Neutralizing monoclonal antibodies against sarbecoviruses

Abstract

The present invention provides neutralizing monoclonal antibodies targeting the spike protein of sarbecoviruses such as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-1 or SARS-CoV-2, such as COVID-19). The monoclonal antibodies of the present invention can inhibit or neutralize SARS-CoV-1 or SARS-CoV-2 activity and, advantageously, due to their significant cross-reactivity between SARS-CoV-2 variants, can be used to treat, prevent or diagnose COVID-19 infection in humans.

Description

[Technical field]

[0001] The Coronaviridae family includes numerous virus species that are further divided into four genera. [Background technology]

[0002] Seven species have been noted in humans and are highly diverse in terms of adaptation, pathogenicity and spread. Indeed, four species are endemic and highly adapted to humans (HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1), two are epidemic (MERS-CoV and extinct SARS-CoV), one is pandemic, and the recently emerged and currently circulating SARS-CoV and SARS-CoV-2 belong to the same genus (betacoronavirus) and subgenus (sarbecovirus), but are phylogenetically distinct and have significantly different characteristics in terms of infectivity and clinical signs. These aspects demonstrate the difficulty of completely sampling the entire Coronaviridae family, which is necessary to implement an effective surveillance program, and therefore highlight the need to have therapeutic strategies with broad activity against CoVs.

[0003] In general, the ideal target for inhibiting viral replication is represented by the protein that mediates entry, since it prevents cell infection.Therefore, entry inhibitors can be used as effective therapeutic agents and for prevention, regardless of whether they have long half-life.The relevance of inhibiting viral entry is demonstrated by the fact that most neutralizing antibodies induced by natural infection or vaccination recognize the basic region of the viral protein involved in entry.

[0004] Also, the long half-life of monoclonal antibodies (mAbs) allows their use in prophylaxis. Although immunization is the most effective approach to limit and possibly eradicate SARS-CoV-2, the identification of neutralizing mAbs (nAbs) is essential to address vaccine-specific limitations regarding delayed response, the proportion of non-responders, and the inability to vaccinate immunocompromised individuals. Also, there are several unresolved questions regarding long-lasting humoral immunity against SARS-CoV-2 and the effect of continuously emerging mutant strains that may partially interfere with the efficacy of vaccination in the long term. In summary, neutralizing mAbs against SARS-CoV-2 are and will be a fundamental therapeutic and prophylactic tool, and the availability of a panel of diverse mAbs will help to address the emergence of new mutant strains. Furthermore, there is the potential to identify cross-reactive mAbs, as a small number of mAbs naturally cross-neutralize SARS-CoV and SARS-CoV-2, and recently an in vitro matured mAb (called ADG-2) that neutralizes several sarbecoviruses has been described.

[0005] The entire entry process of all CoVs into host cells is mediated by the spike (S), a large transmembrane glycoprotein that protrudes from the viral envelope. The spike is a homotrimer in which each monomer is produced as a single protein (S0) that is cleaved into S1 and S2 subunits by host proteases. S1 is located at the apical end and contains a receptor binding domain (RBD), thus mediating the attachment phase by interacting with host proteins. S2 forms the spike stalk, which contains a fusion peptide, a transmembrane domain, and heptad repeats (HR1 and HR2), all of which are required to fuse the viral envelope to the host membrane.

[0006] CoV entry is a multi-step process that involves receptor binding of all three RBDs in each spike trimer, proteolytic cleavage of at least one specific spike site, and subsequent fusion.

[0007] SARS-CoV-2, like SARS-CoV and some animal CoVs, specifically binds to angiotensin-converting enzyme 2 (ACE2). The binding event is necessary but not sufficient to mediate entry, since SARS-CoV-2 S needs to be processed by host proteases that also specifically determine the viral entry site. Indeed, SARS-CoV-2 fusion occurs at the plasma membrane when the spike is cleaved by furin and TMPRSS2, and conversely, the spike enters from the endosomal compartment, where it is cleaved by cathepsins. Upon full binding and cleavage of the spike, S1 dissociates from S2, allowing exposure of the fusion peptide and insertion into the host membrane. The final molecular event that leads to fusion is a dramatic conformational change of S2, including the entire subunit, driven by refolding of HR1 and HR2. Notably, all events from binding to fusion require significant structural rearrangements and therefore could potentially be targets of neutralizing nAbs.

[0008] The spike protein of CoV is the most prominent protein exposed on the virion surface and is the primary target of the humoral response.

[0009] Characterization of sera from SARS-CoV-2-infected individuals has identified several neutralizing epitopes located on the spike protein that either overlap with domains essential for its function or indirectly inhibit the conformational changes necessary to mediate attachment and fusion.

[0010] The majority of neutralizing epitopes identified so far are conformational and located in the RBD, and all nAbs that bind to it can be divided into four classes characterized by different modes of action: class 1 and class 2 antibodies directly interfere with the interaction between ACE2 and spike by recognizing the same subdomain in the RBD, the so-called receptor binding motif (RBM), whereas classes 3 and 4 bind cryptic epitopes that indirectly block downstream conformational changes required for correct RBM exposure or fusion.

[0011] The broadly neutralizing mAb ADG-2, despite having a unique binding mode, recognizes an epitope partially shared with class 1 nAbs, highlighting that even small differences in terms of involved residues and approach angles can dramatically affect the biological activity of nAbs. At a lower frequency, nAbs recognizing other epitopes have been identified as well. Indeed, potent neutralization has been reported for mAbs binding to a region of S1 not directly involved in entry (the N-terminal domain - NTD).

[0012] In addition, neutralizing antibodies that recognize linear epitopes located downstream of the RBD or overlapping with the fusion peptide have been identified in patient sera. See Table 1 in Lanying Du et al., 2021[1], which summarizes representative human neutralizing monoclonal antibodies (nAbs) against SARS-CoV-2 and their relative target antigens (i.e., RBD, NTD, etc.).

[0013] Overall, neutralizing responses in patient sera are dominated by antibodies specific for the RBD, although several other spike domains can be effectively targeted. It is worth mentioning that the RBD and S1 are generally the most variable domains of the spike, both in view of the ongoing emergence of SARS-CoV-2 variants and among CoVs belonging to the same subgenus.

[0014] Therefore, nAbs targeting the conserved spike protein domain are urgently needed and of great interest as they would reasonably have a broader spectrum of neutralizable isolates / species.

[0015] Therapeutic mAbs for the treatment of COVID-19 are being developed at an accelerated pace that is unprecedented for any disease.

[0016] Currently, eight potent SARS-CoV-2 RBD-specific nAbs have been approved by the US Food and Drug Administration (FDA) under Emergency Use Authorization (EUA) to treat non-hospitalized COVID-19 patients at high risk for severe disease.

[0017] The following COVID-19 mAbs are in clinical use: bamlanivimab (LY-CoV555) [2], the combination of bamlanivimab (LY-CoV555) and etesevimab (LY-CoV016 or JS016) from Eli Lilly [3], the combination of casirivimab (REGN10933) and imdevimab (REGN10987) from Regeneron [4] (REGEN-COV), the combination of silgavimab (COV2-2130 or AZD1061) and tixagevimab (COV2-2196 or AZD8955) from AstraZeneca [5], the monotherapy-based nAb sotrovimab (VIR-7831) from GSK and Vir Biotechnology [6], and regdanvimab (CT-P59) from Celltrion [7]. Another set of Nab-based monotherapy and combination therapies are in Phase III trials: 2B04[8] and 47D11[9] from AbbVie, BRII-196 and BRII-198 from Brii Biosciences

[10] , and Tychan's TY027, also in Phase III trials

[10] .

[0018] A comprehensive list of nAbs currently in Phase I, II, and III trials and in the clinic is summarized in Figure 2A of Kumar S et al., 2021

[11] , and is incorporated herein by reference.

[0019] Several SARS-CoV-2 variants have been reported from different parts of the world. According to the World Health Organization (WHO), recognized variants are upgraded to "variants of concern" (VOC) if the acquisition of new mutations increases viral transmission, increases mortality, and significantly reduces the efficacy of treatments and vaccines. "Variants of interest" (VOI) are variants with new mutations that may affect disease severity, transmissibility, immune evasion, and diagnostic evasion.

[0020] The current variants of concern are the alpha (B.1.1.7, identified in the UK)

[12] , beta (B.1.351, identified in South Africa)

[13] , gamma (P.1, identified in Brazil)

[14] , delta (B.1.617.2, identified in India)

[15] , and sub- and descendant lineages of Omicron (B.1.1.529)

[16] , Omicron BA.2 (B.1.1.529+BA.2), Omicron BA.4 (B.1.1.529+BA.4) and Omicron BA.5.2 (B.1.1.529+BA.5)). Current notable variants are Eta (B.1.525, identified in the UK / Nigeria), Iota (B.1.526, identified in the US)

[17] , Kappa (B.1.617.1, identified in India)

[15] , and Lambda (C.37, identified in Peru)

[18] . For reference, see Figure 2B in Kumar S et al., 2021

[11] , which provides a comprehensive list of mutations present in the current SARS-Cov-2 VOCs and VOIs.

[0021] Ideally, an effective antiviral treatment strategy should have the ability to prevent infection / disease caused by new variants while simultaneously maintaining breadth against multiple existing viral strains / mutants. Recent studies have reported that many NTD-specific NAbs are relatively less effective against all emerging variants, while RBD-specific NAbs are variably effective against emerging variants and VOCs. Most of the potent therapeutic nAbs as monotherapy showed complete abrogation or reduced neutralizing activity against SARS-CoV-2 emerging variants containing E484K / Q or L452R mutations.

