EP4175982A1 - Neutralizing antibodies to sars coronavirus-2 - Google Patents

Neutralizing antibodies to sars coronavirus-2

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Publication number
EP4175982A1
EP4175982A1 EP21735766.4A EP21735766A EP4175982A1 EP 4175982 A1 EP4175982 A1 EP 4175982A1 EP 21735766 A EP21735766 A EP 21735766A EP 4175982 A1 EP4175982 A1 EP 4175982A1
Authority
EP
European Patent Office
Prior art keywords
seq
antibody
sars
cov
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21735766.4A
Other languages
German (de)
French (fr)
Inventor
Emanuele ANDREANO
Anna Kabanova
Dario CARDAMONE
Concetta DE SANTI
Ida PACIELLO
Noemi MANGANARO
Elisa PANTANO
Piero Pileri
Claudia SALA
Rino Rappuoli
Marco TROISI
Fabioa VACCA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fondazione Toscana Life Sciences
Original Assignee
Fondazione Toscana Life Sciences
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Filing date
Publication date
Priority claimed from IT102020000015754A external-priority patent/IT202000015754A1/en
Application filed by Fondazione Toscana Life Sciences filed Critical Fondazione Toscana Life Sciences
Publication of EP4175982A1 publication Critical patent/EP4175982A1/en
Pending legal-status Critical Current

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Classifications

    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • C07K2317/70—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

Definitions

  • the present invention relates to monoclonal antibodies or antigen-binding portion thereof that have a potent neutralizing activity against Coronavirus, in particular against SARS- CoV-2.
  • the invention relates also to the use of such monoclonal antibodies or antigen binding portion thereof in therapy, prophylaxis, and diagnosis of Coronavirus, in particular SARS-CoV-2 dependent diseases.
  • mAbs Human monoclonal antibodies
  • mAbs Human monoclonal antibodies
  • the well-established safety profile and the large experience for their development make mAbs ideal candidates for rapid development especially in epidemic and pandemic settings. So far mAbs have rarely been used in the field of infectious diseases, mostly because the large quantities needed for therapy made them not cost effective.
  • the enormous technological progress in isolating and screening memory B cells allowed identification of highly potent neutralizing mAbs and further improvement of their potency by several orders of magnitude through established engineering procedures. This possibility resulted in a decreased quantity of antibodies necessary for therapy thus making non- intravenous delivery of potent neutralizing mAbs possible.
  • mAbs have the possibility to become one of the first drugs that can be used for immediate therapy of any patient testing positive for the virus, and even to provide immediate protection from infection in high-risk populations.
  • Preliminary evidences show that plasma from infected subjects improves the outcome of patients with severe disease, therefore it is highly possible that a mAb-based therapy and/or prophylaxis be highly effective.
  • vaccination strategies inducing neutralizing antibodies have already shown to protect non-human primates from infection.
  • mAbs offer a series of advantages.
  • they are the ones that can be developed in the shortest period of time.
  • the extensive clinical experience with the safety of more than 50 commercial mAbs approved to treat cancer, inflammation and autoimmunity provides high confidence on their safety, support the possibility of having an accelerated regulatory pathway.
  • the long industrial experience in developing and manufacturing mAbs decreases the risks usually associated with technical development of investigational products.
  • the enormous technical progress in the field allows to shorten the conventional timelines and go from discovery to proof of concept trials in 5-6 months.
  • Several candidates are presently under development in the field of HIV, pandemic influenza, RSV and many other infectious diseases.
  • SARS-CoV-2 entry into host cells is mediated by the interaction between S-protein and the human angiotensin converting enzyme 2 (ACE2).
  • ACE2 human angiotensin converting enzyme 2
  • the S-protein is a trimeric class I viral fusion protein which exists as a metastable pre-fusion conformation and as a stable post-fusion conformation.
  • Each S-protein monomer is composed of two distinct regions, the SI and S2 subunits. Structural rearrangement occurs when the receptor binding domain (RBD) present in the SI subunit binds to the host cell membrane. This interaction destabilizes the pre-fusion state of the S-protein triggering the transition into the post-fusion conformation which in turn results in the entry of the virus particle into the host cell.
  • RBD receptor binding domain
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds to a region of human severe acute respiratory syndrome (SARS) Corona Virus 2 (SARS-CoV-2) Spike (S) protein.
  • said region is i) in the SI domain of SARS-CoV-2 S-protein; or (ii) in the S2 domain of SARS-CoV-2 S-protein; or (iii) in the SARS-CoV-2 S-protein trimer in its pre-fusion conformation or (iv) in the SARS-CoV-2 S-protein trimer in its post-fusion conformation or (v) in the receptor binding domain (RBD) of SARS-CoV-2 S-protein or in a combination thereof.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 Corona Virus 2
  • S receptor binding domain
  • the invention provides a human monoclonal antibody or antigen-binding portion that specifically binds to human severe acute respiratory syndrome (SARS) Corona Virus (SARS-CoV-2) S-protein, wherein said antibody or antigen-binding portion thereof provides more than 25% inhibition of the binding between the human ACE2 receptor and the viral Spike protein as measured by the neutralization of binding (NOB) assay.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 Corona Virus
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds to human severe acute respiratory syndrome (SARS) Corona Virus (SARS-CoV-2) S-protein, wherein said antibody or antigen-binding portion thereof has a neutralizing activity.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 Corona Virus
  • ICIOO inhibitory concentration
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising the light chain variable domain (VL) and heavy chain variable domain (VH) of a monoclonal antibody selected from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising the CDRs of a monoclonal antibody selected from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02,
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising the VL and VH domains that are at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to the VL and VH domains, respectively, of a monoclonal antibody selected from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
  • the invention provides human monoclonal antibodies or antigen-binding portion thereof that compete for the SARS-CoV-2 S-protein with any of the antibodies herein disclosed.
  • the invention provides human monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed, for use in a prophylactic or therapeutic treatment of a viral infection or conditions or disorders resulting from such infection.
  • the invention provides human monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed, for use in a prophylactic or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular Coronavirus disease 2019 (COVID-19).
  • the invention provides a method of preventing or treating the SARS-CoV- 2 infection or conditions or disorders resulting from such infection, in particular Coronavirus disease 2019 (COVID-19), comprising administering a human monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed, to a subject in need thereof.
  • the invention further provides human monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed for use in the diagnosis, prophylaxis and/or treatment of a subject having, or at risk of developing, a virus infection, in particular a coronavirus infection, more in particular SARS-CoV-2 infection. Furthermore, the invention pertains to the use of the human binding molecules and/or the nucleic acid molecules of the invention in the diagnosis/detection of such viral infections.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one or more human monoclonal antibodies or antigen-binding portions thereof according to any one of the embodiments herein disclosed and a pharmaceutically acceptable carrier and its use in the prevention and/or treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular Coronavirus disease 2019 (COVID-19).
  • the invention provides an isolated cell line that produces the antibody or antigen-binding portion thereof according to any one of the embodiments herein disclosed.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the antibody or antigen-binding portion thereof according to any one of the embodiments herein disclosed.
  • the invention provides a vector comprising the nucleic acid molecule encoding the antibody or antigen-binding portion thereof embodiments according to any one of the embodiments herein disclosed, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule.
  • the invention provides a non-human transgenic animal or transgenic plant comprising the nucleic acid according to any one of the preceding embodiments, wherein the non human transgenic animal or transgenic plant expresses said nucleic acid.
  • said non-human transgenic animal is a mammal.
  • the invention provides the use of the human monoclonal antibody or an antigen-binding portion thereof according to any one of the embodiments herein disclosed in the diagnosis of the SARS-CoV-2 infection.
  • the invention provides an in vitro method for revealing the presence of the SARS-CoV-2 in a sample comprising the following steps: i) Contacting the antibody or an antigen-binding portion thereof according to any one of the embodiments herein disclosed; ii) Detecting the binding of said antibody or an antigen-binding portion thereof to the S-protein of the SARS-CoV-2.
  • the invention provides an in vitro method for the diagnosis of the SARS- CoV-2 infection in a subject comprising the following steps: i) Contacting the antibody or an antigen-binding portion thereof according to any one of the embodiments herein disclosed with a biological sample of said subject; ii) Detecting the binding of said antibody or an antigen-binding portion thereof to the S-protein of the SARS-CoV-2.
  • Fig. 1 S-protein binding and neutralization titration of SARS-CoV-2 convalescent donors’ plasma.
  • A Plasma samples were two-fold diluted starting at 1 :80 to test their ability to bind the S-protein trimer in its pre-fusion state by ELISA. Results were considered as positive when the OD405 value was at least two times higher than the blank.
  • B Plasma samples were two-fold diluted starting at 1 : 10 to test their ability to neutralize SARS-CoV- 2 in vitro. Results were considered as positive when no cytopathic effect (-) was observed on Vero E6 cells.
  • the graph shows on the Y axis the Logio S-protein binding titer and on the X axis the Logio neutralization of binding (NOB) titer of plasma collected from COVID-19 convalescent patients.
  • the graph shows on the Y axis the Logio S-protein binding titer and on the X axis the Logio neutralization titer of plasma collected from COVID-19 convalescent patients.
  • Fig. 2 Gating strategy for S-protein specific MBC single cell sorting. Starting from top left to the right panel, the gating strategy shows: Live/Dead; Morphology; Singlets; CD19 + B cells; CD19 + CD27 + IgD ; CD19 + CD27 + IgDTgM ; CD19 + CD27 + IgDTgM S-protein + B cells.
  • FIG. 3 Identification of SARS-CoV-2 S-protein specific mAbs isolated from convalescent donors.
  • A The graph shows supernatants tested for binding to the SARS- CoV-2 S-protein SI + S2 subunits. Threshold of positivity has been set as two times the value of the blank (dotted line) Darker dots represent mAbs which bind to the SI + S2 while light yellow dots represent mAbs which do not bind. The total number (N) of single cell sorted B cell supernatants screened for binding is also shown for each donor.
  • B The graph shows supernatants tested for binding to the SARS-CoV-2 S-protein stabilized in its pre fusion conformation.
  • Threshold of positivity has been set as two times the value of the blank (dotted line). Red dots represent mAbs which bind to the S-protein while pink dots represent mAbs which do not bind.
  • the total number (N) of single cell sorted B cell supernatants screened for binding is also shown for each donor.
  • Fig. 4 Neutralization of S-protein binding to Vero E6 cell receptors by S-protein specific mAbs.
  • A Schematic representation of the neutralization of binding (NOB) assay used to screen isolated S-protein specific mAbs for their ability to abrogate the interaction between SARS-CoV-2 and Vero E6 cell receptors.
  • B The graph shows supernatants tested by NOB assay. Threshold of positivity has been set as 50% of binding neutralization (dotted line). Dark blue dots represent mAbs able to neutralize the binding between SARS-CoV-2 and receptors on Vero E6 cells, while light blue dots represent non-neutralizing mAbs. The total number (N) of S-protein specific supernatants screened by NOB assay is shown for each donor.
  • FIG. 5 SARS-CoV-2 neutralization assay for S-protein specific mAbs.
  • A Schematic representation of the virus neutralization assay used in this study to assess functional activities of S-protein specific mAbs.
  • B Representative microscope images showing the cytopathic effect of SARS-CoV-2 or the protective efficacy of the screened supernatants on Vero E6 cells.
  • Fig. 6 Workflow and timeline for SARS-CoV-2 neutralizing antibodies identification.
  • the overall scheme shows three different phases for the identification of SARS-CoV-2 neutralizing antibodies (nAbs).
  • Fig. 7 Identification of SARS-CoV-2 S-protein specific neutralizing antibodies (nAbs).
  • the graph shows supernatants tested for binding to the SARS-CoV-2 S-protein stabilized in its prefusion conformation. Threshold of positivity has been set as two times the value of the blank (dotted line). Red dots represent mAbs which bind to the S-protein while pink dots represent mAbs which do not bind.
  • the bar graph shows the percentage of not-neutralizing (gray), partially neutralizing (pale yellow) and neutralizing mAbs (dark red) identified per each donor. The total number (N) of antibodies tested per individual is shown on top of each bar.
  • C The graph shows the neutralization potency of each nAb tested once expressed as recombinant full-length IgGl.
  • Dashed lines show different ranges of neutralization potency (500 - 100 - 10 ng/mL). Dots were colored based on their neutralization potency and were classified as weakly neutralizing (>500 ng/mL; pale orange), medium neutralizing (100 - 500 ng/mL; orange), highly neutralizing (10 - 100 ng/mL; dark orange) and extremely neutralizing (1 - 10 ng/mL; dark red). The total number (N) of antibodies tested per individual is shown on top of each graph.
  • Fig. 8 Functional characterization of potent SARS-CoV-2 S-protein specific nAbs.
  • a - B - C Graphs show binding curves to the S-protein in its trimeric conformation, S 1 -domain and S2-domain. Mean ⁇ SD of technical triplicates are shown. Dashed lines represent the threshold of positivity;
  • D Neutralization of binding (NoB) curves for selected antibodies were shown as percentage of reduction of signal emitted by a fluorescently labled S-protein incubated with Vero E6 cells. Mean ⁇ SD of technical duplicates are shown.
  • Dashed lines represent the threshold of positivity;
  • E - F Neutralization curves for selected antibodies were shown as percentage of viral neutralization against the authentic SARS- CoV-2 wild type and D614G strains. Data are representative of technical triplicates.
  • G - H Neutralization potency of fourteen selected antibodies against the authentic SARS-CoV-2 wild type and D614G strains. Dashed lines show different ranges of neutralization potency (500 - 100 - 10 ng/mL). In all graphs selected antibodies are shown in dark red, pink, gray and light blue based on their ability to recognize the SARS-CoV-2 Sl-RBD, SI -domain, S- protein trimer only and S2-domain respectively.
  • Fig. 9 Identification of four different sites of pathogen vulnerability on the S-protein surface.
  • A Representative cytometer peaks per each of the four antibody groups are shown. Positive (only primary antibody) and negative (un-conjugated beads) controls are shown as green and red peaks respectively. Competing and not-competing nAbs are shown in blue and green peaks.
  • B The heatmap shows the competition matrix observed among the 14 nAbs tested. Threshold of competition was set at 50% of fluorescent signal reduction. A speculative representation of the vulnerability sites are shown on the S-protein surface.
  • FIG. 10 Heavy and light chain analyses of selected nAbs.
  • a - B Bar graphs show the heavy and light chains usage for neutralizing antibodies against SARS-CoV-2 in the public repertoire compared to the antibodies identified in this study. Our and public antibodies are shown in dark and light colors respectively.
  • C - D The heavy and light chain percentage of identity to the inferred germline and amino acidic CDR3 length are shown as violin and distribution plot respectively.
  • E The heatmap shows the frequency of heavy and light chain pairing for SARS-CoV-2 neutralizing human monoclonal antibodies already published. The number within the heatmap cells represent the amount of nAbs described in this manuscript showing a novel heavy and light chain rearrangement.
  • FIG. 11 EM epitope mapping of RBD mAbs.
  • A Negative stain, 200 nm scale bar is shown;
  • B binding on the RBD;
  • C epitope/paratope interaction residues;
  • D Epitope region recognized on the receptor binding moif (RBM) and shared residues.
  • FIG. 12 Prophylactic efficacy of MAD0004J08 in the golden Syrian hamster model of SARS-CoV-2 infection.
  • A Schematic representation of the prophylactic study performed in golden Syrian hamster.
  • B The figure shows the impact of three different doses of J08- MUT on body weight loss change upon SARS-CoV-2 infection.
  • Statistical analyses were performed among hamsters that received J08-MUT and the IgGl isotype control. Statistically significant differences were calculated with the two-way analysis of variance (ANOVA) and significances are shown as * (p ⁇ 0.05), ** (p ⁇ 0.01), *** (p ⁇ 0.001) and ****
  • FIG. 13 Therapeutic efficacy of MAD0004J08 in the golden Syrian hamster model of SARS-CoV-2 infection.
  • A Schematic representation of the therapeutic study performed in golden Syrian hamster.
  • B The figure shows the impact on body weight loss change upon SARS-CoV-2 infection.
  • Statistical analyses were performed among hamsters that received J08 and the IgGl isotype control. Statistically significant differences were calculated with the two-way analysis of variance (ANOVA) and significances are shown as * (p ⁇ 0.05), ** (p£0.01), *** (p ⁇ 0.001) and **** (p ⁇ 0.0001).
  • FIG. 14 Neutralization activity of human monoclonal antibodies against SARS-CoV-2 PT188-EM.
  • a - B Neutralization curves against the SARS-CoV-2 wild type and SARS- CoV-2 PT188-EM are shown respectively.
  • C Summary of neutralization potency observed for the fourteen selected antibodies. Colours as dark red, pink, gray and light blue represent antibodies in the Sl-RBD, SI, S-protein and S2 Groups respectively.
  • FIG. 15 J08 neutralization activity against SARS-CoV-2 variants by CPE-based assay.
  • a - D Graphs show the neutralization activity of MAD0004J08 against the wild type virus (WT) (A), UK (B), BZ (C) and SA (D) variants.
  • WT wild type virus
  • BZ BZ
  • SA D
  • ICIOO 100% inhibitory concentration
  • FIG. 1 J08 neutralization activity against SARS-CoV-2 variants by S-fusion neutralization assay.
  • the graph shows the neutralization activity of J08 against the D614G, UK, IN, SA and BZ variants.
  • the table below reports the 100% inhibitory concentration (ICIOO) observed per each variant.
  • FIG. 17 J08 binding to different states of the SARS-CoV-2 RBD.
  • A The graph shows the binding of J08 to the RBD-down tight (state 1), loose (state 2) and up (state 3) position.
  • J08 Fab is shown in dark and light blue for the heavy and light chain respectively while the whole SARS-CoV-2 spike is shown in gray.
  • Red spheres show three highly mutated residues in SARS-CoV-2 VoCs which are K417, E484 and N501.
  • B Shows the overall binding region of J08 to the RBD in the three different states.
  • C - E The three panel show the amino acidic interaction of the heavy and light chain to the RBD in its state 1 (C), state 2 (D) and state 3 (E).
  • FIG. 18 J08 footprint on the RBD.
  • A This panel shows the spike protein (in gray) with the RBD in its up state. Red spheres show three highly mutated residues in SARS-CoV-2 VoCs which are K417, E484 and N501. This panel also show J08, S2E12, CV07-250 and ACE2 position in respect to the RBD. In brackets the immunoglobulin heavy chain variable region (VH) for each antibody is reported.
  • VH immunoglobulin heavy chain variable region
  • FIG. 19 Evolution of an authentic SARS-CoV-2 escape mutant.
  • A The graph shows J08 neutralization titer after each mutation acquired by the authentic virus.
  • B SARS-CoV-2 S- protein gene showing type, position of mutations and frequency of mutations.
  • Figure 20 The impact of SARS-CoV-2 J08 escape mutant RBD and NTD mutations using a lentiviral pseudotype platform.
  • A The graph shows the ND50 curves of J08 against the lentiviral pseudotype particles developed to mimic the evolution of the authentic SARS- CoV-2 escape mutant of J08.
  • B The graph shows the ND50 curves of J08 against the lentiviral pseudotype particles developed to assess the impact of single RBD and NTD mutations, or the combination of E484D + Q493H, on J08.
  • FIG. 21 Assessment of hPBMC activation by J08-WT and MUT.
  • A the graph shows the production of IFN-a by hPBMC co-incubated with SARS-CoV-2 in presence of J08 WT and MUT.
  • B the graph shows the production of IL-6 by hPBMC co-incubated with SARS- CoV-2 in presence of J08 WT and MUT
  • polypeptide encompasses native or artificial proteins, protein fragments and polypeptide analogues of a protein sequence.
  • a polypeptide may be monomeric or polymeric.
  • isolated protein is a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a protein may also be rendered substantially free of naturally-associated components by isolation, using protein purification techniques well known in the art.
  • isolated antibodies include an anti- SARS- CoV-2 S-protein antibody that has been affinity purified using SARS-CoV-2 S-protein or a portion thereof, an anti- SARS-CoV-2 S-protein antibody that has been synthesized by a hybridoma or other cell line in vitro , and a human anti- SARS-CoV-2 S-protein antibody derived from a transgenic animal.
  • a protein or polypeptide is "substantially pure", “substantially homogeneous", or “substantially purified” when at least about 60 to 75% of a sample exhibits a single polypeptide.
  • the polypeptide or protein may be monomeric or multimeric.
  • a substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% WAV of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.
  • polypeptide fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence.
  • fragments are at least 5, 6, 8 or 10 amino acids long.
  • the fragments are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150 or 200 amino acids long.
  • polypeptide analogue refers to a polypeptide that comprises a segment that has substantial identity to a portion of an amino acid sequence and that has at least one of the following properties: (1) specific binding to SARS-CoV-2 S-protein under suitable binding conditions, (2) ability to inhibit SARS-CoV-2 S-protein.
  • polypeptide analogues comprise a conservative amino acid substitution (or insertion or deletion) with respect to the native sequence.
  • Analogues typically are at least 20 or 25 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as a full- length polypeptide.
  • polypeptide fragments or polypeptide analogue antibodies with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 substitutions from the germline amino acid sequence.
  • amino acid substitutions to an anti- SARS-CoV-2 S-protein antibody or antigen-binding portion thereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogues, but still retain specific binding to SARS-CoV-2 S-protein.
  • Analogues can include various muteins of a sequence other than the normally occurring peptide sequence.
  • single or multiple amino acid substitutions may be made in the normally occurring sequence, preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence; e.g., a replacement amino acid should not alter the anti-parallel [beta]-sheet that makes up the immunoglobulin binding domain that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence.
  • glycine and proline would not be used in an anti-parallel [beta]-sheet.
  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), the type of coronavirus that causes coronavirus disease 2019 (COVID-19), where an "antibody” is referred to herein with respect to the invention, it is normally understood that an antigen-binding portion thereof may also be used.
  • An antigen-binding portion competes with the intact antibody for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., second ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, nanobodies and any polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.
  • CDR complementarity determining region
  • an antibody that is referred to by number is the same as a monoclonal antibody that is obtained from the human peripheral blood mononuclear cells (PBMCs) isolated from the donor of the same number.
  • PBMCs peripheral blood mononuclear cells
  • monoclonal antibody MAD0004J08 is the same antibody as one obtained from PBMCs isolated from subject identified by the code 004, or a subclone thereof.
  • a Fd fragment means an antibody fragment that consists of the VH and CH 1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al, Nature 341:544-546 (1989)) consists of a VH domain.
  • the antibody is a single-chain antibody (scFv) in which a VL and VH domains are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain.
  • scFv single-chain antibody
  • the antibodies are diabodies, i.e., are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.
  • a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.
  • the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated noncovalently.
  • the binding sites may be identical to one another or may be different.
  • human antibody means any antibody in which the variable and constant domain sequences are human sequences or any of the CDRs of the variable domain sequences are human sequences.
  • the term encompasses antibodies with sequences derived from human genes, but which have been changed, e.g. to decrease possible immunogenicity, increase affinity, eliminate cysteines that might cause undesirable folding, etc.
  • the term encompasses such antibodies produced recombinantly in non-human cells, which might impart glycosylation not typical of human cells.
  • chimeric antibody as used herein means an antibody that comprises regions from two or more different antibodies.
  • epitope includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule.
  • Epitopes or antigenic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three- dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope may be "linear” or “conformational.” In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another.
  • a “neutralizing antibody”, an antibody with “neutralizing activity”, as used herein means an antibody that neutralizes a biological effect that its target (e.g., a pathogen or an infectious particle) may have.
  • a “neutralizing antibody”, an antibody with “neutralizing activity”, as used herein is for example an antibody or antigen-binding portion thereof showing a 100% inhibitory concentration (ICIOO) of at least less than 100 ng/ml, preferably less than 50 ng/ml, more preferably less than 25 ng/ml when tested in an in vitro neutralization assay against the SARS-CoV-2 vims, performed for example as disclosed herein in the examples.
  • ICIOO inhibitory concentration
  • an antibody is said to specifically bind an antigen when the dissociation constant is for example ⁇ 1 mM, preferably ⁇ 100 nM and most preferably ⁇ 10 nM.
  • the dissociation constant may be measured by any of the methods available in the state of the art as for example using enzyme-linked immunosorbent assay (ELISAs), radioimmunoassay (RIAs), flow cytometry, surface plasmon resonance, such as BIACORE(TM).
  • the expression “specifically binds to a region of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike (S) protein” as herein means that the antibody or its antigen-binding portion provokes more than 50% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay as described in the examples.
  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
  • polynucleotide as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms.
  • isolated polynucleotide as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotides with which the "isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
  • nucleotides as used herein includes deoxyribonucleotides and ribonucleotides.
  • modified nucleotides as used herein includes nucleotides with modified or substituted sugar groups and the like.
  • oligonucleotide linkages referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et ah, Nucl. Acids Res.
  • oligonucleotide can include a label for detection, if desired.
  • “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • expression control sequence means polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • vector as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a plasmid, i.e., a circular double stranded piece of DNA into which additional DNA segments may be ligated.
  • the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • the vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").
  • recombinant host cell means a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell” and “host cell” mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • sequence identity in the context of nucleotide or aminoacidic sequences means the residues in two sequences that are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides.
  • polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs available, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods MoF Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J MoF Biol 276:71-84 (1998); incorporated herein by reference).
  • nucleic acid or fragment thereof, or aminoacidic when referring to a nucleic acid or fragment thereof, or aminoacidic means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights as supplied with the programs, share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity.
  • 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., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well- known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994).
  • Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulphur-containing side chains: cysteine and methionine.
  • Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al, Science 256:1443-45 (1992), incorporated herein by reference.
  • a “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix. 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 amino acid substitutions.
  • GCG contains programs such as "Gap” and "Bestfit” which can be used with default parameters as specified by the programs to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof.
  • the terms “label” or “labelled” refers to incorporation of another molecule in the antibody.
  • the label is a detectable marker, e.g., incorporation of a radiolabelled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods).
  • the label or marker can be therapeutic, e.g., a drug conjugate or toxin.
  • Various methods of labelling polypeptides and glycoproteins are known in the art and may be used.
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds to a region of human severe acute respiratory syndrome (SARS) Corona Virus 2 (SARS-CoV-2) Spike (S) protein and at least partially inhibits the S-protein binding to a receptor.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 Corona Virus 2
  • S Spike
  • said region is i) in the SI domain of SARS-CoV-2 S-protein; or (ii) in the S2 domain of SARS-CoV-2 S-protein; or (iii) in the SARS-CoV-2 S-protein trimer in its pre-fusion conformation or in its post-fusion conformation or a combination of i) with ii) or in a combination of i) with iii) or in a combination of ii) with iii) or a combination of i) with iv) or (v) in the receptor binding domain (RBD) of SARS-CoV-2 S-protein.
