were chosen, named VHH6 (or VHH72mut6), VHH65 (or VHH72mut65), VHH66 (or VHH72mut66), VHH71 (or VHH72mut71), VHH76 (or VHH72mut76), respectively, whose respective sequences are SEQ ID NO: 2-6). These were cloned with a constant fragment of human immunoglobulin (having the sequence SEQ ID NO: 8) for expression in the form of VHH-Fc in HEK293 cells.

In addition, a humanized version of the VHH76 (or VHH72mut76) variant was generated using the so-called "best fit" technique. The sequence IGHV3-23*01, corresponding to the germinal sequence (i.e., devoid of mutation linked to the maturation of the antibody) encoded by the human V gene closest to that of VHH72mut76, was used. The humanized VHH72mut76 variant thus obtained has the sequence SEQ ID NO:7.

The affinity constants of the VHH-Fc molecules were measured by BioLayer Interferometry (OctetRED96) for the SARS-CoV1 and SARS-CoV2 RBD antigens, as well as for the SARS-CoV2 variants (alpha, beta, gamma and delta) ( and Table 4). The five improved clones tested show a significant gain in affinity towards the RBD SARS-CoV2 antigen compared to the parental antibody VHH72. The dissociation constants of these molecules are notably slower and explain most of the gain in affinity. In addition, the presence of substitutions within the RBD SARS-CoV2 antigen corresponding to the natural variants of the SARS-CoV2 virus do not affect recognition by VHH-Fc molecules. Interestingly, the five VHH-Fc molecules show excellent affinity for the RBD-SARS-CoV1 antigen, without any selection pressure having been introduced to maintain this "cross-reactivity".

Table 4 : Affinity constants of VHH-Fc molecules measured for SARS-CoV1 and SARS-CoV2 RBD antigens, as well as for SARS-CoV2 variants (alpha, beta, gamma and delta)

RBD SARS-CoV2 RBD SARS-CoV1 RBD SARS-CoV2 Alpha RBD SARS-CoV2 Beta RBD SARS-CoV2 Gamma RBD SARS-CoV2 Delta mutant K _D app (pm) K _we (1/Ms) K _off (1/s) K _D app (pm) VHH6 69.4 5.48E+05 3.80E-05 43.6 205 236 172 121 VHH65 87.8 4.93E+05 4.33E-05 147 77.5 127 192 160 VHH66 13.1 1.06E+06 1.38E-05 111 85.3 ≤10 121 ≤10 VHH71 29.8 7.19E+05 2.02E-05 337 ≤10 ≤10 21.4 ≤10 VHH76 101 4.60E+05 4.66E-05 95.6 190 158 163 ≤10

To complete the characterization of the improved molecules, an ELISA neutralization test was implemented to verify the ability of these molecules to inhibit the interaction between the RBD protein and the soluble receptor angiotensin-converting enzyme 2 (ACE2) ( ). The improved VHH-Fc molecules show much lower IC50 inhibitory values than the parent molecule which correlates well with the measured affinity increase.

Finally, a whole virus neutralization test on human cells indicates that each of the improved VHH-Fc variants is able to effectively neutralize the SARS-CoV2 virus and different variants (data not shown). The neutralizing potency of each of the enhanced VHH-Fc variants is significantly greater than that of the parental VHH72.

The VHH-Fc clones selected have the S57G, T103V and V104W substitutions in common. To better understand the involvement of the different common substitutions, the affinity constants of clones incorporating these mutations alone or in combination were measured. The obtained Octet data (Table 5) show that each of these substitutions individually enhances the affinity for the SARS-CoV2 RBD protein, with affinities between 2 and 7 nM for monomutants. The S57G/V104W as well as S57G/T103V/V104W combinations show some additivity, with affinities measured below the nanomolar (respectively 0.53 nM and 0.18 nM). On the other hand, the T103V/V104W mutations do not seem to have a synergistic effect, the measured affinity of 5.5 nM being of the same order of magnitude as for the mutations tested individually.