[0022] For example, bamlanivimab (LY-CoV555) was no longer considered for EUA because it was ineffective against all VOCs.

[0023] Currently, combination therapies including a cocktail of nAbs targeting different non-overlapping epitopes on the RBD have demonstrated very good efficacy and promising correlates of protection against SARS-CoV-2 and its variants (see references). Furthermore, the newly identified RBD core-binding nAbs SARS2-38 and LY-CoV1404 as monotherapies potently neutralize all variants of concern of SARS-CoV-2.

[0024] The present authors identified neutralizing antibodies targeting conserved regions of the spike proteins of SARS-CoV-1 and SARS-CoV-2 as promising and attractive therapeutic candidates to be used alone or in combination to address the disease burden caused by SARS-CoV-2 in all VOCs and VOIs known to date. Summary of the Invention

[0025] Therefore, the object of the present invention is to provide a method for producing a a) i) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:93; ii) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:94; iii) a heavy chain variable sequence comprising a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:95; and / or b) i) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:96; ii) a CDR2 comprising an amino acid sequence selected from the group consisting of GAS, AAS and DAS; iii) a light chain variable sequence comprising: a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:97; An isolated antibody or antibody fragment that binds to a conserved region of a sarbecovirus spike protein, characterized by an antigen-binding site comprising:

[0026] The complementarity determining regions (CDRs; CDR1-CDR3) and framework regions (FRs; FR1-FR4) of the heavy chain and constant region variable domains that characterize the sequence of the isolated antibody or antibody fragment of the present invention have been determined according to the IMGT nomenclature system.

[0027] According to a preferred embodiment, the heavy chain variable sequence of the antibody or antibody fragment of the invention further comprises: a) a framework region (FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:98; b) a framework region (FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 99; c) a framework region (FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 100; d) a framework region (FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 101; and / or The light chain variable sequence may further comprise: a) a framework region (FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 102; b) a framework region (FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 103; c) a framework region (FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:104; d) a framework region (FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:105.

[0028] According to a preferred embodiment of the invention, the antibody or antibody fragment comprises: a) a heavy chain variable sequence comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 1; CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and / or and a light chain variable sequence comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 13, CDR2 comprising the amino acid sequence of GAS, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20. According to another preferred embodiment, the heavy chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 25, a framework region FR2 comprising SEQ ID NO: 29, a framework region FR3 comprising SEQ ID NO: 33, and a framework region FR4 comprising SEQ ID NO: 37, and the light chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 41, a framework region FR2 comprising SEQ ID NO: 46, a framework region FR3 comprising SEQ ID NO: 51, and a framework region FR4 comprising SEQ ID NO: 56, b) the heavy chain variable sequence comprises CDR1 comprising the amino acid sequence of SEQ ID NO:1, CDR2 comprising the amino acid sequence of SEQ ID NO:5, and CDR3 comprising the amino acid sequence of SEQ ID NO:9; and / or The light chain variable sequence comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of GAS, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 21. According to another preferred embodiment, the heavy chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 25, a framework region FR2 comprising SEQ ID NO: 29, a framework region FR3 comprising SEQ ID NO: 33, and a framework region FR4 comprising SEQ ID NO: 38, and the light chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 42, a framework region FR2 comprising SEQ ID NO: 47, a framework region FR3 comprising SEQ ID NO: 52, and a framework region FR4 comprising SEQ ID NO: 57; c) the heavy chain variable sequence comprises CDR1 comprising the amino acid sequence of SEQ ID NO:2, CDR2 comprising the amino acid sequence of SEQ ID NO:6, and CDR3 comprising the amino acid sequence of SEQ ID NO:10; and / or The light chain variable sequence comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of AAS, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22. According to another preferred embodiment, the heavy chain variable sequence may further comprise a framework region FR1 consisting of SEQ ID NO: 26, a framework region FR2 consisting of SEQ ID NO: 30, a framework region FR3 comprising SEQ ID NO: 34, and a framework region FR4 comprising SEQ ID NO: 39, and the light chain variable sequence may further comprise a framework region FR1 consisting of SEQ ID NO: 43, a framework region FR2 comprising SEQ ID NO: 48, a framework region FR3 comprising SEQ ID NO: 53, and a framework region FR4 comprising SEQ ID NO: 58; d) the heavy chain variable sequence comprises CDR1 comprising the amino acid sequence of SEQ ID NO:3, CDR2 comprising the amino acid sequence of SEQ ID NO:7, and CDR3 comprising the amino acid sequence of SEQ ID NO:11; and / or The light chain variable sequence comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 comprising the amino acid sequence of AAS, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23. According to another preferred embodiment, the heavy chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 27, a framework region FR2 comprising SEQ ID NO: 31, a framework region FR3 comprising SEQ ID NO: 35, and a framework region FR4 consisting of SEQ ID NO: 40, and the light chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 44, a framework region FR2 comprising SEQ ID NO: 49, a framework region FR3 comprising SEQ ID NO: 54, and a framework region FR4 comprising SEQ ID NO: 56, e) the heavy chain variable sequence comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and / or The light chain variable sequence comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising the amino acid sequence of DAS, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 24. According to another preferred embodiment, the heavy chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 28, a framework region FR2 comprising SEQ ID NO: 32, a framework region FR3 comprising SEQ ID NO: 36, and a framework region FR4 comprising SEQ ID NO: 39, and the light chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 45, a framework region FR2 comprising SEQ ID NO: 50, a framework region FR3 comprising SEQ ID NO: 55, and a framework region FR4 comprising SEQ ID NO: 59, f) the heavy chain variable sequence comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 93, CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and CDR3 comprising the amino acid sequence of SEQ ID NO: 95; and / or The light chain variable sequence comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 96, a CDR2 comprising the amino acid sequence of AAS, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 97. According to another preferred embodiment, the heavy chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 98, a framework region FR2 comprising SEQ ID NO: 99, a framework region FR3 comprising SEQ ID NO: 100, and a framework region FR4 comprising SEQ ID NO: 101, and the light chain variable sequence may further comprise a framework region FR1 comprising SEQ ID NO: 102, a framework region FR2 comprising SEQ ID NO: 103, a framework region FR3 comprising SEQ ID NO: 104, and a framework region FR4 comprising SEQ ID NO: 105.

[0029] According to a preferred embodiment of the invention, the antibody or antibody fragment of the invention (clone Fab 5#) LESGGGLVKPGGSLRLSCAASGFNFNTYTMNWVRQAPGKGLEWVSSISSSSSYIDNADSVKGRFTIYRDNAKKSLYLRMIGLRVEDSGVYYCTRVNPQAKGSDWLDPPINQYYGMDVWGQGTTVTVSS (SEQ ID NO: 60) and / or ELTLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 61) and a light chain variable sequence comprising the amino acid sequence of:

[0030] In another preferred embodiment, the antibody or antibody fragment of the invention (clone Fab BU.2) LESGGGLVKPGGSLRLSCAASGFNFNTYTMNWVRQAPGKGLEWVSSISSSSSYIDNADSVKGRFTIYRDNAKKSLYLRMIGLRVEDSGVYYCTRVNPQAKGSDWLDPPINQYYGMDVWGQRDHGHRSP (SEQ ID NO: 62) and / or ELTLTQSPATLSLSPGDRATLSCRASQSVSSSYLAWYQHKPGQPPRLLIYGASSRAAGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYVTFGPGTKVDIK★ (SEQ ID NO: 63) and a light chain variable sequence comprising the amino acid sequence of:

[0031] According to another alternative preferred embodiment of the invention, the isolated antibody or antibody fragment of the invention (clone Fab BU.7) comprises: LEWGPGLVKPSETLSLTCTVSGGSISSINYYWVWIRQPPGKGLEWIGNINYSGTTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQTYYYDRRGYYRPEPIEHWGQGTLVTVSS (SEQ ID NO: 64) and / or ELVMTQSPSFLSASVGDRVTITCRASQDVRSFLHWYQQRPGKAPKLLIYAASMVSSEVPSRFSGSGSETDFTLTIDGLQPEDVATYFCQQTYDTPLTFGGGTAVDIK (SEQ ID NO: 65) and a light chain variable sequence comprising the amino acid sequence of:

[0032] In another embodiment of the invention, the isolated antibody or antibody fragment of the invention (clone Fab BU.11) comprises: LESGPGLLKPSQSLSLTCAISGDSVSRRSVAWNWIRQSPSRGLEWLGRTYYRSKWFSEYGVSVRGRITISPDTTKNQFSLQLNSVTPEDTAVYYCRKSKGRQQLAESTSSVWTWGQGTTVIVYS (SEQ ID NO: 66) and / or ELVMTQSPSSLSAFVGDRVTLTCRASQGIRNDLNWYQQKPGQPPKLLIYAASALQSGVPSRFSGSGFGTDFTLTISSLQPEDIATYYCLQDYNFPRTFGQGTKVEIK (SEQ ID NO: 67) and a light chain variable sequence comprising the amino acid sequence of:

[0033] In yet another alternative preferred embodiment of the invention, the isolated antibody or antibody fragment of the invention (clone Fab BU.54) comprises: LEWGPGLVKASQTLSLTCTVSGGSISSRNFYWSWIRQPGGKGLEWIGRIYTSGSTNYNPSLKSRVTISLDTSKSQFSLKLSSVTAADTAVYYCARGTFYYDRSGNGRLDPLDYWGQGTLVTVS (SEQ ID NO: 68) and / or ELVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGFGTHFTLTISSLQPEDFATYYCQQHDNLVTFGGGTKVEIK (SEQ ID NO: 69) and a light chain variable sequence comprising the amino acid sequence of:

[0034] According to a final alternative preferred embodiment of the invention, the isolated antibody or antibody fragment (clone Fab BS.70) of the invention comprises LESGGGVVQPGTSLRLSCAASGFTFNNFGMHWVRQAPGKGLEWVAMISYEGSKDFYADSVKGRFTISKDHARNTVYLQMNSLRAEDTAEYYCAKDKAIFMISAGRTLDFWGQGTLVTVSS (SEQ ID NO: 70) and / or ELVMTQSPSSLSASVGDRVTITCRASQNIGIYLNWYQQKPGKAPKLLIYAASSLQNGVPSRFSGSGSGTDFTLTISTLQPEDFATYWCQQGYSTPLTFGGGTKVEIR (SEQ ID NO: 71) and a light chain variable sequence comprising the amino acid sequence of:

[0035] A further object of the present invention is a nucleotide sequence encoding the heavy and / or light chain variable region of the above-listed antibodies or antibody fragments of the invention.