  • RBD receptor binding domain
  • said region is in the SI domain of SARS-CoV-2 S-protein and said human monoclonal antibody or antigen-binding portion thereof is selected from J08, 114, F05, G12, C14, B07, 121, J13 and D14.
  • said region is in the receptor binding domain (RBD) of SARS-CoV-2 S-protein and said human monoclonal antibody or antigen-binding portion thereof is selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12 and MAD0008C14.
  • the receptor-binding domain (RBD) is an independently folded domain of the S-protein known in the art (see for example Wrapp D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Science 367: 1260-1263).
  • the invention provides a human monoclonal antibody or antigen-binding portion that specifically binds to human severe acute respiratory syndrome (SARS) Corona Virus (SARS-CoV-2) S-protein, wherein said antibody or antigen-binding portion thereof elicits equal to or more than 25%, 50%, 60%, 70%, 80%, 90%, 95% or 99% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay, performed for example as disclosed in the examples.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 Corona Virus
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds to human severe acute respiratory syndrome (SARS) Corona Virus (SARS-CoV-2) S-protein, wherein said antibody or antigen-binding portion thereof has a neutralizing activity.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 Corona Virus
  • such human monoclonal antibody or antigen binding portion thereof shows a 100% inhibitory concentration (ICIOO) of less than 100 ng/ml, preferably less than 50, 25, 20, 10, 8, 6, 5, 4, 3, 2, or 1 ng/ml, when tested in an in vitro neutralization assay against the SARS-CoV-2 virus, for example against the 2019-nCoV strain 2019-nCov/Italy-INMIl, at 100TCID50 viral dose or against some vims mutants such as for example against the mutant D614G or E484K or the escape mutant (SARS-CoV-2 PT188- EM.
  • ICIOO inhibitory concentration
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof which specifically binds to human severe acute respiratory syndrome (SARS) Corona Vims (SARS-CoV-2) S-protein with an affinity constant (KD) of equal to or less than about 1000 pM, preferably with a KD of equal to or less than about 500, 250, 200, 100, 50, 20, 10 pM, as measured by surface plasmon resonance (SPR), for example measured by SPR as disclosed in the present description.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 corthelial protein
  • KD affinity constant
  • SPR surface plasmon resonance
  • the invention provides the nucleic acids encoding the full-length, or variable domain-comprising portions, of heavy and light chains, and the corresponding deduced amino acid sequences can be found in the sequence listing herein enclosed in the description.
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising: (a) a heavy chain variable domain amino acid sequence that comprises the amino acid sequence of the heavy chain variable domain of an antibody selected from: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02,
  • the invention provides a monoclonal antibody or an antigen-binding portion thereof that specifically binds human SARS-CoV-2 S-protein, comprising: (a) a heavy chain variable domain amino acid sequence that comprises the heavy chain CDR1 , CDR2 and CDR3 amino acid sequences of an antibody selected from: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22,
  • MAD0041M02 MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07,
  • MAD0041M02 MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20,
  • the invention provides a monoclonal antibody or an antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein, wherein the antibody comprises FR1, FR2, FR3 and FR4 amino acid sequences from an antibody selected from: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
  • the invention provides a monoclonal antibody that specifically binds SARS-CoV-2 S-protein, wherein said antibody comprises a heavy chain of an antibody selected from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10,
  • the invention provides a monoclonal antibody that specifically binds SARS-CoV-2 S-protein, wherein said antibody comprises a light chain of an antibody selected from the group consisting of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02,
  • the invention provides a monoclonal antibody that specifically binds SARS-CoV-2 S-protein, wherein said antibody comprises a heavy chain and a light chain of the same antibody which is selected from the group consisting of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising VL and VH domains that are at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to the VL and VH domains, respectively, of a monoclonal antibody selected from the group consisting of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising the light chain and the heavy chain that are at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to the light chain and the heavy chain, respectively, of a monoclonal antibody selected from the group consisting of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10,
  • One type of amino acid substitution that may be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine.
  • the substitution can be made in a CDR or framework region of a variable domain or in the constant domain of an antibody.
  • the cysteine is canonical.
  • Another type of amino acid substitution that may be made is to change any potential proteolytic sites in the antibody. Such sites may occur in a CDR or framework region of a variable domain or in the constant domain of an antibody. Substitution of cysteine residues and removal of proteolytic sites may decrease the risk of any heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is to eliminate asparagine-glycine pairs, which form potential deamidation sites, by altering one or both of the residues.
  • the C-terminal lysine of the heavy chain of the anti SARS-CoV-2 S-protein antibody of the invention is cleaved.
  • the heavy and light chains of the anti-SARS-CoV-2 S-protein antibodies may optionally include a signal sequence.
  • the class and subclass of anti-SARS-CoV-2 S-protein antibodies may be determined by any method known in the art.
  • the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are commercially available.
  • the class and subclass can be determined by ELISA, or Western blot (immunoblot) as well as other techniques.
  • the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various class and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.
  • the human anti-SARS-CoV-2 S-protein antibody is an IgG, an IgM, an IgE, an IgA, or an IgD molecule.
  • the human anti-SARS-CoV-2 S- protein antibody is an IgG and is an IgGl, IgG2, IgG3, IgG4 subclass. In still another embodiment, the human antibody subclass is IgGl.
  • the anti-SARS-CoV-2 S-protein antibodies bind to SARS-CoV-2 S-protein with high affinity. In some embodiments, the anti-SARS-CoV-2 S- protein antibodies bind with high affinity to the SI domain of SARS-CoV-2 S-protein. In some embodiments, the anti-SARS-CoV-2 S-protein antibodies bind to the S2 domain of SARS-CoV-2 S-protein. In another embodiment, the anti-SARS-CoV-2 S-protein antibody binds to SARS-CoV S-protein.
  • the binding affinity and dissociation rate of an anti-SARS- CoV-2 S-protein antibody to SARS-CoV-2 S-protein can be determined by methods known in the art.
  • the binding affinity can be measured by ELISAs, RIAs, flow cytometry, surface plasmon resonance, such as BIACORE(TM).
  • the dissociate rate can be measured by surface plasmon resonance.
  • the binding affinity and dissociation rate is measured by surface plasmon resonance. More preferably, the binding affinity and dissociation rate are measured using BIACORE(TM).
  • Example V exemplifies a method for determining affinity constants of anti-SARS-CoV- 2 S-protein monoclonal antibodies.
  • the invention provides, an antigen binding protein" (“ABP”) that binds to an epitope on the receptor binding domain (RBD) of SARS-CoV-2 S-protein, wherein said epitope comprises the sequence SEQ ID NO: 343 or a sequence that is identical at least 80, 90, 95% to SEQ ID NO: 343.
  • An "antigen binding protein” (“ABP") as used herein means any protein that binds a specified target antigen.
  • the specified target antigen is the receptor binding domain (RBD) of SARS-CoV-2 S-protein or fragment thereof, the sequence.
  • Antigen binding protein includes but is not limited to antibodies and binding parts thereof, preferably human monoclonal antibody or fragment thereof.
  • Examples of the epitope comprising the amino acid sequence shown in SEQ ID NO: 343 includes an epitope consisting of a continuous partial sequence of the amino acid sequence in the receptor binding domain (RBD) of SARS-CoV-2 S-protein SEQ ID NO:344, which comprises the amino acid sequence shown in SEQ ID NO: 343, and preferably has an amino acid length of 20 or less, for example 19, 18, 17, 16, 15 or less amino acid of SEQ ID NO:344.
  • the invention comprises a neutralizing antigen binding protein that binds to said epitope, wherein the antigen binding protein binds to is positioned 10, 9, 8 angstroms or less from at least one of the following residues 477 to 489 in the receptor binding domain (RBD) of SARS-CoV-2 S-protein, preferably is positioned 10, 9, 8 angstroms or less from all residues 477 to 489 in the receptor binding domain (RBD) of SARS-CoV-2 S-protein.
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof able to specifically bind the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 both in its up and down state.
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds the epitopes of the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 herein disclosed with a footprint of less than 1000 A, preferably equal to about 400 - 700A.
  • RBD receptor binding domain
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof neutralizing all the variants alfa, beta, gamma and delta of SARS-CoV-2, in particular showing in the CPE-based assay a 100% inhibitory concentration (ICIOO) of less than 10 ng/mL for all the variants alfa, beta, gamma and delta and/or in the S-fusion neutralization assay a 50% inhibitory concentration (IC50) of less than 1 ng/ml against all the variants alfa, beta, gamma and delta.
  • ICIOO inhibitory concentration
  • IC50 50% inhibitory concentration
  • the invention provides a human monoclonal antibody or antigen-binding portion thereof that does not induce the production of pro-inflammatory cytokines by human PBMC in presence of SARS-CoV-2.
  • the invention provides a human anti-SARS-CoV-2 S-protein monoclonal antibody that binds to SARS-CoV-2 S-protein and competes or cross-competes with and/or binds the same epitope as an antibody selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10,
  • test antibody if the test antibody is not able to bind to SARS-CoV-2 S-protein at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the human anti-SARS-CoV-2 S-protein antibody, or the binding of the human anti-SARS-CoV-2 S-protein antibody may induce a conformational change in the SARS-CoV-2 S-protein that prevents or reduces binding of the test antibody.
  • This experiment can be performed using ELISA, RIA, BIACORE(TM), flow cytometry or other methods known in the art.
  • the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases SARS-CoV-2 S-protein binding to a receptor, in particular, to angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases SARS-CoV-2 S- protein-mediated viral entry into cells.
  • the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases fusion of viral and cell membranes.
  • the invention provides an anti-SARS-CoV-2 S- protein antibody that decreases viral load.
  • the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases in severity for any period of time symptoms or conditions resulting from SARS-CoV-2 infection.
  • the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases in severity for a day, a week, a month, 6 months, a year, or for the remainder of the subjects’ life symptoms or conditions resulting from SARS-CoV-2 infection by 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%.
  • the invention provides an anti-SARS-CoV-2 S-protein antibody that may perform any combination of the preceding embodiments.
  • the mAh constant region of the antibodies is modified for half-life extension and reduced risk of Antibody-Dependent Enhancement (ADE) of disease.
  • AD Antibody-Dependent Enhancement
  • two different and alternative sets of mutations into their constant domains may be applied.
  • mutations that abrogate binding to Fc receptors will be introduced in the Fc part of the IgGl molecule as previously described (L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G LALA as in Schlothauer et al., 2016). All of these modifications may be carried out by means of site-directed mutagenesis, for example using the Agilent Quick-Change II Site-Directed Mutagenesis Kit, according to the manufacturer’s recommendations.
  • the antibody comprises the variable regions of an antibody selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MADOIOOFIO, MAD0100L19, MAD0101H20,
  • the antibody comprises the variable regions of an antibody selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22,
  • MAD0041M02 MADOIOOFIO, MAD0100L19, MAD0101H20, MAD0102F20,
  • the present invention also encompasses nucleic acid molecules encoding anti- SARS-CoV- 2 S-protein antibodies or antigen-binding portions thereof.
  • different nucleic acid molecules encode a heavy chain and a light chain of an anti-SARS-CoV-2 S- protein immunoglobulin.
  • the same nucleic acid molecule encodes a heavy chain and a light chain of an anti-SARS-CoV-2 S-protein immunoglobulin.
  • the nucleic acid encodes a SARS-CoV-2 S-protein antibody, or antigen- binding portion thereof, of the invention.
  • the nucleic acid molecule comprises a nucleotide sequence that encodes a VL amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions and/or 1, 2, or 3 non- conservative substitutions compared to germline. Substitutions may be in the CDR regions, the framework regions, or in the constant domain.
  • the nucleic acid molecule encodes a VL amino acid sequence comprising one or more variants compared to germline sequence that are identical to the variations found in the VL of one of the antibodies selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
  • the nucleic acid molecule encodes at least three amino acid substitutions compared to the germline sequence found in the VL of one of the antibodies selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
  • the nucleic acid molecule comprises a nucleotide sequence that encodes the VL amino acid sequence of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04,
  • the nucleic acid encodes an amino acid sequence comprising the light chain CDRs of one of said above-listed antibodies.
  • said portion is a contiguous portion comprising CDR1- CDR3.
  • the nucleic acid encodes the amino acid sequence of the light chain CDRs of said antibody.
  • said portion encodes a contiguous region from CDR1-CDR3 of the light chain of an anti-SARS-CoV-2 S-protein antibody.
  • the nucleic acid molecule encodes a VL amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a VL amino acid sequence of a VL region of any one of antibodies MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22,
  • Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, to a nucleotide sequence encoding the amino acid sequence of a VL region.
  • the nucleic acid encodes a full-length light chain of an antibody selected MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
  • the nucleic acid molecule encodes the variable domain of the heavy chain (VH) that comprises a human VH1, VH3 or VH4 family gene sequence or a sequence derived therefrom.
  • VH variable domain of the heavy chain
  • the nucleic acid molecule encodes one or more amino acid mutations compared to the germline sequence that are identical to amino acid mutations found in the VH of one of monoclonal antibodies MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22,
  • MAD0041M02 MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20,
  • the nucleic acid molecule comprises a nucleotide sequence that encodes at least a portion of the VH amino acid sequence of a monoclonal antibody selected from monoclonal antibodies MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
  • the nucleic acid molecule comprises a nucleotide sequence that encodes the amino acid sequence of one of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22,
  • MAD0041M02 MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20,
  • the nucleic acid molecule comprises at least a portion of the nucleotide sequence of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
  • said portion encodes the VH region (with or without a signal sequence), a CDR3 region, all three CDR regions, or a contiguous region including CDR1-CDR3.
  • the nucleic acid molecule encodes a VH amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the VH amino acid sequences of any one of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
  • Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, to a nucleotide sequence encoding the amino acid sequence of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
  • the nucleic acid encodes a full-length heavy chain of an antibody selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
  • nucleic acid may comprise the nucleotide sequence of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
  • a nucleic acid molecule encoding the heavy or light chain of an anti-SARS-CoV-2 S-protein antibody or portions thereof can be isolated from any source that produces such antibody.
  • the nucleic acid molecules are isolated from a B cell isolated from an animal immunized with SARS-CoV-2 S-protein or from an immortalized cell derived from such a B cell that expresses or encodes an anti-SARS-CoV-2 S-protein antibody.
  • Methods of isolating mRNA encoding an antibody are well known in the art. See, e.g., Sambrook et al. The mRNA may be used to produce cDNA for use in the polymerase chain reaction (PCR) or cDNA cloning of antibody genes.
  • the nucleic acid molecule is isolated from a hybridoma that has as one of its fusion partners a human immunoglobulin- producing cell from a non-human transgenic animal.
  • the human immunoglobulin producing cell is isolated from a XENOMOUSE animal.
  • the human immunoglobulin-producing cell is from a non-human, non mouse transgenic animal, as described above.
  • the nucleic acid is isolated from a non-human, non-transgenic animal.
  • the nucleic acid molecules isolated from a non-human, non-transgenic animal may be used, e.g., for humanized antibodies.
  • a nucleic acid encoding a heavy chain of an anti-SARS-CoV-2 S-protein antibody of the invention can comprise a nucleotide sequence encoding a VH domain of the invention joined in-frame to a nucleotide sequence encoding a heavy chain constant domain from any source.
  • a nucleic acid molecule encoding a light chain of an anti-SARS- CoV-2 S-protein antibody of the invention can comprise a nucleotide sequence encoding a VL domain of the invention joined in-frame to a nucleotide sequence encoding a light chain constant domain from any source.
  • nucleic acid molecules encoding the variable domain of the heavy (VH) and/or light (VL) chains are "converted" to full-length antibody genes.
  • nucleic acid molecules encoding the VH or VL domains are converted to full-length antibody genes by insertion into an expression vector already encoding heavy chain constant (CH) or light chain constant (CL) domains, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector, and/or the VL segment is operatively linked to the CL segment within the vector.
  • CH heavy chain constant
  • CL light chain constant
  • nucleic acid molecules encoding the VH and/or VL domains are converted into full-length antibody genes by linking, e.g., ligating, a nucleic acid molecule encoding a VH and/or VL domains to a nucleic acid molecule encoding a CH and/or CL domain using standard molecular biological techniques.
  • Nucleotide sequences of human heavy and light chain immunoglobulin constant domain genes are known in the art. See, e.g., Kabat et ah, Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publ. No. 91- 3242, 1991.
  • Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed from a cell into which they have been introduced and the anti-SARS-CoV- 2 S-protein antibody isolated.
  • the nucleic acid molecules may be used to recombinantly express large quantities of anti- SARS-CoV-2 S-protein antibodies.
  • the nucleic acid molecules also may be used to produce chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives, as described further below. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for antibody humanization, also as described below.
  • a nucleic acid molecule of the invention is used as a probe or PCR primer for a specific antibody sequence.
  • the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify regions of DNA that could be used, inter alia, to isolate additional nucleic acid molecules encoding variable domains of anti- SARS-CoV-2 S-protein antibodies.
  • the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are from highly variable domains of the heavy and light chains of the antibody of interest.
  • the oligonucleotides encode all or a part of one or more of the CDRs of antibodies MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
  • nucleic acid molecules herein disclosed may be DNA or RNA molecules.
  • the invention provides vectors comprising nucleic acid molecules that encode the heavy chain of an anti-SARS-CoV-2 S-protein antibody of the invention or an antigen-binding portion thereof.
  • the invention also provides vectors comprising nucleic acid molecules that encode the light chain of such antibodies or antigen-binding portion thereof.
  • the invention further provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.
  • the anti- SARS-CoV-2 S-protein antibodies or antigen-binding portions of the invention are expressed by inserting DNAs encoding partial or full-length light and heavy chains, obtained as described above, into expression vectors such that the genes are operatively linked to necessary expression control sequences such as transcriptional and translational control sequences.
  • Expression vectors include plasmids, retroviruses, adenoviruses, adeno- associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like.
  • the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In one embodiment, both genes are inserted into the same expression vector.
  • the antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
  • a convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can easily be inserted and expressed, as described above.
  • splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions.
  • the recombinant expression vector also can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non-immunoglobulin protein).
  • the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell.
  • regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • adenovirus e.g.
  • AdMLP adenovirus major late promoter
  • polyoma adenovirus major late promoter
  • strong mammalian promoters such as native immunoglobulin and actin promoters.
  • AdMLP adenovirus major late promoter
  • Methods for expressing antibodies in plants, including a description of promoters and vectors, as well as transformation of plants is known in the art. See, e.g., United States Patent 6,517,529, incorporated herein by reference.
  • the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, incorporated herein by reference).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.
  • DHFR dihydrofolate reductase
  • said vector is a selected from RNA virus vectors, DNA virus vectors, plasmid viral vectors, adenovirus vectors, adenovirus associated virus vectors, herpes virus vectors and retrovirus vectors.
  • the invention provides compositions (e.g., pharmaceutical compositions), methods, kits and reagents, comprising an isolated nucleic acid molecules according or a vectors according to any one of the embodiments herein disclosed for use in the prevention and/or treatment of a SARS-CoV-2 infections, in particular in humans and other mammals.
  • nucleic acid molecules and vectors are formulated in a nanoparticle, for example in lipid nanoparticle, cationic lipid nanoparticle, examples of such formulations can be found in US2020197510 herein incorporated by reference.
  • Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein Nucleic acid molecules encoding anti-SARS-CoV-2 S-protein antibodies and vectors comprising these nucleic acid molecules can be used for transfection or transformation of a suitable mammalian, plant, bacterial or yeast host cell. Transfection or transformation can be by any known method for introducing polynucleotides into a host cell.
  • Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene- mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art (see, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein by reference).
  • Methods for transforming plant cells are well known in the art, including, e.g., Agrobacterium- mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • CHO cells include, inter alia, Chinese hamster ovary (CHO) cells, N50 cells, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines.
  • Cell lines of particular preference are selected through determining which cell lines have high expression levels.
  • Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells.
  • the antibodies When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
  • Plant host cells include, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc.
  • Bacterial host cells include E. coli and Streptomyces species.
  • Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.
  • the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions.
  • the GS system is discussed in whole or part in connection with European Patent Nos. 0216 846, 0256055, 0323 997 and 0338 841. It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation of the antibodies.
  • Anti-SARS-CoV-2 S-protein antibodies of the invention also can be produced transgenically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom.
  • anti-SARS-CoV-2 S- protein antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957, incorporated herein by reference.
  • non- human transgenic animals that comprise human immunoglobulin loci are immunized with SARS-CoV-2 S-protein or an immunogenic portion thereof, as described above.
  • Methods for making antibodies in plants are described, e.g., in U.S. patents 6,046,037 and 5,959,177, incorporated herein by reference.
  • non-human transgenic animals or plants are produced by introducing one or more nucleic acid molecules encoding an anti-SARS-CoV-2 S-protein antibody of the invention into the animal or plant by standard transgenic techniques. See Hogan and United States Patent 6,417,429, supra.
  • the transgenic cells used for making the transgenic animal can be embryonic stem cells or somatic cells or a fertilized egg.
  • the transgenic non human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See, e.g., Hofian et al.
  • the transgenic non-human animals have a targeted disruption and replacement by a targeting construct that encodes a heavy chain and/or a light chain of interest.
  • the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains that specifically bind to SARS-CoV-2 S-protein, and preferably to (i) the SI domain of SARS-CoV-2 S-protein; (ii) the S2 domain of SARS-CoV-2 S-protein; or (iii) both (i) and (ii).
  • the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains that specifically bind to human SARS-CoV-2 S-protein.
  • the transgenic animals comprise nucleic acid molecules encoding a modified antibody such as a single-chain antibody, a chimeric antibody or a humanized antibody.
  • the anti-SARS-CoV- 2 S-protein antibodies may be made in any transgenic animal.
  • the non human animals are mice, rats, sheep, pigs, goats, cattle or horses.
  • the non-human transgenic animal expresses said encoded polypeptides in blood, milk, urine, saliva, tears, mucus and other bodily fluids.
  • Another aspect of the invention provides a method for converting the class or subclass of an anti-SARS-CoV-2 S-protein antibody to another class or subclass.
  • a nucleic acid molecule encoding a VL or VH that does not include sequences encoding CL or CH is isolated using methods well-known in the art.
  • the nucleic acid molecule then is operatively linked to a nucleotide sequence encoding a CL or CH from a desired immunoglobulin class or subclass. This can be achieved using a vector or nucleic acid molecule that comprises a CL or CH chain, as described above.
  • an anti- SARS- CoV-2 S-protein antibody that was originally IgM can be class switched to an IgG.
  • Another method for producing an antibody of the invention comprising a desired isotype comprises the steps of isolating a nucleic acid encoding a heavy chain of an anti- SARS-CoV-2 S-protein antibody and a nucleic acid encoding a light chain of an anti- SARS- CoV-2 S-protein antibody, isolating the sequence encoding the VH region, ligating the VH sequence to a sequence encoding a heavy chain constant domain of the desired isotype, expressing the light chain gene and the heavy chain construct in a cell, and collecting the anti-SARS-CoV-2 S-protein antibody with the desired isotype.
  • a fusion antibody or immunoadhesin may be made that comprises all or a portion of an anti-SARS-CoV-2 S-protein antibody of the invention linked to another polypeptide.
  • only the variable domains of the anti-SARS-CoV-2 S- protein antibody are linked to the polypeptide.
  • the VH domain of an anti-SARS-CoV-2 S-protein antibody is linked to a first polypeptide, while the VL domain of an anti-SARS-CoV-2 S-protein antibody is linked to a second polypeptide that associates with the first polypeptide in a manner such that the VH and VL domains can interact with one another to form an antigen binding site.
  • the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with one another (see below under Single Chain Antibodies).
  • the VH-linker- VL antibody is then linked to the polypeptide of interest.
  • the fusion antibody is useful for directing a polypeptide to a SARS-CoV-2 S-protein -expressing cell or tissue.
  • the polypeptide may be a therapeutic agent, such as a toxin, chemokine or other regulatory protein, or may be a diagnostic agent, such as an enzyme that may be easily visualized, such as horseradish peroxidase.
  • fusion antibodies can be created in which two (or more) single-chain antibodies are linked to one another.
  • VH- and VL- encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (GIy4 -Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker.
  • a flexible linker e.g., encoding the amino acid sequence (GIy4 -Ser)3
  • the single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used.
  • Bispecific or polyvalent antibodies may be generated that bind specifically to SARS-CoV-2 S-protein and to another molecule.
  • Bispecific antibodies or antigen-binding fragments can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.
  • bispecific antibodies may be formed as “diabodies” or "Janusins".
  • the bispecific antibody binds to two different epitopes of SARS-CoV-2 S-protein.
  • the bispecific antibody has a first heavy chain and a first light chain from monoclonal antibody MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
  • the additional light chain and heavy chain also are from one of the above-identified monoclonal antibodies, but are different from the first heavy and light chains.
  • the modified antibodies described above are prepared using one or more of the variable domains or CDR regions from a human anti-SARS-CoV-2 S-protein monoclonal antibody provided herein.
  • An anti-SARS-CoV-2 S-protein antibody or antigen-binding portion of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein).
  • another molecule e.g., another peptide or protein.
  • the antibodies or portion thereof are derivatized such that the SARS-CoV-2 S-protein binding is not affected adversely by the derivatization or labelling.
  • the antibodies and antibody portions of the invention are intended to include both intact and modified forms of the human anti-SARS-CoV-2 S-protein antibodies described herein.
  • an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detection agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • another antibody e.g., a bispecific antibody or a diabody
  • a detection agent e.g., a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • a cytotoxic agent e.g., a cytotoxic agent
  • a pharmaceutical agent e.g., a protein or peptid
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, II. [0179] Another type of derivatized antibody is a labelled antibody.
  • Useful detection agents with which an antibody or antigen-binding portion of the invention may be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, phycoerythrin, 5-dimethylamine-l-napthalenesulfonyl chloride, lanthanide phosphors and the like.
  • An antibody can also be labelled with enzymes that are useful for detection, such as horseradish peroxidase, [beta]-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • an antibody When an antibody is labelled with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a coloured reaction product, which is detectable.
  • An antibody can also be labelled with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • An antibody can also be labelled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • An anti-SARS-CoV-2 S-protein antibody can also be labelled with a radiolabelled amino acid.