Table 5 : Affinity constants measured for clones incorporating different substitutions alone or in combination

RBD SARS-CoV 2 Mutation K _D app (pm) k _we (1/Ms) k _off (1/s) S57A 3460 1.07E+06 3.70E-03 S57G 3430 8.89E+05 3.05E-03 T103V 2970 1.64E+06 4.87E-03 V104W 2200 1.79E+06 3.94E-03 S57G / T103V 1550 9.21E+05 1.43E-03 S57G/V104W 528 1.08E+06 5.72E-04 T103V / V104W 3220 1.42E+06 4.58E-03 S57G / T103V /V104W 526 8.31E+05 4.37E-04 S57A/V104W 5090 6.44E+05 3.24E-03 VHH72 (WT) 38600 1.40E+06 8.80 E-02

In order to better understand the structural modifications and the new molecular interactions induced by these substitutions, a model of interaction between the RBD domain of SARS-CoV2 and VHH72 incorporating the 3 substitutions S57G/T103V/V104W was established by homology, from of the previously published three-dimensional structure of the SARS-CoV1/VHH72 RBD complex (22). The model suggests a slight modification of the CDR2 loop linked to the S57G substitution. The presence of glycine allows greater flexibility and frees space for the side chain of the Y369 residue. This residue indeed has a different conformation in the structure of the SARS-CoV-1 antigen. Moreover, the V104W mutation seems to better accommodate a hydrophobic cavity, delimited by residues F377, P384 and C379 ( ).

3. Discussion

The molecular engineering approach described here has made it possible to obtain highly affinity VHH72 variants with improved affinity constants of almost a factor of 1000 compared to the parental molecule. These molecules are also broad-spectrum since they are both able to recognize the antigens of the SARS-CoV1 and SARS-CoV2 viruses as well as the different natural variants observed for SARS-CoV2.

The increase in affinity of these molecules for the RBD domain of the Spike protein has made it possible to increase their power of neutralizing the virus significantly. Treatment of patients with these antibodies could therefore prove to be more effective than with the non-mutated parental molecule VHH72. Furthermore, the effective dose for the treatment could possibly be reduced without altering the therapeutic efficacy. Finally, through the broad-spectrum recognition of the different RBD variants, these new molecules will be potentially effective for the different strains of the virus, current or likely to be discovered in the future. These molecules therefore have great potential for therapeutic application in humans.

Claims (15)

Linker comprising the amino acid sequence SEQ ID NO:1, comprising at least one substitution of an amino acid residue selected from the group consisting of:

1. The residue at position 57 of SEQ ID NO:1, substituted with an amino acid selected from G, N, and M; preferably by an amino acid G;
2. The residue at position 58 of SEQ ID NO:1, substituted with an amino acid selected from: S, W, R, and H; preferably by an S-amino acid;
3. The residue at position 60 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, L, I, T, M, H, Q, Y, and F;
4. The residue at position 61 of SEQ ID NO:1, substituted with an amino acid selected from: S, R, P, Q, I, M, and F; preferably by an amino acid selected from S, and R;
5. The residue at position 62 of SEQ ID NO:1, substituted with an amino acid selected from any amino acid except D, C, and Q; in particular substituted with an amino acid preferably selected from: F, L, V, P, I, M, W, Y, A, G, S, T, N, E, H, K, and R;
6. The residue at position 98 of SEQ ID NO:1, substituted with an amino acid preferably selected from any amino acid except A, P, and D;
7. The residue at position 101 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, F, Y, C, E, and W;
8. The residue at position 102 of SEQ ID NO:1, substituted by an amino acid preferably selected from: E, Y, and N;
9. The residue at position 103 of SEQ ID NO:1, preferably substituted with an amino acid V;
10. The residue at position 104 of SEQ ID NO:1, substituted with an amino acid selected from: W, and Y; And
11. Any combination of these.

Binding agent according to claim 1, comprising at least one substitution of an amino acid residue selected from: the substitution of the residue at position 57 of SEQ ID NO: 1 by an amino acid selected from G, N, and M ; substitution of the residue at position 103 of SEQ ID NO:1; the substitution of the residue at position 104 of SEQ ID NO:1 by an amino acid selected from: W, and Y; and any combination thereof. Linker comprising the amino acid sequence SEQ ID NO:1, comprising a combination of at least two amino acid residue substitutions, the substituted amino acid residue being selected from the group consisting of:

1. The residue at position 57 of SEQ ID NO:1, substituted by an amino acid preferably selected from: G, N and M;
2. The residue at position 58 of SEQ ID NO:1, substituted by an amino acid preferably selected from: S, W, R, and H;
3. The residue at position 60 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, L, I, T, M, H, Q, Y, and F;
4. The residue at position 61 of SEQ ID NO:1, substituted with an amino acid preferably selected from: S, R, P, Q, I, M, W, and F;
5. The residue at position 62 of SEQ ID NO:1, substituted with an amino acid preferably selected from any amino acid except D, C, and Q; in particular substituted with an amino acid preferably selected from: F, L, V, P, I, M, W, Y, A, G, S, T, N, E, H, K, and R;
6. The residue at position 98 of SEQ ID NO:1, substituted with an amino acid preferably selected from any amino acid except A, P, and D;
7. The residue at position 101 of SEQ ID NO:1, substituted with an amino acid preferably selected from: V, F, Y, C, E, and W;
8. The residue at position 102 of SEQ ID NO:1, substituted by an amino acid preferably selected from: E, Y, and N;
9. The residue at position 103 of SEQ ID NO:1, preferably substituted with an amino acid V; And
10. The residue at position 104 of SEQ ID NO:1, substituted by an amino acid preferably selected from: W, and Y; And
11. Any combination of these.

A binding agent according to any preceding claim comprising at least one combination of amino acid residue substitutions selected from:

1. the combination of the substitution of the residue at position 57 of SEQ ID NO:1 and the substitution of the residue at position 103 of SEQ ID NO:1;
2. the combination of the substitution of the residue at position 57 of SEQ ID NO:1 and the substitution of the residue at position 104 of SEQ ID NO:1; And
3. the combination of the substitution of the residue at position 57 of SEQ ID NO: 1, and of the substitution of the residue at position 103 of SEQ ID NO: 1, and of the substitution of the residue at position 104 of SEQ ID NO: 1.

Binding agent according to any one of the preceding claims, comprising an amino acid sequence chosen from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO: 6. Binding agent according to any one of the preceding claims, comprising, or consisting of, at least one binding agent chosen from: an antibody, a functional derivative of an antibody, and a functional fragment of an antibody. A binding agent according to any preceding claim, wherein the binding agent is humanized. A binding agent according to any preceding claim, further comprising an antibody heavy chain fragment. Binding agent according to any one of the preceding claims, capable of recognizing:

• an RBD domain (Receptor Binding Domain) of a sarbecovirus Spike protein; and or
• an RBD domain of a Spike protein of the original SARS-CoV2 virus, and an RBD domain of a Spike protein of at least one variant of the original SARS-CoV2 virus.

Binding agent according to any one of the preceding claims, in which the dissociation constant K _{d(VHH72 substituted)} measured for an RBD domain of a Spike protein of the SARS-CoV2 virus is lower than the dissociation constant K _{d(VHH72 not substituted)} of the original VHH72 single domain antibody, comprising the amino acid sequence SEQ ID NO:1, measured for an RBD domain of a Spike protein of SARS-CoV2 virus. Nucleic acid molecule encoding a binding agent according to any one of the preceding claims, preferably comprising a nucleic acid sequence having at least 85% identity with a sequence chosen from SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. A vector comprising the nucleic acid molecule according to claim 11. A host cell comprising the nucleic acid molecule according to claim 11 and/or the vector according to claim 12. Binding agent according to any one of Claims 1 to 10, nucleic acid molecule according to Claim 11, vector according to Claim 12, or host cell according to Claim 13, for its use in the prevention or treatment of a disease, the disease being preferably selected from diseases linked to sarbecoviruses. Usein vitroat least one binding agent according to any one of claims 1 to 10, for:

1. diagnosing a sarbecovirus-related disease in a subject suspected of suffering from a sarbecovirus-related disease;
2. follow-up in a subject suffering from a disease linked to sarbecoviruses;
3. stratification of a subject suffering from a sarbecovirus-related disease;
4. the evaluation of the effectiveness of a treatment (in particular curative) administered to a subject suffering from a disease linked to sarbecoviruses;
5. detecting the presence or absence of at least one sarbecovirus in a sample;
6. determining the amount of at least one sarbecovirus in a sample;
7. screening compounds/molecules capable of detecting and/or recognizing at least one sarbecovirus; preferably screening compounds/molecules capable of neutralizing at least one sarbecovirus; Or
8. any combination of i. to vii. ;

the disease linked to sarbecoviruses being preferably chosen from infections by a sarbecovirus.