[0036] In a preferred embodiment, the nucleotide sequence of the present invention is selected from the group consisting of:

[0037] Heavy Chain (Clone Fab#5): ctcgagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaacttcaatacctataccatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtagtagtagttacatagacaacgcagactcagtgaagggccgattcacca tctacagagacaacgccaagaagtcactgtatctgcgaatgatcggcctgagagtcgaggactccggtgtgtattactgtacgcgagtgaacccccag gccaaagggtcggactggttggatccccccatcaaccaatactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 72)

[0038] Light chain (Fab#5): gagctcacactcacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcagctatttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtagctcaccgtggacgttcggccaagggaccaaggttgaaatcaaa (SEQ ID NO: 73)

[0039] Heavy chain (Fab BU.2) ctcgagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaacttcaatacctataccatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtagtagtagtagttacatagacaacgcagactcagtgaagggccgattcaccatctacagagacaacgccaagaagtcactgtatctgcgaatgatcggcctgagagtcgaggactccggtgtgtattactgtacgcgagtgaacccccaggccaaagggtcggactggttggatccccccatcaaccaatactacggtatggacgtctggggccaaagggaccacggtcaccggtctcct (SEQ ID NO: 74)

[0040] Light chain (Fab BU.2) gagctcacactcacgcagtctccagccaccctgtctttgtctccaggggatagagccaccctctcctgcagggccagtcagagtgttagcagcagttacttagcctggtaccagcacaaacctggccagccccccaggctcctcatctatggtgcatccagcagggccgccggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtacgtcactttcggccctgggaccaaagtggatatcaaa(SEQ ID NO: 75)

[0041] Heavy chain (Fab BU.7) ctcgagtggggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccatcagcagcataaattactactgggtctggattcgccagcccccagggaaggggctggagtggattgggaatatcaattacagtgggaccaccaactacaacccgtccctcaagagtcgagtcaccatatccgtggacacgtccaagaaccagttctccctgaagctgagctctgtgaccgccgcagacacggctgtctattactgtgcgagacaaacgtattactatgataggcgtggttattaccgcccggagcccattgagcactggggccagggaaccctggtcaccgtctcctca(SEQ ID NO: 76)

[0042] Light chain (Fab BU.7) gagctcgtgatgacacagtctccatccttcctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcaagatgttagaagttttttacattggtatcaacaaagaccagggaaagcccctaagttgttgatctatgctgcatccatggtgtcgagtgaggtcccgtcaaggttcagtggcagtggatctgagacagatttcactctcaccatcgacggtctgcaacctgaagatgttgcaacttacttctgtcaacagacttacgacacgccgctcaccttcggcggcgggaccgcggttgacatcaaa(SEQ ID NO:77)

[0043] Heavy chain (Fab BU.11) ctcgagtcaggtccaggactgctgaagccctcgcagagcctctcactcacctgtgccatctccggagacagtgtctctaggagaagtgttgcttggaactggatcagacagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggttttctgagtatggcgtatctgtgagaggtcgaataaccatcagtccagacacaaccaagaaccagttctccctgcagctgaactccgtgactcccgaggacacggctgtctattactgtcgaaagtcgaaagggaggcagcagttggcagagtctacgagttcggtatggacgtggggccaagggaccacggtcatcgtctactca(SEQ ID NO:78)

[0044] Light chain (Fab BU.11) gagctcgtgatgacacagtctccatcctccctgtctgcatttgtgggagatagagtcaccctcacttgccgggcaagtcagggcattagaaatgatttaaactggtatcagcagaaaccagggcaaccccctaaactcctgatctatgctgcatccgctttacaaagtggcgtcccatcaaggttcagcggcagtgggtttggcacagatttcactctcaccatcagcagcctgcagcctgaagatattgcaacttattactgtctacaagattacaatttccctcggacgttcggccaagggaccaaggtggaaatcaaa (SEQ ID NO: 79)

[0045] Heavy chain (Fab BU.54) ctcgagtggggcccaggactggtgaaggcttcacagaccctgtccctcacctgcactgtctctggtggctccatcagcagtaggaatttctattggagttggatccggcagcccggcgggaagggactggagtggattgggcgtatctatacaagtgggagcaccaattacaatccctccctcaagagtcgagtcactatatcattagacacgtccaagagtcagttctccctgaagctgagctctgtgaccgccgcagacacggccgtgtattattgtgcgagagggacgttttattatgataggagtggtaatggtcgattagatccgcttgactactggggccagggaaccctggtcaccgtctcctca(SEQ ID NO: 80)

[0046] Light chain (Fab BU.54) gagctcgtgatgacacagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatttgggacacattttacgttaaccatcagcagcctgcagcctgaagattttgcaacatattactgtcaacagcatgataatctcgtcactttcggcggagggaccaaggtggagatcaaa(SEQ ID NO: 81)

[0047] Heavy chain (BS.70): ctcgagtctgggggaggcgtggtccagcctgggacgtccctgagactctcctgtgcggcctctggattcaccttcaataattttgggatgcactgggtccgccaggctccaggcaagggtctggagtgggtggcaatgatctcatatgaaggaagtaaggatttctatgcagactccgtgaagggccgattcaccatctccaaagaccatgccaggaatacggtctatctgcaaatgaacagcctgagagctgaggacacggcagaatattactgtgcgaaagataaggctatatttatgatttctgccggacggactttggacttctggggccagggaaccctggtcaccgtctcctcag(SEQ ID NO: 82)

[0048] Light chain (Fab BS.70): gagctcgtgatgacacagtctccatcctccctgtctgcatctgtaggagacagagtcaccattacttgccgggcaagtcagaacattggcatctatttgaattggtatcagcagaaacctgggaaagccccaaagctcctgatctatgctgcatccagtttgcaa aatggggtcccatcgaggttcagtggcagtggatctgggacagacttcactctcaccatcagcactctgcaacctgaagattttgcaacttactggtgtcaacagggttacagtaccccactcactttcggcggagggaccaaggtagagatcagac (SEQ ID NO: 83)

[0049] Furthermore, the present invention relates to an expression vector comprising at least one nucleotide sequence encoding the heavy chain variable region and / or the light chain variable region of the antibody or antibody fragment of the present invention.

[0050] A further object of the present invention contemplates a host cell containing the vector outlined above.

[0051] Another object of the invention is a hybridoma comprising at least one nucleotide sequence encoding the heavy chain variable region and / or the light chain variable region of an antibody or antibody fragment of the invention.

[0052] Furthermore, the present invention contemplates a formulation for passive immunoprophylaxis based on a molecule / vector comprising at least one of the nucleotide sequences or expression vectors of the present invention together with one or more pharma- ceutically acceptable formulation excipients and / or adjuvants. The vaccine formulation may be advantageously used in the prevention of sarbecovirus-mediated diseases in a subject, in particular severe acute respiratory syndrome mediated by SARS-CoV-1 or SARS-CoV-2.

[0053] Preferably, the antibody or antibody fragment of the invention is a monoclonal antibody. In another preferred embodiment, the antibody fragment is a Fab fragment.

[0054] Even more preferably, the antibody or antibody fragment of the invention is a human antibody.

[0055] Preferably, the antibody or antibody fragment is human IgG or an IgG fragment selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or a recombinant IgG containing a mutated Fc portion to improve its binding and neutralizing activity or all Fc-mediated immune functions.

[0056] In alternative embodiments, the antibodies or antibody fragments of the invention are in a format selected from the group comprising single chain antibodies (scFv), nanobodies, bispecific antibodies, monoclonal antibodies conjugated to drugs and / or molecules that affect their pharmacokinetics and / or their delivery.

[0057] According to a further embodiment, the antibody or antibody fragment of the present invention is labeled with a marker or conjugated to a drug.

[0058] As an example, antitumor activity is achieved by conjugating antibodies with different effector molecules that achieve cell death after antibody binding and internalization. Such effector molecules include cytotoxic agents [19, 20], bacterial or plant protein toxins (immunotoxins) [21, 22], and radiopharmaceuticals

[23] .

[0059] A further object of the present invention is the use of the antibody or antibody fragment of the present invention for detecting the presence of a sarbecovirus, preferably SARS-CoV-1 or SARS-CoV-2, in a biological sample.

[0060] The present invention further contemplates the antibody or antibody fragment of the present invention for use in the medical field for the treatment and / or prevention of a sarbecovirus mediated disease in a subject, in particular severe acute respiratory syndrome mediated by SARS-CoV-1 or SARS-CoV-2.

