WO2022053188A1 - Nouvelles séquences d'adn encodant des anticorps humains scfv à activité neutralisante contre le sars-coronavirus-2 et leurs utilisations - Google Patents

Nouvelles séquences d'adn encodant des anticorps humains scfv à activité neutralisante contre le sars-coronavirus-2 et leurs utilisations Download PDF

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WO2022053188A1
WO2022053188A1 PCT/EP2021/064161 EP2021064161W WO2022053188A1 WO 2022053188 A1 WO2022053188 A1 WO 2022053188A1 EP 2021064161 W EP2021064161 W EP 2021064161W WO 2022053188 A1 WO2022053188 A1 WO 2022053188A1
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sars
cov
virus
antibody fragment
antibody
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Rita De Santis
Olga Minenkova
Emilio Merlo Pich
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Alfasigma S.P.A.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention relates to human antibody molecules in the format of single chain variable region fragments (scFv) capable of specifically binding to and neutralizing SARS- coronavirus 2 (SARS-CoV-2) and its variants, wherein the VH CDR3 sequence is SEQ ID NO:44.
  • the antibodies of such group are named scFv76-cluster and comprise the sequences as disclosed below.
  • the invention relates also to the compositions comprising human antibody molecules in the format of single chain variable region fragments (scFv), which are useful in the prophylaxis and/or treatment of a condition resulting from SARS-CoV 2 infection.
  • the invention relates to the corresponding DNA sequences, proteins encoded by such sequences and related protein compositions.
  • SARS-CoV-2 novel pathogenic SARS-coronavirus 2
  • Human convalescent serum is an option for prevention and treatment of COVID-19 disease based on the presence of high titer of specific antibodies for SARS-CoV-2 and it is now experimentally used for some COVID-19 patient populations or as prevention of infection in medical staff exposed to patients. It is considered a form of passive immunization, different from vaccination that typically requires an active clonal selection of specific antibodies occurring in the immune system.
  • Passive antibody therapy involves the administration of antibodies against a given agent (i.e. , a virus) to a susceptible individual for the purpose of preventing or treating an infectious disease due to that agent, thus providing immediate immunity to these persons compared to vaccination that requires time for the patient to develop its own immune response.
  • Passive antibody therapy has history going back to the 1890s and, at that time, before development of antibiotics, was the only mean of treating certain infectious diseases.
  • SARS-CoV-1 shows that convalescent sera contain neutralizing antibodies to the relevant virus.
  • SARS-CoV-2 the anticipated mechanism of action by which passive antibody therapy would mediate protection is viral neutralization.
  • Possible sources of antibodies for SARS-CoV-2 are human convalescent sera from individuals who have recovered from COVID-19, monoclonal antibodies, or recombinant antibody fragments.
  • Gattinger P and colleagues 1 reported that not always such antibodies prevent virus receptor binding and, finally, an immune related paradoxical phenomenon known as antibody dependent enhancement (ADE), that has already been described in other viral infections (i.e. Ebola), would not only be non-protective but even enhance infection and/or worsen the clinical course thus posing a note of caution when treating COVID-19 patients with convalescent sera 2 .
  • ADE antibody dependent enhancement
  • Yuan M and colleagues 3 that human anti-SARS-CoV-2 antibodies or antibody fragments can be generated and tested for virus neutralization. Yuan M and colleagues 3 , disclosed whole size human antibodies CC12.1 , CC12.3 and B38 also binding RBD and inhibiting viral infectivity.
  • caplacizumab for acquired thrombotic thrombocytopenic purpura (aTTP), a life-threatening autoimmune rare disorder (EMA/490172/2018), while it may not be considered acceptable for prevention and treatment of SARS-CoV-2 infection in a large population of otherwise healthy individuals.
  • aTTP acquired thrombotic thrombocytopenic purpura
  • EMA/490172/2018 life-threatening autoimmune rare disorder
  • hACE2 human angiotensin-converting enzyme 2
  • S protein uses its receptor-binding domain (RBD) to bind hACE2 and mediates viral protein to fuse with cellular membrane of epithelial cells as a step preceding viral internalization and infection 8 .
  • RBD-targeting antibodies as instrument for virus neutralization useful for treatment SARS-CoV-2 infection.
  • human single chain variable antibody fragments that specifically recognize an epitope within the human ACE2 receptor binding motif (RBM) of the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 have a neutralizing activity against SARS-CoV-2, wherein said antibody fragments comprise the VH CDR3 sequence ARDLSVAGAFDI (SEQ ID NO:44) and at least one of the protein with a sequence selected from amino acid sequences having any of SEQ ID NOs:2, 4, 6, 8, 10 or 12 can be used in the treatment and I or prevention of SARS-CoV 2 infection.
  • the antibody fragment has the ability to interfere with the binding of the virus spike protein to ACE2 receptor which results in a virus neutralization potency.
  • the present invention relates to human single chain variable antibody fragments (scFv) that specifically bind to the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 and have neutralizing activity against SARS-CoV-2 virus and variants of the virus having one or more mutations in the spike protein, wherein said antibody fragments comprise at least one of the sequences selected from amino acid sequences having any of SEQ ID NOs:2, 4, 6, 8, 10 or 12 or a sequence being at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, still more preferably at least 98% identical to any of said sequences, wherein the VH CDR3 sequence is ARDLSVAGAFDI (SEQ ID NO:44), wherein the VH and the VL variable regions are connected by a linker and wherein the antibody fragment binds at least to the following residues within the human ACE2 receptor binding motif of the RBD of the spike protein of the SARS-CoV-2 virus L455, F456,Y473, N487 and Y489
  • the invention moreover relates to a human single chain variable antigen-binding antibody fragment (scFv) that comprises an antigen-binding site specifically binding to the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 virus and variants of the virus having one or more mutations in the spike protein at the amino acids L455, F456, Y473, N487 and Y489 and wherein the antibody has an affinity to the spike protein of the SARS- CoV-2 virus or a variant thereof of 0.1 to 14.9 nM and an IC50 value for the SARS-CoV-2 virus between 16 and 1000 ng/ml when numbered according to SEQ ID NO:54.
  • scFv human single chain variable antigen-binding antibody fragment
  • Another object of the invention is a DNA sequence, wherein the sequence is selected from SEQ ID NOs:1 , 3, 5, 7, 9 or 11 or a degeneracy related sequence thereof or a codon optimized sequence thereof for expression in host cells suitable for industrial production. Processes of codon optimization are known in the art. They are based on sequence features considered to influence protein expression level, such as the use of frequent codons in a host organism or the introduction of stabilizing codons.
  • Another object of the invention is a pharmaceutical composition comprising at least one antibody fragment SEQ ID NOs:2, 4, 6, 8, 10 or 12 or a sequence being at least 85% identical, together with pharmaceutically acceptable excipients.
  • the present invention provides novel human single chain antigen-binding antibody fragments (scFv) made by VL and VH immunoglobulin variable regions connected by a flexible linker as defined above.
  • scFv being able to specifically bind with high affinity to the receptor binding domain (RBD) of the ‘spike’ protein of SARS-CoV-2.
  • the human single chain antigen binding antibody fragments (scFv) of the invention are useful for diagnosis, prevention and/or treatment of conditions related to SARS-CoV-2 infection and complications thereof in humans.