  • the radiolabel can be used for both diagnostic and therapeutic purposes. For instance, the radiolabel can be used to detect SARS-CoV-2 S-protein-expressing tumours by x-ray or other diagnostic techniques. Further, the radiolabel can be used therapeutically as a toxin for cancerous cells or tumours.
  • the anti-SARS-CoV-2 S-protein antibody can be labelled with a paramagnetic, radioactive or florigenic ion that is detectable upon imaging.
  • the paramagnetic ion is chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III).
  • the radioactive ion is iodine 123, technetium 99, indium 111, rhenium 188, rhenium 186, copper 67, iodine 131, yttrium90, iodine 125, astatine 211, and gallium 67.
  • the anti-SARS-CoV-2 S-protein antibody is labelled with an X-ray imaging agent such as lanthanum (III), gold (III) lead (II) and bismuth (III).
  • compositions and Kits The invention relates to compositions comprising the human anti-SARS-CoV-2 S-protein antibody of the invention and one or more pharmaceutical acceptable excipients and/or carriers.
  • the composition may comprise antibodies or a binding portion thereof of any of the preceding embodiments.
  • the subject of treatment is a human.
  • the subject is a veterinary subject.
  • an antagonist anti-SARS-CoV-2 S-protein antibody that binds to the SI domain and one that binds to the S2 domain or antigen-binding portions of either or both are both administered to a subject, either together or separately.
  • the antibodies are in a composition comprising a pharmaceutically acceptable carrier.
  • one or more of the antagonist SARS-CoV-2 S-protein antibodies of the invention are administered in combination with one or more additional antagonistic antibodies that bind different epitopes on the S-protein, that bind the S-protein from different isolates of SARS- CoV-2 and/or that bind different stages of SARS-CoV-2 (i.e., early, middle or late stage virus).
  • pharmaceutically acceptable carrier means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
  • compositions of this invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans.
  • the preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the antibody is administered by intravenous infusion or injection.
  • the antibody is administered by intramuscular or subcutaneous injection.
  • compositions are typically sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the anti-SARS-CoV-2 S-protein antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the antibodies of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, or intravenous infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Other modes of administration include intraperitoneal, intrabronchial, transmucosal, intraspinal, intrasynovial, intraaortic, intranasal, ocular, otic, topical and buccal.
  • the active compound of the antibody compositions may be prepared with a carrier that will protect the antibody against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).
  • the invention also provides compositions suitable for administration by inhalation, which comprise the anti-SARS-CoV-2 S-protein antibodies described herein.
  • the anti-SARS- CoV-2 S-protein antibodies may be conveniently delivered to a subject in the form of an aerosol spray presentation from pressurized packs or from a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions, suitable for administration through the oral mucosa which comprise the anti-SARS-CoV-2 S-protein antibody described herein.
  • Oral transmucosal delivery refers to the delivery of a delivery vehicle across a mucous membrane in the oral cavity, pharyngeal cavity, or esophagus, and may be contrasted, for example, with traditional oral delivery, in which absorption of a drug occurs in the intestine.
  • oral transmucosal delivery routes of administration in which the anti-SARS-CoV- 2 S-protein antibodies are absorbed through the buccal, sublingual, gingival, pharyngeal, and/or esophageal mucosa are all encompassed within "oral transmucosal delivery," as that term is used herein.
  • the anti-SARS- CoV-2 S-protein antibody may be formulated, for example, into chewing gums (see U.S. Pat No. 5,711,961) or buccal patches (see e.g. U.S. Patent No. 5,298,256).
  • the invention also provides compositions suitable for administration through the vaginal mucosa, which comprise the anti-SARS-CoV-2 S-protein antibodies described herein.
  • the anti-SARS- CoV-2 S-protein antibodies of the invention may be formulated into a vaginal suppository, foam, cream, tablet, capsule, ointment, or gel.
  • the compositions comprising the anti-SARS-CoV-2 S-protein antibodies are formulated with permeants appropriate to the transmucosal barrier to be permeated.
  • penetrants are generally known in the art, and include, for example, for trans mucosal administration bile salts and fusidic acid derivatives.
  • an anti-SARS-CoV-2 S-protein antibody of the invention can be orally administered, for example, with an inert diluent or an assailable edible carrier.
  • the compound (and other ingredients, if desired) can also be enclosed in a hard- or soft-shell gelatine capsule, compressed into tablets, or incorporated directly into the subject's diet.
  • the anti-SARS-CoV-2 S-protein antibodies can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • an inhibitory anti-SARS-CoV-2 S-protein antibody of the invention is co-formulated with and/or co-administered with one or more additional therapeutic agents, particularly anti-viral agents.
  • These therapeutic agents include, without limitation, antibodies that bind other targets, photosensitizers, androgen, oestrogen, nonsteroidal anti-inflammatory agents, antihypertensive agents, analgesic agents, antidepressants, antibiotics, anticancer agents, anaesthetics, antiemetics, anti-infectants, contraceptives, antidiabetic agents, steroids, anti-allergy agents, chemotherapeutic agents, anti-migraine agents, agents for smoking cessation, anti-viral agents, immunosuppressants, thrombolytic agent, cholesterol-lowering agents and anti-obesity agents.
  • Therapeutic agents also include peptide analogues that inhibit SARS-CoV-2 S-protein activity, antibodies or other molecules that prevent SARS-CoV-2 entry into a cell, including but not limited to preventing S-protein binding to a receptor such as the ACE2 receptor, and agents that inhibit SARS-CoV-2 S-protein expression.
  • the additional agents that inhibit SARS-CoV-2 S-protein expression comprise an antisense nucleic acid capable of hybridizing to a SARS-CoV-2 S-protein mRNA, such as a hairpin RNA or siRNA, locked nucleic acids (LNA) or ribozymes. Sequence-specific nucleic acids capable of inhibiting gene function by RNA interference are well-known in the art.
  • the therapeutic agent(s) that is co-formulated with and/or co-administered with an inhibitory anti-SARS-CoV-2 S- protein antibody of the invention is an antimicrobial agent.
  • Antimicrobial agents include antibiotics (e.g. antibacterial), antiviral agents, antifungal agents, and anti -protozoan agents.
  • Non-limiting examples of antimicrobial agents are sulfonamides, trimethoprim- sulfamethoxazole, quinolones, penicillins, and cephalosporins.
  • the compositions of the invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antigen-binding portion of the invention.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.
  • the dosage proposed for the monoclonal antibodies against Covidl9 is calculated in order to achieve a neutralizing titer in the serum of 1/100.
  • the 100 - 400 mgs dosages will allow to move from intravenous to intramuscular injection. This route of administration could be a key advantage in emergency scenarios as it will allow to administer the antibodies herein disclosed, such for example MAD0004J08 in non-hospital care settings increasing the number of people that can quickly benefit from its foreseen therapeutic effect.
  • Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the pharmaceutical composition comprising equal or less than 400 mg for dosage unit of the human monoclonal antibody or antigen-binding portion thereof, more preferably less than 400, 350, 300, 250, 200, 150, 100, 50, 25, 10 mg for dosage unit.
  • the pharmaceutical composition comprising such dosage unit is preferably for parental administration, for example for intravenous, subcutaneous, intraperitoneal or intramuscular administration.
  • the pharmaceutical composition is in the liquid form in a concentration between 20 and 200 mg/ml, more preferably between 40 and 80 mg/ml.
  • An exemplary, non-limiting range for a therapeutically or prophylactically- effective amount of an antibody or antibody portion of the invention is 0.025 to 50 mg/kg, more preferably 0.1 to 5 mg/kg, more preferably 0.1-5, 0.1 to 4 or 0.25 to 3 mg/kg.
  • the invention provides human monoclonal antibody or an antigen-binding portion according to any one of the embodiments herein disclosed, for use in a method for prophylactic or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular COVID-19, wherein said method comprising the step of administering to a patient between 0.025 to 50 mg/kg, more preferably 0.1 to 5 mg/kg, more preferably 0.1-5, 0.1 to 4 or 0.25 to 3 mg/kg once a day, for example for at least one, two, three, four, five, six, seven, eight, nine, ten, eleven days.
  • Such patient is a mammal, preferably a human.
  • a formulation contains 5 mg/ml of antibody in a buffer of 20mM sodium citrate, pH 5.5, 140mM NaCl, and 0.2mg/ml polysorbate 80. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • kits comprising an anti-SARS-CoV-2 S-protein, or antigen-binding portion, of the invention or a composition comprising such an antibody or antigen-binding fragment.
  • a kit may include, in addition to the antibody or composition, diagnostic or therapeutic agents.
  • a kit can also include instructions for use in a diagnostic or therapeutic method, as well as packaging material such as, but not limited to, ice, dry ice, styrofoam, foam, plastic, cellophane, shrink wrap, bubble wrap, cardboard and starch peanuts.
  • the kit includes the antibody or a composition comprising it and a diagnostic agent that can be used in a method described below.
  • the kit includes the antibody or a composition comprising it and one or more therapeutic agents that can be used in a method described below.
  • the antibodies or binding portion thereof or composition comprising such antibodies according to any one of the embodiments herein disclosed are for use in the prevention or the treatment of patients infected with Coronavirus, in particular infected with SARS-CoV-2, for example infected with SARS-CoV-2 wild type, SARS-CoV-2 mutant D615G and/or SARS-CoV-2 mutant E484K or the escape mutant (SARS-CoV-2 PT188-EM or others mutants.
  • the use of such antibodies and compositions of SARS-CoV-2-specific mAbs include, but are not limited to passive immunization in persons at risk of contracting the infection (e.g. professionally exposed personnel, people living in endemic areas) and therapy of acute cases, either hospitalized or not.
  • the invention also relates to compositions for inhibiting viral infection, and in particular Coronavirus infection, more in particular SARS-CoV-2 infection in a mammal comprising an amount of an antibody of the invention in combination with an amount of an antiviral agent, wherein the amounts of the anti-SARS- CoV-2 S-protein antibody and of antiviral agent are together effective in inhibiting viral replication, viral infection of new cells or viral loads.
  • the antibodies according to the invention may use also as diagnostic tools for rapid detection of SARS-CoV-2 infection.
  • the invention provides diagnostic methods.
  • the anti-SARS-CoV-2 S-protein antibodies can be used to detect SARS-CoV-2 S-protein in a biological sample in vitro or in vivo.
  • the invention provides a method for diagnosing the presence or location of SARS-CoV-2 viruses in a subject in need thereof.
  • the anti-SARS-CoV-2 S-protein antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, flow cytometry, tissue immunohistochemistry, Western blot (immunoblot) or immunoprecipitation.
  • the anti- SARS-CoV-2 S-protein antibodies of the invention can be used to detect SARS-CoV-2 S- protein from humans.
  • the invention provides a method for detecting SARS-CoV-2 S-protein in a biological sample comprising contacting the biological sample with an anti-SARS-CoV- 2 S-protein antibody of the invention and detecting the bound antibody.
  • the anti-SARS-CoV-2 S-protein antibody is directly labelled with a detectable label.
  • the anti-SARS-CoV-2 S-protein antibody (the first antibody) is unlabelled and a second antibody or other molecule that can bind the anti-SARS-CoV-2 S- protein antibody is labelled.
  • a second antibody is chosen that is able to specifically bind the particular species and class of the first antibody.
  • the anti-SARS-CoV-2 S-protein antibody is a human IgG
  • the secondary antibody could be an anti-human-IgG.
  • Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially, e.g., from Pierce Chemical Co.
  • Example of biological samples to use in the diagnostic methods herein disclosed are urine, stool, blood, saliva, biopsies, cerebrospinal fluid, nasopharyngeal and oropharyngeal wash, sputum, endotracheal aspirate, bronchoalveolar lavage or other biological samples obtainable from a human subject.
  • Suitable labels for the antibody or secondary antibody have been disclosed supra, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, [b eta] -gal actosi dase, or acetylcholinesterase
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin
  • an example of a luminescent material includes luminol.
  • SARS-CoV-2 S-protein can be assayed in a biological sample by a competition immunoassay utilizing SARS-CoV-2 S-protein standards labelled with a detectable substance and an unlabelled anti-SARS-CoV-2 S-protein antibody.
  • a competition immunoassay utilizing SARS-CoV-2 S-protein standards labelled with a detectable substance and an unlabelled anti-SARS-CoV-2 S-protein antibody.
  • the biological sample, the labelled SARS-CoV-2 S-protein standards and the anti-SARS-CoV-2 S-protein antibody are combined and the amount of labelled SARS-CoV-2 S-protein standard bound to the unlabelled antibody is determined.
  • the amount of SARS-CoV-2 S-protein in the biological sample is inversely proportional to the amount of labelled SARS-CoV-2 S-protein standard bound to the anti-SARS-CoV-2 S-protein antibody.
  • the anti-SARS-CoV-2 S-protein antibodies can be used to detect SARS-CoV-2 S-protein in cultured cells or as a diagnostic assay in samples from a subject.
  • the diagnostic methods according to any embodiments herein disclosed may be followed by a further step of the administration in the positive subject of an anti-SARS-CoV-2 drugs, for example according to any of the Therapeutic Methods herein disclosed.
  • the invention provides a method for neutralizing SARS- CoV-2 by administering an anti-SARS-CoV-2 S-protein antibody to a patient in need thereof.
  • an anti-SARS-CoV-2 S-protein antibody is a human antibody.
  • the antibody, or antigen-binding portion thereof binds to the SI domain of SARS-CoV-2 S-protein.
  • the patient is a human patient.
  • the patient may be a mammal infected with SARS-CoV-2.
  • the invention provides methods of treating, aiding in the treatment, preventing or aiding in the prevention of, SARS-CoV-2 infection and conditions or disorders resulting from such infection, in a subject by administering to the subject a therapeutically-effective or prophylactically effective amount of an anti-SARS-CoV-2 S-protein antibody of the invention.
  • Antibodies and antigen-binding fragments thereof which are antagonists of SARS-CoV-2 S-protein can be used as therapeutics for SARS-CoV-2 infection.
  • the antibody may be administered locally or systemically.
  • compositions comprising anti-SARS-CoV-2 S- protein antibodies may be administered to the subject, for example, orally, nasally, vaginally, buccally, rectally, via the eye, or via the pulmonary route, in a variety of pharmaceutically acceptable dosing forms, which will be familiar to those skilled in the art.
  • the anti-SARS-CoV-2 S-protein antibodies may be administered via the nasal route using a nasal insufflator device.
  • the anti-SARS-CoV-2 S-protein antibodies can also be administered to the eye in a gel formulation.
  • a formulation containing the anti- SARS-CoV-2 S-protein antibodies may be conveniently contained in a two- compartment unit dose container, one compartment containing a freeze-dried anti-SARS- CoV-2 S-protein antibody preparation and the other compartment containing normal saline.
  • the serum concentration of the antibody may be measured by any method known in the art.
  • the antibodies of the present invention are administered to the subject in combination with other therapeutic agents.
  • the additional therapeutic agents may be treating the symptoms of the SARS-CoV-2 infection on their own, and may optionally synergize with the effects of the antibodies.
  • the additional agent that is administered may be selected by one skilled in the art for treating the infection.
  • Co administration of the antibody with an additional therapeutic agent encompasses administering a composition comprising the anti-SARS-CoV-2 S-protein antibody and the additional therapeutic agent as well as administering two or more separate compositions, one comprising the anti-SARS-CoV-2 S-protein antibody and the other(s) comprising the additional therapeutic agent(s).
  • co-administration or combination therapy generally means that the antibody and additional therapeutic agents are administered at the same time as one another, it also encompasses instances in which the antibody and additional therapeutic agents are administered at different times. For instance, the antibody may be administered once every three days, while the additional therapeutic agent is administered once daily. Alternatively, the antibody may be administered prior to or subsequent to treatment with the additional therapeutic agent, for example after a patient has failed therapy with the additional agent. Similarly, administration of the anti-SARS- CoV-2 S-protein antibody may be administered prior to or subsequent to other therapy.
  • the antibody and one or more additional therapeutic agents may be administered once, twice or at least the period of time until the condition is treated, palliated or cured.
  • the combination therapy is administered multiple times.
  • the combination therapy may be administered from three times daily to once every six months.
  • the administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months, or may be administered continuously via a minipump.
  • the combination therapy may be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, or parenteral.
  • the invention provides a method for treating, preventing or alleviating the symptoms of a SARS-CoV-2 mediated disorder in a subject in need thereof, comprising the step of administering to said subject an antibody or antigen binding portion according to any one of the preceding embodiments, further comprising at least one additional therapeutic agent selected from the group consisting of: (a) one or more antibodies from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10,
  • the invention provides a kit for treating, preventing or alleviating the symptoms of a SARS-CoV-2 mediated disorder in a subject in need thereof, comprising a) one or more antibodies from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02,
  • the human monoclonal antibody or antigen-binding portion thereof herein disclosed may also be used advantageously as a diagnostic reagent in an in vitro method for detecting in a biological sample previously obtained from a patient (such as for example a serum, plasma, blood sample or any other suitable biological material, obtained from the patient, preferably a human being) anti-Coronavirus antibodies, in particular SARS-Cov-2 antibodies.
  • a biological sample previously obtained from a patient
  • a patient such as for example a serum, plasma, blood sample or any other suitable biological material, obtained from the patient, preferably a human being
  • anti-Coronavirus antibodies in particular SARS-Cov-2 antibodies.
  • a diagnostic kit comprising the human monoclonal antibody or antigen binding portion thereof herein disclosed of the invention, as a specific reagent, also falls within the scope of the invention, said kit being in particular designed for the detection and/or quantification, in a biological sample previously obtained from a patient, of anti-coronavirus antibodies.
  • the human monoclonal antibody or antigen-binding portion thereof herein disclosed may also be used advantageously for the design of a vaccine against coronavirus.
  • Rappuoli, Rino et al. “ Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design. ”
  • human mAh may be used to identify protective antigens/epitopes.
  • Structural characterization of the Ab-antigen complex may be used to instruct antigen design.
  • the human monoclonal antibody or antigen-binding portion thereof herein disclosed for the design of a vaccine against a coronavirus, in particular against the SARS-Cov-2 virus is within the scope of the invention.
  • the human monoclonal antibody or antigen-binding portion thereof herein disclosed may be used for the preparation of mimotopes, such as for example anti-idiotype antibodies, peptides, S-protein truncated or artificial forms or others, endowed with the ability of evoking the antibodies herein disclosed.
  • the anti-idiotype antibodies are preferred.
  • the anti-idiotype antibodies are antibodies specifically directed against the idiotype of the neutralizing antibodies used for the manufacture thereof, and thus are able to mimic the key epitopes that they recognize.
  • the manufacture of anti-idiotype antibodies is carried out by per se known methodologies that do not need further detailed explanations here.
  • mimotopes, preferably anti-idiotype antibodies, directed against an antibody of the invention fall within the scope of the invention.
  • the human monoclonal antibody or antigen-binding portion thereof herein disclosed may be used for the manufacture of anti idiotype antibodies according to methods per se known.
  • Anti-idiotype antibodies are antibodies specifically directed towards the idiotype of the broad-range neutralizing antibodies used to prepare them, and as such are able to mimic the key epitopes they recognize.
  • anti-idiotype antibodies directed against a monoclonal antibody of the invention are also included in the scope of the invention.
  • the following experimental section is provided solely by way of illustration and not limitation and does not intend to restrict the scope of the invention as defined in the appended claims.
  • the claims are an integral part of the description.
  • PBMCs Human peripheral blood mononuclear cells isolation from SARS-CoV-2 convalescent donors
  • PBMCs Peripheral blood mononuclear cells
  • the protein was purified from filtered cell supernatants using NiNTA resin (GE Healthcare #11-0004-58), eluted with 250 mM Imidazole (Sigma Aldrich #56750), dialyzed against PBS, and then stored at 4°C prior to use.
  • PBMCs peripheral blood mononuclear cells
  • RT room temperature
  • CD19 V421 (BD cat# 562440), IgM PerCP-Cy5.5 (BD cat# 561285), CD27 PE (BD cat# 340425), IgD-A700 (BD cat# 561302), CD3 PE-Cy7 (BioLegend cat# 300420), CD14 PE-Cy7 (BioLegend cat# 301814), CD56 PE-Cy7 (BioLegend cat# 318318) and cells were incubated at 4°C for additional 30 min.
  • 384-well flat-bottom microtiter plates (Nunc MaxiSorp 384-well plates; Sigma-Aldrich) were coated with 25 m ⁇ /well of antigen (1:1 mix of SI and S2 subunits, 1 pg/ml each; The Native Antigen Company, Oxford, United Kingdom) diluted in coating buffer (0.05 M carbonate-bicarbonate solution, pH 9.6), and incubated overnight at 4°C.
  • the plates were then washed three times with 100 m ⁇ /well washing buffer (50 mM Tris Buffered Saline (TBS) pH 8.0, 0.05% Tween-20) and saturated with 50 m ⁇ /well blocking buffer containing Bovine Serum Albumin (BSA) (50 mM TBS pH 8.0, 1% BSA, 0.05% Tween-20) for 1 hour (h) at 37°C. After further washing, samples diluted 1 :5 in blocking buffer were added to the plate. Blocking buffer was used as a blank.
  • TBS Tris Buffered Saline
  • BSA Bovine Serum Albumin
  • HRP horseradish peroxidase
  • ELISA assay was used to detect SARS-CoV-2 S-protein specific mAbs and to screen plasma from SARS-CoV-2 convalescent donors.
  • 384-well plates Nunc MaxiSorp 384 well plates; Sigma Aldrich
  • PBS/BSA 1%) 50 pL/well of saturation buffer (PBS/BSA 1%) was used to saturate unspecific binding and plates were incubated at 37°C for lh without CO2.
  • PNPP p- nitrophenyl phosphate
  • Thermo Fisher was used as soluble substrate to detect SARS-CoV- 2 S-protein specific monoclonal antibodies and the final reaction was measured by using the Varioskan Lux Reader (Thermo Fisher Scientific) at a wavelength of 405 nm. Samples were considered as positive if optical density at 405 nm (OD405) was two times the blank.
  • African green monkey kidney cell line Vero E6 cells (American Type Culture Collection [ATCC] #CRL-1586) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) - High Glucose (Euroclone, Pero, Italy) supplemented with 2 mM L- Glutamine (Lonza, Milano, Italy), penicillin (100 U/mL) - streptomycin (100 pg/mL) mixture (Lonza, Milano, Italy) and 10% Foetal Bovine Serum (FBS) (Euroclone, Pero, Italy). Cells were maintained at 37°C, in a 5% CO2 humidified environment and passaged every 3-4 days.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Foetal Bovine Serum
  • Wild type SARS CoV-2 2019 2019-nCoV strain 2019-nCov/Italy-INMIl virus was purchased from the European Virus Archive goes Global (EVAg, Spallanzani Institute, Rome).
  • EVAg European Virus Archive goes Global
  • Vero E6 cell monolayers were prepared in T175 flasks (Sarstedt) containing supplemented D-MEM high glucose medium.
  • Vero E6 were seeded in 96-well plates (Sarstedt) at a density of l,5xl0 4 cells/well the day before the assay.
  • the stabilized Spike protein was coupled to Streptavidin-PE (eBioscience # 12- 4317-87, Thermo Fisher) for 30 min at 4°C and then incubated with VERO E6 cells. Binding was assessed by flow cytometry. The stabilized Spike protein bound VERO E6 cells with high affinity (data not shown).
  • the SARS-CoV-2 virus was propagated in Vero E6 cells cultured in DMEM high Glucose supplemented with 2% FBS, 100 U/mL penicillin, 100 pg/mL streptomycin. Cells were seeded at a density of lxlO 6 cells/mL in T175 flasks and incubated at 37°C, 5% CO2 for 18- 20 hours. The sub-confluent cell monolayer was then washed twice with sterile Dulbecco’s phosphate buffered saline (DPBS).
  • DPBS sterile Dulbecco’s phosphate buffered saline
  • Cells were inoculated with 3.5 ml of the virus properly diluted in DMEM 2% FBS at a multiplicity of infection (MOI) of 0.001, and incubated for lh at 37°C in a humidified environment with 5% CO2. At the end of the incubation, 50 mL of DMEM 2% FBS were added to the flasks. The infected cultures were incubated at 37°C, 5% CO2 and monitored daily until approximately 80-90% of the cells exhibited cytopathic effect (CPE). Culture supernatants were then collected, centrifuged at 4°C at 1,600 rpm for 8 minutes to allow removal of cell debris, aliquoted and stored at -80°C as the harvested viral stock.
  • MOI multiplicity of infection
  • Viral titers were determined in confluent monolayers of Vero E6 cells seeded in 96-well plates using a 50% tissue culture infectious dose assay (TCID50). Cells were infected with serial 1:10 dilutions (from 10-1 to 10-11) of the virus and incubated at 37°C, in a humidified atmosphere with 5% CO2. Plates were monitored daily for the presence of SARS-CoV-2 induced CPE for 4 days using an inverted optical microscope. The virus titer was estimated according to Spearman-Karber formula (14) and defined as the reciprocal of the highest viral dilution leading to at least 50% CPE in inoculated wells. Semi-quantitative live SARS-CoV-2-based neutralization assay
  • Plasma samples were heat-inactivated for 30 minutes at 56°C and 2-fold serially diluted starting from 1:10 to 1:2,560 dilution, then mixed with an equal volume of viral solution containing 100 TCID50 of SARS-CoV-2 diluted in D-MEM high Glucose 2% FBS. After 1-hour incubation at 37°C, 5% C02, 100 m ⁇ of the virus-plasma mixture at each dilution was passed to a cell plate containing a sub-confluent Vero E6 cell monolayer.
  • the neutralization activity of culture supernatants from monoclonal S-protein-specific memory B cells was evaluated by means of a qualitative live-virus based neutralization assay against a one-point dilution of the samples.
  • Supernatants were mixed in a 1:3 ratio with a SARS-CoV-2 viral solution containing 25 TCID50 of virus (final volume: 30 m ⁇ ). After 1- hour incubation at 37°C, 5% CO2, 25 m ⁇ of each virus-supernatant mixture was added to the wells of a 96-well plate containing a sub-confluent Vero E6 cell monolayer.
  • Selected SARS-CoV-2 neutralizing mAbs are cloned and expressed as described (Tiller et al, 2008; Giuliani et al., 2018). Briefly, the heavy and light chain variable region sequences are recovered from lysed single cell sorted B cells. Recovered amplicons are cloned into suitable Igyl, IgK and Ig vectors for Escherichia coli DH5a transformation. Purified plasmids are used to transiently express full-length IgGs in Expi293 cells. Supernatants of Expi293 cell cultures are collected and tested to assess mAb expression by means of ELISA assays and mAb potency by means of neutralization assays.