[0061] The term "SARS-Cov-2" encompasses any of the previously known variants of concern (VOCs) alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), D614 G (University of Pavia) and omicron (B.1.1.529), or variants of interest (VOIs) eta (B.1.525), iota (B.1.526), ​​kappa (B.1.617.1) and lambda (C.37).

[0062] The antibody or antibody fragment of the present invention can be used alone or in combination with another antibody or antibody fragment against a different conserved region of the spike protein. The combination of the antibody or antibody fragment of the present invention increases the cross-reactivity of the therapeutic treatment against different VOCs of SARS-CoV-2. The second antibody can also be an antibody different from the claimed antibody. The combination of antibodies targeting different epitopes can lead to improved therapeutic efficacy, i.e., overcoming drug-related side effects of already approved molecules or eliminating drug resistance of possible new circulating mutant strains.

[0063] According to a preferred embodiment, the subject is an immunocompromised patient or a patient with a history of cardiovascular and / or respiratory disease. Alternatively, the patient may be an elderly patient (over 70 years old) or a vaccine-averse subject.

[0064] The antibodies or antibody fragments according to the invention may also be advantageously administered therapeutically in humans in combination antibody cocktails.

[0065] Preferably, the antibody or antibody fragment of the invention is administered 5 to 10 days after the onset of SARS-CoV-1 or SARS-CoV-2 infection.

[0066] An object of the present invention is a pharmaceutical composition comprising at least one antibody or antibody fragment of the invention as an active ingredient, together with one or more pharma- ceutically acceptable excpients and / or adjuvants. In a preferred embodiment, the antibody or antibody fragment is adapted for systemic administration by the intravenous route. Alternatively, the invention contemplates intramuscular or subcutaneous administration routes.

[0067] The present invention further relates to a pharmaceutical composition comprising at least one antibody or antibody fragment of the present invention for use in the treatment or prevention of a sarbecovirus-mediated disease in a subject, in particular severe acute respiratory syndrome mediated by SARS-CoV-1 or SARS-CoV-2.

[0068] A further object of the present invention is a composition or a kit of parts, comprising a first antibody or antibody fragment binding to a targeted conserved region in the spike protein of SARS-CoV-1 or SARS-CoV-2 according to the invention, and a second antibody or antibody fragment binding to a different targeted conserved region in the spike protein of SARS-CoV-1 or SARS-CoV-2 than the first antibody, for simultaneous, separate or sequential administration in a subject suffering from a sarbecovirus mediated disease, in particular severe acute respiratory syndrome mediated by SARS-CoV-1 or SARS-CoV-2. Preferably, the first antibody or antibody fragment and the second antibody or antibody fragment are in separate containers.

[0069] The invention will now be described, by way of non-limiting example, in accordance with preferred embodiments thereof with particular reference to the accompanying drawings, in which: FIG. [Brief description of the drawings]

[0070] [Figure 1]Figure 1 shows a general scheme for the preparation and selection of a naive recombinant antibody library. The diagram shows the general steps involved in the construction and selection of recombinant antibodies from a phage display library. B lymphocytes are first isolated from the blood of a donor. Total mRNA is extracted from the isolated lymphocyte cells and reverse transcribed into complementary DNA (cDNA). Variable heavy (VH) and variable light (VL) antibody genes are amplified by polymerase chain reaction using gene-specific primers and assembled before cloning into a phage display vector. The resulting phages display antibody fragments on their surface. During selection, the filamentous phage display library is incubated with antigen immobilized on a solid phase. Antigen-binding phages remain bound to the antigen, while unbound phages are washed away. Bound phages are then eluted and amplified by infection with bacterial cells. By changing the expression host to a non-suppressor bacterial strain, soluble antibody fragments can be expressed and secreted into the medium. [Figure 2A] FIG. 1 shows the complete amino acid (Panel A) and nucleotide sequences (Panel B) of the heavy and light chain variable regions in each clone, specifying both the framework regions FR1 to FR4 and the complementarity determining regions CDR1 to CDR3. [Figure 2B] FIG. 1 shows the complete amino acid (Panel A) and nucleotide sequences (Panel B) of the heavy and light chain variable regions in each clone, specifying both the framework regions FR1 to FR4 and the complementarity determining regions CDR1 to CDR3. [Figure 2C] FIG. 1 shows the complete amino acid (Panel A) and nucleotide sequences (Panel B) of the heavy and light chain variable regions in each clone, specifying both the framework regions FR1 to FR4 and the complementarity determining regions CDR1 to CDR3. [Figure 3-1]Figure 1 shows the binding activity of selected clones (#5, BU.2, BU.7, BU.11, BU.54, BS.70) in both Fab and IgG1 format assessed by ELISA assay. Selected Fabs and IgGs were tested for their binding to recombinant proteins of different VOCs. [Figure 3-2] Figure 1 shows the binding activity of selected clones (#5, BU.2, BU.7, BU.11, BU.54, BS.70) in both Fab and IgG1 format assessed by ELISA assay. Selected Fabs and IgGs were tested for their binding to recombinant proteins of different VOCs. [Figure 3-3] Figure 1 shows the binding activity of selected clones (#5, BU.2, BU.7, BU.11, BU.54, BS.70) in both Fab and IgG1 format assessed by ELISA assay. Selected Fabs and IgGs were tested for their binding to recombinant proteins of different VOCs. [Diagram 3-4] Figure 1 shows the binding activity of selected clones (#5, BU.2, BU.7, BU.11, BU.54, BS.70) in both Fab and IgG1 format assessed by ELISA assay. Selected Fabs and IgGs were tested for their binding to recombinant proteins of different VOCs. [Figure 4-1] Figure 1 shows the results of immunofluorescence analysis of the binding activity of selected clones, both in Fab and IgG1 format. The selected Fabs are able to detect VeroE6 cells infected with D614G, alpha_B.1.1.7, beta_B.1.135, gamma P.1, delta_B.1.617.1, Omicron_B.1.529 (BA.1), Omicron_B.1.529 (BA.2), Omicron_B.1.529 (BA.4) and Omicron_B.1.529 (BA.5) virus variants. [Figure 4-2]Figure 1 shows the results of immunofluorescence analysis of the binding activity of selected clones, both in Fab and IgG1 format. The selected Fabs are able to detect VeroE6 cells infected with D614G, alpha_B.1.1.7, beta_B.1.135, gamma P.1, delta_B.1.617.1, Omicron_B.1.529 (BA.1), Omicron_B.1.529 (BA.2), Omicron_B.1.529 (BA.4) and Omicron_B.1.529 (BA.5) virus variants. [Figure 4-3] Figure 1 shows the results of immunofluorescence analysis of the binding activity of selected clones, both in Fab and IgG1 format. The selected Fabs are able to detect VeroE6 cells infected with D614G, alpha_B.1.1.7, beta_B.1.135, gamma P.1, delta_B.1.617.1, Omicron_B.1.529 (BA.1), Omicron_B.1.529 (BA.2), Omicron_B.1.529 (BA.4) and Omicron_B.1.529 (BA.5) virus variants. [Figure 4-4] Figure 1 shows the results of immunofluorescence analysis of the binding activity of selected clones, both in Fab and IgG1 format. The selected Fabs are able to detect VeroE6 cells infected with D614G, alpha_B.1.1.7, beta_B.1.135, gamma P.1, delta_B.1.617.1, Omicron_B.1.529 (BA.1), Omicron_B.1.529 (BA.2), Omicron_B.1.529 (BA.4) and Omicron_B.1.529 (BA.5) virus variants. [Figure 5A]FIG. 1 shows the neutralizing activity of clone Fab#5 in both Fab and IgG1 formats against the VOCs tested (D614G, B.1.1.7, P.1 and B.1.135) (Panel A) and the neutralizing activity of clones Fab BU.2, BU.7, BU.11, BU.54 and BS.70 in both Fab and IgG1 formats against the VOCs tested (D614G, alpha_B.1.1.7, beta_B.1.135, delta B.1.617.1, gamma_P.1, Omicron B.1.1.529 (BA.1), Omicron B.1.1.529 (BA.2)) (Panel B). [Figure 5B-1] FIG. 1 shows the neutralizing activity of clone Fab#5 in both Fab and IgG1 formats against the VOCs tested (D614G, B.1.1.7, P.1 and B.1.135) (Panel A) and the neutralizing activity of clones Fab BU.2, BU.7, BU.11, BU.54 and BS.70 in both Fab and IgG1 formats against the VOCs tested (D614G, alpha_B.1.1.7, beta_B.1.135, delta B.1.617.1, gamma_P.1, Omicron B.1.1.529 (BA.1), Omicron B.1.1.529 (BA.2)) (Panel B). [Figure 5B-2] FIG. 1 shows the neutralizing activity of clone Fab#5 in both Fab and IgG1 formats against the VOCs tested (D614G, B.1.1.7, P.1 and B.1.135) (Panel A) and the neutralizing activity of clones Fab BU.2, BU.7, BU.11, BU.54 and BS.70 in both Fab and IgG1 formats against the VOCs tested (D614G, alpha_B.1.1.7, beta_B.1.135, delta B.1.617.1, gamma_P.1, Omicron B.1.1.529 (BA.1), Omicron B.1.1.529 (BA.2)) (Panel B). [Figure 5B-3]FIG. 1 shows the neutralizing activity of clone Fab#5 in both Fab and IgG1 formats against the VOCs tested (D614G, B.1.1.7, P.1 and B.1.135) (Panel A) and the neutralizing activity of clones Fab BU.2, BU.7, BU.11, BU.54 and BS.70 in both Fab and IgG1 formats against the VOCs tested (D614G, alpha_B.1.1.7, beta_B.1.135, delta B.1.617.1, gamma_P.1, Omicron B.1.1.529 (BA.1), Omicron B.1.1.529 (BA.2)) (Panel B). [Figure 5B-4] FIG. 1 shows the neutralizing activity of clone Fab#5 in both Fab and IgG1 formats against the VOCs tested (D614G, B.1.1.7, P.1 and B.1.135) (Panel A) and the neutralizing activity of clones Fab BU.2, BU.7, BU.11, BU.54 and BS.70 in both Fab and IgG1 formats against the VOCs tested (D614G, alpha_B.1.1.7, beta_B.1.135, delta B.1.617.1, gamma_P.1, Omicron B.1.1.529 (BA.1), Omicron B.1.1.529 (BA.2)) (Panel B). [Figure 6] FIG. 1 shows the pseudovirus-based neutralizing activity of clone Fabs BU.7 and BU.54 against Omicron VOCs of SARS-CoV-1 and SARS-CoV-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] For the purpose of better illustrating the present invention, the following examples are provided here, which should be considered as illustrative and non-limiting examples thereof. The neutralizing nAbs of the present invention have been identified based on their binding affinity to SARS-CoV1 and SARS-CoV-2 spikes or specific domains and characterized with respect to neutralizing activity and epitope specificity, as described in the following examples. These aspects are the basis for selecting the most promising nAbs as those that provide the highest potency and / or recognize conserved spike epitopes, which are the main parameters that determine potential cross-reactive treatments effective against all VOIs and VOIcs of SARS-Cov-2 known so far or that will emerge in the future.