  • the invention provides novel DNA sequences encoding human scFv immunoglobulin fragments and compositions of molecules, encoded by such sequences, capable of specifically binding to SARS-CoV-2 and having SARS-CoV-2 neutralizing activity.
  • said binding molecules are human scFv antibody fragments selected from phage display libraries obtained by recombinant DNA methods starting from RNA of COVID-19 convalescent subjects.
  • the invention further provides for the use of the compositions of the invention in the prophylaxis and/or treatment of subjects having, or at risk of developing a condition resulting from SARS-CoV-2 exposure, by administering such compositions by nasal lavage, throat spray, drops or by aerosol or by oral route or intravenous infusion.
  • the RBD antigenic epitope recognized by the human scFv antibody fragments object of the present invention has been mapped in the receptor binding motif (RBM) of RBD at residues L455, F456, Y473, N487 and Y489 (numbering according to SEQ ID NO: 54, Figure 18), none of which has so far been involved in the RBD mutations of SARS-CoV-2 emerging variants of concern (VOCs).
  • RBM receptor binding motif
  • VOCs SARS-CoV-2 emerging variants of concern
  • the invention moreover relates to a probe based on SEQ ID NO:44 to isolate antibodies having a neutralizing activity against SARS-CoV2 as well as the antibodies isolated by said probe.
  • Figure 1 shows the binding activity of sera of convalescent COVID-19 individuals against spike-SARS-CoV-2, inhibition of spike- hACE2 interaction and inhibitory activity of viral infectivity.
  • Figure 2 shows SDS-PAGE (panel A) and Western Blot (panel B) analyses of scFv76-58 and scFv76 anti-SARS-CoV-2.
  • Figure 3 shows specific recognition of SARS-CoV-2 spike by scFv76 in spike transfected cells, through HCS imaging (panel A) and cytofluorimetry (panel B) analysis.
  • Figure 4 shows representative sensorgrams of scFv76 and scFv76-77 on RBD-Fc immobilized chip (Surface Plasmon Resonance, Biacore).
  • Figure 5 shows inhibition of spike-induced syncytia formation (cell-cell fusion) by scFv76.
  • Figure 6 shows the chromatographic profile after size exclusion chromatography of scFv76 before and after nebulization.
  • Figure 7 shows temperature resistance of scFv antibodies measured by binding ELISA on SARS-CoV-2 spike protein.
  • Figure 8 shows resistance to the trypsin digestion of scFv antibodies.
  • Figure 9 shows critical residues for scFv76 binding to spike monomer (epitope mapping).
  • Figure 10 shows average of bioluminescence signal expressed in photons/sec from mice infected with luciferase-expressing SARS-CoV-2 (D614G) pseudotyped virus and treated with scFv76.
  • Figure 11 shows the nucleotide and deduced amino acid sequence of scFv76.
  • Figure 12 shows the nucleotide and deduced amino acid sequence of scFv76-58.
  • Figure 13 shows the nucleotide and deduced amino acid sequence of scFv76-55.
  • Figure 14 shows the nucleotide and deduced amino acid sequence of scFv76-57.
  • Figure 15 shows the nucleotide and deduced amino acid sequence of scFv76-77.
  • Figure 16 shows the nucleotide and deduced amino acid sequence of scFv76-46.
  • Figure 17 shows the alignment of amino acid sequence of scFv76-cluster antibodies.
  • Figure 18 shows as SEQ ID NO:54 the spike RBD sequence of the wild type SARS-CoV2- Wuhan strain (PDB#6Z2M).
  • Bold residues are the residues described in SARS-CoV2- variants. Changes amino acids are shown.
  • the amino acids of the RBD epitope recognized by the scFv76 cluster antibodies at position 455, 456,473,487 and 489 are shown in black background. This numbering is the basis for the referenced sequence positions.
  • Figure 19 shows a Surface Plasmon Resonance experiment to investigate epitope recognition.
  • the present invention provides novel sequences encoding single chain variable antibody fragments (scFv) and pharmaceutical compositions comprising said proteins specifically binding to the Receptor Binding Domain (RBD) of the ‘spike’ protein of the SARS-CoV-2.
  • scFv single chain variable antibody fragments
  • RBD Receptor Binding Domain
  • scFv human single chain variable antibody fragments
  • VOCs spike protein variants of SARS- CoV-2 and neutralize variants of concern (VOCs) (www.cdc.gov/coronavirus/2019- ncov/variants/index.html) bearing mutations K417N, L452R, E484K and N501Y in relation to the wildtype strain (Wuhan strain PDB ID #6Z2M, SEQ ID NO:54, Figure 18).
  • the RBD epitope recognized by scFv antibodies of the present invention is composed by residues within the receptor binding motif (RBM) at positions L455, F456, Y473, N487 and Y489 none of which described as mutated in VOCs.
  • RBM receptor binding motif
  • Whole molecule IgG were previously described to recognize RBD. Yuan Met colleagues 3 , Science 2020 (htps://pubmed.ncbi.nlm.nih.gov/32661058 show that whole size human antibodies CC12.1 , CC12.3 and B38 also bind RBD and inhibit viral infectivity.
  • these antibodies have a shorter VH CDR3 (7-9 amino acids) and contact RBD in a much higher number of residues, also including residues known to be mutated in SARS-CoV-2 variants (i.e. A475, K417, N501 , Y453), compared to the scFv antibodies object of the present invention.
  • Cao Y et colleagues 13 Cell 2020 (htps://pubmed.ncbi.nlm.nih.gov/32425270 describe that 12 whole size neutralizing human antibodies also recognize RBD and inhibit interaction with human ACE2 but no information on the epitope recognized are provided and therefore foreseeing the implications of viral mutations on their reactivity is not possible.
  • the epitope amino acids within the ACE2 receptor binding motif of the SARS-CoV2-virus as identified according to the present invention are surprisingly associated with unique therapeutic properties of the antibody fragments binding to them as detailed below.
  • the antibody fragments of the invention are surprisingly useful against all presently known mutations of the virus.
  • the biochemical and viral neutralization properties of the scFv antibodies herein described are compatible with the development of pharmaceutical preparations to prevent and counteract the infection of SARS-CoV-2 and its related variants by intranasal and/or aerosol delivery and/or oral administration. To our knowledge, this is the first example of drug candidates in the human scFv antibody format for COVID-19 treatment.
  • the claimed six human scFv antibodies recognize an epitope within the human ACE2 receptor binding motif (RBM) which is spanning from residue 438 to residue 506 of the spike RBD (residue 333-527) (htps://doi.Org/10.3390/cells9112343).
  • RBM human ACE2 receptor binding motif
  • the epitope to which the antibodies of the invention bind consists of residues L455, F456, Y473, N487, Y489. It is to note that the scFv76-cluster antibody reactivity is not significantly affected by mutations at residues K417, L452, E484, N501 and D614 which are found singularly or combined in the most threatening VoCs and lineages of SARS-CoV-2 variants including B.1.427 and B.1.429 found in California (USA) and B1.1.7, P1 , B1.351 found in South Africa, Brazil and UK. Mutation L452R found combined with E484Q in the recently identified variant under investigation (VUI) B.1.617 in India.