  • SARS-CoV-2 convalescent donors plasma analyses. Plasma S-protein binding titers for each subject were measured by ELISA assays. Neutralization activity was detected by NOB and by neutralization of SARS-CoV-2 infection of Vero cells. Table 2. SARS-CoV-2 convalescent donors S-protein specific MBCs analyses. The Table reports the number of S-protein-specific MBCs that were sorted and screened (for binding by ELISA and for functionality by NOB and viral neutralization) for each subject enrolled in this study.
  • RNA from single cells was reverse transcribed in 25 pL of reaction volume composed by 1 pL of random hexamer primers (50 ng/pL), 1 pL of dNTP-Mix (10 mM), 2 pL 0.1 M DTT, 40U/pL RNAse OUT, MgC12 (25 mM), 5x FS buffer and Superscript® IV reverse transcriptase (Invitrogen). Final volume was reached by adding nuclease-free water (DEPC).
  • DEPC nuclease-free water
  • RT Reverse transcription
  • All PCR reactions were performed in a nuclease-free water (DEPC) in a total volume of 25 pL/well. Briefly, 4 pL of cDNA were used for the first round of PCR (PCRI).
  • PCRI-master mix contained 10 pM of VH and 10 pM VL primer-mix ,10mM dNTP mix, 0,125 pL of Kapa Long Range Polymerase (Sigma), 1,5 pL MgC12 and 5 pL of 5x Kapa Long Range Buffer.
  • PCRI reaction was performed at 95°/3’, 5 cycles at 95°C/30”, 57°C/30”, 72°C/30” and 30 cycles at 95°C/30”, 60°C/30”, 72°C/30” and a final extension of 72 2’. All nested PCR reactions (PCRII) were performed using 3,5 pL of unpurified PCRI product using the same cycle conditions. PCRII products were then purified by Millipore MultiScreen® PCRp96 plate according to manufacture instructions. Samples were eluted with 30 pi nuclease-free water (DEPC) into 96-well plates and quantify by Qubit Fluorometric Quantitation assay (Invitrogen). Characterization of SARS-CoV-2 RBD-Antibodies binding by Flow cytometry
  • Flow cytometry analysis was performed to define antibodies interaction with S-protein- receptor-binding domain (RBD). Briefly, APEXTM Antibody Labeling Kits (Invitrogen) was used to conjugate 20 pg of selected antibodies to Alexa fluor 647, according to the manufacturer instructions. Then, 1 mg of magnetic bead (DynabeadsTM His-Tag, Invitrogen) were coated with 70 pg of histidine tagged RBD. To assess the ability of each antibody to bind the RBD domain, 20 pg/mL of labelled antibody were incubated with 40 pg/mL of beads-bound RBD for 1 hour on ice.
  • APEXTM Antibody Labeling Kits Invitrogen
  • Antibodies specificity to bind SARS-CoV-2 S-protein and their possible competition was analysed performing a Flow cytometer-based assay.
  • 200 pg of stabilized histidine tagged S-protein were coated with 1 mg of magnetic beads (DynabeadsTM His-Tag, invitrogen).
  • 20 pg of each antibody was labelled with Alexa fluor 647 working with the APEXTM Antibody Labeling Kits (invitrogen).
  • beads-bound S-protein 40 pg/mL
  • unlabeled antibodies 40 pg/mL
  • Beads-antibody complex was washed with Phosphate-buffered saline (PBS) and incubated with labelled antibodies (20 pg/mL) for 1 hour on ice. After incubation, the mix Beads-antibodies were washed, resuspended in 150 pL of PBS and read using FACScantoII flow cytometer (Becton Dickinson). Beads-bound and non-bound S-protein incubated with labelled antibodies were used as positive and negative control, respectively. Population gating and analysis was carried out using FlowJo (version 10).
  • Fluorescently coded microspheres were used to profile the ability of selected antibodies to interact with Fc receptors (Boudreau et ak, 2020).
  • the antigen of interest SARS -CoV-2 S- protein RBD
  • the beads were incubated with diluted antibody (diluted in PBS), allowing “on bead” affinity purification of antigen-specific antibodies.
  • the bound antibodies were subsequently probed with tetramerized recombinant human FCyR2A and FcRN and analyzed using Luminex. The data is reported as the median fluorescence intensity of PE for a specific bead channel.
  • ADNP Antibody-dependent neutrophil phagocytosis
  • the antibody:bead complexes are added to primary neutrophils isolated from healthy blood donors using negative selection (StemCell EasySep Direct Human Neutrophil Isolation Kit), and phagocytosis was allowed to proceed for 1 hour. The cells were then washed and fixed, and the extent of phagocytosis was measured by flow cytometry. The data is reported as a phagocytic score, which takes into account the proportion of effector cells that phagocytosed and the degree of phagocytosis. Each sample is run in biological duplicate using neutrophils isolated from two distinct donors. The monoclonal antibodies were tested for ADNP activity at a range of 30 pg/mL to 137.17 ng/ml.
  • ADNKA Antibody-dependent NK cell activation
  • Stabilized SARS-CoV-2 Spike trimer was used to coat ELISA plates, which were then washed and blocked. Diluted antibody (diluted in PBS) was added to the antigen coated plates, and unbound antibodies were washed away.
  • NK cells purified from healthy blood donor leukopaks using commercially available negative selection kits (StemCell EasySep Human NK Cell Isolation Kit) were added, and the levels of IFN-g was measured after 5 hours using flow cytometry.
  • the data is reported as the percent of cells positive for IFN-g. Each sample is tested with at least two different NK cell donors, with all samples tested with each donor. The monoclonal antibodies were tested for ADNKA activity at a range of 20 pg/mL to 9.1449 ng/mL. Affinity evaluation of SARS-CoV-2 neutralizing antibodies
  • Anti -Human IgG Polyclonal Antibody (Southern Biotech 2040-01) was immobilized via amine group on two flow cells of a CM5 sensor chip.
  • anti-human IgG Ab diluted in lOmM Na acetate pH 5.0 at the concentration of 25 pg/ml was injected for 360 sec over the dextran matrix, which had been previously activated with a mixture of 0.1M 1 -ethyl-3 (3 -dimethylaminopropyl)-carbodiimide (EDC) and 0.4M N-hydroxyl succinimide (NHS) for 420 sec. After injection of the antibody, Ethanolamine 1M was injected to neutralize activated group.
  • Anti-SPIKE protein human mAbs were diluted in HBS-EP+ (Hepes 10 mM, NaCl 150 mM, EDTA 3.4 mM, 0.05% p20, pH 7.4) and injected for 120 sec at 10 pl/min flow rate over one of the two flow cells containing the immobilized Anti-Human IgG Antibody, while running buffer (HBS-EP+) was injected over the other flow cell to be taken as blank. Dilution of each mAb was adjusted in order to have comparable levels of RU for each capture mAb.
  • the NOVA Lite HEp-2 ANA Kit (Inova Diagnostics) was used in accordance to the manufacturer’s instructions to test antibodies the autoreactivity of selected antibodies which were tested at a concentration of 100 pg/mL. Kit positive and negative controls were used at three different dilutions (1:1 - 1:10 - 1:100). Images were acquired using a DMI3000 B microscope (Leica) and an exposure time of 300 ms, channel intensity of 2000 and a gamma of 2.
  • the SARS-2 CoV-GSAS-6P-Mut7 and Fab J08 complex was formed by mixing trimer to fab at a 1 :3 molar ratio for 30 minutes at room temperature.
  • the complex was briefly mixed with fluorinated octyl-maltoside (final concentration 0.02% w/v) and deposited on Quantifoil Au 1.2/1.3-300 mesh grids that had been plasma cleaned for 7 seconds. Grid freezing was facilitated by a Vitrobot Mark IV set to 4*C, 100% humidity, 3 s blot time, 6 s wait time, and blot force 0.
  • the Leginon software was used once again for data collection automation on a FEI Talos Arctica (200 kEV) paired with a Gatan K2 (4k x 4k) camera.
  • >MAD0004J08 shows a 100% inhibitory concentration (ICIOO) of 7.2 ng/mL when tested for an in vitro neutralization assay against the authentic SARS-CoV-2 virus (2019-nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • >MAD0004J08 shows specific binding to the SARS-CoV-2 Spike protein SI domain.
  • >MAD0004J08 shows 53% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • >MAD0004J08 heavy chain is composed of the rearrangement of IGHV1-69 and IGHJ4 immunoglobulin genes. The MAD0004J08 heavy chain shows less than 4% mutation rate with respect to germline genes.
  • MAD0004J08 light chain is composed of the rearrangement of IGKV3-11 and IGKJ4 immunoglobulin genes.
  • the MAD0004J08 light chain shows less than 2% mutation rate with respect to germline genes.
  • >MAD0100I14 shows a 100% inhibitory concentration (ICIOO) of 23.7 ng/mL when tested for an in vitro neutralization assay against the authentic SARS-CoV-2 virus (2019-nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • >MAD0100I14 shows specific binding to the SARS-CoV-2 Spike protein SI domain.
  • >MAD0100I14 shows 98% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0100I14 heavy chain is composed of the rearrangement of IGHV1-58 and IGHJ3 immunoglobulin genes.
  • the MAD0100I14 heavy chain shows less than 4% mutation rate with respect to germline genes.
  • MAD0100I14 light chain is composed of the rearrangement of IGKV3-20 and IGKJ1 immunoglobulin genes.
  • the MAD0100I14 light chain shows less than 2% mutation rate with respect to germline genes.
  • >MAD0102F05 shows a 100% inhibitory concentration (ICIOO) of 8.1 ng/mL when tested for an in vitro neutralization assay against the authentic SARS-CoV-2 virus (2019-nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • ICIOO inhibitory concentration
  • >MAD0102F05 shows specific binding to the SARS-CoV-2 Spike protein SI domain.
  • >MAD0102F05 shows 89% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0102F05 heavy chain is composed of the rearrangement of IGHV3-53 and IGHJ6 immunoglobulin genes.
  • the MAD0102F05 heavy chain shows less than 3% mutation rate with respect to germline genes.
  • MAD0102F05 light chain is composed of the rearrangement of IGKV1-17 and IGKJ1 immunoglobulin genes.
  • the MAD0102F05 light chain shows less than 5% mutation rate with respect to germline genes.
  • MAD0041G12 shows a 100% inhibitory concentration (ICIOO) of 23.62 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0041G12 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
  • MAD0041G12 shows 100% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0041G12 heavy chain is composed of the rearrangement of IGHV1-69 and IGHJ4 immunoglobulin genes.
  • the MAD0041G12 heavy chain shows 95.59% identity to the germline genes.
  • MAD0041G12 light chain is composed of the rearrangement of IGKV3-15 and IGKJ4 immunoglobulin genes.
  • MAD0041I21 shows a 100% inhibitory concentration (ICIOO) of 48.25 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0041I21 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
  • MAD0041I21 heavy chain is composed of the rearrangement of IGHV3-30 and IGHJ4 immunoglobulin genes.
  • the MAD0041I21 heavy chain shows 98.28% identity to the germline genes.
  • MAD0041I21 light chain is composed of the rearrangement of IGKV1-9 and IGKJ4 immunoglobulin genes.
  • mutant IgGl constant region backbone which contains the following three groups of mutations: L234A/L235A as in Hezareh et ah, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ah, 2016; M428L/N434S as in Zalevsky et ah, 2010.
  • L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0041K22 shows a 100% inhibitory concentration (ICIOO) of 48.42 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0041K22 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
  • MAD0041K22 shows 63% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0041K22 heavy chain is composed of the rearrangement of IGHV3-30 and IGHJ6 immunoglobulin genes.
  • the MAD0041K22 heavy chain shows 97.30% identity to the germline genes.
  • MAD0041K22 light chain is composed of the rearrangement of IGKV3-20 and IGKJ4 immunoglobulin genes.
  • variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ah, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ah, 2016; M428L/N434S as in Zalevsky et ah, 2010.
  • the L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0041M02 shows a 100% inhibitory concentration (ICIOO) of 72.71 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0041M02 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
  • MAD0041M02 shows 31% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0041M02 heavy chain is composed of the rearrangement of IGHV3-30 and IGHJ6 immunoglobulin genes. The MAD0041M02 heavy chain shows 96.97% identity to the germline genes.
  • MAD0041M02 light chain is composed of the rearrangement of IGKV2-40 and IGKJ1 immunoglobulin genes.
  • mutant IgGl constant region backbone which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0100F10 shows a 100% inhibitory concentration (ICIOO) of 234.77 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0100F10 shows 45% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0100F10 heavy chain is composed of the rearrangement of IGHV1-24 and IGHJ6 immunoglobulin genes.
  • the MAD0100F10 heavy chain shows 96.61% identity to the germline genes.
  • MAD0100F10 light chain is composed of the rearrangement of IGKV2-24 and IGKJ2 immunoglobulin genes.
  • MAD0100F10 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • the L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0100L19 shows a 100% inhibitory concentration (ICIOO) of 1473.67 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0100L19 shows specific binding to the SARS-CoV-2 Spike protein S-2 domain.
  • MAD0100L19 shows 15% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0100L19 heavy chain is composed of the rearrangement of IGHV3-11 and IGHJ5 immunoglobulin genes.
  • the MAD0100L19 heavy chain shows 96.26% identity to the germline genes.
  • MAD0100L19 light chain is composed of the rearrangement of IGKV1-33 and IGKJ5 immunoglobulin genes.
  • variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ah, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ah, 2016; M428L/N434S as in Zalevsky et ah, 2010.
  • the L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0101H20 shows a 100% inhibitory concentration (ICIOO) of 93.11 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0101H20 heavy chain is composed of the rearrangement of IGHV1-69D and IGHJ2 immunoglobulin genes.
  • the MAD0101H20 heavy chain shows 97.30% identity to the germline genes.
  • MAD0101H20 light chain is composed of the rearrangement of IGKV3-11 and IGKJ1 immunoglobulin genes.
  • MAD0101H20 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • the L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0102F20 shows a 100% inhibitory concentration (ICIOO) of 450.17 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • ICIOO inhibitory concentration
  • MAD0102F20 shows 21% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0102F20 heavy chain is composed of the rearrangement of IGHV1-24 and IGHJ5 immunoglobulin genes.
  • the MAD0102F20 heavy chain shows 96.93% identity to the germline genes.
  • MAD0102F20 light chain is composed of the rearrangement of IGKV3-15 and IGKJ2 immunoglobulin genes.
  • variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0102F22 SUMMARY > MAD0102F22 shows a 100% inhibitory concentration (ICIOO) of 1531.49 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • ICIOO inhibitory concentration
  • MAD0102F22 shows specific binding to the SARS-CoV-2 Spike protein S-2 domain.
  • MAD0102F22 heavy chain is composed of the rearrangement of IGHV4-59 and IGHJ2 immunoglobulin genes.
  • the MAD0102F22 heavy chain shows 96.58% identity to the germline genes.
  • MAD0102F22 light chain is composed of the rearrangement of IGKV3-20 and IGKJ2 immunoglobulin genes.
  • variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ak, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ak, 2016; M428L/N434S as in Zalevsky et ak, 2010.
  • L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0102G04 shows a 100% inhibitory concentration (ICIOO) of 2889.47 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0102G04 shows specific binding to the SARS-CoV-2 Spike protein S-2 domain.
  • MAD0102G04 shows 51% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0102G04 heavy chain is composed of the rearrangement of IGHV1-69D and IGHJ3 immunoglobulin genes.
  • the MAD0102G04 heavy chain shows 96.52% identity to the germline genes.
  • MAD0102G04 light chain is composed of the rearrangement of IGKV1-27 and IGKJ3 immunoglobulin genes.
  • MAD0102G04 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • the L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0008C14 shows a 100% inhibitory concentration (ICIOO) of 56.19 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0008C14 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
  • MAD0008C14 heavy chain is composed of the rearrangement of IGHV3-53 and IGHJ6 immunoglobulin genes.
  • the MAD0008C14 heavy chain shows 96.64% identity to the germline genes.
  • MAD0008C14 light chain is composed of the rearrangement of IGKV1-9 and IGKJ5 immunoglobulin genes.
  • variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0008D14 shows a 100% inhibitory concentration (ICIOO) of 57.25 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0008D14 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
  • MAD0008D14 heavy chain is composed of the rearrangement of IGHV3-30 and IGHJ4 immunoglobulin genes. The MAD0008D14 heavy chain shows 96.61% identity to the germline genes.
  • MAD0008D14 light chain is composed of the rearrangement of IGKV1-39 and IGKJ1 immunoglobulin genes.
  • variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0008B07 shows a 100% inhibitory concentration (ICIOO) of 24.71 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0008B07 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
  • MAD0008B07 shows 3% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0008B07 heavy chain is composed of the rearrangement of IGHV1-46 and IGHJ4 immunoglobulin genes.
  • the MAD0008B07 heavy chain shows 97.59% identity to the germline genes.
  • MAD0008B07 light chain is composed of the rearrangement of IGKV1-16 and IGKJ5 immunoglobulin genes.
  • mutant IgGl constant region backbone which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • the L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0008D12 shows a 100% inhibitory concentration (ICIOO) of 92.94 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0008D12 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
  • MAD0008D12 heavy chain is composed of the rearrangement of IGHV3-53 and IGHJ6 immunoglobulin genes.
  • the MAD0008D12 heavy chain shows 95.95% identity to the germline genes.
  • MAD0008D12 light chain is composed of the rearrangement of IGKV1-9 and IGKJ5 immunoglobulin genes.
  • MAD0102I15 shows a 100% inhibitory concentration (ICIOO) of 123.18 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0102I15 shows 46% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
  • MAD0102I15 heavy chain is composed of the rearrangement of IGHV1-24 and IGHJ4 immunoglobulin genes.
  • the MAD0102I15 heavy chain shows 96.93% identity to the germline genes.
  • MAD0102I15 light chain is composed of the rearrangement of IGKV1-9 and IGKJ2 immunoglobulin genes.
  • variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0103J13 shows a 100% inhibitory concentration (ICIOO) of 444.26 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
  • MAD0103J13 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
  • MAD0103J13 heavy chain is composed of the rearrangement of IGHV1-69D and IGHJ6 immunoglobulin genes.
  • the MAD0103J13 heavy chain shows 99.32% identity to the germline genes.
  • MAD0103J13 light chain is composed of the rearrangement of IGKV3-11 and IGKJ3 immunoglobulin genes.
  • variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010.
  • L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life.
  • This mutant version is referred to as "the mutant" herein.
  • MAD0103J13 are also respectively abbreviated with J08, 114, F05, G12, 121, K22, M02,
  • SARS-CoV-2 neutralizing antibodies can be classified in four groups into four groups
  • nAbs were selected for further characterization. All nAbs were able to bind the SARS-CoV-2 S-protein in its trimeric conformation.
  • the mAbs named J08, 114, F05, G12, C14, B07, 121, J13 and D14 were also able to specifically bind the SI domain.
  • the nAbs named H20, 115, F10 and F20 were not able to bind single SI or S2 domains but only the S-protein in its trimeric state, while the nAb LI 9 bound only the S2 subunit.
  • the first group (Group I) is composed by Sl-RBD specific nAbs (J08, 114, F05, G12 and Cl 4) and showed extremely high neutralization potency against both the WT and D614G live viruses ranging from 3,91 to 157,49 ng/mL.
  • the second group (Group II) is composed by SI nAbs that did not bind the RBD (B07, 121, J13 and D14). These antibodies also showed good neutralization potency ranging from 49,61 to 500 ng/mL but inferior to Sl-RBD directed nAbs.
  • Antibodies belonging to Group I and II showed picomolar affinity to the S-protein with a KD ranging from 0.78 to 6.30 E-10M.
  • the third group (Group III) is composed by antibodies able to bind the S-protein only in its whole trimeric conformation (H20, 115, F10 and F20). Antibodies belonging to this group showed lower affinity to the S- protein (KD 7.57 E-8M - 6.40 E-9M) compared to Group I and II nAbs and medium neutralization potencies ranging from 155,02 to 492,16 ng/mL.
  • the fourth and final group (Group IV) composed only by L19 nAb and recognized exclusively the S2 domain of the S- protein showing the lowest neutralization potency with 19,8 pg/mL and 12,5 pg/mL for the authentic WT and D614G strain respectively.
  • nAbs were further characterized by a competition assay that allowed us to speculate on the regions recognized by these antibodies. Briefly, beads were coated with SARS-CoV-2 trimeric S-protein and incubated with a primary unlabeled antibody in order to saturate the binding site on the antigen surface. Following the first incubation step a secondary Alexa-647 labeled antibody was incubated with the antigen/unlabeled-mAb complex. If the secondary labeled-antibody did not recognize the same epitope of the primary unlabeled-mAb a fluorescent signal was detected when tested by flowcytometry.
  • the 14 selected nAbs were genetically characterized and their IGHV and IGKV genes compared with publicly available SARS-CoV-2 neutralizing antibody sequences.
  • IGHV1-69 one of the most predominant heavy chain gene for SARS-CoV-2 nAbs
  • IGHV1-24 one of the least representative heavy chain V gene
  • Tested antibodies also showed to use the most common germline observed for SARS-CoV-2 nAbs which is the IGHV3-53 (Yuan et al., 2020).
  • IGHV1-69 and IGHV1-24 accommodate IGHJ diversity
  • nAbs belonging to the IGHV3-53 gene family showed to strictly use the IGHJ6 gene.
  • H-CDR3 heavy chain V gene somatic hypermutation level and complementary determining region 3
  • IGHV1-69 derived nAbs rearrange exclusively with IGKV3 gene family while IGHV1-24 derived nAbs accommodate light chain diversity.
  • some of our candidates showed unique heavy and light chain pairing when compared to the public SARS-CoV-2 nAb repertoire.
  • five different heavy and light chain rearrangements not previously described for nAbs against SARS-CoV-2 were identified. These include the IGHV1-24; IGKV1-9, IGH V 1 -24 ; IGKV 3-15, IGHV1-46;IGKV1-16, IGHV3-30;IGKVl-9, IGHV3-53;IGKV1- 17.
  • Antibody-dependent enhancement (ADE) disease has been previously shown to be a potential clinical risk following coronaviruses infection (Lee et al., 2020). Therefore, to optimize the suitability for clinical development and reduce the risk of ADE, five different point mutations were introduced in the constant region (Fc) of the three most potent nAbs (J08, 114 and F05) which were renamed J08-MUT, I14-MUT and F05-MUT.
  • the first two point mutations (M428L/N434S) were introduced to enhance antibody half-life and to increase tissue distribution and persistence (Zalevsky et al., 2010, Gaudinski et ak, 2018, Pegu et ak, 2017).
  • the remaining three point mutations (L234A/L235A/ P329G) were introduced to reduce antibody dependent functions such as binding to FCyRs and cell-based activities (Schlothauer et ak, 2016).
  • a bead based Luminex assay was performed. Briefly the beads were coated with SARS-CoV-2 S-protein receptor binding domain (RBD). Antibodies were tested at eight-point dilutions and the binding was detected with FCyR2A and FcRn (Neonatal Fc receptor) at pH6.2 and 7.4.
  • FCyR2A and FcRn Neonatal Fc receptor
  • the FOyR2A was selected as it is predominantly expressed on the surface of phagocytic cells (such as monocytes, macrophages and neutrophils) and are associated with phagocytosis of immune complexes and antibody opsonized targets (Ackerman et ak, 2013).
  • FcRn which is highly expressed on endothelial cells and circulating monocytes, was selected as it is responsible for the recycling and serum half-life of IgG in the circulation (Mackness et ak, 2019).
  • This latter receptor was shown to possess a tighter binding at lower pH (eg. pH 6.2) compared to physiological pHs (eg. pH 7.4) (Booth et ak, 2018).
  • the results shown demonstrate that the binding to the FOyR2A was completely abrogated for the mutated version of candidate nAbs (J08-MUT, I14-MUT and F05-MUT) compared to their respective wild type (WT) versions (J08, 114 and F05) (Fig.
  • ADNP antibody-dependent neutrophil phagocytosis
  • ADNK antibody-dependent NK cell activation
  • ADNP assay primary human neutrophils were used to detect the antibody binding to SARS-CoV-2 S-protein RBD coated beads, while ADNK activity was evaluated by using primary human NK cells and detecting the release of the proinflammatory cytokine IFN-g. Complete abrogation of both ADNP and ADNK was observed for all three Fc-engineered candidate nAbs compared to their WT versions and control antibody (CR3022) confirming the lack of Fc-mediated cellular activities. Potency and autoreactivity evaluation of Fc-engineered candidates
  • the three engineered antibodies were tested to confirm their binding specificity, NoB ability and neutralization potency against both the WT (SARS-CoV-2/INMIl-Isolate/2020/Italy: MT066156) and the widespread SARS-CoV-2 D614G mutated strain (SARS-CoV- 2/human/ITA/INMI4/2020, clade GR, D614G (S): MT527178) to evaluate their cross neutralization ability.
  • the three engineered nAbs maintained their Sl-doamin binding specificity and an extremely high NoB ability showing an half maximal effective concentration (EC50) of 78, 6, 15,6 and 68,5 ng/mL for J08, 114 and F05 respectively.
  • the three engineered candidate nAbs showed an extremely high neutralization potency with J08 and F05 able to neutralize both strains with an ICIOO inferior to 10 ng/mL (both at 3,91 ng/mL for the WT and the D616G strains).
  • nsEM Single particle negative stain electron microscopy
  • Model docking of PDB 7BYR shows that the fabs overlap with the receptor binding motif (RBM), and therefore are positioned to sterically block receptor hACE2 engagement.
  • RBM receptor binding motif
  • HC heavy chain
  • LC light chain
  • STPCNGVEGFNCY residues 477 to 489
  • Table 3 Characteristics of selected neutralizing antibodies. The table summarizes the binding specificity, affinity, NoB and neutralization features for the fourteen neutralizing antibodies selected in this study.
  • Table 5 Genetic analyses of fourteen selected SARS-CoV-2 nAbs. The table describes the heavy and light chain V-J gene usage, heacy complementary determining region 3 (H- CDR3) length and percentage of nucleotide germline identity for all the fourteen antibodies characterized in this study. Neutralization activity of selected antibodies against SARS-CoV-2 E484K escape mutant
  • J08-MUT provides protection in golden Syrian hamster model of SARS-CoV-2 infection.
  • the golden Syrian hamster model has been widely used to assess monoclonal antibody prophylactic and therapeutic activities against SARS-CoV-2 infection.
  • This model has shown to manifest severe forms of SARS-CoV-2 infection mimicking more closely the clinical disease observed in humans (Baum et al., 2020, Imai et al., 2020, Rogers et al., 2020b, Sia et al., 2020).