[0072] Example 1: Generation of a new human antibody phage display library and selection of antibodies capable of binding with high affinity to the spike protein of SARS-CoV-2 by a biopanning procedure

[0073] Materials and Methods Phage Display Technology This technology focuses on the construction of libraries of peptide or antibody variants, which are then fused to a phage-coat protein and selected for their affinity to a target of interest (see FIG. 1 for reference).

[0074] All surface proteins of bacteriophages can be engineered for display, but the most used are pVIII and pIII from M13 filamentous phage. Each virion contains about 2700 copies of the former proteins, representing almost 87% of its mass, which are half exposed to the environment and therefore allow efficient display of only short sequences of peptides due to the virion structure. On the other hand, pIII can be used for larger peptides such as "functional parts" of antibodies, resulting in only a small loss of infectivity in a few cases. Since each library is constituted by a phagemid vector containing only the sequence of the phage coat protein fused to the peptide of interest, a helper phage with reduced packaging efficiency is required to obtain a population of phages that are infectious and constituted by the modified coating protein. A biopanning procedure is then performed to select phages for their ability to bind the antigen of interest. Many factors must be taken into account, such as the variability of the library of molecules exposed on the phage, the target conformation, the affinity and the avidity. As mentioned, phage display has been widely used to discover novel therapeutic agents against pathogens, particularly mAbs.

[0075] This has been made possible by two different biopanning strategies: using specific molecular targets such as membrane receptors (S protein) or using whole viruses or infected cells. However, because surface antigens often present motifs that can elicit non-neutralizing mAbs and evade the host immune response, a screening procedure of the biopanning results must be performed appropriately to identify only a small number of effective molecules that can selectively target the antigen of interest.

[0076] Generation of a novel human antibody phage display library Peripheral (for Fab#5) and bone marrow (for Fabs BU.2, BU.7, BU.11, BU.54) blood samples were collected from subjects previously infected with SARS-CoV-2 and subsequently vaccinated with COVID-19 vaccine (2 doses of Comirnaty by Pfizer) and used to collect PBMCs and B cell precursors, respectively.

[0077] PBMC extraction was performed according to the Histopaque®-1077 (SIGMA-ALDRICH) protocol. At the end of the procedure, 10 mL of Trizol Reagent (SIGMA-ALDRICH) was used to extract PBMC. 6 Cells were lysed and then cellular RNA was extracted using the RNeasy Mini Kit (QIAGEN). Antibody phagemid IgG libraries were generated by reverse transcription of B cell-mRNA into cDNA using SuperScript™ IV reverse transcriptase (Thermo Fisher Scientific). Heavy and light chains were amplified from the resulting cDNA using PFU Native Polymerase enzyme (Agilent) and the primer pairs listed in the table below.

[0078] [Table 1] Both the heavy and light variable antibody chains were then cloned into the phagemid vector pComb3XSS (PVT10572, DBA).

[0079] The phagemid library was then converted to a phage library by transforming electrocompetent Gram-negative cells expressing sex pili (XL1-Blue, Agilent) and superinfecting them with helper phage (M13K07, NEB).

[0080] With the aim of maximizing the selection of cross-reactive antibodies in the Fab format (antibodies recognizing a greater number of SARS-CoV-2 clinical isolates), we screened the new library by using different biopanning procedures.

[0081] Recombinant spike (S) proteins of different SARS-CoV-2 mutants were used: D614G (University of Pavia) in Fab#5, and B.1.1.7 (40589-V08B6, Sino Biological Co.), P.1 (40589-V08B10, Sino Biological Co.) or B.1.135 (40589-V08B7, Sino Biological Co.) in Fab BU.2, BU.7, BU.11, BU.54.

[0082] Five rounds of biopanning of the phage display antibody library were performed on recombinant spike proteins expressed in their full-length format. A cross-selection strategy based on the sequential use of different antigens was implemented.

[0083] Bacteriophage carrying on their capsid monoclonal antibodies (expressed as Fab fragments), selected by biopanning, were used to obtain DNA encoding the antibody expressed on their surface.

[0084] DNA encoding the variable antibody regions present on the phage surface was sequenced and used for protein purification using an E. coli expression system (XL1-Blue, Agilent) and immunoaffinity chromatography (HiTrap® Protein L, Merck). The IgG1 format of Fab#5 (IgG#5) was obtained from Genscript through their high throughput antibody production service (GenScript).

[0085] result Five distinct clones were identified: Fab#5 and Fabs BU.2, BU.7, BU.11, and BU.54.

[0086] The amino acid sequences of the heavy and light chain variable regions are as follows:

[0087] 1. Fab#5 Hc: LESGGGLVKPGGSLRLSCAASGFNFNTYTMNWVRQAPGKGLEWVSSISSSSSYIDNADSVKGRFTIYRDNAKKSLYLRMIGLRVEDSGVYYCTRVNPQAKGSDWLDPPINQYYGMDVWGQGTTVTVSS (SEQ ID NO: 60)

[0088] Lc: ELTLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 61)

[0089] 2.Fab BU.2 Hc: LESGGGLVKPGGSLRLSCAASGFNFNTYTMNWVRQAPGKGLEWVSSISSSSSYIDNADSVKGRFTIYRDNAKKSLYLRMIGLRVEDSGVYYCTRVNPQAKGSDWLDPPINQYYGMDVWGQRDHGHRSP (SEQ ID NO: 62)

[0090] Lc: ELTLTQSPATLSLSPGDRATLSCRASQSVSSSYLAWYQHKPGQPPRLLIYGASSRAAGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYVTFGPGTKVDIK (SEQ ID NO: 63)

[0091] 3.Fab BU.7 Hc: LEWGPGLVKPSETLSLTCTVSGGSISSINYYWVWIRQPPGKGLEWIGNINYSGTTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQTYYYDRRGYYRPEPIEHWGQGTLVTVSS (SEQ ID NO: 64)

[0092] Lc: ELVMTQSPSFLSASVGDRVTITCRASQDVRSFLHWYQQRPGKAPKLLIYAASMVSSEVPSRFSGSGSETDFTLTIDGLQPEDVATYFCQQTYDTPLTFGGGTAVDIK (SEQ ID NO: 65)

[0093] 4.Fab BU.11 Hc: LESGPGLLKPSQSLSLTCAISGDSVSRRSVAWNWIRQSPSRGLEWLGRTYYRSKWFSEYGVSVRGRITISPDTTKNQFSLQLNSVTPEDTAVYYCRKSKGRQQLAESTSSVWTWGQGTTVIVYS (SEQ ID NO: 66)

[0094] Lc: ELVMTQSPSSLSAFVGDRVTLTCRASQGIRNDLNWYQQKPGQPPKLLIYAASALQSGVPSRFSGSGFGTDFTLTISSLQPEDIATYYCLQDYNFPRTFGQGTKVEIK (SEQ ID NO: 67)

[0095] 5.Fab BU.54 Hc: LEWGPGLVKASQTLSLTCTVSGGSISSRNFYWSWIRQPGGKGLEWIGRIYTSGSTNYNPSLKSRVTISLDTSKSQFSLKLSSVTAADTAVYYCARGTFYYDRSGNGRLDPLDYWGQGTLVTVSS (SEQ ID NO: 68)

[0096] Lc: ELVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGFGTHFTLTISSLQPEDFATYYCQQHDNLVTFGGGTKVEIK (SEQ ID NO: 69)

[0097] 6. Fab BS.70 Hc: LESGGGVVQPGTSLRLSCAASGFTFNNFGMHWVRQAPGKGLEWVAMISYEGSKDFYADSVKGRFTISKDHARNTVYLQMNSLRAEDTAEYYCAKDKAIFMISAGRTLDFWGQGTLVTVSS (SEQ ID NO: 70)

[0098] Lc: ELVMTQSPSSLSASVGDRVTITCRASQNIGIYLNWYQQKPGKAPKLLIYAASSLQNGVPSRFSGSGSGTDFTLTISTLQPEDFATYWCQQGYSTPLTFGGGTKVEIR (SEQ ID NO: 71)