  • the human scFv antibodies of the present invention are able to compete the SARS-CoV-2 spike-hACE2 binding in vitro.
  • the scFv76 antibody exhibits a potent neutralization activity against SARS-CoV-2 infectivity, together with surprisingly high resistance to physical/chemical stresses.
  • the scFv76-cluster antibodies herein described are derived from the IGHV3-53 germ line as many other monoclonal antibodies previously described. scFv76-cluster antibodies, except for scFv76-57 and scFv76-55 that use different IGKV sequences, pair with IGKV 3-20 germ line-derived light chains (Table 1 ).
  • ScFv58 uses the same IGHV germline-derived heavy chain with a different CDR3 sequence of scFv76 exhibiting lower viral neutralization activity. These referential scFv5, scFv50 and scFv86 all recognize RBD, but do not show viral neutralization properties. Analysis of sequence alignment of scFv76-cluster antibodies as shown in Figure 17, indicates that the biochemical and biological features of the claimed scFv antibodies, are surprisingly determined by a novel and unique VH CD3 as such properties are substantially maintained in the presence of mutations in the remaining VH region and independently of VL pairing.
  • Said VH CD3 region has SEQ ID NO:44.
  • the scFv76-cluster antibodies herein described show resistance to various stressing conditions (i.e., high temperature, proteolysis, nebulization, various pH, lyophilization), low risk of triggering excessive immune response or antibody-dependent enhancement (ADE) due to the absence of the Fc domain, and less expected immunogenicity than camelid nanobodies due to their origin from a human repertoire. Moreover, scFv76-cluster antibodies recognize an antigenic epitope surprisingly not affected by emerging SARS-CoV-2 mutations.
  • the antibody library used to select scFv76-cluster antibodies was specifically obtained by COVID-19 convalescent subjects selected for their high titer of anti-SARS-CoV- 2 immunoglobulins, therefore enriching for antibody fragment sequences that have neutralizing properties.
  • scFv antibodies described in the present invention appear to be among the most suitable products for their intended uses and not described by other so far.
  • the invention also describes the process for obtaining the proteins and the use in the prevention and treatment in patients of SARS-CoV-2 infections.
  • Such scFv proteins are capable of neutralizing infections caused by the virus by blocking its entrance into the human cells harboring hACE2 receptors.
  • the compositions are suitable for diagnosing, preventing and/or treating conditions resulting from SARS-CoV-2 infection.
  • Administration routes of the compositions, object of the present invention include but are not limited to addressing SARS-Cov-2 infection in the respiratory, olfactory and gastroenteric systems using various topical formulations, for example nasal spray solutions, aerosol and/or oral formulations.
  • the present invention describes fully human scFv antibodies that are soluble in aqueous solutions and exhibit higher affinity for the SARS-CoV-2 RBM within the RBD than most published human monoclonal antibodies particularly in the recognition of RBD mutations.
  • High binding properties, affinity and neutralization of RBD interaction with ACE2 are also maintained for mutated RBD proteins (Table 2, 3, 4).
  • the binding properties of scFv76-cluster antibodies correlate with high neutralization capacity of viral infectivity (Table 5, 6).
  • Figure 5 shows that scFv antibodies of the present invention, could be useful to prevent/counteract the effect of cell-cell fusion (syncytia formation) induced by SARS-CoV-2. This effect is expected to translate into beneficial therapeutic effect by reducing lung damages in COVID-19 patients.
  • scFv antibodies herein described correlates with their potent virus neutralization capacity in vitro (Table 5, 6) despite their monovalent format.
  • All scFv antibodies herein described were identified from phage display libraries obtained from blood- derived RNA of COVID-19 convalescent donors with very high anti-SARS-CoV-2 immunoglobulin titers. Phage display antibody libraries were obtained from RNA of selected convalescent subjects with the highest antibody response against the virus.
  • scFv antibodies object of the present invention, are surprisingly resistant to physical stressing conditions (i.e., high temperature, nebulization procedures, proteolytic digestion, variable pH, lyophilization) making these molecules ideal candidates for a variety of administration routes, such as nasal spray, aerosol, oral or intravenous routes ( Figure 6, Table 7, 8).
  • physical stressing conditions i.e., high temperature, nebulization procedures, proteolytic digestion, variable pH, lyophilization
  • the present invention provides means and methods for use of such scFv antibodies in prevention and/or treatment of SARS-CoV-2 infections based on the use of high affinity human scFv antibody fragments neutralizing viral infectivity by competing for the binding of SARS-CoV-2 spike RBD to the human ACE2 receptor without inducing potentially dangerous Fc-dependent immune activation that could be observed upon vaccination or passive antibody treatments in the COVID-19 syndrome.
  • the molecules of the present invention have lower immunogenic properties when compared with camelid antibodies. This is a critical safety advantage when the intended use is prevention of infection in otherwise healthy subjects.
  • the present invention provides single chain variable antibody fragments (scFv) that specifically bind to the receptor binding motif within the receptor binding domain of the spike protein of SARS-CoV-2 and have a neutralizing activity against SARS-CoV-2, thereby preventing or treating a SARS-CoV-2-related disorder, preferably COVID-19.
  • scFv single chain variable antibody fragments
  • the antibody fragments of the invention comprise at least one sequence selected from an amino acid sequence having any of SEQ ID NOs: 2, 4, 6, 8, 10 or 12 or a sequence being at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, still more preferably 98% identical to any of said sequences, wherein the VH CDR3 sequence is SEQ ID NO:44.
  • the antibody fragment of the invention may consist of only one of said sequences or may comprise two, three, four, five or six of said sequences connected to each other in variable order and optionally separated by a spacer.
  • the antibody fragment of the invention may also contain repetitions of the individual sequences in variable numbers or may contain combinations of identical sequences.
  • CDR regions are complementarity determining regions of an antibody and refer to the three hypervariable regions in each chain of the antibody that interrupt the four framework regions established by the light and heavy chain variable regions.
  • the CDRs are primarily responsible for antibody binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1 , CDR2 and CDR3 numbered sequentially starting from the N-terminus and are also typically identified by the chain in which the particular CDR is located.
  • VH CDR3 or CDR-H3 is located in a variable region of the heavy chain of the antibody in which it is found
  • a VL CDR1 or CDR-L1 is the CDR1 from the variable region of the light chain of the antibody in which it is found.
  • Phage display is a powerful tool for isolating peptides or antibodies binding to a given target.
  • a single chain antibody contains an antigen binding domain in one polypeptide instead of two as in monoclonal antibodies.
  • the above single chain antibody fragments may be genetically or chemically fused to effector moieties including, for example, antiviral drugs, anti-inflammatory drugs or half-life extender moieties such as human serum albumin or PEG or an avidity increasing molecule.
  • the above antibody fragments may be bispecific molecules being genetically or chemically fused to a second antibody recognizing an independent epitope of the SARS- CoV-2 receptor binding domain.
  • the bispecific molecules may also comprise two or more of the above single chain variable antibody fragments, optionally also fused to a second antibody recognizing an independent epitope of the SARS-CoV-2 receptor binding domain.
  • the respective parts of the molecule may be connected via a suitable linker or spacer.