  • the monoclonal antibody J08-MUT was administered at three different concentrations (4 - 1 - 0.25 mg/kg) via intraperitoneal injection.
  • Placebo and IgGl isotype control groups were included in the study which received a saline solution and an anti-influenza antibody at the concentration of 4 mg/kg respectively.
  • the J08-MUT 4 mg/kg group and the 1 and 0.25 mg/kg groups were tested in two independent experiments.
  • the IgGl isotype control group was tested in parallel with the J08-MUT 4 mg/kg group while the placebo is an average of the two experiments.
  • the day after, hamsters were anesthetized using 5% isoflurane, and inoculated with 5xl0 5 PFU of SARS-CoV-2 (2019-nCoV/USA-WAl/2020) via the intranasal route, in a final volume of 100 pL. Baseline body weights were measured before infection as well as monitored daily for 12 days post infection.
  • MAD0004J08 was able to treat SARS-CoV-2 infection in golden Syrian hamster when administered at 4 mg/kg.
  • 20 hamsters were divided into 3 arms (six animals each).
  • the monoclonal antibody MAD0004J08 was administered at one concentration (4 mg/kg) via intraperitoneal injection.
  • Placebo and IgGl isotype control groups were included in the study which received a saline solution and an anti-influenza antibody at the concentration of 4 mg/kg respectively.
  • SARS-CoV-2 golden Syrian hamster infection model was previously shown to be a suitable small animal model to support the development of prophylactic and therapeutic tools against Covid-19. Indeed, several characteristics shared between SARS-CoV-2 infected human and hamster lungs, such as severe, bilateral, peripherally distributed, multilobular ground glass opacity, and regions of lung consolidation (DOI:10.1038/s41586-020-2342-5; DOI: 10.1073/pnas.2009799117). Based on the observed efficacy, where 0.25 pg/mL (i.e.
  • J08-MUT 50 pg/animal) of J08-MUT was sufficient to prevent SARS-CoV-2 infection, and the average size of a golden Syrian hamster (approx.150 - 200 g), it’s possible to estimate that 250 pg/Kg will be an adequate dosage for protection and treatment of SARS-CoV-2 infection. If we evaluate 70 kg as average size of an adult human, we can estimate that 17.5 mg would be already sufficient to mediate protection or for treatment of the infection. Therefore, a proposed dosage of 100 mg or 400 mg for clinical studies will result in 6 and 22 times respectively more antibody than the minimum dosage needed.
  • Example of Composition of MAD0004J08 drug product The drug product MAD0004J08 consists of 2.5 ml of a sterile filtered solution containing 40 mg/mL of human monoclonal antibody (humAb) in single-use glass vials.
  • the composition of the drug product is provided in Table 1.
  • the solution is filled in 2R-3 mL glass vials and closed with a 13 mm flurotec rubber stopper.
  • the drug product for early clinical trials was manufactured by Istituto Biochimico Italiani Lorenzini according to cGMP standards.
  • the second method is a S-fuse neutralization assay previously described by Planas et al. Nature Medicine 2021 , 27:917-9242.
  • the CPE-based neutralization assay sees the co-incubation of J08 with a SARS-CoV-2 viral solution containing 100 TCID50 of virus and after 1 hour incubation at 37°C, 5% CO2. The mixture was then added to the wells of a 96-well plate containing a sub-confluent Vero E6 cell monolayer. Plates were incubated for 3 days at 37°C in a humidified environment with 5% CO2, then examined for CPE by means of an inverted optical microscope.
  • J08 was tested at a starting concentration of 1 pg/rnL and diluted step 1 :2 for twelve points.
  • S-fuse neutralization assay U20S-ACE2 GFP1-10 orGFP 11 cells, also termed S-Fuse cells, become GFP + cells when they are productively infected with SARS-CoV-2.
  • the virus was incubated with J08 at a starting concentration of 5 pg/rnL and diluted step 1 :5 for eight points. Then, 18 h later, cells were fixed with 2% paraformaldehyde, washed and stained with Hoechst (1 :1 ,000 dilution; Invitrogen).
  • SARS- CoV-2 viruses were tested: D614G, B.1.1.7 (Alpha; UK), B.1.1.248 (Beta; BZ), B.1.351 (Gamma; SA) and B.1.617 (Delta; IN).
  • a virus-only control and a cell-only control were included in each plate to assist in distinguishing absence or presence of CPE.
  • the content of the well corresponding to the lowest sample dilution that showed complete CPE was diluted 1 :100 and transferred to the antibody-containing wells of the pre-dilution 24-well plate prepared for the subsequent virus passage.
  • both the virus pressured with J08 and the virus-only control were harvested, propagated in 25cm 2 flasks and aliquoted at -80°C to be used for RNA extraction, RT-PCR and sequencing.
  • Cells were treated with R848, a specific TLR7/8 agonist (5mM, Invivogen) as positive control, or with CpG 2395 (3pg/mL, Invivogen) a specific TLR9 agonist as positive control.
  • cell culture supernatants were harvested and treated for 30 minutes at 56°C, then stored at -80°C for later use.
  • SARS-CoV2 inactivation was tested by back titration for each experiment. Release of IFN-a in the supernatant was measured by a specific ELISA kit (PBL assay science). Production of IL-6 in the supernatant was quantified by specific cytometric bead arrays (BD Biosciences) on a FACS Canto (BD Biosciences) and analyzed by FCAP array software (BD Biosciences).
  • SARS-CoV-2 emerging variants also defined as variants of concern (VoC)
  • mAbs monoclonal antibodies
  • the variants most in the spotlight are B.1.1.7 (isolated in the United Kingdom), B.1.351 (isolated in South Africa), B.1.1.248 (isolated in Brazil) and B.1.617 (isolated in India) now re-named by the World Health Organization (WHO) as alpha, beta, gamma and delta variants respectively.
  • WHO World Health Organization
  • SARS-CoV-2 variants have also been listed as variants of concern (VoC).
  • J08 is able to bind the RBD in all conformational states due to a unique footprint
  • J08 is able to bind the RBD in its down tight state (state 1) by using residues S30 and Y32 in the light chain complementary determining region 1 (CDRL1) which interact with the RBD residues S477 and N487 respectively, and residues R50 in the heavy chain CDR2 (CDRH2) and Y100 in the CDRH3 which interact with residues Y489 and K417 respectively ( Figure 17B - C).
  • CDRL1 light chain complementary determining region 1
  • CDRH2 heavy chain CDR2
  • Y100 residues Y489 and K417 respectively
  • J08 uses the CDRH2 residues R50, L54, R56 and M58 to interact with F486, F490 and Q493 on the RBD-1 protomer, while it uses the framework 1 (FW1) residue G26, the CDRH1 residue Y32 and the CDRH3 residue A96 to interact with residues T500, P499 and N440 respectively on the RBD-2 protomer ( Figure 17B and D).
  • J08 interacts with the RBD in its up conformation (state 3), using the CDRH2 residues D55, L54 and R56 which make contact with Y489, Q493, V483 and E484 respectively ( Figure 17B and E).
  • J08 epitope was compared with other two antibodies, named S2E12 and CV07-250, that recognize a similar region on the RBD ( Figure 18A). As shown in Figure 18B, despite these three antibodies recognize the same external loop on the top of the RBD, J08 shows the smallest footprint (dark blue on the RBD shown in gray) among them all ( Figure 18B). Despite this small footprint, J08 epitope still overlaps with the footprint of the angiotensin converting- enzyme 2 (ACE2), therefore interfering with the RBD/ACE2 interaction which is the onset of viral entry into the host cells.
  • ACE2 angiotensin converting- enzyme 2
  • Fc-engineering J08 does not induce the production of pro-inflammatory cytokines
  • hPBMC human peripheral blood mononuclear cells
  • IFN-a Interferon a
  • IL-6 Interleukin 6
  • CpG 2395 and R848 were used as positive and negative control respectively for toll-like receptor 9 (TLR9) activation and IFN-a production. As shown in Figure 21A, CpG 2395 was able to properly activate hPBMC and produce IFN-a while R848 did not stimulate the cells.
  • J08-MUT did not induced the production of IFN-a in presence of SARS-CoV-2 while J08-WT produced higher level than the positive control in a dose-response fashion (Figure 21A).
  • J08-MUT did not induce IL- 6 production which is even lower than the virus alone control (-).
  • J08-WT induced up to 3-fold increase of IL-6 production compared to hPBMC/SARS-CoV-2 alone ( Figure 21 B).
  • J08 is a pan-SARS-CoV-2 variant neutralizing human monoclonal antibody with an extreme neutralization potency against all variants of concern (UK, BZ, SA and IN).
  • J08 shows a unique modality of binding to the RBD as its small epitope footprint allows the binding to all RBD states (up and down).
  • J08-MUT does not induce the production of pro-inflammatory cytokines by human PBMC in presence of SARS-CoV-2.
  • ALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S S V VT VP S S SLGT
  • variable domain of Light chain of MAD0100I14 EIVMTQSPGTLSLAPGERATLSCRASQSVSSSYLGWYQQKPGQAPRLLIYGASSRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGRSPWTFGQGTKVEIKRT >SEQ ID NO:27 Heavy chain of MAD0100I14
  • variable domain of Heavy chain of MAD0102F05 EVQLVESGGGLVQPGGSLRLSCAASGFTVSINYMSWVRQAPGKGLEWVSVIYSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAPLLWADSYYMDV WGKGTTVTVSSAS
  • variable domain of Light chain of MAD0102F05 AIQMTQSPSSLSASVGDRVTITCRASQDIRNNLGWFQQKPGKAPKRLIYAASTLQR GVPSRFSGSGSGTEFTLTIS SLQPEDF ATYY CLQHN S YLWTF GQGTKVEIKRT >SEQ ID NO:45 Heavy chain of MAD0102F05
  • GGT C AAAGGCTTCT ATCCC AGCGAC ATCGCCGT GGAGTGGGAGAGC AAT GGGC

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Abstract

The present invention relates to monoclonal antibodies or antigen-binding portion thereof that have a potent neutralizing activity against Coronavirus, in particular against SARS-CoV-2. The invention relates also to the use of such monoclonal antibodies or antigen-binding portion thereof in therapy, prophylaxis, and diagnosis of Coronavirus, in particular SARS-CoV-2 dependent diseases.

Description

NEUTRALIZING ANTIBODIES TO SARS CORONAVIRUS-2
DESCRIPTION
Technical field of the invention
The present invention relates to monoclonal antibodies or antigen-binding portion thereof that have a potent neutralizing activity against Coronavirus, in particular against SARS- CoV-2. The invention relates also to the use of such monoclonal antibodies or antigen binding portion thereof in therapy, prophylaxis, and diagnosis of Coronavirus, in particular SARS-CoV-2 dependent diseases.
State of the art
Human monoclonal antibodies (mAbs) are an industrially mature technology with more than 50 products already approved in the field of cancer, inflammation and autoimmunity. The well-established safety profile and the large experience for their development make mAbs ideal candidates for rapid development especially in epidemic and pandemic settings. So far mAbs have rarely been used in the field of infectious diseases, mostly because the large quantities needed for therapy made them not cost effective. However, in recent years, the incredible technological progress in isolating and screening memory B cells allowed identification of highly potent neutralizing mAbs and further improvement of their potency by several orders of magnitude through established engineering procedures. This possibility resulted in a decreased quantity of antibodies necessary for therapy thus making non- intravenous delivery of potent neutralizing mAbs possible.
In the case of SARS-CoV-2, where so far there are no effective therapeutic or prophylactic interventions, mAbs have the possibility to become one of the first drugs that can be used for immediate therapy of any patient testing positive for the virus, and even to provide immediate protection from infection in high-risk populations. Preliminary evidences show that plasma from infected subjects improves the outcome of patients with severe disease, therefore it is highly possible that a mAb-based therapy and/or prophylaxis be highly effective. Furthermore, vaccination strategies inducing neutralizing antibodies have already shown to protect non-human primates from infection. These results further stress the importance of mAbs as a measure to target SARS-CoV-2 infection and to contain its circulation.
Among the many therapeutic options available, mAbs offer a series of advantages. First, they are the ones that can be developed in the shortest period of time. In fact, the extensive clinical experience with the safety of more than 50 commercial mAbs approved to treat cancer, inflammation and autoimmunity provides high confidence on their safety, support the possibility of having an accelerated regulatory pathway. In addition, the long industrial experience in developing and manufacturing mAbs decreases the risks usually associated with technical development of investigational products. Finally, the incredible technical progress in the field allows to shorten the conventional timelines and go from discovery to proof of concept trials in 5-6 months. Several candidates are presently under development in the field of HIV, pandemic influenza, RSV and many other infectious diseases. Perhaps the most striking demonstration of the power of mAbs for emerging infections came from the Ebola experience. In this case rapidly developed potent mAbs were among the first drugs to be tested in the Ebola outbreak and showed remarkable efficacy in preventing mortality. Given the striking efficacy of this intervention, potent mAbs became the first, and, so far, the only drug to be recommended for Ebola by the World Health Organization (WHO). Given the pivotal role of the SARS-CoV-2 transmembrane spike glycoprotein (S-protein) for viral pathogenesis, it is considered as the main target to elicit potent neutralizing antibodies and the focus for the development of therapeutic and prophylactic tools against this virus. Indeed, SARS-CoV-2 entry into host cells is mediated by the interaction between S-protein and the human angiotensin converting enzyme 2 (ACE2). The S-protein is a trimeric class I viral fusion protein which exists as a metastable pre-fusion conformation and as a stable post-fusion conformation. Each S-protein monomer is composed of two distinct regions, the SI and S2 subunits. Structural rearrangement occurs when the receptor binding domain (RBD) present in the SI subunit binds to the host cell membrane. This interaction destabilizes the pre-fusion state of the S-protein triggering the transition into the post-fusion conformation which in turn results in the entry of the virus particle into the host cell. Single cell RNA-seq analyses evaluating the expression levels of ACE2 in different human organs has shown that SARS-CoV-2, through S-protein binding, can invade human cells in different major physiological systems including the respiratory, cardiovascular, digestive and urinary systems, enhancing the possibility of spreading and infection. Therefore, it is important to produce neutralizing mAbs or antigen-binding portion thereof that are effective to block the entry process of the virus. Accordingly, there remains an urgent need for potent, broad spectrum antibody therapeutics for use in therapy, prophylaxis, and diagnosis of Coronavirus, in particular SARS-CoV-2, dependent diseases.
Summary of the Invention To identify potent human mAbs against SARS-CoV-2 the inventors isolated thousands of S- protein specific-memory B cells derived from several COVID-19 convalescent donors. The inventors screened naturally produced mAbs against either the S 1 and/or S2 subunits and the S-protein trimer stabilized in its pre-fusion conformation. The screening strategy, disclosed in detail in the examples, allowed identification of human mAbs that have a potent neutralizing activity against Coronavirus, in particular against SARS-CoV-2.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds to a region of human severe acute respiratory syndrome (SARS) Corona Virus 2 (SARS-CoV-2) Spike (S) protein. In one embodiment, said region is i) in the SI domain of SARS-CoV-2 S-protein; or (ii) in the S2 domain of SARS-CoV-2 S-protein; or (iii) in the SARS-CoV-2 S-protein trimer in its pre-fusion conformation or (iv) in the SARS-CoV-2 S-protein trimer in its post-fusion conformation or (v) in the receptor binding domain (RBD) of SARS-CoV-2 S-protein or in a combination thereof.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion that specifically binds to human severe acute respiratory syndrome (SARS) Corona Virus (SARS-CoV-2) S-protein, wherein said antibody or antigen-binding portion thereof provides more than 25% inhibition of the binding between the human ACE2 receptor and the viral Spike protein as measured by the neutralization of binding (NOB) assay.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds to human severe acute respiratory syndrome (SARS) Corona Virus (SARS-CoV-2) S-protein, wherein said antibody or antigen-binding portion thereof has a neutralizing activity. In particular, such human monoclonal antibody or antigen binding portion thereof shows 100% inhibitory concentration (ICIOO) of less than 100 ng/ml when tested in an in vitro neutralization assay against the SARS-CoV-2 vims, for example against the 2019-nCoV strain 2019-nCov/Italy-INMIl, at 100TCID50 viral dose.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising the light chain variable domain (VL) and heavy chain variable domain (VH) of a monoclonal antibody selected from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising the CDRs of a monoclonal antibody selected from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02,
MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22,
MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12,
MAD0102I15, MAD0103J13.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising the VL and VH domains that are at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to the VL and VH domains, respectively, of a monoclonal antibody selected from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
In certain aspects, the invention provides human monoclonal antibodies or antigen-binding portion thereof that compete for the SARS-CoV-2 S-protein with any of the antibodies herein disclosed.
In certain aspects, the invention provides human monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed, for use in a prophylactic or therapeutic treatment of a viral infection or conditions or disorders resulting from such infection.
In certain aspects, the invention provides human monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed, for use in a prophylactic or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular Coronavirus disease 2019 (COVID-19).
In certain aspects, the invention provides a method of preventing or treating the SARS-CoV- 2 infection or conditions or disorders resulting from such infection, in particular Coronavirus disease 2019 (COVID-19), comprising administering a human monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed, to a subject in need thereof.
The invention further provides human monoclonal antibody or an antigen-binding portion according to any embodiments herein disclosed for use in the diagnosis, prophylaxis and/or treatment of a subject having, or at risk of developing, a virus infection, in particular a coronavirus infection, more in particular SARS-CoV-2 infection. Furthermore, the invention pertains to the use of the human binding molecules and/or the nucleic acid molecules of the invention in the diagnosis/detection of such viral infections.
In certain aspects, the invention provides a pharmaceutical composition comprising at least one or more human monoclonal antibodies or antigen-binding portions thereof according to any one of the embodiments herein disclosed and a pharmaceutically acceptable carrier and its use in the prevention and/or treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular Coronavirus disease 2019 (COVID-19).
In certain aspects, the invention provides an isolated cell line that produces the antibody or antigen-binding portion thereof according to any one of the embodiments herein disclosed.
In certain aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the antibody or antigen-binding portion thereof according to any one of the embodiments herein disclosed.
In certain aspects, the invention provides a vector comprising the nucleic acid molecule encoding the antibody or antigen-binding portion thereof embodiments according to any one of the embodiments herein disclosed, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule.
In certain aspects, the invention provides a non-human transgenic animal or transgenic plant comprising the nucleic acid according to any one of the preceding embodiments, wherein the non human transgenic animal or transgenic plant expresses said nucleic acid. In certain embodiments, said non-human transgenic animal is a mammal.
In certain aspects, the invention provides the use of the human monoclonal antibody or an antigen-binding portion thereof according to any one of the embodiments herein disclosed in the diagnosis of the SARS-CoV-2 infection.
In certain aspects, the invention provides an in vitro method for revealing the presence of the SARS-CoV-2 in a sample comprising the following steps: i) Contacting the antibody or an antigen-binding portion thereof according to any one of the embodiments herein disclosed; ii) Detecting the binding of said antibody or an antigen-binding portion thereof to the S-protein of the SARS-CoV-2.
In certain aspects, the invention provides an in vitro method for the diagnosis of the SARS- CoV-2 infection in a subject comprising the following steps: i) Contacting the antibody or an antigen-binding portion thereof according to any one of the embodiments herein disclosed with a biological sample of said subject; ii) Detecting the binding of said antibody or an antigen-binding portion thereof to the S-protein of the SARS-CoV-2.
The invention contemplates combinations of any of the foregoing aspects and embodiments of the invention.
Brief description of the drawings
Fig. 1. S-protein binding and neutralization titration of SARS-CoV-2 convalescent donors’ plasma. (A) Plasma samples were two-fold diluted starting at 1 :80 to test their ability to bind the S-protein trimer in its pre-fusion state by ELISA. Results were considered as positive when the OD405 value was at least two times higher than the blank. (B) Plasma samples were two-fold diluted starting at 1 : 10 to test their ability to neutralize SARS-CoV- 2 in vitro. Results were considered as positive when no cytopathic effect (-) was observed on Vero E6 cells. (C) The graph shows on the Y axis the Logio S-protein binding titer and on the X axis the Logio neutralization of binding (NOB) titer of plasma collected from COVID-19 convalescent patients. (D) The graph shows on the Y axis the Logio S-protein binding titer and on the X axis the Logio neutralization titer of plasma collected from COVID-19 convalescent patients.
Fig. 2. Gating strategy for S-protein specific MBC single cell sorting. Starting from top left to the right panel, the gating strategy shows: Live/Dead; Morphology; Singlets; CD19+ B cells; CD19+CD27+IgD ; CD19+CD27+IgDTgM ; CD19+CD27+IgDTgM S-protein+B cells.
Fig. 3. Identification of SARS-CoV-2 S-protein specific mAbs isolated from convalescent donors. (A) The graph shows supernatants tested for binding to the SARS- CoV-2 S-protein SI + S2 subunits. Threshold of positivity has been set as two times the value of the blank (dotted line) Darker dots represent mAbs which bind to the SI + S2 while light yellow dots represent mAbs which do not bind. The total number (N) of single cell sorted B cell supernatants screened for binding is also shown for each donor. (B) The graph shows supernatants tested for binding to the SARS-CoV-2 S-protein stabilized in its pre fusion conformation. Threshold of positivity has been set as two times the value of the blank (dotted line). Red dots represent mAbs which bind to the S-protein while pink dots represent mAbs which do not bind. The total number (N) of single cell sorted B cell supernatants screened for binding is also shown for each donor.
Fig. 4. Neutralization of S-protein binding to Vero E6 cell receptors by S-protein specific mAbs. (A) Schematic representation of the neutralization of binding (NOB) assay used to screen isolated S-protein specific mAbs for their ability to abrogate the interaction between SARS-CoV-2 and Vero E6 cell receptors. (B) The graph shows supernatants tested by NOB assay. Threshold of positivity has been set as 50% of binding neutralization (dotted line). Dark blue dots represent mAbs able to neutralize the binding between SARS-CoV-2 and receptors on Vero E6 cells, while light blue dots represent non-neutralizing mAbs. The total number (N) of S-protein specific supernatants screened by NOB assay is shown for each donor.
Fig. 5. SARS-CoV-2 neutralization assay for S-protein specific mAbs. (A) Schematic representation of the virus neutralization assay used in this study to assess functional activities of S-protein specific mAbs. (B) Representative microscope images showing the cytopathic effect of SARS-CoV-2 or the protective efficacy of the screened supernatants on Vero E6 cells.
Fig. 6. Workflow and timeline for SARS-CoV-2 neutralizing antibodies identification.
The overall scheme shows three different phases for the identification of SARS-CoV-2 neutralizing antibodies (nAbs). The phase 1 consisted in the enrolment of Covid-19 patients (14) from which PBMC was isolated. Memory B cells were single cell sorted (N= 4,277) and after 2 weeks of incubation antibodies were screened for their binding specificity against the S-protein trimer and S1/S2 domains. Once S-protein specific monoclonal antibodies were identified (N=l,731) the phase 2 started. All specific mAbs were tested in vitro to evaluate their neutralization activity against the live virus and 453 nAbs were identified. nAbs showing different binding profiles on the S-protein surface were selected for further functional characterization and to identify different neutralizing regions on the surface of the S-protein surface. Phase 3 starts with the characterization of the heavy and light chain sequences of selected mAbs (N=14) and the engineering of the Fc-portion of the three most promising candidates. These latter were also selected for structural analyses that allowed the identification of the neutralizing epitopes on the S-protein. Finally, the most potent antibody was tested for its prophylactic effect in a golden Syrian hamster model of SARS-CoV-2 infection.
Fig. 7. Identification of SARS-CoV-2 S-protein specific neutralizing antibodies (nAbs).
(A) The graph shows supernatants tested for binding to the SARS-CoV-2 S-protein stabilized in its prefusion conformation. Threshold of positivity has been set as two times the value of the blank (dotted line). Red dots represent mAbs which bind to the S-protein while pink dots represent mAbs which do not bind. (B) The bar graph shows the percentage of not-neutralizing (gray), partially neutralizing (pale yellow) and neutralizing mAbs (dark red) identified per each donor. The total number (N) of antibodies tested per individual is shown on top of each bar. (C) The graph shows the neutralization potency of each nAb tested once expressed as recombinant full-length IgGl. Dashed lines show different ranges of neutralization potency (500 - 100 - 10 ng/mL). Dots were colored based on their neutralization potency and were classified as weakly neutralizing (>500 ng/mL; pale orange), medium neutralizing (100 - 500 ng/mL; orange), highly neutralizing (10 - 100 ng/mL; dark orange) and extremely neutralizing (1 - 10 ng/mL; dark red). The total number (N) of antibodies tested per individual is shown on top of each graph.
Fig. 8. Functional characterization of potent SARS-CoV-2 S-protein specific nAbs. (A - B - C) Graphs show binding curves to the S-protein in its trimeric conformation, S 1 -domain and S2-domain. Mean ± SD of technical triplicates are shown. Dashed lines represent the threshold of positivity; (D) Neutralization of binding (NoB) curves for selected antibodies were shown as percentage of reduction of signal emitted by a fluorescently labled S-protein incubated with Vero E6 cells. Mean ± SD of technical duplicates are shown. Dashed lines represent the threshold of positivity; (E - F) Neutralization curves for selected antibodies were shown as percentage of viral neutralization against the authentic SARS- CoV-2 wild type and D614G strains. Data are representative of technical triplicates. (G - H) Neutralization potency of fourteen selected antibodies against the authentic SARS-CoV-2 wild type and D614G strains. Dashed lines show different ranges of neutralization potency (500 - 100 - 10 ng/mL). In all graphs selected antibodies are shown in dark red, pink, gray and light blue based on their ability to recognize the SARS-CoV-2 Sl-RBD, SI -domain, S- protein trimer only and S2-domain respectively.
Fig. 9. Identification of four different sites of pathogen vulnerability on the S-protein surface. (A) Representative cytometer peaks per each of the four antibody groups are shown. Positive (only primary antibody) and negative (un-conjugated beads) controls are shown as green and red peaks respectively. Competing and not-competing nAbs are shown in blue and green peaks. (B) The heatmap shows the competition matrix observed among the 14 nAbs tested. Threshold of competition was set at 50% of fluorescent signal reduction. A speculative representation of the vulnerability sites are shown on the S-protein surface.