[0099] The nucleotide sequence encoding this is as follows:

[0100] 1. Fab#5 Hc: ctcgagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaacttcaatacctataccatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtagtagtagtagttacatagacaacgcagactcagtgaagggccgattcaccatctacagagacaacgccaagaagtcactgtatctgcgaatgatcggcctgagagtcgaggactccggtgtgtattactgtacgcgagtgaacccccaggccaaagggtcggactggttggatccccccatcaaccaatactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 72)

[0101] Lc: gagctcacactcacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcagctatttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtagctcaccgtggacgttcggccaagggaccaaggttgaaatcaaa(SEQ ID NO: 73)

[0102] 2.Fab BU.2 Hc: ctcgagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaacttcaatacctataccatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtagtagtagtagttacatagacaacgcagactcagtgaagggccgattcaccatctacagagacaacgccaagaagtcactgtatctgcgaatgatcggcctgagagtcgaggactccggtgtgtattactgtacgcgagtgaacccccaggccaaagggtcggactggttggatccccccatcaaccaatactacggtatggacgtctggggccaaagggaccacggtcaccggtctcct (SEQ ID NO: 74)

[0103] Lc: gagctcacactcacgcagtctccagccaccctgtctttgtctccaggggatagagccaccctctcctgcagggccagtcagagtgttagcagcagttacttagcctggtaccagcacaaacctggccagccccccaggctcctcatctatggtgcatccagcagggccgccggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtacgtcactttcggccctgggaccaaagtggatatcaaa (SEQ ID NO: 75)

[0104] 3.Fab BU.7 Hc: ctcgagtggggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccatcagcagcataaattactactgggtctggattcgccagcccccagggaaggggctggagtggattgggaatatcaattacagtgggaccaccaactacaacccgtccctcaagagtcgagtcaccatatccgtggacacgtccaagaaccagttctccctgaagctgagctctgtgaccgccgcagacacggctgtctattactgtgcgagacaaacgtattactatgataggcgtggttattaccgcccggagcccattgagcactggggccagggaaccctggtcaccgtctcctca(SEQ ID NO: 76)

[0105] Lc: gagctcgtgatgacacagtctccatccttcctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcaagatgttagaagttttttacattggtatcaacaaagaccagggaaagcccctaagttgttgatctatgctgcatccatggtgtcgagtgaggtcccgtcaaggttcagtggcagtggatctgagacagatttcactctcaccatcgacggtctgcaacctgaagatgttgcaacttacttctgtcaacagacttacgacacgccgctcaccttcggcggcgggaccgcggttgacatcaaa(SEQ ID NO: 77)

[0106] 4.Fab BU.11 Hc: ctcgagtcaggtccaggactgctgaagccctcgcagagcctctcactcacctgtgccatctccggagacagtgtctctaggagaagtgttgcttggaactggatcagacagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggttttctgagtatggcgtatctgtgagaggtcgaataaccatcagtccagacacaaccaagaaccagttctccctgcagctgaactccgtgactcccgaggacacggctgtctattactgtcgaaagtcgaaagggaggcagcagttggcagagtctacgagttcggtatggacgtggggccaagggaccacggtcatcgtctactca (SEQ ID NO: 78)

[0107] Lc: gagctcgtgatgacacagtctccatcctccctgtctgcatttgtgggagatagagtcaccctcacttgccgggcaagtcagggcattagaaatgatttaaactggtatcagcagaaaccagggcaaccccctaaactcctgatctatgctgcatccgctttacaaagtggcgtcccatcaaggttcagcggcagtgggtttggcacagatttcactctcaccatcagcagcctgcagcctgaagatattgcaacttattactgtctacaagattacaatttccctcggacgttcggccaagggaccaaggtggaaatcaaa (SEQ ID NO: 79)

[0108] 5.Fab BU.54 Hc: ctcgagtggggcccaggactggtgaaggcttcacagaccctgtccctcacctgcactgtctctggtggctccatcagcagtaggaatttctattggagttggatccggcagcccggcgggaagggactggagtggattgggcgtatctatacaagtgggagcaccaattacaatccctccctcaagagtcgagtcactatatcattagacacgtccaagagtcagttctccctgaagctgagctctgtgaccgccgcagacacggccgtgtattattgtgcgagagggacgttttattatgataggagtggtaatggtcgattagatccgcttgactactggggccagggaaccctggtcaccgtctcctca(SEQ ID NO: 80)

[0109] Lc: gagctcgtgatgacacagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatttgggacacattttacgttaaccatcagcagcctgcagcctgaagattttgcaacatattactgtcaacagcatgataatctcgtcactttcggcggagggaccaaggtggagatcaaa(SEQ ID NO: 81)

[0110] 6.Fab BS.70

[0111] Hc: ctcgagtctgggggaggcgtggtccagcctgggacgtccctgagactctcctgtgcggcctctggattcaccttcaataattttgggatgcactgggtccgccaggctccaggcaagggtctggagtgggtggcaatgatctcatatgaaggaagtaaggatttctatgcagactccgtgaagg gccgattcaccatctccaaagaccatgccaggaatacggtctatctgcaaaatgaacagcctgagagctgaggacacggcagaatattactgtgcgaaagataaggctatatttatgatttctgccggacggactttggacttctggggccagggaaccctggtcaccgtctcctcag (SEQ ID NO: 82)

[0112] Lc: gagctcgtgatgacacagtctccatcctccctgtctgcatctgtaggagacagagtcaccattacttgccgggcaagtcagaacattggcatctatttgaattggtatcagcagaaacctgggaaagccccaaagctcctgatctatgctgcatccagtttgcaa aatggggtcccatcgaggttcagtggcagtggatctgggacagacttcactctcaccatcagcactctgcaacctgaagattttgcaacttactggtgtcaacagggttacagtaccccactcactttcggcggagggaccaaggtagagatcagac (SEQ ID NO: 83)

[0113] FIG. 2 shows the complete amino acid and nucleotide sequences of the heavy and light chain variable regions in each clone, specifying both the framework regions FR1-FR4 and the complementarity determining regions CDR1-CDR3.

[0114] Example 2: Study on the binding activity of the neutralizing mAb of the present invention Fab binding assessment a) Enzyme-linked immunosorbent assay (ELISA) An ELISA assay was used to test the binding efficiency of the Fabs.

[0115] 100 ng of recombinant spike protein D614G (University of Pavia), B.1.1.7 (40589-V08B6, Sino Biological), P.1 (40589-V08B10, Sino Biological), or B.1.135 (40589-V08B7, Sino Biological) was coated onto an ELISA plate (CLS3690-100EA, Merck) and incubated overnight at 4°C. A 1% bovine serum albumin (BSA; Sigma-Aldrich) solution in PBS was added as a nonspecific reactivity control. The next day, the plate was blocked with a 1% BSA solution in PBS to prevent nonspecific binding to the wells, and different dilutions of Fab were incubated with the antigen for 1 h at 37°C. After washing with 0.1% Tween 20 in PBS solution (Sigma-Aldrich), Fab was detected using goat anti-human IgG (Fab specific)-peroxidase antibody (A0293, Sigma-Aldrich), His-tagged proteins with anti-His6-peroxidase antibody (11 2416001, Sigma-Aldrich) as coating control, and Pierce TMB substrate kit (ThermoFisher Scientific). OD450 was measured using Multiskan GO (ThermoFisher Scientific).

[0116] result Fab#5 binds to alpha, gamma and omicron S proteins IgG#5 binds to D614G, alpha, beta, gamma, delta, omicron, and S proteins of SARS-CoV-1 Fab BU.2 binds to the alpha S protein Fab BU.7 binds to the alpha and S proteins of SARS-CoV-1, but not to the gamma and omicron proteins IgG BU.7 hardly binds to the S protein of omicron Fab BU.11 binds to the alpha S protein IgG BU.54 binds to the alpha, delta, omicron, and S proteins of SARS-CoV-1 Fab BS.70 binds to the alpha, delta, omicron, and S proteins of SARS-CoV-1

[0117] The results are shown in Figure 3. The data showed that Fab#5 is able to recognize the B.1.1.7 recombinant protein even when used at low concentrations, but also the P.1 spike when used at high concentrations. IgG#5 binds well to the recombinant spikes of all SARS-CoV-2 mutants tested (D614G, B.1.1.7, B.1.351, P.1, B.1.1.529, B.1.617.1). BU.7 binds to the P.1 protein and even better to the B.1.1.7 spike protein. The IgG format also shows weak binding activity to the B.1.617.1 and B.1.1.529 proteins. Furthermore, the B.1.1.7 spike is well recognized by BU.11 and BU.2 Fabs, and IgG BU.54 recognizes B.11.7, B.1.617.1, P.1, B.1.1.529 and SARS-CoV-1 recombinant proteins well. Fab BS.70 also binds low concentrations of B.1.1.7, B.1.617.1 and B.1.1.529 proteins. Finally, the SARS-CoV-1 spike protein is very well recognized by IgG#5, BU.7, IgG BU.54 and Fab BS.70 in both Fab and IgG formats.