  • an antibody fragment according to the invention is binding specifically to the receptor binding domain of the spike protein of the SARS-CoV-2 when it binds at least to the following residues within the human ACE2 receptor binding motif of the spike RBD of the SARS-CoV-2 virus L455, F456,Y473, N487 and Y489 via its antigen binding domain more readily than it would bind to a random unrelated epitope.
  • the term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
  • neutralizing activity is to mean an activity to stop the virus from infecting the body by affecting how the molecules on the surface of the virus can enter into host cells and/or stop propagation of said virus in the cells.
  • neutralization not only refers to a reduction but also to a prevention, inhibition and delay of the SARS-CoV- 2 virus propagation. Whether or not an antibody has a neutralizing activity against SARS- CoV-2 can be determined by methods known to a person skilled in the art. A corresponding SARS-CoV-2 microneutralization assay is disclosed below.
  • An antibody fragment according to the present invention is considered effective if it neutralizes at least 50% of a tissue culture infectious dose of the SARS-CoV-2 virus at an antibody concentration of 1000 ng/ml or less.
  • the term “being identical” means that two peptide sequences, when optimally aligned, such as by programs GAP or BEST FIT using default GAP ways as supplied with the program, share at least 85%, 90% identity, preferably at least 90% identity, more preferably at least 95% identity, still more preferably at least 98% sequence identity with the identified sequences.
  • residue positions that are not identical differ by conservative amino acid substitutions.
  • a conservative amino acid substitution is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g. a charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • sequence identity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches sequences using measures of similarity assigned to various substitutions, deletions and other modifications including conservative substitutions.
  • the invention relates also to the possibility to combine more than one neutralizing antibody fragment in a novel molecular entity, e.g., dimeric or multimeric peptides, that could target the SARS-CoV-2 in more efficient way, here defined as “ SARS-CoV-2 neutralizing antibody derivatives”.
  • the invention also relates to the use of SEQ ID NO:44 as a probe for identifying neutralizing antibodies against SARS-CoV-2 as well as the antibodies identified by said probe.
  • the invention also relates to pharmaceutical compositions comprising at least one of the above antibody fragments or derivatives thereof, together with pharmaceutically acceptable excipients.
  • the pharmaceutical composition may also be in association with further active ingredients such as antiviral agents, anti-inflammatory drugs, vitamins, minerals, etc. and contains pharmaceutically acceptable excipients.
  • the pharmaceutical composition is adjusted by means of suitable excipients to the application route of the composition.
  • An exemplary but not limiting pharmaceutical formulation for intranasal administration is composed of a solution for a dry powder containing in each puff 0.1 -1 .0 mg of an antibody fragment of the invention with 100-300 mM sucrose, 0.01 -0.07% Tween-80 and 0.1 -0.5% carboxymethyl or hydroxypropylcellulose.
  • the route of administration may be, for example, oral, parenteral, by inhalation or topical, for intranasal and oral cavity delivery.
  • parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration.
  • the preferred application route of the antibody fragments of the invention is an intranasal administration.
  • the formulations may be in any form that is suitable for the desired administration routes.
  • topical intranasal or nasal delivery are nasal spray, nebulizers for aerosol, nasal drops, nasal wash or irrigation solutions, typically containing at least one of the above scFv antibodies or their multimeric derivatives as the active pharmaceutical ingredient at doses of 0.01 to 100 mg.
  • the devices and procedures to obtain optimal delivery of the scFv antibodies and their derivatives may be of any type that is suitable to achieve effective delivery of the scFv antibodies or derivatives thereof to the nasal mucosa of the anterior, intermediate and posterior part of the nasal cavity, as well as to the olfactory epithelium and olfactory nerve endings.
  • oral delivery are powders, water dispersible granules, capsules, soft gel capsules, tablets or syrups for oral delivery, typically containing one of the above scFv antibodies or their derivatives as the active pharmaceutical ingredient at doses of 0.01 to 100 mg.
  • scFv antibodies dry formulations of scFv antibodies as powder for nasal/throat/lung topical application typically containing scFv antibody or derivatives thereof as the Active Pharmaceutical Ingredient (API) at doses of 0.01-100 mg.
  • the antibody may be suitably labelled, for example by coupling the antibody of the invention to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials and non-radioactive paramagnetic metal ions.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials and non-radioactive paramagnetic metal ions.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials and non-radioactive paramagnetic metal ions.
  • the invention also relates to the above antibody fragments or derivatives thereof, their dimeric or multimeric compositions defined as derivatives or to the above pharmaceutical compositions for use in a method of preventing a SARS-CoV-2 virus-mediated disorder, preferably COVID-19.
  • Prevention of SARS-CoV-2 infection typically requires the topical administration of scFv antibodies or derivatives thereof to the nasal cavity and/or oral cavity and related proximal organs of the upper and lower respiratory system (e.g., larynx and trachea and lung) and the gastrointestinal system in individual that may have risk of exposure to affected individuals, at doses of 0.01 -100 mg. This approach will reduce the probability of airborne virus to infect the individuals that choose this treatment.
  • SARS-CoV-2 infection can be by parenteral administration of scFv antibodies at doses that could be ranging from 1 to 15000 mg in case of exposure of a subject to contaminated air, gas, liquid, objects.
  • the invention also relates to the above antibody fragments, their dimeric or multimeric compositions or to the above pharmaceutical compositions for use in a method of treating a SARS-CoV-2 virus-mediated disorder, preferably COVID-19.
  • SARS- CoV-2 can cause severe pneumonia, diarrhea, dyspnea and multiple organ failure in humans.
  • the antibodies of the invention are particularly useful to avoid thromboembolic or inflammatory complications of a SARS-CoV2- infection and/or reducing the damage to the lung caused thereby.
  • SARS-CoV-2 recombinant spike glycoproteins used for analysis of human sera from convalescent individuals and for selection of scFv-displayed library were purchased from different sources to diversify tag sequences and facilitate selection.
  • S1 SARS-CoV-2 spike glycoprotein
  • S1 SARS-CoV-2 spike glycoprotein
  • S1 SARS-CoV-2 spike glycoprotein
  • S1 SARS-CoV-2 spike glycoprotein
  • S1 SARS-CoV-2 spike glycoprotein
  • S1 SARS-CoV-2 spike glycoprotein
  • S1 S1
  • sheep Fc-Tag expressed in HEK293 cells
  • Reative Diagnostics For libraries selection and analysis of phage clones SARS-CoV-2 Spike RBD-human Fc-tag (Sino Biological) and SARS-CoV-2 RBD-mouse Fc- tag (Creative Diagnostics) were used.
  • Human ACE2-Fc (mouse IgG 1 ) expressed in HEK293 cells was purchased from Sino Biological
  • the immune human antibody phage display libraries were prepared by using cDNA obtained from lymphocytes of convalescent COVID-19 individuals chosen based on their serum ability to recognise spike glycoprotein and capacity to neutralize the SARS-CoV-2 infectivity in vitro.
  • Three libraries, CV1 , CV10 and CVMIX, were prepared from the cDNA derived from CV1 , CV10 and pool of CV3, CV6, CV8 and CV9 mRNA.