Fig. 10 Heavy and light chain analyses of selected nAbs. (A - B) Bar graphs show the heavy and light chains usage for neutralizing antibodies against SARS-CoV-2 in the public repertoire compared to the antibodies identified in this study. Our and public antibodies are shown in dark and light colors respectively. (C - D) The heavy and light chain percentage of identity to the inferred germline and amino acidic CDR3 length are shown as violin and distribution plot respectively. (E) The heatmap shows the frequency of heavy and light chain pairing for SARS-CoV-2 neutralizing human monoclonal antibodies already published. The number within the heatmap cells represent the amount of nAbs described in this manuscript showing a novel heavy and light chain rearrangement.
Fig. 11 EM epitope mapping of RBD mAbs. (A) Negative stain, 200 nm scale bar is shown; (B) binding on the RBD; (C) epitope/paratope interaction residues; (D) Epitope region recognized on the receptor binding moif (RBM) and shared residues.
Fig. 12 Prophylactic efficacy of MAD0004J08 in the golden Syrian hamster model of SARS-CoV-2 infection. (A) Schematic representation of the prophylactic study performed in golden Syrian hamster. (B) The figure shows the impact of three different doses of J08- MUT on body weight loss change upon SARS-CoV-2 infection. Statistical analyses were performed among hamsters that received J08-MUT and the IgGl isotype control. Statistically significant differences were calculated with the two-way analysis of variance (ANOVA) and significances are shown as * (p<0.05), ** (p<0.01), *** (p<0.001) and ****
(p<0.0001).
Fig. 13 Therapeutic efficacy of MAD0004J08 in the golden Syrian hamster model of SARS-CoV-2 infection. (A) Schematic representation of the therapeutic study performed in golden Syrian hamster. (B) The figure shows the impact on body weight loss change upon SARS-CoV-2 infection. Statistical analyses were performed among hamsters that received J08 and the IgGl isotype control. Statistically significant differences were calculated with the two-way analysis of variance (ANOVA) and significances are shown as * (p<0.05), ** (p£0.01), *** (p<0.001) and **** (p<0.0001).
Fig. 14 Neutralization activity of human monoclonal antibodies against SARS-CoV-2 PT188-EM. (A - B). Neutralization curves against the SARS-CoV-2 wild type and SARS- CoV-2 PT188-EM are shown respectively. (C) Summary of neutralization potency observed for the fourteen selected antibodies. Colours as dark red, pink, gray and light blue represent antibodies in the Sl-RBD, SI, S-protein and S2 Groups respectively.
Figure 15. J08 neutralization activity against SARS-CoV-2 variants by CPE-based assay. (A - D) Graphs show the neutralization activity of MAD0004J08 against the wild type virus (WT) (A), UK (B), BZ (C) and SA (D) variants. The table below reports the 100% inhibitory concentration (ICIOO) observed per each variant.
Figure 16. J08 neutralization activity against SARS-CoV-2 variants by S-fusion neutralization assay. The graph shows the neutralization activity of J08 against the D614G, UK, IN, SA and BZ variants. The table below reports the 100% inhibitory concentration (ICIOO) observed per each variant.
Figure 17. J08 binding to different states of the SARS-CoV-2 RBD. (A) The graph shows the binding of J08 to the RBD-down tight (state 1), loose (state 2) and up (state 3) position. J08 Fab is shown in dark and light blue for the heavy and light chain respectively while the whole SARS-CoV-2 spike is shown in gray. Red spheres show three highly mutated residues in SARS-CoV-2 VoCs which are K417, E484 and N501. (B) Shows the overall binding region of J08 to the RBD in the three different states. (C - E) The three panel show the amino acidic interaction of the heavy and light chain to the RBD in its state 1 (C), state 2 (D) and state 3 (E).
Figure 18. J08 footprint on the RBD. (A) This panel shows the spike protein (in gray) with the RBD in its up state. Red spheres show three highly mutated residues in SARS-CoV-2 VoCs which are K417, E484 and N501. This panel also show J08, S2E12, CV07-250 and ACE2 position in respect to the RBD. In brackets the immunoglobulin heavy chain variable region (VH) for each antibody is reported. (B) This panel shows the spike protein (in gray) with the RBD in its up state. The foot print of the three antibodies and ACE2 is shown on the RBD and are colored in blue, orange, green and red for J08, S2E12, CV07-250 and ACE2 respectively.
Figure 19. Evolution of an authentic SARS-CoV-2 escape mutant. (A) The graph shows J08 neutralization titer after each mutation acquired by the authentic virus. (B) SARS-CoV-2 S- protein gene showing type, position of mutations and frequency of mutations. Figure 20. The impact of SARS-CoV-2 J08 escape mutant RBD and NTD mutations using a lentiviral pseudotype platform. (A) The graph shows the ND50 curves of J08 against the lentiviral pseudotype particles developed to mimic the evolution of the authentic SARS- CoV-2 escape mutant of J08. (B) The graph shows the ND50 curves of J08 against the lentiviral pseudotype particles developed to assess the impact of single RBD and NTD mutations, or the combination of E484D + Q493H, on J08.
Figure 21. Assessment of hPBMC activation by J08-WT and MUT. (A) the graph shows the production of IFN-a by hPBMC co-incubated with SARS-CoV-2 in presence of J08 WT and MUT. (B) the graph shows the production of IL-6 by hPBMC co-incubated with SARS- CoV-2 in presence of J08 WT and MUT
Detailed description of the invention
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1990), incorporated herein by reference. The following terms, unless otherwise indicated, shall be understood to have the following meanings:
The term "polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide analogues of a protein sequence. A polypeptide may be monomeric or polymeric. The term "isolated protein", "isolated polypeptide" or "isolated antibody" is a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally-associated components by isolation, using protein purification techniques well known in the art. Examples of isolated antibodies include an anti- SARS- CoV-2 S-protein antibody that has been affinity purified using SARS-CoV-2 S-protein or a portion thereof, an anti- SARS-CoV-2 S-protein antibody that has been synthesized by a hybridoma or other cell line in vitro , and a human anti- SARS-CoV-2 S-protein antibody derived from a transgenic animal. A protein or polypeptide is "substantially pure", "substantially homogeneous", or "substantially purified" when at least about 60 to 75% of a sample exhibits a single polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% WAV of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification. The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence. In some embodiments, fragments are at least 5, 6, 8 or 10 amino acids long. In other embodiments, the fragments are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150 or 200 amino acids long. The term "polypeptide analogue" as used herein refers to a polypeptide that comprises a segment that has substantial identity to a portion of an amino acid sequence and that has at least one of the following properties: (1) specific binding to SARS-CoV-2 S-protein under suitable binding conditions, (2) ability to inhibit SARS-CoV-2 S-protein. Typically, polypeptide analogues comprise a conservative amino acid substitution (or insertion or deletion) with respect to the native sequence. Analogues typically are at least 20 or 25 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as a full- length polypeptide. Some embodiments of the invention include polypeptide fragments or polypeptide analogue antibodies with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 substitutions from the germline amino acid sequence. In certain embodiments, amino acid substitutions to an anti- SARS-CoV-2 S-protein antibody or antigen-binding portion thereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogues, but still retain specific binding to SARS-CoV-2 S-protein. Analogues can include various muteins of a sequence other than the normally occurring peptide sequence. For example, single or multiple amino acid substitutions, preferably conservative amino acid substitutions, may be made in the normally occurring sequence, preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence; e.g., a replacement amino acid should not alter the anti-parallel [beta]-sheet that makes up the immunoglobulin binding domain that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence. In general, glycine and proline would not be used in an anti-parallel [beta]-sheet. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et ai, Nature 354:105 (1991), incorporated herein by reference.
The term “SARS-CoV-2” is for Severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), the type of coronavirus that causes coronavirus disease 2019 (COVID-19), where an "antibody" is referred to herein with respect to the invention, it is normally understood that an antigen-binding portion thereof may also be used. An antigen-binding portion competes with the intact antibody for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., second ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, nanobodies and any polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide. From N-terminus to C-terminus, both the mature light and heavy chain variable domains comprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain herein is in accordance with the definitions of IMGT convention described in Lefranc et al. (2003), Developmental & Comparative Immunology 27.1 (2003): 55-77.
As used herein, an antibody that is referred to by number is the same as a monoclonal antibody that is obtained from the human peripheral blood mononuclear cells (PBMCs) isolated from the donor of the same number. For example, monoclonal antibody MAD0004J08 is the same antibody as one obtained from PBMCs isolated from subject identified by the code 004, or a subclone thereof.
As used herein, a Fd fragment means an antibody fragment that consists of the VH and CH 1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al, Nature 341:544-546 (1989)) consists of a VH domain.
In some embodiments, the antibody is a single-chain antibody (scFv) in which a VL and VH domains are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain. (Bird et al, Science 242:423-426 (1988) and Huston et al, Proc. Natl Acad. ScL USA 85:5879-5883 (1988)). In some embodiments, the antibodies are diabodies, i.e., are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites. (See e.g., Holliger P. et al, Proc. Natl. Acad. ScL USA 90:6444-6448 (1993), and Poljak R. J. et al, Structure 2:1121- 1123 (1994)). In such embodiments, the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated noncovalently. In embodiments having one or more binding sites, the binding sites may be identical to one another or may be different.
As used herein, the term "human antibody" means any antibody in which the variable and constant domain sequences are human sequences or any of the CDRs of the variable domain sequences are human sequences. The term encompasses antibodies with sequences derived from human genes, but which have been changed, e.g. to decrease possible immunogenicity, increase affinity, eliminate cysteines that might cause undesirable folding, etc. The term encompasses such antibodies produced recombinantly in non-human cells, which might impart glycosylation not typical of human cells. The term "chimeric antibody" as used herein means an antibody that comprises regions from two or more different antibodies.
The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopes or antigenic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three- dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational." In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another.
A "neutralizing antibody", an antibody with "neutralizing activity", as used herein means an antibody that neutralizes a biological effect that its target (e.g., a pathogen or an infectious particle) may have. A "neutralizing antibody", an antibody with "neutralizing activity", as used herein is for example an antibody or antigen-binding portion thereof showing a 100% inhibitory concentration (ICIOO) of at least less than 100 ng/ml, preferably less than 50 ng/ml, more preferably less than 25 ng/ml when tested in an in vitro neutralization assay against the SARS-CoV-2 vims, performed for example as disclosed herein in the examples.
An antibody is said to specifically bind an antigen when the dissociation constant is for example < 1 mM, preferably < 100 nM and most preferably < 10 nM. The dissociation constant may be measured by any of the methods available in the state of the art as for example using enzyme-linked immunosorbent assay (ELISAs), radioimmunoassay (RIAs), flow cytometry, surface plasmon resonance, such as BIACORE(TM). For example, the expression “specifically binds to a region of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike (S) protein” as herein means that the antibody or its antigen-binding portion provokes more than 50% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay as described in the examples.
The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms.
The term "isolated polynucleotide" as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotides with which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
The term "naturally occurring nucleotides" as used herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" as used herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et ah, Nucl. Acids Res. 14:9081 (1986); Stec et al, J. Am. Chem. Soc. 106:6077 (1984); Stein et ah, Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design 6:539 (1991); Zon et al.. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990), the disclosures of which are hereby incorporated by reference. An oligonucleotide can include a label for detection, if desired. "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" as used herein means polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The term "vector", as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").
The term "recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell" and "host cell" mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.
The term "percent sequence identity" in the context of nucleotide or aminoacidic sequences means the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs available, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods MoF Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J MoF Biol 276:71-84 (1998); incorporated herein by reference). The term "substantial similarity" or "substantial sequence similarity," when referring to a nucleic acid or fragment thereof, or aminoacidic means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above. As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights as supplied with the programs, share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity. In certain embodiments, 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., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well- known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulphur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al, Science 256:1443-45 (1992), incorporated herein by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix. 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 amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters as specified by the programs to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof.
As used herein, the terms "label" or "labelled" refers to incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabelled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labelling polypeptides and glycoproteins are known in the art and may be used.
Human Anti-SARS-CoV-2 S-protein Antibodies and Characterization Thereof In one embodiment, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds to a region of human severe acute respiratory syndrome (SARS) Corona Virus 2 (SARS-CoV-2) Spike (S) protein and at least partially inhibits the S-protein binding to a receptor.
In one embodiment said region is i) in the SI domain of SARS-CoV-2 S-protein; or (ii) in the S2 domain of SARS-CoV-2 S-protein; or (iii) in the SARS-CoV-2 S-protein trimer in its pre-fusion conformation or in its post-fusion conformation or a combination of i) with ii) or in a combination of i) with iii) or in a combination of ii) with iii) or a combination of i) with iv) or (v) in the receptor binding domain (RBD) of SARS-CoV-2 S-protein.
In one embodiment said region is in the SI domain of SARS-CoV-2 S-protein and said human monoclonal antibody or antigen-binding portion thereof is selected from J08, 114, F05, G12, C14, B07, 121, J13 and D14. In one preferred embodiment said region is in the receptor binding domain (RBD) of SARS-CoV-2 S-protein and said human monoclonal antibody or antigen-binding portion thereof is selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12 and MAD0008C14. The receptor-binding domain (RBD) is an independently folded domain of the S-protein known in the art (see for example Wrapp D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Science 367: 1260-1263).
In one embodiment, the invention provides a human monoclonal antibody or antigen-binding portion that specifically binds to human severe acute respiratory syndrome (SARS) Corona Virus (SARS-CoV-2) S-protein, wherein said antibody or antigen-binding portion thereof elicits equal to or more than 25%, 50%, 60%, 70%, 80%, 90%, 95% or 99% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay, performed for example as disclosed in the examples.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds to human severe acute respiratory syndrome (SARS) Corona Virus (SARS-CoV-2) S-protein, wherein said antibody or antigen-binding portion thereof has a neutralizing activity. In particular, such human monoclonal antibody or antigen binding portion thereof shows a 100% inhibitory concentration (ICIOO) of less than 100 ng/ml, preferably less than 50, 25, 20, 10, 8, 6, 5, 4, 3, 2, or 1 ng/ml, when tested in an in vitro neutralization assay against the SARS-CoV-2 virus, for example against the 2019-nCoV strain 2019-nCov/Italy-INMIl, at 100TCID50 viral dose or against some vims mutants such as for example against the mutant D614G or E484K or the escape mutant (SARS-CoV-2 PT188- EM.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof which specifically binds to human severe acute respiratory syndrome (SARS) Corona Vims (SARS-CoV-2) S-protein with an affinity constant (KD) of equal to or less than about 1000 pM, preferably with a KD of equal to or less than about 500, 250, 200, 100, 50, 20, 10 pM, as measured by surface plasmon resonance (SPR), for example measured by SPR as disclosed in the present description.
In certain aspects, the invention provides the nucleic acids encoding the full-length, or variable domain-comprising portions, of heavy and light chains, and the corresponding deduced amino acid sequences can be found in the sequence listing herein enclosed in the description.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising: (a) a heavy chain variable domain amino acid sequence that comprises the amino acid sequence of the heavy chain variable domain of an antibody selected from: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02,
MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22,
MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07,MAD0008D12,
MAD0102I15, MAD0103J13; (b) a light chain variable domain amino acid sequence that comprises the amino acid sequence of the light chain variable domain of an antibody selected from:MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13; (c) a heavy chain variable domain of (a) and a light chain variable domain of (b); or (d) heavy chain and light chain variable domain amino acid sequences comprising the heavy chain and light chain variable domain amino acid sequences, respectively, from the same antibody selected from: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07,MAD0008D12, MAD0102I15, MAD0103J13.
In certain aspects, the invention provides a monoclonal antibody or an antigen-binding portion thereof that specifically binds human SARS-CoV-2 S-protein, comprising: (a) a heavy chain variable domain amino acid sequence that comprises the heavy chain CDR1 , CDR2 and CDR3 amino acid sequences of an antibody selected from: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22,
MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07,
MAD0008D12, MAD0102I15, MAD0103J13; (b) a light chain variable domain amino acid sequence that comprises the light chain CDR1 , CDR2 and CDR3 amino acid sequences of an antibody selected from: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14,
MAD0008D14, MAD0008B07,MAD0008D12, MAD0102I15, MAD0103J13: (c) a heavy chain variable domain of (a) and a light chain variable domain of (b); or (d) the heavy chain variable domain and light chain variable domain of (c), comprising heavy chain and light chain CDR amino acid sequences from the same antibody selected from: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22,
MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20,
MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07,
MAD0008D12, MAD0102I15, MAD0103J13.
In certain aspects, the invention provides a monoclonal antibody or an antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein, wherein the antibody comprises FR1, FR2, FR3 and FR4 amino acid sequences from an antibody selected from: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
In certain aspects, the invention provides a monoclonal antibody that specifically binds SARS-CoV-2 S-protein, wherein said antibody comprises a heavy chain of an antibody selected from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10,
MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04,
MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15,
MAD0103J13. In certain aspects, the invention provides a monoclonal antibody that specifically binds SARS-CoV-2 S-protein, wherein said antibody comprises a light chain of an antibody selected from the group consisting of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02,
MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12,
MAD0102I15, MAD0103J13. In certain aspects, the invention provides a monoclonal antibody that specifically binds SARS-CoV-2 S-protein, wherein said antibody comprises a heavy chain and a light chain of the same antibody which is selected from the group consisting of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising VL and VH domains that are at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to the VL and VH domains, respectively, of a monoclonal antibody selected from the group consisting of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds SARS-CoV-2 S-protein comprising the light chain and the heavy chain that are at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to the light chain and the heavy chain, respectively, of a monoclonal antibody selected from the group consisting of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10,
MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04,
MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
One type of amino acid substitution that may be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine. In one embodiment, there is a substitution of a non-canonical cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant domain of an antibody. In some embodiments, the cysteine is canonical.
Another type of amino acid substitution that may be made is to change any potential proteolytic sites in the antibody. Such sites may occur in a CDR or framework region of a variable domain or in the constant domain of an antibody. Substitution of cysteine residues and removal of proteolytic sites may decrease the risk of any heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is to eliminate asparagine-glycine pairs, which form potential deamidation sites, by altering one or both of the residues. In some embodiments, the C-terminal lysine of the heavy chain of the anti SARS-CoV-2 S-protein antibody of the invention is cleaved. In various embodiments of the invention, the heavy and light chains of the anti-SARS-CoV-2 S-protein antibodies may optionally include a signal sequence.
The class and subclass of anti-SARS-CoV-2 S-protein antibodies may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA, or Western blot (immunoblot) as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various class and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.
In some embodiments, the human anti-SARS-CoV-2 S-protein antibody is an IgG, an IgM, an IgE, an IgA, or an IgD molecule. In one embodiment, the human anti-SARS-CoV-2 S- protein antibody is an IgG and is an IgGl, IgG2, IgG3, IgG4 subclass. In still another embodiment, the human antibody subclass is IgGl.
Binding Affinity of Anti-SARS-CoV-2 S-protein Antibodies to SARS-CoV-2 S-protein.
In some embodiments of the invention, the anti-SARS-CoV-2 S-protein antibodies bind to SARS-CoV-2 S-protein with high affinity. In some embodiments, the anti-SARS-CoV-2 S- protein antibodies bind with high affinity to the SI domain of SARS-CoV-2 S-protein. In some embodiments, the anti-SARS-CoV-2 S-protein antibodies bind to the S2 domain of SARS-CoV-2 S-protein. In another embodiment, the anti-SARS-CoV-2 S-protein antibody binds to SARS-CoV S-protein. The binding affinity and dissociation rate of an anti-SARS- CoV-2 S-protein antibody to SARS-CoV-2 S-protein can be determined by methods known in the art. The binding affinity can be measured by ELISAs, RIAs, flow cytometry, surface plasmon resonance, such as BIACORE(TM). The dissociate rate can be measured by surface plasmon resonance. Preferably, the binding affinity and dissociation rate is measured by surface plasmon resonance. More preferably, the binding affinity and dissociation rate are measured using BIACORE(TM). One can determine whether an antibody has substantially the same KD as an anti-SARS-CoV-2 S-protein antibody by using methods known in the art. Example V exemplifies a method for determining affinity constants of anti-SARS-CoV- 2 S-protein monoclonal antibodies.
Identification of SARS-CoV-2 S-protein Epitopes Recognized by Anti-SARS-CoV-2 S- protein Antibodies.
As disclosed in more details in the Examples, the inventors found that the most potent neutralizing antibodies MAD0004J08, MAD0100I14 and MAD0102F05 specifically bind an epitope comprising the loop containing residues 477 to 489 in the receptor binding domain (RBD) of SARS-CoV-2 S-protein (SEQ ID NO:344), these residues correspond to the amino acid sequence STPCNGVEGFNCY (SEQ ID NO: 343). In certain aspects, the invention provides, an antigen binding protein" ("ABP") that binds to an epitope on the receptor binding domain (RBD) of SARS-CoV-2 S-protein, wherein said epitope comprises the sequence SEQ ID NO: 343 or a sequence that is identical at least 80, 90, 95% to SEQ ID NO: 343. An "antigen binding protein" ("ABP") as used herein means any protein that binds a specified target antigen. In the present application, the specified target antigen is the receptor binding domain (RBD) of SARS-CoV-2 S-protein or fragment thereof, the sequence. "Antigen binding protein" includes but is not limited to antibodies and binding parts thereof, preferably human monoclonal antibody or fragment thereof. Examples of the epitope comprising the amino acid sequence shown in SEQ ID NO: 343 includes an epitope consisting of a continuous partial sequence of the amino acid sequence in the receptor binding domain (RBD) of SARS-CoV-2 S-protein SEQ ID NO:344, which comprises the amino acid sequence shown in SEQ ID NO: 343, and preferably has an amino acid length of 20 or less, for example 19, 18, 17, 16, 15 or less amino acid of SEQ ID NO:344. In some aspects, the invention comprises a neutralizing antigen binding protein that binds to said epitope, wherein the antigen binding protein binds to is positioned 10, 9, 8 angstroms or less from at least one of the following residues 477 to 489 in the receptor binding domain (RBD) of SARS-CoV-2 S-protein, preferably is positioned 10, 9, 8 angstroms or less from all residues 477 to 489 in the receptor binding domain (RBD) of SARS-CoV-2 S-protein. In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof able to specifically bind the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 both in its up and down state.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that specifically binds the epitopes of the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 herein disclosed with a footprint of less than 1000 A, preferably equal to about 400 - 700A.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof neutralizing all the variants alfa, beta, gamma and delta of SARS-CoV-2, in particular showing in the CPE-based assay a 100% inhibitory concentration (ICIOO) of less than 10 ng/mL for all the variants alfa, beta, gamma and delta and/or in the S-fusion neutralization assay a 50% inhibitory concentration (IC50) of less than 1 ng/ml against all the variants alfa, beta, gamma and delta.
In certain aspects, the invention provides a human monoclonal antibody or antigen-binding portion thereof that does not induce the production of pro-inflammatory cytokines by human PBMC in presence of SARS-CoV-2.
The invention provides a human anti-SARS-CoV-2 S-protein monoclonal antibody that binds to SARS-CoV-2 S-protein and competes or cross-competes with and/or binds the same epitope as an antibody selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10,
MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04,
MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15,
MAD0103J13. If two antibodies reciprocally compete with each other for binding to SARS- CoV-2 S-protein, they are said to cross-compete.
One can determine whether an antibody binds to the same epitope or cross competes for binding with an anti-SARS-CoV-2 S-protein antibody by using methods known in the art. In one embodiment, one allows the anti-SARS-CoV-2 S-protein antibody of the invention to bind to SARS-CoV-2 S-protein under saturating conditions and then measures the ability of the test antibody to bind to SARS-CoV-2 S-protein. If the test antibody is able to bind to SARS-CoV-2 S-protein at the same time as the anti-SARS-CoV-2 S-protein antibody, then the test antibody binds to a different epitope as the anti-SARS-CoV-2 S-protein antibody. However, if the test antibody is not able to bind to SARS-CoV-2 S-protein at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the human anti-SARS-CoV-2 S-protein antibody, or the binding of the human anti-SARS-CoV-2 S-protein antibody may induce a conformational change in the SARS-CoV-2 S-protein that prevents or reduces binding of the test antibody. This experiment can be performed using ELISA, RIA, BIACORE(TM), flow cytometry or other methods known in the art.
To test whether an anti-SARS-CoV-2 S-protein antibody cross-competes with another anti- SARS-CoV-2 S-protein antibody, one may use the competition method described above in two directions i.e. determining if the reference antibody blocks the test antibody and vice versa. In one embodiment, the experiment is performed using ELISA. Methods of determining KD are discussed further below.
In another embodiment, the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases SARS-CoV-2 S-protein binding to a receptor, in particular, to angiotensin-converting enzyme 2 (ACE2). In another embodiment, the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases SARS-CoV-2 S- protein-mediated viral entry into cells. In another embodiment, the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases fusion of viral and cell membranes. In another embodiment, the invention provides an anti-SARS-CoV-2 S- protein antibody that decreases viral load. In another embodiment, the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases in severity for any period of time symptoms or conditions resulting from SARS-CoV-2 infection. In certain embodiments, the invention provides an anti-SARS-CoV-2 S-protein antibody that inhibits, blocks, or decreases in severity for a day, a week, a month, 6 months, a year, or for the remainder of the subjects’ life symptoms or conditions resulting from SARS-CoV-2 infection by 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In certain embodiments, the invention provides an anti-SARS-CoV-2 S-protein antibody that may perform any combination of the preceding embodiments.
In certain embodiments, the mAh constant region of the antibodies is modified for half-life extension and reduced risk of Antibody-Dependent Enhancement (ADE) of disease. For example, to enhance the therapeutic activity of mAbs two different and alternative sets of mutations into their constant domains (M252Y/S254T/T256E according to Dall’Acqua et al., 2006; M428L/N434S as reported by Zalevsky et al., 2010) may be applied.
In certain embodiments, in order to reduce the risk of Antibody-Dependent Enhancement (ADE) of disease, mutations that abrogate binding to Fc receptors will be introduced in the Fc part of the IgGl molecule as previously described (L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G LALA as in Schlothauer et al., 2016). All of these modifications may be carried out by means of site-directed mutagenesis, for example using the Agilent Quick-Change II Site-Directed Mutagenesis Kit, according to the manufacturer’s recommendations.
In certain embodiments, the antibody comprises the variable regions of an antibody selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MADOIOOFIO, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13 and a mutant IgGl constant region backbone, which contains one or more of the following groups of mutations: L234A/L235A (as in Hezareh et al., 2001; Beltramello et al., 2010), P329G (as in Schlothauer et al., 2016); M428L/N434S (as in Zalevsky et al., 2010). Preferably the antibody comprises the variable regions of an antibody selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22,
MAD0041M02, MADOIOOFIO, MAD0100L19, MAD0101H20, MAD0102F20,
MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07,
MAD0008D12, MAD0102I15, MAD0103J13and a mutant IgGl constant region backbone, which contains all three groups of such mutations.
Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of Making Antibodies Nucleic Acids
The present invention also encompasses nucleic acid molecules encoding anti- SARS-CoV- 2 S-protein antibodies or antigen-binding portions thereof. In some embodiments, different nucleic acid molecules encode a heavy chain and a light chain of an anti-SARS-CoV-2 S- protein immunoglobulin. In other embodiments, the same nucleic acid molecule encodes a heavy chain and a light chain of an anti-SARS-CoV-2 S-protein immunoglobulin. In one embodiment, the nucleic acid encodes a SARS-CoV-2 S-protein antibody, or antigen- binding portion thereof, of the invention. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a VL amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions and/or 1, 2, or 3 non- conservative substitutions compared to germline. Substitutions may be in the CDR regions, the framework regions, or in the constant domain. In some embodiments, the nucleic acid molecule encodes a VL amino acid sequence comprising one or more variants compared to germline sequence that are identical to the variations found in the VL of one of the antibodies selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
In some embodiments, the nucleic acid molecule encodes at least three amino acid substitutions compared to the germline sequence found in the VL of one of the antibodies selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes the VL amino acid sequence of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04,
MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15,
MAD0103J13 or a variant or portion thereof. In some embodiments, the nucleic acid encodes an amino acid sequence comprising the light chain CDRs of one of said above-listed antibodies. In some embodiments, said portion is a contiguous portion comprising CDR1- CDR3. In some embodiments, the nucleic acid encodes the amino acid sequence of the light chain CDRs of said antibody. In some embodiments, said portion encodes a contiguous region from CDR1-CDR3 of the light chain of an anti-SARS-CoV-2 S-protein antibody.
In some embodiments, the nucleic acid molecule encodes a VL amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a VL amino acid sequence of a VL region of any one of antibodies MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22,
MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12,
MAD0102I15, MAD0103J13. Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, to a nucleotide sequence encoding the amino acid sequence of a VL region.
In another embodiment, the nucleic acid encodes a full-length light chain of an antibody selected MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13 or a light chain comprising a mutation, such as one disclosed herein.
In still another embodiment, the nucleic acid molecule encodes the variable domain of the heavy chain (VH) that comprises a human VH1, VH3 or VH4 family gene sequence or a sequence derived therefrom. In some embodiments, the nucleic acid molecule encodes one or more amino acid mutations compared to the germline sequence that are identical to amino acid mutations found in the VH of one of monoclonal antibodies MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22,
MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20,
MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07 or MAD0008D12, MAD0102I15, MAD0103J13.
In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes at least a portion of the VH amino acid sequence of a monoclonal antibody selected from monoclonal antibodies MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14,
MAD0008D14, MAD0008B07, MAD0008D12 , MAD0102I15, MAD0103J13 all three CDR regions, a contiguous portion including CDR1 -CDR3, or the entire VH region, with or without a signal sequence. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes the amino acid sequence of one of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22,
MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20,
MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13 or said sequence lacking the signal sequence. In some preferred embodiments, the nucleic acid molecule comprises at least a portion of the nucleotide sequence of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14,
MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13 or said sequence lacking the signal sequence. In some embodiments, said portion encodes the VH region (with or without a signal sequence), a CDR3 region, all three CDR regions, or a contiguous region including CDR1-CDR3.
In some embodiments, the nucleic acid molecule encodes a VH amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the VH amino acid sequences of any one of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14,
MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13.
Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions, such as those described above, to a nucleotide sequence encoding the amino acid sequence of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14,
MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13 or that encodes a VH region thereof.
In another embodiment, the nucleic acid encodes a full-length heavy chain of an antibody selected from MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07,MAD0008D12, MAD0102I15, MAD0103J13 or a heavy chain having the amino acid sequence of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19,
MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14,
MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13 with or without a signal sequence, or a heavy chain comprising a mutation, such as one of the variants discussed herein. Further, the nucleic acid may comprise the nucleotide sequence of MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13 with or without a signal sequence, or a nucleic acid molecule encoding a heavy chain comprising a mutation, such as one of the variants discussed herein.
A nucleic acid molecule encoding the heavy or light chain of an anti-SARS-CoV-2 S-protein antibody or portions thereof can be isolated from any source that produces such antibody. In various embodiments, the nucleic acid molecules are isolated from a B cell isolated from an animal immunized with SARS-CoV-2 S-protein or from an immortalized cell derived from such a B cell that expresses or encodes an anti-SARS-CoV-2 S-protein antibody. Methods of isolating mRNA encoding an antibody are well known in the art. See, e.g., Sambrook et al. The mRNA may be used to produce cDNA for use in the polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In one embodiment, the nucleic acid molecule is isolated from a hybridoma that has as one of its fusion partners a human immunoglobulin- producing cell from a non-human transgenic animal. In an even more preferred embodiment, the human immunoglobulin producing cell is isolated from a XENOMOUSE animal. In another embodiment, the human immunoglobulin-producing cell is from a non-human, non mouse transgenic animal, as described above. In another embodiment, the nucleic acid is isolated from a non-human, non-transgenic animal. The nucleic acid molecules isolated from a non-human, non-transgenic animal may be used, e.g., for humanized antibodies. In some embodiments, a nucleic acid encoding a heavy chain of an anti-SARS-CoV-2 S-protein antibody of the invention can comprise a nucleotide sequence encoding a VH domain of the invention joined in-frame to a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, a nucleic acid molecule encoding a light chain of an anti-SARS- CoV-2 S-protein antibody of the invention can comprise a nucleotide sequence encoding a VL domain of the invention joined in-frame to a nucleotide sequence encoding a light chain constant domain from any source. In a further aspect of the invention, nucleic acid molecules encoding the variable domain of the heavy (VH) and/or light (VL) chains are "converted" to full-length antibody genes. In one embodiment, nucleic acid molecules encoding the VH or VL domains are converted to full-length antibody genes by insertion into an expression vector already encoding heavy chain constant (CH) or light chain constant (CL) domains, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector, and/or the VL segment is operatively linked to the CL segment within the vector. In another embodiment, nucleic acid molecules encoding the VH and/or VL domains are converted into full-length antibody genes by linking, e.g., ligating, a nucleic acid molecule encoding a VH and/or VL domains to a nucleic acid molecule encoding a CH and/or CL domain using standard molecular biological techniques. Nucleotide sequences of human heavy and light chain immunoglobulin constant domain genes are known in the art. See, e.g., Kabat et ah, Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publ. No. 91- 3242, 1991. Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed from a cell into which they have been introduced and the anti-SARS-CoV- 2 S-protein antibody isolated.
The nucleic acid molecules may be used to recombinantly express large quantities of anti- SARS-CoV-2 S-protein antibodies. The nucleic acid molecules also may be used to produce chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives, as described further below. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for antibody humanization, also as described below.
In another embodiment, a nucleic acid molecule of the invention is used as a probe or PCR primer for a specific antibody sequence. For instance, the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify regions of DNA that could be used, inter alia, to isolate additional nucleic acid molecules encoding variable domains of anti- SARS-CoV-2 S-protein antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are from highly variable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotides encode all or a part of one or more of the CDRs of antibodies MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13 or variants thereof as described herein. The nucleic acid molecules herein disclosed may be DNA or RNA molecules. Vectors
The invention provides vectors comprising nucleic acid molecules that encode the heavy chain of an anti-SARS-CoV-2 S-protein antibody of the invention or an antigen-binding portion thereof. The invention also provides vectors comprising nucleic acid molecules that encode the light chain of such antibodies or antigen-binding portion thereof. The invention further provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof. In some embodiments, the anti- SARS-CoV-2 S-protein antibodies or antigen-binding portions of the invention are expressed by inserting DNAs encoding partial or full-length light and heavy chains, obtained as described above, into expression vectors such that the genes are operatively linked to necessary expression control sequences such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno- associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. The antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In one embodiment, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can easily be inserted and expressed, as described above. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector also can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non-immunoglobulin protein). In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g. the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Patent No. 5,168,062, U.S. Patent No. 4,510,245 and U.S. Patent No. 4,968,615. Methods for expressing antibodies in plants, including a description of promoters and vectors, as well as transformation of plants is known in the art. See, e.g., United States Patent 6,517,529, incorporated herein by reference. Methods of expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells, are also well known in the art. In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, incorporated herein by reference). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.
In some embodiments said vector is a selected from RNA virus vectors, DNA virus vectors, plasmid viral vectors, adenovirus vectors, adenovirus associated virus vectors, herpes virus vectors and retrovirus vectors. In some aspects, the invention provides compositions (e.g., pharmaceutical compositions), methods, kits and reagents, comprising an isolated nucleic acid molecules according or a vectors according to any one of the embodiments herein disclosed for use in the prevention and/or treatment of a SARS-CoV-2 infections, in particular in humans and other mammals. In some embodiments such nucleic acid molecules and vectors are formulated in a nanoparticle, for example in lipid nanoparticle, cationic lipid nanoparticle, examples of such formulations can be found in US2020197510 herein incorporated by reference. Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein Nucleic acid molecules encoding anti-SARS-CoV-2 S-protein antibodies and vectors comprising these nucleic acid molecules can be used for transfection or transformation of a suitable mammalian, plant, bacterial or yeast host cell. Transfection or transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene- mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art (see, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein by reference). Methods for transforming plant cells are well known in the art, including, e.g., Agrobacterium- mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, N50 cells, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris. Further, expression of antibodies of the invention from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0216 846, 0256055, 0323 997 and 0338 841. It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation of the antibodies. Transgenic Animals and Plants
Anti-SARS-CoV-2 S-protein antibodies of the invention also can be produced transgenically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. In connection with the transgenic production in mammals, anti-SARS-CoV-2 S- protein antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957, incorporated herein by reference. In some embodiments, non- human transgenic animals that comprise human immunoglobulin loci are immunized with SARS-CoV-2 S-protein or an immunogenic portion thereof, as described above. Methods for making antibodies in plants are described, e.g., in U.S. patents 6,046,037 and 5,959,177, incorporated herein by reference.
In some embodiments, non-human transgenic animals or plants are produced by introducing one or more nucleic acid molecules encoding an anti-SARS-CoV-2 S-protein antibody of the invention into the animal or plant by standard transgenic techniques. See Hogan and United States Patent 6,417,429, supra. The transgenic cells used for making the transgenic animal can be embryonic stem cells or somatic cells or a fertilized egg. The transgenic non human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See, e.g., Hofian et al. Manipulating the Mouse Embryo: A Laboratory Manual second ed., Cold Spring Harbor Press (1999); Jackson et al, Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999), all incorporated herein by reference. In some embodiments, the transgenic non-human animals have a targeted disruption and replacement by a targeting construct that encodes a heavy chain and/or a light chain of interest. In one embodiment, the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains that specifically bind to SARS-CoV-2 S-protein, and preferably to (i) the SI domain of SARS-CoV-2 S-protein; (ii) the S2 domain of SARS-CoV-2 S-protein; or (iii) both (i) and (ii). In one embodiment, the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains that specifically bind to human SARS-CoV-2 S-protein. In some embodiments, the transgenic animals comprise nucleic acid molecules encoding a modified antibody such as a single-chain antibody, a chimeric antibody or a humanized antibody. The anti-SARS-CoV- 2 S-protein antibodies may be made in any transgenic animal. In one embodiment, the non human animals are mice, rats, sheep, pigs, goats, cattle or horses. The non-human transgenic animal expresses said encoded polypeptides in blood, milk, urine, saliva, tears, mucus and other bodily fluids.
Class switching
Another aspect of the invention provides a method for converting the class or subclass of an anti-SARS-CoV-2 S-protein antibody to another class or subclass. In some embodiments, a nucleic acid molecule encoding a VL or VH that does not include sequences encoding CL or CH is isolated using methods well-known in the art. The nucleic acid molecule then is operatively linked to a nucleotide sequence encoding a CL or CH from a desired immunoglobulin class or subclass. This can be achieved using a vector or nucleic acid molecule that comprises a CL or CH chain, as described above. For example, an anti- SARS- CoV-2 S-protein antibody that was originally IgM can be class switched to an IgG. Further, the class switching may be used to convert one IgG subclass to another, e.g., from IgGl to IgG2. Another method for producing an antibody of the invention comprising a desired isotype comprises the steps of isolating a nucleic acid encoding a heavy chain of an anti- SARS-CoV-2 S-protein antibody and a nucleic acid encoding a light chain of an anti- SARS- CoV-2 S-protein antibody, isolating the sequence encoding the VH region, ligating the VH sequence to a sequence encoding a heavy chain constant domain of the desired isotype, expressing the light chain gene and the heavy chain construct in a cell, and collecting the anti-SARS-CoV-2 S-protein antibody with the desired isotype.
Modified Antibodies
In another embodiment, a fusion antibody or immunoadhesin may be made that comprises all or a portion of an anti-SARS-CoV-2 S-protein antibody of the invention linked to another polypeptide. In one embodiment, only the variable domains of the anti-SARS-CoV-2 S- protein antibody are linked to the polypeptide. In still another embodiment, the VH domain of an anti-SARS-CoV-2 S-protein antibody is linked to a first polypeptide, while the VL domain of an anti-SARS-CoV-2 S-protein antibody is linked to a second polypeptide that associates with the first polypeptide in a manner such that the VH and VL domains can interact with one another to form an antigen binding site. In still another embodiment, the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with one another (see below under Single Chain Antibodies). The VH-linker- VL antibody is then linked to the polypeptide of interest. The fusion antibody is useful for directing a polypeptide to a SARS-CoV-2 S-protein -expressing cell or tissue. The polypeptide may be a therapeutic agent, such as a toxin, chemokine or other regulatory protein, or may be a diagnostic agent, such as an enzyme that may be easily visualized, such as horseradish peroxidase. In addition, fusion antibodies can be created in which two (or more) single-chain antibodies are linked to one another. This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one wants to create a bispecific antibody or nanobody. To create a single chain antibody, (scFv) the VH- and VL- encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (GIy4 -Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker. See, e.g., Bird et al, Science 242:423-426 (1988); Huston et al, Proc. Natl. Acad. ScL USA 85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990). The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to SARS-CoV-2 S-protein and to another molecule. Bispecific antibodies or antigen-binding fragments can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al, J. Immunol. 148:1547-1553 (1992). In addition, bispecific antibodies may be formed as "diabodies" or "Janusins". In some embodiments, the bispecific antibody binds to two different epitopes of SARS-CoV-2 S-protein. In some embodiments, the bispecific antibody has a first heavy chain and a first light chain from monoclonal antibody MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21,
MAD0041K22, MAD0041M02, MAD0100F10, MAD0100L19, MAD0101H20,
MAD0102F20, MAD0102F22, MAD0102G04, MAD0008C14, MAD0008D14,
MAD0008B07, MAD0008D12, MAD0102I15, MAD0103J13 and an additional antibody heavy chain and light chain. In some embodiments, the additional light chain and heavy chain also are from one of the above-identified monoclonal antibodies, but are different from the first heavy and light chains. In some embodiments, the modified antibodies described above are prepared using one or more of the variable domains or CDR regions from a human anti-SARS-CoV-2 S-protein monoclonal antibody provided herein.
Derivatized and Labelled Antibodies
An anti-SARS-CoV-2 S-protein antibody or antigen-binding portion of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein). In general, the antibodies or portion thereof are derivatized such that the SARS-CoV-2 S-protein binding is not affected adversely by the derivatization or labelling. Accordingly, the antibodies and antibody portions of the invention are intended to include both intact and modified forms of the human anti-SARS-CoV-2 S-protein antibodies described herein. For example, an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detection agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag). One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, II. [0179] Another type of derivatized antibody is a labelled antibody. Useful detection agents with which an antibody or antigen-binding portion of the invention may be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, phycoerythrin, 5-dimethylamine-l-napthalenesulfonyl chloride, lanthanide phosphors and the like. An antibody can also be labelled with enzymes that are useful for detection, such as horseradish peroxidase, [beta]-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody is labelled with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a coloured reaction product, which is detectable. An antibody can also be labelled with biotin, and detected through indirect measurement of avidin or streptavidin binding. An antibody can also be labelled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. An anti-SARS-CoV-2 S-protein antibody can also be labelled with a radiolabelled amino acid. The radiolabel can be used for both diagnostic and therapeutic purposes. For instance, the radiolabel can be used to detect SARS-CoV-2 S-protein-expressing tumours by x-ray or other diagnostic techniques. Further, the radiolabel can be used therapeutically as a toxin for cancerous cells or tumours. In some embodiments, the anti-SARS-CoV-2 S-protein antibody can be labelled with a paramagnetic, radioactive or florigenic ion that is detectable upon imaging. In some embodiments, the paramagnetic ion is chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III). In other embodiments, the radioactive ion is iodine 123, technetium 99, indium 111, rhenium 188, rhenium 186, copper 67, iodine 131, yttrium90, iodine 125, astatine 211, and gallium 67. In other embodiments, the anti-SARS-CoV-2 S-protein antibody is labelled with an X-ray imaging agent such as lanthanum (III), gold (III) lead (II) and bismuth (III). Compositions and Kits The invention relates to compositions comprising the human anti-SARS-CoV-2 S-protein antibody of the invention and one or more pharmaceutical acceptable excipients and/or carriers.
In certain embodiments, the composition may comprise antibodies or a binding portion thereof of any of the preceding embodiments. In some embodiments, the subject of treatment is a human. In other embodiments, the subject is a veterinary subject. In some embodiments, an antagonist anti-SARS-CoV-2 S-protein antibody that binds to the SI domain and one that binds to the S2 domain or antigen-binding portions of either or both, are both administered to a subject, either together or separately. In certain embodiments the antibodies are in a composition comprising a pharmaceutically acceptable carrier. In another embodiment, one or more of the antagonist SARS-CoV-2 S-protein antibodies of the invention are administered in combination with one or more additional antagonistic antibodies that bind different epitopes on the S-protein, that bind the S-protein from different isolates of SARS- CoV-2 and/or that bind different stages of SARS-CoV-2 (i.e., early, middle or late stage virus). As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. The compositions of this invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one embodiment, the antibody is administered by intravenous infusion or injection. In still another embodiment, the antibody is administered by intramuscular or subcutaneous injection. Therapeutic compositions are typically sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the anti-SARS-CoV-2 S-protein antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. The antibodies of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, or intravenous infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Other modes of administration include intraperitoneal, intrabronchial, transmucosal, intraspinal, intrasynovial, intraaortic, intranasal, ocular, otic, topical and buccal. In certain embodiments, the active compound of the antibody compositions may be prepared with a carrier that will protect the antibody against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). The invention also provides compositions suitable for administration by inhalation, which comprise the anti-SARS-CoV-2 S-protein antibodies described herein. The anti-SARS- CoV-2 S-protein antibodies may be conveniently delivered to a subject in the form of an aerosol spray presentation from pressurized packs or from a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Dellamary et al. (2004) J Control Release. ;95(3): 489-500 describes formulations for the pulmonary delivery of antibodies. The invention also provides compositions, suitable for administration through the oral mucosa, which comprise the anti-SARS-CoV-2 S-protein antibody described herein. Oral transmucosal delivery refers to the delivery of a delivery vehicle across a mucous membrane in the oral cavity, pharyngeal cavity, or esophagus, and may be contrasted, for example, with traditional oral delivery, in which absorption of a drug occurs in the intestine. Accordingly, routes of administration in which the anti-SARS-CoV- 2 S-protein antibodies are absorbed through the buccal, sublingual, gingival, pharyngeal, and/or esophageal mucosa are all encompassed within "oral transmucosal delivery," as that term is used herein. For administration through the transmucosal mucosa, the anti-SARS- CoV-2 S-protein antibody may be formulated, for example, into chewing gums (see U.S. Pat No. 5,711,961) or buccal patches (see e.g. U.S. Patent No. 5,298,256). The invention also provides compositions suitable for administration through the vaginal mucosa, which comprise the anti-SARS-CoV-2 S-protein antibodies described herein. The anti-SARS- CoV-2 S-protein antibodies of the invention may be formulated into a vaginal suppository, foam, cream, tablet, capsule, ointment, or gel. In certain embodiments, the compositions comprising the anti-SARS-CoV-2 S-protein antibodies are formulated with permeants appropriate to the transmucosal barrier to be permeated. Such penetrants are generally known in the art, and include, for example, for trans mucosal administration bile salts and fusidic acid derivatives. In certain embodiments, an anti-SARS-CoV-2 S-protein antibody of the invention can be orally administered, for example, with an inert diluent or an assailable edible carrier. The compound (and other ingredients, if desired) can also be enclosed in a hard- or soft-shell gelatine capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the anti-SARS-CoV-2 S-protein antibodies can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. Additional active compounds also can be incorporated into the compositions. In certain embodiments, an inhibitory anti-SARS-CoV-2 S-protein antibody of the invention is co-formulated with and/or co-administered with one or more additional therapeutic agents, particularly anti-viral agents. These therapeutic agents include, without limitation, antibodies that bind other targets, photosensitizers, androgen, oestrogen, nonsteroidal anti-inflammatory agents, antihypertensive agents, analgesic agents, antidepressants, antibiotics, anticancer agents, anaesthetics, antiemetics, anti-infectants, contraceptives, antidiabetic agents, steroids, anti-allergy agents, chemotherapeutic agents, anti-migraine agents, agents for smoking cessation, anti-viral agents, immunosuppressants, thrombolytic agent, cholesterol-lowering agents and anti-obesity agents. Therapeutic agents also include peptide analogues that inhibit SARS-CoV-2 S-protein activity, antibodies or other molecules that prevent SARS-CoV-2 entry into a cell, including but not limited to preventing S-protein binding to a receptor such as the ACE2 receptor, and agents that inhibit SARS-CoV-2 S-protein expression. In one embodiment, the additional agents that inhibit SARS-CoV-2 S-protein expression comprise an antisense nucleic acid capable of hybridizing to a SARS-CoV-2 S-protein mRNA, such as a hairpin RNA or siRNA, locked nucleic acids (LNA) or ribozymes. Sequence-specific nucleic acids capable of inhibiting gene function by RNA interference are well-known in the art. Such combination therapies may require lower dosages of the inhibitory anti-SARS-CoV-2 S-protein antibody as well as the co-administered agents, thus avoiding possible toxicities or complications associated with the various monotherapies. In certain specific embodiments, the therapeutic agent(s) that is co-formulated with and/or co-administered with an inhibitory anti-SARS-CoV-2 S- protein antibody of the invention is an antimicrobial agent. Antimicrobial agents include antibiotics (e.g. antibacterial), antiviral agents, antifungal agents, and anti -protozoan agents. Non-limiting examples of antimicrobial agents are sulfonamides, trimethoprim- sulfamethoxazole, quinolones, penicillins, and cephalosporins. The compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding portion of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount. For example, the dosage proposed for the monoclonal antibodies against Covidl9 (mAbCol9) is calculated in order to achieve a neutralizing titer in the serum of 1/100. Since most Covid-19 convalescent people have a neutralizing titer ranging from 1/20 to 1/320, it is assumed that a titer exceeding 1/100 will provide sufficient neutralizing potency to eliminate the virus from the blood and the lungs. Considering for example the MAD0004J08 neutralization potency of 3 ng/mL (i.e. 3 pg/L or 3 pg/Kg) we can assume that we can achieve a neutralization titer of 1/100 with 0.3 mg/kg. Therefore, 21 mgs for a person of 70 kg would be sufficient to achieve a titer of 1/100. A proposed dose of 100 mg exceeds the titer of 1/100 by 5fold, while a proposed titer of 400 mg exceeds the titer of 1/100 by 20 fold. Advantageously, the 100 - 400 mgs dosages will allow to move from intravenous to intramuscular injection. This route of administration could be a key advantage in emergency scenarios as it will allow to administer the antibodies herein disclosed, such for example MAD0004J08 in non-hospital care settings increasing the number of people that can quickly benefit from its foreseen therapeutic effect.
Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the anti-SARS-CoV-2 S-protein antibody or portion thereof and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an antibody for the treatment of sensitivity in individuals.
In one preferred embodiment, the pharmaceutical composition comprising equal or less than 400 mg for dosage unit of the human monoclonal antibody or antigen-binding portion thereof, more preferably less than 400, 350, 300, 250, 200, 150, 100, 50, 25, 10 mg for dosage unit.
The pharmaceutical composition comprising such dosage unit is preferably for parental administration, for example for intravenous, subcutaneous, intraperitoneal or intramuscular administration. In one preferred embodiment, the pharmaceutical composition is in the liquid form in a concentration between 20 and 200 mg/ml, more preferably between 40 and 80 mg/ml. An exemplary, non-limiting range for a therapeutically or prophylactically- effective amount of an antibody or antibody portion of the invention is 0.025 to 50 mg/kg, more preferably 0.1 to 5 mg/kg, more preferably 0.1-5, 0.1 to 4 or 0.25 to 3 mg/kg. In one embodiment, the invention provides human monoclonal antibody or an antigen-binding portion according to any one of the embodiments herein disclosed, for use in a method for prophylactic or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, in particular COVID-19, wherein said method comprising the step of administering to a patient between 0.025 to 50 mg/kg, more preferably 0.1 to 5 mg/kg, more preferably 0.1-5, 0.1 to 4 or 0.25 to 3 mg/kg once a day, for example for at least one, two, three, four, five, six, seven, eight, nine, ten, eleven days. Such patient is a mammal, preferably a human.
In some embodiments, a formulation contains 5 mg/ml of antibody in a buffer of 20mM sodium citrate, pH 5.5, 140mM NaCl, and 0.2mg/ml polysorbate 80. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Another aspect of the present invention provides kits comprising an anti-SARS-CoV-2 S-protein, or antigen-binding portion, of the invention or a composition comprising such an antibody or antigen-binding fragment. A kit may include, in addition to the antibody or composition, diagnostic or therapeutic agents. A kit can also include instructions for use in a diagnostic or therapeutic method, as well as packaging material such as, but not limited to, ice, dry ice, styrofoam, foam, plastic, cellophane, shrink wrap, bubble wrap, cardboard and starch peanuts. In one embodiment, the kit includes the antibody or a composition comprising it and a diagnostic agent that can be used in a method described below. In still another embodiment, the kit includes the antibody or a composition comprising it and one or more therapeutic agents that can be used in a method described below.