[0118] b) Immunofluorescence analysis Viruses and cells Vero E6 (Vero C1008, clone E6-CRL-1586, ATCC) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with non-essential amino acids (NEAA), penicillin / streptomycin (P / S), Hepes buffer, and 10% (v / v) fetal bovine serum (FBS). Four clinical isolates of SARS-CoV-2 were obtained and propagated in Vero E6 cells: D614G (hCoV-19 / Italy / UniSR1 / 2020, GSAID accession ID: EPI_ISL_413489), B.1.1.7(alpha) (19 / Italy / LOM-UniSR7 / 2021, GSAID accession ID: EPI_ISL_1924880), C.36_3 (hCoV-19 / Italy / LOM-UnINSU / 2021, GSAID accession ID: EPI_ISL_1924880), and C.36_3 (hCoV-19 / Italy / LOM-UnINSU / 2021, GSAID accession ID: EPI_ISL_1924880). GISAID Accession ID: EPI_ISL_1509923), B.1.351(beta) (hCoV-19 / Italy / LOM-UniSR6 / 2021, GISAID Accession ID: EPI_ISL_1599180), P.1(gamma) (hCoV-19 / Italy / LOM-UniSR8 / 2021, GISAID Accession ID: EPI_ISL_1925323) delta (hCoV-19 / Italy / LOM-UniSR12 / 2021, GSAID accession ID: EPI_ISL_4198505), Omicron BA.1 (hCoV-19 / Italy / LOM-UniSR14 / 2021, GSAID accession ID: EPI_ISL_12188061), Omicron BA.2 (hCoV-19 / Italy / LOM-UniSR15 / 2022.GISAI D accession ID: EPI_ISL_13285445), omicron BA.4 (hCoV-19 / Italy / LOM-UniSR17 / 2022.GISAID accession ID: EPI_ISL_13878328), and omicron BA.5.2 (hCoV-19 / Italy / LOM-UniSR16 / 2022.GISAID accession ID: EPI_ISL_13878326).

[0119] Virus titration Viral stocks were titrated using both plaque reduction assay (PRA, PFU / ml) and end point dilution assay (EDA, TCID50 / ml). In PRA, confluent monolayers of Vero E6 cells were infected with eight 10-fold dilutions of the viral stock. After 1 h of adsorption at 37°C, cell-free virus was removed. The cells were then incubated for 48 h in DMEM containing 2% FBS and 0.5% agarose. Cells were fixed, stained, and viral plaques were counted. In EDA, Vero E6 cells were seeded in 96-well plates and infected at 95% cell area coverage with a base 10 dilution of the viral stock. After 1 h of adsorption at 37°C, cell-free virus was removed, cells were washed once with PBS, and complete medium was added to the cells. After 48 h, cells were observed to assess the presence of cytopathic effect (CPE). The TCID50 / ml of viral stock was then determined by applying the Reed-Muench formula.

[0120] Immunofluorescence assay Vero E6 cells were seeded in 96-well plates and infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for 1 h at 37 °C 24 h before performing the experiment with a cell occupancy rate of 95% for each well. The cells were then washed once with PBS to remove cell-free virus particles and virus-containing mixtures, and the controls were replaced with complete DMEM supplemented with 2% FBS. The plates were incubated for 72 h at 37 °C in the presence of CO2. They were then washed once with PBS, fixed with MeOH:Ac (1:1) for 15 min, and stored at -20 °C. After rinsing the cells twice with PBS (1 wash), the selected Fab (1:200 dilution) was applied to the cells as the primary antibody. Anti-spike antibody (0150-R007, Sino Biological Co., Ltd.) was added as a control. After washing once with PBS, Fab was detected using a goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 488 (A-11008, Thermo Scientific). Hoechst 33258 (Sigma-Aldrich) was used for nuclear staining.

[0121] result All selected Fabs detected infected cells, with minor differences among the VOCs tested (Fig. 4 ).

[0122] Fab#5 binds to cells infected with D614G, alpha, beta, gamma, delta, omicron (BA.1), and omicron (BA.2). IgG#5 binds to cells infected with D614G, alpha, beta, gamma, delta, omicron (BA.1), omicron (BA.2), and omicron (BA.5), but not significantly to omicron (BA.4). Fab BU.2 binds to cells infected with D614G, alpha, gamma, delta, and omicron (BA.2), but not significantly to beta, and does not bind to omicron (BA.1) Fab BU.7 binds to cells infected with D614G, alpha, beta, gamma, delta, omicron (BA.1), and omicron (BA.2). IgG BU.7 binds to cells infected with D614G, alpha, beta, gamma, delta, omicron (BA.1), omicron (BA.2), omicron (BA.5), and omicron (BA.4). Fab BU.11 binds to cells infected with D614G, alpha, gamma, delta, and beta, but poorly to omicron (BA.2) and does not bind to omicron (BA.1) IgG BU.54 binds to cells infected with D614G, alpha, beta, gamma, delta, omicron (BA.1), omicron (BA.2), and omicron (BA.5), but not significantly to omicron (BA.4). Fab BS.70 binds to cells infected with D614G, alpha, beta, gamma, delta, and omicron (BA.1), as well as omicron (BA.2) and (BA.4), but not significantly to (BA.5).

[0123] The B.1.1.7 variant is better recognized by Fab#5, BU.2, BU.11 and IgG BU.54, but poorly recognized by BS.70 and BU.7 Fab. However, the IgG format of BU.7 shows improved binding activity for this variant. The B.1.135 variant is only well recognized by clone#5 in both Fab and IgG formats, IgG BU.7, IgG BU.54 and Fab BS.70, while only a faint green signal is observed using the other Fabs. Binding to the P.1 variant is well observed with all Fabs tested, although Fab#5 signal was the least intense. The delta variant is well recognized by all Fabs and IgGs tested, while Omicron (BA.1) is not recognized by only BU.2 and BU.11 Fabs. Finally, Omicron (BA.2) is recognized by all but Fab BU.11, albeit with a weaker signal than BA.1. Fab BS.70 is the clone showing the strongest binding signal to all mutants tested, followed by IgG#5, IgG BU.7 and IgG BU.54. Both Omicron BA.4 and BA.5 are well recognized by IgGBU.7 on infected cells. IgG#5 and IgG BU.54 strongly recognize the Omicron BA.5 sublineage and recognize BA.4 with a lower binding signal.

[0124] (Example 3): Neutralizing activity evaluation Microneutralization experiment Vero E6 cells were seeded in 96-well plates 24 hours before the experiment with 95% cell occupancy for each well. Two-fold serial dilutions of selected clones were incubated with SARS-CoV-2 at an MOI of 0.01 for 1 hour at 37°C. After washing the cells once with PBS, the virus-serum mixtures and positive infection controls were applied to the Vero E6 monolayers and virus adsorption was carried out for 1 hour at 37°C. The cells were then washed once with PBS to remove cell-free virus particles and virus-containing mixtures, and the controls were replaced with complete DMEM supplemented with 2% FBS. The plates were incubated for 72 hours at 37°C in the presence of CO2. Experiments were performed in triplicate. Neutralizing activity was assessed by the presence of cytopathic effect (CPE) detected in the presence of virus-serum mixtures compared to the positive infection controls.

[0125] result Clone #5 showed significant neutralizing activity against the D614G variant both as Fab format and as IgG1. However, IgG1 showed enhanced activity against the other tested VOCs compared to the Fab fragment, except for the B.1.135 variant (Figure 5A). All tested BU clones are able to efficiently inhibit D614G, but only BU.7 retains its activity against all tested VOCs. Fab BU.2 is able to neutralize the B.1.1.7 variant better than the other two, whereas BU.11 showed significant activity against P.1 (Figure 5B).

[0126] For more details, Fab#5 neutralizes D614G and to a lesser extent alpha and gamma IgG#5 neutralizes D614G, alpha, gamma, and less beta Fab BU.2 neutralizes D614G, alpha, delta, and less gamma & beta Fab BU.7 neutralizes D614G, alpha, gamma, beta, delta, and to a lesser extent omicron IgG BU.7 neutralizes D614G, alpha, gamma, beta, delta, omicron, and omicron (BA.2) Fab BU.11 neutralizes D614G and less alpha and gamma IgG BU.54 neutralizes D614G, alpha, gamma, beta, delta, omicron, and omicron (BA.2) Fab BS.70 neutralizes gamma and less omicrons

[0127] Table 2-3-4 below shows the IC values ​​shown by selected clones Fab#5, IgG#5, Fab BU.2, Fab BU.7, Fab BU.11, Fab BS.70, IgG BU.7, and IgG BU.54 against different VOCs of SARS-CoV-2. 50 Neutralizing activity expressed as μg / ml is summarized.

[0128] [Table 2] [Table 3] [Table 4]

[0129] Example 4: Neutralization assay using pseudoviruses method SERS-CoV-2-pseudotyped particles were prepared and titrated.

[0130] Briefly, HEK293 T cells were co-transfected with S plasmids from either SARS-CoV-1 or SARS-CoV-2 Omicron, HIV gag-pol and Luc-reporter plasmids.

[0131] Supernatants were harvested 72 hours after transfection, clarified, and filtered. Viral titers were assessed on HEK293T ACE2-TMPRSS2 cells using 2-fold serial dilutions of mock virus stocks. Luciferase expression was quantified after 48 hours (37°C in the presence of 5% CO2). Titers were calculated as RLU / ml.

[0132] Surrogate neutralization assay Neutralization assays were performed by incubating pseudovirus (106 RLU) with endpoint 2-fold serial dilutions of monoclonal antibodies for 1 h (37°C in the presence of 5% CO2) before adding the neutralization mixture to 104 HEK 293T-ACE2-TMPRSS2 cells per well. Luciferase activity was quantified 72 h post-infection (37°C).

[0133] result Neutralization assays using this alternative strategy show that monoclonal antibodies IgG BU.7 and IgG BU.54 of the invention neutralize the Omicron variants of SARS-CoV-1 and SARS CoV-2. Figure 6 shows the results of the neutralization assay using pseudoviruses.