  • These libraries were prepared in scFv format by inserting assembled scFv genes in proprietary vector pKM19 (WO 2007/074496 A2 and Pavoni E et al, 2007) 10 .
  • the libraries had average size about of 3- 5x10E7 independent clones each.
  • the libraries were preincubated with mouse serum for 30 min at 37°C and then panned on 2 pg of mouse Fc-fused RBD protein adsorbed on Dynabeads conjugated with Protein G (Dynal) for 1 h at 37 °C.
  • the phages bound to RBD antigen were separated by magnetic concentrator (Dynal).
  • PBST PBS containing 0.05% Tween 20
  • bound phages were eluted by using 0.1 M glycine-HCI, pH 2.2, and neutralized with 2 M Tris, pH 9.0.
  • Second round of selection was performed by coating a well of ELISA plate with 0.5 pg of RBD protein.
  • scFv genes of selected antibodies were cloned in pKM16 for expression 15 .
  • Suspensions of bacteria transformed with single scFv expression plasmid were keep at -80°C in medium with 10% glycerol and plated when necessary to get single colonies.
  • the samples were further purified from endotoxin by using Triton X-114. To purify solution from residual Triton X-114 Pierce Detergent Removal Spin Columns were used, according to manufacturer’s instruction. Samples were filter sterilized and stored in aliquots at -80 °C. Protein identity was estimated by SDS-PAGE and Western blot analyses. Protein concentration was measured spectrophotometrically by using Nano Drop 2000c (Thermo Scientific).
  • phage ELISA multiwell plates (Immunoplate Maxisorb, Nunc) were coated overnight at 4 °C with SARS-CoV-2 RBD-human Fc-tag solution in PBS or unrelated proteins at a concentration of 0.5 pg/mL in PBS. After discarding coating solution, plates were blocked 1 hour at 37 °C with blocking buffer (5% non-fat dry milk, 0.05% Tween 20 in PBS). Then, plates were washed several times with washing buffer (0.05% Tween 20 in PBS). Phage supernatants in blocking buffer (1 :1 ) were added to each well and incubated 1 hour at 37 °C.
  • blocking buffer 5% non-fat dry milk, 0.05% Tween 20 in PBS
  • the plates were washed and bound phages detected by an anti-M13 HRP-conjugated (Amersham Biosciences, Uppsala, Sweden) antibody.
  • the immunoreaction was developed by incubation with TMB liquid substrate (Sigma) 15 minutes and stopped by the addition of 25 pL 2 M H2SO4. The results were expressed as the difference between absorbance at 450 and 620 nm, determined by an automated ELISA reader.
  • ELISA with soluble scFv was developed by using an anti-FLAG HRP-conjugated antibody (A8592, Sigma Aldrich).
  • Multiwell plates (Immunoplate Maxisorb, Nunc) were coated overnight at 4 °C with SARS- CoV-2 spike glycoprotein (S1 ) with sheep Fc-Tag at a concentration of 0.5 pg/mL in PBS. The plates were blocked with 5% non-fat dry milk, 0.05% Tween 20 in PBS. Sera diluted in blocking buffer were added to the wells and incubated for 1 hour at 37 °C. The plates were washed, and the bound immunoglobulins detected by anti-human Fc polyclonal antibodies produced in goat (A0170, Sigma-Aldrich). The serum dilution giving value of 0.5 at A450nm was calculated.
  • the plates were coated with SARS-CoV-2 spike glycoprotein (S1 ) with sheep Fc-Tag as above. After washing and blocking, 50 pL of different sera dilutions or scFv (2x concentration) in blocking buffer were added to the wells and incubated for 30 min. Then 50 pL of ACE2-mouse Fc at the concentration of 2 pg/mL (2x concentration) were added to the wells and incubated further 60 min. Human ACE-mouse Fc binding to spike was detected by using an anti-mouse alkaline phosphatase-conjugated secondary antibody (A2429, Sigma-Aldrich) preincubated with 5% of sheep serum in blocking buffer.
  • S1 SARS-CoV-2 spike glycoprotein
  • sheep Fc-Tag sheep Fc-Tag
  • the VERO cells suspensions in culture medium at the concentration of 10E5 cells/mL were distributed at 100 pL/wells in the 96-well plates and checked under the microscope to confirm homogeneous cell distribution.
  • the plates were incubated overnight at 37 °C.
  • Serial sera or scFv dilutions were incubated with virus for 1 h at 37 °C and added to the cells. Cytopathogenic effect was read 4 days after infection.
  • Micro-Neutralization 50 antibody titer neutralizing sample according to the Reed- Muench method.
  • the MN50 corresponds to the highest dilution of sample which completely protects the cells from CPE (no lysis) in at least half of the replicates (i.e., 2 wells or more out of 4 wells).
  • HCS High Content Screening
  • SPR analysis was performed by a Biacore T200 (GE).
  • SARS-CoV-2 RBD-Fc was immobilized on a CM5 chip (Cytiva) to a response level of 1000 Rll.
  • ScFvs were fluxed on the chip at six different concentrations at a flow rate of 30 mL/min with a contact time of 480 s and a dissociation time of 900 s.
  • Sensorgrams were recorded by subtraction of a reference cell, and data obtained were fitted by Biacore Evaluation Software 3.2 with a 1 :1 fitting model.
  • 96-well maxisorp plate was coated with 100 pL/well of 0.5 pg/ml S1 protein or RBD domain solution in PBS at 4°C, ON. Coating solution was discarded, wells were washed with washing solution, PBS, 0.1 % Tween20 and blocked for 2 h with blocking solution, 1 % BSA in PBS, 0.1 % Tween20. ScFv antibodies at double concentration: 600, 200, 66, 22 ng/mL were dispensed in blocking buffer 50 pL/well and incubated for 30 min at 37 °C.
  • hACE2 50 pL/well of hACE2 at double concentration 2 pg/mL were added to the well without discarding scFv solution and incubated for further 60 min at 37°C. Plate was washed and bound hACE2 was detected by using an anti-mouse Fc alkaline phosphatase-conjugated antibody.
  • 293T cells were co-transfected with plasmids encoding Flag-tagged SARS-CoV-2 S-glycoprotein (Sino Biological) and GFP (pCMV-GFP, Clontech) using Lipofectamine 2000 (Life Technologies). 293T cells, co-transfected with the GFP- encoding plasmid and the empty vector, were used as negative control. At 40 hours post transfection cells were detached with trypsin and incubated with different concentrations of neutralizing scFv76 or non-neutralizing scFv5 antibodies at 37°C.
  • lentiviral SARS-CoV-2 S-pseudotyped particles (LV) carrying a firefly luciferase reporter gene 293T cells were co-transfected with the SARS-2 S-CF plasmid (or alternatively the mutants N501Y, N501Y+E484K and K417N+E484K+N501Y or the pCAGGS-SARS2-S-G614 plasmid), the pCMVR 8.74 lentiviral packaging plasmid and the pLenti CMV Puro LUC plasmid.
  • Caco-2 hACE2-expressing cells were plated in white clear-bottom 96-well plates. After 24 h, 100 JJL of DMEM containing 5% FCS and 8 pg/mL polybrene were dispensed into the wells, and cells were incubated for 30 min. Serial dilutions of scFv were prepared in medium. Each dilution was tested in duplicate. Aliquots (60 JJ L) of the virus particle suspension were added to an equal amount of each antibody dilution (60 JJL) and incubated for 1 hour at 37°C. Pseudovirus:scFv mixtures (50yL) were then added to Caco-2 hACE2 cells after medium removal.
  • Luciferase activity (Relative Luciferase Units or RLU) was detected at 72 h post-infection by using Bright-Glo Luciferase Assay System Kit (Promega) in a Microplate Luminometer (Wallac-Perkin Elmer).
  • Shotgun Mutagenesis epitope mapping services were provided by Integral Molecular (Philadelphia, PA).
  • a mutation library of the spike protein was created by high-throughput, site-directed mutagenesis. Each residue was individually mutated to alanine, with alanine codons mutated to serine.
  • the mutant library was arrayed in 384-well microplates and transiently transfected into HEK 293-T cells. Following transfection, cells were incubated with the indicated antibodies at concentrations pre-determined using an independent immunofluorescence titration curve on wild type protein. ScFv was detected using an Alexa Fluor 488-conjugated secondary antibody and mean cellular fluorescence was determined using Intellicyt iQue flow cytometry platform.
  • Mutated residues were identified as being critical to the scFv76 epitope if they did not support the reactivity of the test antibody but did support the reactivity of the reference MAb. This counterscreen strategy facilitates the exclusion of mutants that are locally misfolded or that have an expression defect.
  • mice Female 4-6 weeks old, were infected via the intranasal route (10 pl/nostril) with a SARS-Cov2-Spike-Luc pseudotyped virus (Lenti-LUC-Spike D614G). A luciferase-expressing lentivirus was used (10 pl/nostril) as negative control. Two h before and four h after the Lenti-LUC-Spike D614G infection, scFv76 antibody (3,7 mg/ml in PBS) was intranasally (10 pl/nostril) administered to mice.
  • SARS-Cov2-Spike-Luc pseudotyped virus Lenti-LUC-Spike D614G
  • scFv76 antibody 3,7 mg/ml in PBS
  • Lenti-LUC-Spike D614G bioluminescence was quantitatively measured through the IVIS200 optical imaging system. Optical imaging mice total body was performed with anesthetized mice. Explanted lungs and nasal turbinates were imaged after euthanasia.
  • Example 1 SELECTION OF BEST IMMUNOGLOBULIN DONORS AMONG COVID-19 CONVALESCENT SUBJECTS
  • MN50 corresponds to the serum dilution inhibiting 50% of cells from viral cytopathogenic effect in at least half of the replicates.
  • Amplification of the antibody variable region genes was performed on cDNA from RNA selected from the best immune COVID-19 responders.
  • Amplified scFv immunoglobulin repertoires were used to construct phage-displayed scFv libraries then used for selection of high affinity antibodies.
  • Amplified scFvs genes were cloned in phage display vector pKM19 for efficient selection and/or maturation of antibodies as in W02007/074496 A2 and Pavoni E et al, 2007 10 . This vector allows to reduce biological bias for scFv expression in E. coli and achieve more complete presentation of functional antibody repertoire in the phage display library.
  • Affinity maturation was also performed in some cases with the purpose to improve features of the selected scFvs. From 6 donors among the best responders were generated 3 separate scFv-diplayed libraries by using amplified VH and VL gene fragments from cDNA obtained from patients CV1 (LibCVI ), CV10 (LibCVI O), mixture of cDNA from CV3, CV6, CV8 and CV9 (LibMIX) with complexity of about 3-5x10E7 clones each.
  • scFv antibodies Three maturation libraries from 50, 58 and 76 phage clones were then generated by error-prone PCR amplification of heavy chains and by shuffling them with light chains from immune and naive light chain repertoires. Average library’s complexity was about 2x10E8 clones. Several high- affinity clones were selected from the maturation libraries.
  • the most active scFv antibodies described in the present invention utilize the IGHV3-53 germ line which is known to be relatively invariant and to be present in several other high affinity antibodies against SARS- CoV-2 3 .
  • the germ line analysis of scFv antibodies of the present invention was performed by alignment on data base https://www.ncbi. nlm. nih. qc wiqbl t //. This analysis revealed identity of the selected antibodies to FR1 -CDR1 -FR2-CDR2-FR3 of the IGHV3-53-1 and new CDR3 region responsible for antigen recognition.
  • Example 3 Construction of scFv libraries from RNA extracted from peripheral blood lymphocytes of convalescent COVID-19 individuals
  • RNAs from lymphocytes of 10 COVID-19 convalescent patients were used to synthesize cDNA by Super Script IV VILO master Mix (Thermo Fisher). About 2.5 pg of RNA was used for each reaction. For each sample of RNA 2-3 reactions were done. Gene fragments encoding for heavy chains were PCR amplified with primers:
  • VHJ5 ACC AGA GCC GCC GCC GCT ACC ACC ACC ACC TGA AGA GAC GGT GAC CAT TGT CCC
  • VHJ6 ACC AGA GCC GCC GCC GCC GCT ACC ACC ACC ACC TGA GGA GAC GGT GAC CGT GGT CCC
  • VKJ2/4 CGG CAC CGG CGC ACC TGC GGC CGC ACG TTT GAT CTC CAS CTT GGT CCC
  • VKJ3 CGG CAC CGG CGC ACC TGC GGC CGC ACG TTT GAT ATC CAC TTT GGT CCC
  • VL3b AGC GGC GGC GGC GGC TCT GGT GGT GGT GGA TCC TCT TCT GAG CTG ACT CAG GAC CC
  • K means mixture of 50% G and 50% T nucleotides
  • Y means mixture of 50% T and 50% C
  • S means mixture of 50% G and 50% C.
  • Primers of SEQID N° 27-30 for heavy chain and primers of SEQID N° 31-37 for light chain have overlapping sequences and encode for standard flexible linker (SerGlyGlyGlyGly)3 as described by Huston JS et al, 11 . Amplified antibody DNA fragments were purified from agarose gel and assembled in single chain sequences by 20 cycles of PCR-like process without primers.
  • Assembled scFv gene fragments were purified by PCR-purification kit (Quiagen), digested with Notl and Ncol endonucleases and cloned in phagemid pKM19, digested with Notl and Ncol and dephosphorylated with Calf Intestinal Phosphatase (CIP).
  • the scFv gene fragments were ligated with vector by using T4 DNA ligase. All enzymes were purchased from New England BioLabs.
  • Ligated DNA plasmids were used to transform E. coli DH5aF’ electrocom petent cells. Transformed bacteria were plated into large LA plates, containing ampicillin and glucose. Next day, bacteria colonies were collected with small quantity of LB. Glycerol was added to have final concentration 10-15%. Aliquots of libraries were stored at -80 °C.
  • the beads were washed 10 times with PBS containing 0.05% Tween 20.
  • the bound phage was eluted with 100 pl of elution buffer (0.1 M glycine pH 2.2 adjusted with HCI) 10 min at room temperature and then neutralized with 10 pl of 2M Tris, pH 9.6.
  • the eluted phage was used to infect 0.6 ml of E. coli DH5aF’ cells.