[0134] Table 5 below shows the ICs exhibited by the selected clones IgG BU.7 and IgG BU.54. 50 Neutralizing activities against omicron variants of SARS-CoV-1 and SARS CoV-2, expressed as μg / ml, are summarized.

[0135] [Table 5]

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Claims

**Claim 1** An antibody or antibody fragment that binds to a conserved region of the spike protein of Sarbecovirus, a) i) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 93, ii) CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 94, and iii) CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 95, comprising a heavy chain variable sequence, and / or b) i) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 96, ii) CDR2 comprising an amino acid sequence selected from the group consisting of GAS, AAS, and DAS, and iii) CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 97, comprising a light chain variable sequence, characterized by an antigen-binding site comprising an antibody or antibody fragment. **Claim 2** The heavy chain variable sequence is 1) a framework region (FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 98, 2) a framework region (FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 99, 3) a framework region (FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 100, and 4) a framework region (FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 101, further comprising, and / or the light chain variable sequence is 5) a framework region (FR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 102, 6) a framework region (FR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 103, 7) A framework region (FR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 104, and 8) A framework region (FR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 105, further comprising The antibody or antibody fragment according to claim 1.

3. The heavy chain variable sequence and / or the light chain sequence are as follows: a) For the heavy chain variable sequence, a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9; for the light chain variable sequence, a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising or consisting of the amino acid sequence GAS, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:

20. b) For the heavy chain variable sequence, a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9; for the light chain variable sequence, a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising or consisting of the amino acid sequence GAS, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:

21. c) For the heavy chain variable sequence, a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 10; for the light chain variable sequence, a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence AAS, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

22. d) For the heavy chain variable array, CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 11; for the light chain variable array, CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 15, CDR2 comprising or consisting of the amino acid sequence AAS, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:

23. e) For the heavy chain variable array, CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 12; for the light chain variable array, CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 16, CDR2 comprising or consisting of the amino acid sequence DAS, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, or f) For the heavy chain variable array, CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 93, CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 94, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 95; for the light chain variable array, CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 96, CDR2 comprising or consisting of the amino acid sequence AAS, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:

97. The antibody or antibody fragment according to claim 1, comprising a combination of the above CDRs.

4. The heavy chain variable array and / or the light chain variable array is as follows: a) For the heavy chain variable array, FR1 comprising or consisting of SEQ ID NO: 25, FR2 comprising or consisting of SEQ ID NO: 29, FR3 comprising or consisting of SEQ ID NO: 33, and FR4 comprising or consisting of SEQ ID NO: 37; for the light chain variable array, FR1 comprising or consisting of SEQ ID NO: 41, FR2 comprising or consisting of SEQ ID NO: 46, FR3 comprising or consisting of SEQ ID NO: 51, and framework region FR4 comprising or consisting of SEQ ID NO:

56. b) For the heavy chain variable array, FR1 comprising or consisting of SEQ ID NO: 25, FR2 comprising or consisting of SEQ ID NO: 29, FR3 comprising or consisting of SEQ ID NO: 33, and FR4 comprising or consisting of SEQ ID NO: 38; for the light chain variable array, FR1 comprising or consisting of SEQ ID NO: 42, FR2 comprising or consisting of SEQ ID NO: 47, FR3 comprising or consisting of SEQ ID NO: 52, and FR4 comprising or consisting of SEQ ID NO:

57. c) For the heavy chain variable array, FR1 comprising or consisting of SEQ ID NO: 26, FR2 comprising or consisting of SEQ ID NO: 30, FR3 comprising or consisting of SEQ ID NO: 34, and FR4 comprising or consisting of SEQ ID NO: 39; for the light chain variable array, FR1 comprising or consisting of SEQ ID NO: 43, FR2 comprising or consisting of SEQ ID NO: 48, FR3 comprising or consisting of SEQ ID NO: 53, and FR4 comprising or consisting of SEQ ID NO:

58. d) For the heavy chain variable array, FR1 comprising or consisting of SEQ ID NO: 27, FR2 comprising or consisting of SEQ ID NO: 31, FR3 comprising or consisting of SEQ ID NO: 35, and FR4 comprising or consisting of SEQ ID NO: 40; for the light chain variable array, FR1 comprising or consisting of SEQ ID NO: 44, FR2 comprising or consisting of SEQ ID NO: 49, FR3 comprising or consisting of SEQ ID NO: 54, and FR4 comprising or consisting of SEQ ID NO:

56. e) For the heavy chain variable array, FR1 comprising or consisting of SEQ ID NO: 28, FR2 comprising or consisting of SEQ ID NO: 32, FR3 comprising or consisting of SEQ ID NO: 36, FR4 comprising or consisting of SEQ ID NO: 39; for the light chain variable array, FR1 comprising or consisting of SEQ ID NO: 45, FR2 comprising or consisting of SEQ ID NO: 50, FR3 comprising or consisting of SEQ ID NO: 55, and FR4 comprising or consisting of SEQ ID NO: 59, or, f) For the heavy chain variable array, FR1 comprising or consisting of SEQ ID NO: 98, FR2 comprising or consisting of SEQ ID NO: 99, FR3 comprising or consisting of SEQ ID NO: 100, FR4 comprising or consisting of SEQ ID NO: 101; for the light chain variable array, FR1 comprising or consisting of SEQ ID NO: 102, FR2 comprising or consisting of SEQ ID NO: 103, FR3 comprising or consisting of SEQ ID NO: 104, and FR4 comprising or consisting of SEQ ID NO: 105, and further comprising a combination of the FRs, the antibody or antibody fragment according to claim 3.

5. The antibody or antibody fragment according to claim 1, a) a heavy chain variable array comprising or consisting of the amino acid sequence of SEQ ID NO: 60 and / or a light chain variable array comprising or consisting of the amino acid sequence of SEQ ID NO: 61, or b) a heavy chain variable array comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and / or a light chain variable array comprising or consisting of the amino acid sequence of SEQ ID NO: 63, c) a heavy chain variable array comprising or consisting of the amino acid sequence of SEQ ID NO: 64 and / or a light chain variable array comprising or consisting of the amino acid sequence of SEQ ID NO: 65, d) a heavy chain variable array comprising or consisting of the amino acid sequence of SEQ ID NO: 66 and / or a light chain variable array comprising or consisting of the amino acid sequence of SEQ ID NO: 67, e) A heavy chain variable sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 68, and / or a light chain variable sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 69, or f) A heavy chain variable sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 70, and / or a light chain variable sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 71, An antibody or antibody fragment comprising or consisting of them. **Claim 6** The antibody or antibody fragment according to claim 1, which is a monoclonal antibody. **Claim 7** The antibody or antibody fragment according to claim 6, wherein the monoclonal antibody is a human monoclonal antibody. **Claim 8** An IgG selected from the group consisting of IgG1, IgG2, IgG3, IgG4, a Fab fragment, a single-chain antibody (scFv), a bispecific antibody, optionally conjugated to a drug or a label, the antibody or antibody fragment according to claim 1. **Claim 9** A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region and / or the light chain variable region of the antibody or antibody fragment according to claim 1. **Claim 10** An expression vector comprising at least one of the nucleotide sequences according to claim 9. **Claim 11** A host cell comprising the expression vector according to claim 10. **Claim 12** A hybridoma comprising at least one of the nucleotide sequences according to claim 9 for producing the antibody or antibody fragment according to any one of claims 1 to 7. **Claim 13** At least one of the nucleotide sequences or expression vectors according to claim 8 or claim 9, together with One or more pharmaceutically acceptable excipients and / or adjuvants, a formulation for passive immunoprevention based on molecules / vectors. **Claim 14** The antibody or antibody fragment according to claim 1 for use in the medical field for the treatment and / or prevention of a sarbecovirus-mediated disease in a subject. **Claim 15** The antibody or antibody fragment according to claim 14, wherein the sarbecovirus-mediated disease is severe acute respiratory syndrome mediated by SARS-CoV-1 or SARS-CoV-2 and their variants of concern. **Claim 16** The antibody or antibody fragment for use according to claim 15, wherein the mutant strain in question is selected from the group comprising the alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), D614G (University of Pavia), and omicron (B.1.1.529) mutant strains of SARS-CoV-2.

17. The antibody or antibody fragment for use according to claim 14, wherein the subject to be treated is an immunocompromised patient, a patient with a history of cardiovascular disease and / or respiratory disease, an elderly patient, or a subject who avoids vaccines.

18. The antibody or antibody fragment for use according to claim 14, wherein the first antibody or antibody fragment is used alone or in combination with a second antibody or antibody fragment against a different conserved region of the spike protein.

19. A pharmaceutical composition comprising at least one of the antibody or antibody fragment of the present invention as an active ingredient, together with one or more pharmaceutically acceptable excipients and / or adjuvants.

20. The pharmaceutical composition according to claim 19, which is suitable for intravenous, intramuscular, or subcutaneous administration.

21. A composition or kit consisting of parts for simultaneous, separate, or sequential administration in a subject suffering from a sarbecovirus-mediated disease, particularly severe acute respiratory syndrome mediated by SARS-CoV-1 or SARS-CoV-2, comprising a first antibody or antibody fragment that binds to a target conserved region in the spike protein of SARS-CoV-1 or SARS-CoV-2, and a second antibody or antibody fragment that binds to a target conserved region different from the first antibody in the spike protein of SARS-CoV-1 or SARS-CoV-2.

22. Use of the antibody or antibody fragment according to claim 1 for detecting the presence of a sarbecovirus in a biological sample.

23. Use of the antibody or antibody fragment according to claim 22, wherein the sarbecovirus is SARS-CoV-1 or SARS-CoV-2.

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