  • the bacteria were plated into a large Luria broth (LA) plate, containing ampicillin and glucose. Next day, bacteria colonies were collected with a small quantity of LB. Glycerol was added to have final concentration 10- 15%.
  • Example 6 Affinity maturation of scFv antibodies
  • Heavy chain DNA fragments of the chosen clones 50 and 76 were amplified with upstream vector primer KM167 GGC TCG TAT GTT GTG TGG AAT TG (SEQ ID NQ:40) and reverse primer VHJ1 - 2-4-5 ACC AGA GCC GCC GCC GCC GCT ACC ACC ACC TGA GGA GAC GGT GAC CAG GGT KCC (SEQ ID NO: 41 ).
  • Heavy chain DNA of 58 was amplified with KM167 and VKJ5 CGG CAC CGG CGC ACC TGC GGC CGC ACG TTT AAT CTC CAG TCG TGT CCC (SEQ ID NO: 42).
  • error-prone conditions were used:
  • Amplified heavy chain DNA fragments were purified from agarose gel and assembled with light chains from immune and naive light chain repertoires in single chain antibody sequences by 20 cycles of PCR-like process without primers as described in Example 2. Assembled scFv gene fragments were cloned in the pKM19 phagemid, as described in Example 2. Three maturation libraries with complexity of about 2x10E8 clones each were constructed. First round of selection of maturation libraries was performed by using RBD mouse Fc-bound dynabeads as described in Example 3. For second round of selection one well of ELISA plate was coated with 0.5 pg/well of SARS-CoV-2 RBD mouse Fc in PBS overnight at 4°C.
  • Conditions of the second round of selection were designed to be stringent.
  • pool of phage after first round of selection was amplified by standard procedure as described in Example 2.
  • the phage suspension was incubated for 1 h at 63 °C before adding to the antigen coated well. After binding the well with bound phage was intensively washed with washing buffer 10 times and then once with 0.1 M glycine pH 4.0 adjusted with HCI for 10 min. The remaining bound phage was eluted, amplified and stored as in Example 3 and 4.
  • Stringent conditions for second round of selection facilitated the selection of scFv particularly resistant to high temperature.
  • a single colony was inoculated in 50 ml LB containing ampicillin and 2% glucose and incubated at 37°C up to OD 0.8. The cells were centrifuged. Obtained pellet was resuspend in 50 ml of fresh LB containing ampicillin and 20 mM MgCl2. The cells were incubated overnight at 30°C. Bacteria were collected by centrifugation. The pellet was resuspended pellet in 2 ml PBS containing anti-protease cocktail. After three cycles of freeze and thaw procedure, cell debris were pelleted by centrifugation. The scFv antibody fragments from crude periplasm ic extract were affinity purified on Ni-NTA resin (Merck) according to manufacturer’s instruction.
  • Imidazole-eluted fractions containing soluble scFv antibodies were pooled and imidazole removed by using Amicon Ultra, 3 kDa cutoff.
  • Amicon Ultra 3 kDa cutoff.
  • 5 pl of triton X-114 were added. The sample was intensively mixed by Vortex, then placed on ice for 2-3 min. The procedure was repeated 3 times. The tube was placed in water bath at 40°C for 2- 3 min, and then mixed. The procedure was repeated 3 times. The sample was centrifuged 30 sec. The triton was found as a drop at the bottom of the tube. The upper phase was carefully collected avoiding contaminating with the triton. The purification procedure was repeated totally 3 times. Residual triton was removed from the scFv solution by using Pierce Detergent Removal Spin Column according to manufacturer’s instruction.
  • scFv76 showing specific spike recognition are given in Figure 3 that shows specific recognition of SARS-CoV-2 spike in spike transfected cells by HCS fluorescence imaging (panel A) and by cytofluorimetry (panel B) by scFv76.
  • panel A spike transfected cells show staining in the cytoplasm and on the surface membrane when incubated with scFv76 followed by FITC-conjugated anti-Flag antibody. Not transfected cells and spike transfected cells without scFv are negative indicating specific recognition of the spike by scFv.
  • VOCs Recombinant spike proteins representative of SARS-CoV-2 variants of concern
  • D614G protein S1 D614G
  • UOCs Recombinant spike proteins representative of SARS-CoV-2 variants of concern
  • results in Table 2 show that all scFv antibodies, except for scFv76-55 which loses some reactivity against S1 (SA-BR), are highly resilient in their binding to the spike variants. Binding analysis of scFv antibodies was extended to single RBD mutations. Results shown on the right side of the Table 2 indicate that single mutations affect differently the binding capacity of antibodies with scFv76-55, scFv5 and scFv86 being the most sensitive to RBD mutations.
  • Data are expressed as ng/mL to have 1.0 OD and are the erage of 2-3 independent experiments ( ⁇ SD).
  • WT Wild strain
  • Example 11 Affinity of anti-SARS-CoV-2 scFv antibodies for the RBD protein
  • the affinity of anti-SARS-CoV-2 scFv antibodies of the present invention for spike protein was analyzed by Surface Plasmon Resonance (SPR). Representative sensorgrams of scFv76 against spike variants S1 (D614G), S1 (UK) and S1 (SA_BR) are shown in Figure 4. The graph shows association and dissociation curves of the scFv at different concentrations indicating particularly low dissociation rate consistent with high affinity. In fact, clone 76 exhibits a KD of 1.08 ⁇ 0.26 nM and clone 76-77 exhibits a KD of 0.44 ⁇ 0.07 nM. (Table 3).
  • Example 12 Capacity of scFv antibodies to inhibit the binding of the viral spike to the human
  • scFv antibodies were evaluated for their ability to inhibit the binding of the viral spike and related variants to the human ACE2 receptor.
  • IC50 values (expressed as ng/mL) are the mean ( ⁇ SE) from 2-4 independent experiments.
  • results in Table 4 show that scFv76-cluster antibodies but not scFv5 and scFv86 inhibit the interaction of S1 wild type (WT), S1 (D614G), S1 (UK) or S1 (SA-BR) with ACE2. Consistently with binding data, inhibition activity of scFv76-55 is the most affected by SA-BR mutations. Effect of single RBD mutations on inhibition by scFv antibodies of binding to ACE2 is also shown in the Table 4. Boxed number indicates fold increase > 10x versus WT (Wuhan strain).
  • Results show A475V and K417N as the most critical mutations for scFv76-55 reactivity.
  • concentration (ng/mL) of antibodies to inhibit by 50% the interaction of all spike variants as well as single RBD mutations with ACE2 is reported in Table 4.
  • results indicate that the scFv76-cluster antibodies, except for scFv76-55, maintain the capacity to strongly inhibit the binding of spike variants and mutated RBD to ACE2.
  • Example 13 Viral neutralization of anti-SARS-CoV-2 scFv antibodies
  • Example 14 Neutralization of pseudovirus bearing spike mutations by anti-SARS-CoV-2 scFv76-cluster antibodies against pseudovirus bearing spike mutations
  • ACE2-transfected CaCo2 cells were infected with luciferase expressing pseudotyped SARS-CoV-2 virus bearing single (D614G, N501Y) or triple (K417N, E484K, N501Y) mutations.
  • Results in Table 6 confirm that all scFv76-cluster antibodies, but not scFv5, inhibit pseudovirus variants and that, consistently with previous data, scFv76-55 antibody is the most sensitive to mutations.
  • Data, expressed as ng/mL to inhibit by 50% viral infection, are the average ( ⁇ SD) of 2 independent replicates of one representative experiment.
  • IC50 ng/mL viral infectivity of luciferase-expressing pseudotyped SARS-CoV-2 virus with spike mutations
  • Example 15 Anti-SARS-CoV-2 scFv antibodies inhibit syncytia formation
  • the effect of the scFv76 antibody on Virus-induced syncytia has been evaluated by a cellfusion assay.
  • the scFv76 antibody proved to be strongly effective at inhibiting cell-cell fusion between Wuhan or D614G spike-expressing human HEK293T cells and human ACE2- transfected A549 cells.
  • HEK293T cells co-transfected with plasmid encoding the SARS- CoV-2 Spike (D614G) and plasmid encoding GFP, or empty vector and GFP (Mock) were detached at 40 h post transfection and incubated 1 h with 10 pg/mL scFv76 or scFv5 antibodies in culture medium.
  • Example 16 Suitability of anti-SARS-CoV-2 scFv76-cluster antibodies to nebulization
  • FIG. 6 shows representative data of scFv76.
  • the antibody (2mg/mL in PBS) was nebulized 5 minutes by means of an AirFamily apparatus for aerosol (Piclndolor) and the nebulized mist recovered into a Falcon tube.
  • HPLC analysis was performed loading 50 pl of before and after nebulization samples on a TSKgel G3000SWXL (7.8x300 mm) column (Tosoh Bioscience).
  • Example 17 Resistance of anti-SARS-CoV-2 scFv76-cluster antibodies to thermal stress
  • Selected scFv76-cluster antibodies were subjected to physical stress by heating. Crude extracts were incubated 1 h at the temperatures 60 and 65 °C. Treated scFv antibodies were analyzed by ELISA against SARS-CoV-2 RBD compared to untreated extracts. ELISA multiwell plates (Immunoplate Maxisorb, Nunc) were coated overnight at 4 °C with SARS- CoV-2 RBD-human Fc-tag. Crude periplasmic extracts were incubated 1 h at the indicated temperatures, plated in the coated wells and incubated 1 hour at 37 °C. Binding of scFv antibody fragments to the antigen was developed by using an anti-FLAG HRP-conjugated antibody.
  • Figure 7 and Table 7 show that scFv76and scFv76-58 antibodies are surprisingly stable to heating.
  • scFv50 antibody and related scFv50-81 B are not thermal resistant; note that these clones do not have the same CDR3 sequence of scFv76.
  • Table 7 indicates the absorbance ELISA values at 450 nM.
  • Example 18 Resistance of anti-SARS-CoV-2 scFv antibodies to trypsin digestion
  • Example 19 Epitope mapping of anti-SARS-CoV-2 scFv76 antibody
  • Results in Table 9 indicate reactivity loss of scFv76 antibody but not of mAb CoV16 (directed against an independent RBD epitope), towards 5 mutated SARS-CoV-2 spike proteins with alanine substituted to residues L455, F456, Y473, N487 and Y489. These residues are within the spike RBM with F456 being the most critical mutation for the scFv76 antibody spike recognition.
  • Figure 9 visualizes the critical residues (spheres) on a crystal of the SARS-CoV- 2 spike protein monomer.
  • Table 9 shows the identification of critical residues for scFv76 RBD binding. Binding to each mutant protein in the alanine scanning library was determined, in duplicate, by high- throughput flow cytometry. For each mutant RBD protein, the mean binding value was plotted as a function of expression (represented by control reactivity of CoV16 antibody). Table 9
  • Example 20 In vivo experiment of neutralization of SARS-CoV-2 using intranasal or aerosol formulations of scFv antibodies in transgenic mice
  • Transgenic human ACE2 mice were intranasally infected with 4x10E5 PFU of SARS-CoV- 2. High viral titers as measured by gPCR were found in lung, brain, trachea, 6 days after infection. High titers or inflammatory cytokines were also detected in bronchoalveolar lavage (BAL) fluid and serum.
  • BAL bronchoalveolar lavage
  • scFv herein described in PBS solution at doses between 0.5-10 mg/Kg, one day before or 3 h after infection, by nasal instillation or by aerosol with nebulization devices as described by De Santis R et al 2014 12 resulted in marked reduction of viral titers as well as inflammatory cytokines in all relevant tissues. Histopathological observation also confirmed the reduction of viral-induced alveolar damage in scFv-treated mice compared to controls.
  • Example 21 In vivo experiment of neutralization of luciferase expressing pseudotyped virus by scFv76 antibody
  • the following experiment shows that the scFv76cluster antibodies basically all recognize the same epitope on the RBD binding motif of the SARS CoV2 spike protein, i.e. bind to the VH CDR3 seguence.
  • RBD mFc-tagged (Sino Biological, 10 pg/mL in acetate buffer pH 5) was immobilized at 300 Rll level on the surface of a flow cell of a Series S sensor chip CM5 (Cytiva) by classical amine coupling procedure, while another flow cell surface was blank-immobilized with ethanolamine to use it as control surface.

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Abstract

La présente invention concerne un fragment d'anticorps humain à chaîne unique du fragment variable (scFv) se liant à l'antigène qui se lie spécifiquement au domaine de liaison au récepteur de la protéine de spicule du virus SARS-CoV-2 et des variants du virus ayant une ou plusieurs mutations dans la protéine de spicule, et qui présente une activité neutralisante contre le SARS-CoV-2 et ses variants de la protéine de spicule, ledit fragment d'anticorps comprenant au moins une des séquences choisies parmi une séquence d'acides aminés comportant l'une quelconque des SEQ ID NOS : 2, 4, 6, 8, 10 ou 12 ou une séquence étant au moins 85 % identique, de préférence au moins 90 % identique, plus préférablement au moins 95 % identique, encore plus préférablement au moins 98 % identique à l'une quelconque desdites séquences, dans laquelle la séquence CDR3 de VH est ARDLSVAGAFDI (SEQ ID NO : 44), dans laquelle les régions variables VH et VL sont reliées par un lieur, et le fragment d'anticorps se liant au moins aux résidus suivants dans le motif de liaison du récepteur ACE2 humain du domaine de liaison au récepteur de la protéine de spicule du virus SARS-CoV-2 L455, F456, Y473, N487 et Y489 et à une séquence d'ADN codant pour lesdits fragments, des compositions pharmaceutiques comprenant lesdits fragments d'anticorps et leurs dérivés, ainsi que lesdits fragments d'anticorps et les compositions pharmaceutiques les comprenant, pour une utilisation dans une méthode de prévention ou de traitement d'une affection provoquée par le virus SARS-CoV-2, de préférence la COVID-19.
PCT/EP2021/064161 2020-09-11 2021-05-27 Nouvelles séquences d'adn encodant des anticorps humains scfv à activité neutralisante contre le sars-coronavirus-2 et leurs utilisations WO2022053188A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2024019110A1 (fr) * 2022-07-20 2024-01-25 国立感染症研究所長が代表する日本国 Anticorps contre le sars-cov-2

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