In one embodiment, the antibodies or binding portion thereof or composition comprising such antibodies according to any one of the embodiments herein disclosed are for use in the prevention or the treatment of patients infected with Coronavirus, in particular infected with SARS-CoV-2, for example infected with SARS-CoV-2 wild type, SARS-CoV-2 mutant D615G and/or SARS-CoV-2 mutant E484K or the escape mutant (SARS-CoV-2 PT188-EM or others mutants. The use of such antibodies and compositions of SARS-CoV-2-specific mAbs include, but are not limited to passive immunization in persons at risk of contracting the infection (e.g. professionally exposed personnel, people living in endemic areas) and therapy of acute cases, either hospitalized or not. The invention also relates to compositions for inhibiting viral infection, and in particular Coronavirus infection, more in particular SARS-CoV-2 infection in a mammal comprising an amount of an antibody of the invention in combination with an amount of an antiviral agent, wherein the amounts of the anti-SARS- CoV-2 S-protein antibody and of antiviral agent are together effective in inhibiting viral replication, viral infection of new cells or viral loads.
Diagnostic Methods of Use
The antibodies according to the invention may use also as diagnostic tools for rapid detection of SARS-CoV-2 infection. In another aspect, the invention provides diagnostic methods. The anti-SARS-CoV-2 S-protein antibodies can be used to detect SARS-CoV-2 S-protein in a biological sample in vitro or in vivo. In one embodiment, the invention provides a method for diagnosing the presence or location of SARS-CoV-2 viruses in a subject in need thereof. The anti-SARS-CoV-2 S-protein antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, flow cytometry, tissue immunohistochemistry, Western blot (immunoblot) or immunoprecipitation. The anti- SARS-CoV-2 S-protein antibodies of the invention can be used to detect SARS-CoV-2 S- protein from humans. The invention provides a method for detecting SARS-CoV-2 S-protein in a biological sample comprising contacting the biological sample with an anti-SARS-CoV- 2 S-protein antibody of the invention and detecting the bound antibody. In one embodiment, the anti-SARS-CoV-2 S-protein antibody is directly labelled with a detectable label. In another embodiment, the anti-SARS-CoV-2 S-protein antibody (the first antibody) is unlabelled and a second antibody or other molecule that can bind the anti-SARS-CoV-2 S- protein antibody is labelled. As is well known to one of skill in the art, a second antibody is chosen that is able to specifically bind the particular species and class of the first antibody. For example, if the anti-SARS-CoV-2 S-protein antibody is a human IgG, then the secondary antibody could be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially, e.g., from Pierce Chemical Co. Example of biological samples to use in the diagnostic methods herein disclosed are urine, stool, blood, saliva, biopsies, cerebrospinal fluid, nasopharyngeal and oropharyngeal wash, sputum, endotracheal aspirate, bronchoalveolar lavage or other biological samples obtainable from a human subject.
Suitable labels for the antibody or secondary antibody have been disclosed supra, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, [b eta] -gal actosi dase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol. In other embodiments, SARS-CoV-2 S-protein can be assayed in a biological sample by a competition immunoassay utilizing SARS-CoV-2 S-protein standards labelled with a detectable substance and an unlabelled anti-SARS-CoV-2 S-protein antibody. In this assay, the biological sample, the labelled SARS-CoV-2 S-protein standards and the anti-SARS-CoV-2 S-protein antibody are combined and the amount of labelled SARS-CoV-2 S-protein standard bound to the unlabelled antibody is determined. The amount of SARS-CoV-2 S-protein in the biological sample is inversely proportional to the amount of labelled SARS-CoV-2 S-protein standard bound to the anti-SARS-CoV-2 S-protein antibody. One can use the immunoassays disclosed above for a number of purposes. For example, the anti-SARS-CoV-2 S-protein antibodies can be used to detect SARS-CoV-2 S-protein in cultured cells or as a diagnostic assay in samples from a subject. The diagnostic methods according to any embodiments herein disclosed may be followed by a further step of the administration in the positive subject of an anti-SARS-CoV-2 drugs, for example according to any of the Therapeutic Methods herein disclosed.
Therapeutic Methods of Use
In another embodiment, the invention provides a method for neutralizing SARS- CoV-2 by administering an anti-SARS-CoV-2 S-protein antibody to a patient in need thereof. Any of the types of antibodies described herein may be used therapeutically. In various embodiments, the anti-SARS-CoV-2 S-protein antibody is a human antibody. In some embodiments, the antibody, or antigen-binding portion thereof, binds to the SI domain of SARS-CoV-2 S-protein. In some embodiments, the patient is a human patient. Alternatively, the patient may be a mammal infected with SARS-CoV-2. In one embodiment, the invention provides methods of treating, aiding in the treatment, preventing or aiding in the prevention of, SARS-CoV-2 infection and conditions or disorders resulting from such infection, in a subject by administering to the subject a therapeutically-effective or prophylactically effective amount of an anti-SARS-CoV-2 S-protein antibody of the invention. Antibodies and antigen-binding fragments thereof which are antagonists of SARS-CoV-2 S-protein can be used as therapeutics for SARS-CoV-2 infection. The antibody may be administered locally or systemically. The therapeutic compositions comprising anti-SARS-CoV-2 S- protein antibodies may be administered to the subject, for example, orally, nasally, vaginally, buccally, rectally, via the eye, or via the pulmonary route, in a variety of pharmaceutically acceptable dosing forms, which will be familiar to those skilled in the art. For example, the anti-SARS-CoV-2 S-protein antibodies may be administered via the nasal route using a nasal insufflator device. The anti-SARS-CoV-2 S-protein antibodies can also be administered to the eye in a gel formulation. For example, before administration, a formulation containing the anti- SARS-CoV-2 S-protein antibodies may be conveniently contained in a two- compartment unit dose container, one compartment containing a freeze-dried anti-SARS- CoV-2 S-protein antibody preparation and the other compartment containing normal saline. The serum concentration of the antibody may be measured by any method known in the art. In another embodiment, the antibodies of the present invention are administered to the subject in combination with other therapeutic agents. In one embodiment, the additional therapeutic agents may be treating the symptoms of the SARS-CoV-2 infection on their own, and may optionally synergize with the effects of the antibodies. The additional agent that is administered may be selected by one skilled in the art for treating the infection. Co administration of the antibody with an additional therapeutic agent (combination therapy) encompasses administering a composition comprising the anti-SARS-CoV-2 S-protein antibody and the additional therapeutic agent as well as administering two or more separate compositions, one comprising the anti-SARS-CoV-2 S-protein antibody and the other(s) comprising the additional therapeutic agent(s). Further, although co-administration or combination therapy generally means that the antibody and additional therapeutic agents are administered at the same time as one another, it also encompasses instances in which the antibody and additional therapeutic agents are administered at different times. For instance, the antibody may be administered once every three days, while the additional therapeutic agent is administered once daily. Alternatively, the antibody may be administered prior to or subsequent to treatment with the additional therapeutic agent, for example after a patient has failed therapy with the additional agent. Similarly, administration of the anti-SARS- CoV-2 S-protein antibody may be administered prior to or subsequent to other therapy.
The antibody and one or more additional therapeutic agents (the combination therapy) may be administered once, twice or at least the period of time until the condition is treated, palliated or cured. Preferably, the combination therapy is administered multiple times. The combination therapy may be administered from three times daily to once every six months. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months, or may be administered continuously via a minipump. The combination therapy may be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, or parenteral. In certain aspects, the invention provides a method for treating, preventing or alleviating the symptoms of a SARS-CoV-2 mediated disorder in a subject in need thereof, comprising the step of administering to said subject an antibody or antigen binding portion according to any one of the preceding embodiments, further comprising at least one additional therapeutic agent selected from the group consisting of: (a) one or more antibodies from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10,
MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04,
MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15,
MAD0103J13: and
(b) one or more antibodies that specifically bind SARS-CoV-2 S-protein of a plurality of SARS-CoV-2 strains; and/or
(c) one or more neutralizing antibodies that do not bind SARS-CoV-2 S-protein; and/or
(d) one or more agents that bind SARS-CoV-2 S-protein receptor; and/or
(e) one or more anti-viral agents.
In certain aspects, the invention provides a kit for treating, preventing or alleviating the symptoms of a SARS-CoV-2 mediated disorder in a subject in need thereof, comprising a) one or more antibodies from the group consisting of: MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02,
MAD0100F10, MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22,
MAD0102G04, MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12,
MAD0102I15, MAD0103J13; and
(b) one or more antibodies that specifically bind SARS-CoV-2 S-protein of a plurality of SARS-CoV-2 strains; and/or
(c) one or more neutralizing antibodies that do not bind SARS-CoV-2 S-protein; and/or
(d) one or more agents that bind SARS-CoV-2 S-protein receptor; and/or
(e) one or more anti-viral agents.
The human monoclonal antibody or antigen-binding portion thereof herein disclosed may also be used advantageously as a diagnostic reagent in an in vitro method for detecting in a biological sample previously obtained from a patient (such as for example a serum, plasma, blood sample or any other suitable biological material, obtained from the patient, preferably a human being) anti-Coronavirus antibodies, in particular SARS-Cov-2 antibodies. These antibodies may be found in the biological sample obtained from the patient for instance as a result of a previous exposure to the virus, or because a monoclonal antibody of the invention had been previously administered to the patient for therapeutic or prophylactic or research purposes. Thus, a diagnostic kit comprising the human monoclonal antibody or antigen binding portion thereof herein disclosed of the invention, as a specific reagent, also falls within the scope of the invention, said kit being in particular designed for the detection and/or quantification, in a biological sample previously obtained from a patient, of anti-coronavirus antibodies.
The human monoclonal antibody or antigen-binding portion thereof herein disclosed may also be used advantageously for the design of a vaccine against coronavirus. As disclosed in Rappuoli, Rino et al. “ Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design. ” The Journal of experimental medicine vol. 213,4 (2016): 469-81. doi: 10.1084/jem.20151960”, human mAh may be used to identify protective antigens/epitopes. Structural characterization of the Ab-antigen complex may be used to instruct antigen design. Thus, also a method or the use of the human monoclonal antibody or antigen-binding portion thereof herein disclosed for the design of a vaccine against a coronavirus, in particular against the SARS-Cov-2 virus is within the scope of the invention. The human monoclonal antibody or antigen-binding portion thereof herein disclosed may be used for the preparation of mimotopes, such as for example anti-idiotype antibodies, peptides, S-protein truncated or artificial forms or others, endowed with the ability of evoking the antibodies herein disclosed. Among these, the anti-idiotype antibodies are preferred. The anti-idiotype antibodies are antibodies specifically directed against the idiotype of the neutralizing antibodies used for the manufacture thereof, and thus are able to mimic the key epitopes that they recognize. The manufacture of anti-idiotype antibodies is carried out by per se known methodologies that do not need further detailed explanations here. Thus, also mimotopes, preferably anti-idiotype antibodies, directed against an antibody of the invention fall within the scope of the invention. The human monoclonal antibody or antigen-binding portion thereof herein disclosed may be used for the manufacture of anti idiotype antibodies according to methods per se known. Anti-idiotype antibodies are antibodies specifically directed towards the idiotype of the broad-range neutralizing antibodies used to prepare them, and as such are able to mimic the key epitopes they recognize. Therefore, anti-idiotype antibodies directed against a monoclonal antibody of the invention are also included in the scope of the invention. The following experimental section is provided solely by way of illustration and not limitation and does not intend to restrict the scope of the invention as defined in the appended claims. The claims are an integral part of the description.
EXAMPLES
1. Materials and Methods
Enrollment of SARS-COV-2 convalescent donors and human sample collection
This work results from a collaboration with the National Institute for Infectious Diseases, IRCCS - Lazzaro Spallanzani Rome (IT) and Azienda Ospedaliera Universitaria Senese, Siena (IT) that provided samples from SARS-CoV-2 convalescent donors who gave their written consent. The study was approved by local ethics committees (Parere 18 2020 in Rome and Parere 17065 in Siena) and conducted according to good clinical practice in accordance with the declaration of Helsinki (European Council 2001, US Code of Federal Regulations, ICH 1997). This study was unblinded and not randomized.
Human peripheral blood mononuclear cells (PBMCs) isolation from SARS-CoV-2 convalescent donors
Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-treated whole blood by density gradient centrifugation (Lympholyte-H; Cedarlane). After separation, PBMC were: i) frozen in liquid nitrogen at concentration of 10 x 106 PBMC/vial using 10% DMSO in heat-inactivated fetal bovine serum (FBS) or ii) resuspended in RPMI 1640 (EuroClone) supplemented with 10% FBS (EuroClone), 2 mmol/L L-glutamine, 2 mmol/L penicillin, and 50 pg/mL streptomycin (EuroClone). Cells were cultured for 18 hours at 37°C with 5% CO2. Blood samples were screened for SARS-CoV-2 RNA and for antibodies against HIV, HB V and HC V.
Expression and purification of SARS-CoV-2 S-kok
The expression vector coding for pre-fusion S ectodomain (Wrapp, Daniel et al. “Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.” Science (New York, N. Y.) vol. 367,6483 (2020): 1260-1263. doi: 10.1126/science. abb250) was used to transiently transfect Expi293F cells (Thermo Fisher #A14527) using Expifectamine (Thermo Fisher # A14525). The protein was purified from filtered cell supernatants using NiNTA resin (GE Healthcare #11-0004-58), eluted with 250 mM Imidazole (Sigma Aldrich #56750), dialyzed against PBS, and then stored at 4°C prior to use.
Single cell sorting of SARS-CoV-2 S-protein+ memory B cells Human peripheral blood mononuclear cells (PBMCs) from SARS-CoV-2 convalescent donors were stained with Live/Dead Fixable Aqua (Invitrogen; Thermo Scientific) in 100 pL final volume diluted 1 :500 at room temperature (RT). After 20 min incubation cells were washed with phosphate buffered saline (PBS) and unspecific bindings were saturated with 50 pL of 20% rabbit serum in PBS. Following 20 min incubation at 4°C cells were washed with PBS and stained with SARS-CoV-2 S-protein labelled with Strep-Tactin®XT DY-488 (iba-lifesciences cat# 2-1562-050) for 30 min at 4°C. After incubation the following staining mix was used CD19 V421 (BD cat# 562440), IgM PerCP-Cy5.5 (BD cat# 561285), CD27 PE (BD cat# 340425), IgD-A700 (BD cat# 561302), CD3 PE-Cy7 (BioLegend cat# 300420), CD14 PE-Cy7 (BioLegend cat# 301814), CD56 PE-Cy7 (BioLegend cat# 318318) and cells were incubated at 4°C for additional 30 min. Stained MBCs were single cell-sorted with a BD FACSAria III (BD Biosciences) into 384-well plates containing 3T3-CD40L feeder cells and were incubated with IL-2 and IL-21 for 14 days a described in the state of the art. ELISA assay with SI and S2 subunits of SARS-CoV-2 S-protein The presence of SI- and S2-binding antibodies in culture supernatants of monoclonal S- protein-specific memory B cells was assessed by means of an ELISA assay implemented with the use of a commercial kit (ELISA Starter Accessory Kit, Catalogue No. E101; Bethyl Laboratories, Montgomery, TX, USA). Briefly, 384-well flat-bottom microtiter plates (Nunc MaxiSorp 384-well plates; Sigma-Aldrich) were coated with 25 mΐ/well of antigen (1:1 mix of SI and S2 subunits, 1 pg/ml each; The Native Antigen Company, Oxford, United Kingdom) diluted in coating buffer (0.05 M carbonate-bicarbonate solution, pH 9.6), and incubated overnight at 4°C. The plates were then washed three times with 100 mΐ/well washing buffer (50 mM Tris Buffered Saline (TBS) pH 8.0, 0.05% Tween-20) and saturated with 50 mΐ/well blocking buffer containing Bovine Serum Albumin (BSA) (50 mM TBS pH 8.0, 1% BSA, 0.05% Tween-20) for 1 hour (h) at 37°C. After further washing, samples diluted 1 :5 in blocking buffer were added to the plate. Blocking buffer was used as a blank. After an incubation of 1 h at 37°C, plates were washed and incubated with 25 mΐ/well secondary antibody (horseradish peroxidase (HRP)-conjugated goat anti-human IgG-Fc Fragment polyclonal antibody, diluted 1:10,000 in blocking buffer, Catalogue No. A80- 104P; (Bethyl Laboratories, Montgomery, TX, USA) for 1 h at 37°C. After three washes, 25 mΐ/well TMB One Component HRP Microwell Substrate (Bethyl Laboratories, Montgomery, TX, USA) was added and incubated 10-15 minutes at RT in the dark. Color development was terminated by addition of 25 mΐ/well 0.2 M H2SO4. Absorbance was measured at 450 nm in a Varioskan Lux microplate reader (Thermo Fisher Scientific). The threshold for sample positivity was set at twice the OD of the blank. ELISA assay with SARS-CoV-2 S-protein pre-fusion trimer
ELISA assay was used to detect SARS-CoV-2 S-protein specific mAbs and to screen plasma from SARS-CoV-2 convalescent donors. 384-well plates (Nunc MaxiSorp 384 well plates; Sigma Aldrich) were coated with 3pg/mL of streptavidin diluted in PBS and incubated at RT overnight. Plates were then coated with SARS-CoV-2 S-protein at 3pg/mL and incubated for lh at room temperature. 50 pL/well of saturation buffer (PBS/BSA 1%) was used to saturate unspecific binding and plates were incubated at 37°C for lh without CO2. Supernatants were diluted 1:5 in PBS/BSA 1%/Tween20 0,05% in 25 pL/well final volume and incubated for lh at 37°C without CO2. 25 pL/well of alkaline phosphatase-conjugated goat anti-human IgG (Sigma-Aldrich) and IgA (Jackson Immuno Research) were used as secondary antibodies. In addition, twelve two-fold serial dilutions of plasma from SARS- CoV-2 infected patients were analyzed in duplicate. Plasma samples were diluted in PBS/BSA 1%/Tween20 0,05% (25 pL/well final volume; Starting Dilution 1:80) and incubated for lh at 37°C without CO2. Next, 25 pL/well of alkaline phosphatase-conjugated goat anti-human IgG (Sigma-Aldrich) was added for lh at 37°C without CO2. Wells were washed three times between each step with PBS/BSA 1%/Tween20 0.05%. PNPP (p- nitrophenyl phosphate) (Thermo Fisher) was used as soluble substrate to detect SARS-CoV- 2 S-protein specific monoclonal antibodies and the final reaction was measured by using the Varioskan Lux Reader (Thermo Fisher Scientific) at a wavelength of 405 nm. Samples were considered as positive if optical density at 405 nm (OD405) was two times the blank. SARS-CoV-2 virus and cell infection
African green monkey kidney cell line Vero E6 cells (American Type Culture Collection [ATCC] #CRL-1586) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) - High Glucose (Euroclone, Pero, Italy) supplemented with 2 mM L- Glutamine (Lonza, Milano, Italy), penicillin (100 U/mL) - streptomycin (100 pg/mL) mixture (Lonza, Milano, Italy) and 10% Foetal Bovine Serum (FBS) (Euroclone, Pero, Italy). Cells were maintained at 37°C, in a 5% CO2 humidified environment and passaged every 3-4 days.
Wild type SARS CoV-2 2019 (2019-nCoV strain 2019-nCov/Italy-INMIl) virus was purchased from the European Virus Archive goes Global (EVAg, Spallanzani Institute, Rome). For virus propagation, sub-confluent Vero E6 cell monolayers were prepared in T175 flasks (Sarstedt) containing supplemented D-MEM high glucose medium. For titration and neutralization tests of SARS-CoV-2, Vero E6 were seeded in 96-well plates (Sarstedt) at a density of l,5xl04 cells/well the day before the assay.
Neutralization of Binding (NOB) Assay
To study the binding of the COVID-19 Spike protein to cell-surface receptor(s) we developed an assay to assess recombinant S-protein specific binding to target cells and neutralization thereof.
To this aim the stabilized Spike protein was coupled to Streptavidin-PE (eBioscience # 12- 4317-87, Thermo Fisher) for 30 min at 4°C and then incubated with VERO E6 cells. Binding was assessed by flow cytometry. The stabilized Spike protein bound VERO E6 cells with high affinity (data not shown).
To assess the content of neutralizing antibodies in sera or in B-cell culture supernatants, two microliters of SARS-CoV-2 Spike-Streptavidin-PE at 15-30 pg/ml in PBS-1%FCS were mixed with two microliters of various dilutions of sera or B-cell culture supernatants in U bottom 96-well plates. After incubation at 37°C for 1 hr, 25xl03 Vero E6 cells suspended in two microliters of PBS 1% FCS were added and incubated for additional 1 hr at 4°C. Non bound protein and antibodies were removed and cell-bound PE-fluorescence was analyzed with a FACScantoII flow cytometer (Becton Dickinson). Data were analyzed using the FlowJo data analysis software package (TreeStar, USA). The specific neutralization was calculated as follows: NOB (%) = 1 - (Sample MFI value - background MFI value) / (Negative Control MFI value - background MFI value).
Viral propagation and titration
The SARS-CoV-2 virus was propagated in Vero E6 cells cultured in DMEM high Glucose supplemented with 2% FBS, 100 U/mL penicillin, 100 pg/mL streptomycin. Cells were seeded at a density of lxlO6 cells/mL in T175 flasks and incubated at 37°C, 5% CO2 for 18- 20 hours. The sub-confluent cell monolayer was then washed twice with sterile Dulbecco’s phosphate buffered saline (DPBS). Cells were inoculated with 3.5 ml of the virus properly diluted in DMEM 2% FBS at a multiplicity of infection (MOI) of 0.001, and incubated for lh at 37°C in a humidified environment with 5% CO2. At the end of the incubation, 50 mL of DMEM 2% FBS were added to the flasks. The infected cultures were incubated at 37°C, 5% CO2 and monitored daily until approximately 80-90% of the cells exhibited cytopathic effect (CPE). Culture supernatants were then collected, centrifuged at 4°C at 1,600 rpm for 8 minutes to allow removal of cell debris, aliquoted and stored at -80°C as the harvested viral stock. Viral titers were determined in confluent monolayers of Vero E6 cells seeded in 96-well plates using a 50% tissue culture infectious dose assay (TCID50). Cells were infected with serial 1:10 dilutions (from 10-1 to 10-11) of the virus and incubated at 37°C, in a humidified atmosphere with 5% CO2. Plates were monitored daily for the presence of SARS-CoV-2 induced CPE for 4 days using an inverted optical microscope. The virus titer was estimated according to Spearman-Karber formula (14) and defined as the reciprocal of the highest viral dilution leading to at least 50% CPE in inoculated wells. Semi-quantitative live SARS-CoV-2-based neutralization assay
To assess the neutralization titer of anti-SARS-CoV-2 plasma samples from COVID-19 convalescent donors, a semi-quantitative neutralization method was used. Plasma samples were heat-inactivated for 30 minutes at 56°C and 2-fold serially diluted starting from 1:10 to 1:2,560 dilution, then mixed with an equal volume of viral solution containing 100 TCID50 of SARS-CoV-2 diluted in D-MEM high Glucose 2% FBS. After 1-hour incubation at 37°C, 5% C02, 100 mΐ of the virus-plasma mixture at each dilution was passed to a cell plate containing a sub-confluent Vero E6 cell monolayer. Plates were incubated for 3 days at 37°C in a humidified environment with 5% CO2, then checked for development of CPE by means of an inverted optical microscope. The reciprocal of the highest plasma dilution that resulted in more than 50% inhibition of CPE was defined as the neutralization titer. Qualitative live SARS-CoV-2-based neutralization assay
The neutralization activity of culture supernatants from monoclonal S-protein-specific memory B cells was evaluated by means of a qualitative live-virus based neutralization assay against a one-point dilution of the samples. Supernatants were mixed in a 1:3 ratio with a SARS-CoV-2 viral solution containing 25 TCID50 of virus (final volume: 30 mΐ). After 1- hour incubation at 37°C, 5% CO2, 25 mΐ of each virus-supernatant mixture was added to the wells of a 96-well plate containing a sub-confluent Vero E6 cell monolayer. Following a 2- hour incubation at 37°C, the virus-serum mixture was removed and 100 mΐ of DMEM 2% FBS were added to each well. Plates were incubated for 3 days at 37°C in a humidified environment with 5% CO2, then examined for CPE by means of an inverted optical microscope. Absence or presence of CPE was defined by comparison of each well with the positive control (plasma sample showing high neutralizing activity of SARS-CoV-2 in infected Vero E6 cells) and negative control (human serum sample negative for SARS-CoV- 2 in ELISA and neutralization assays). Recombinant expression of neutralizing mAbs
Selected SARS-CoV-2 neutralizing mAbs are cloned and expressed as described (Tiller et al, 2008; Giuliani et al., 2018). Briefly, the heavy and light chain variable region sequences are recovered from lysed single cell sorted B cells. Recovered amplicons are cloned into suitable Igyl, IgK and Ig vectors for Escherichia coli DH5a transformation. Purified plasmids are used to transiently express full-length IgGs in Expi293 cells. Supernatants of Expi293 cell cultures are collected and tested to assess mAb expression by means of ELISA assays and mAb potency by means of neutralization assays.
Sequencing of selected mAbs The DNA sequences encoding the neutralizing mAbs identified by means of the NOB and neutralization assays described above are recovered by reverse transcription and Polymerase Chain Reaction (PCR) amplification as outlined in Tiller et al., 2008. The amplicons are subjected to Sanger sequencing of the forward and of the reverse strand in order to obtain the coding region of the variable part of the heavy and light chains.
Predicted Amino Acid Sequences:
TABLES
Table 1. SARS-CoV-2 convalescent donors plasma analyses. Plasma S-protein binding titers for each subject were measured by ELISA assays. Neutralization activity was detected by NOB and by neutralization of SARS-CoV-2 infection of Vero cells. Table 2. SARS-CoV-2 convalescent donors S-protein specific MBCs analyses. The Table reports the number of S-protein-specific MBCs that were sorted and screened (for binding by ELISA and for functionality by NOB and viral neutralization) for each subject enrolled in this study.
Single cell RT-PCR and Ig gene amplification
From the original 384-well sorting plate, 5 pL of cell lysate was used to perform RT-PCR. Total RNA from single cells was reverse transcribed in 25 pL of reaction volume composed by 1 pL of random hexamer primers (50 ng/pL), 1 pL of dNTP-Mix (10 mM), 2 pL 0.1 M DTT, 40U/pL RNAse OUT, MgC12 (25 mM), 5x FS buffer and Superscript® IV reverse transcriptase (Invitrogen). Final volume was reached by adding nuclease-free water (DEPC). Reverse transcription (RT) reaction was performed at 42°C/10’, 25°C/10’, 50°C/60’ and 94°/5\ Heavy (VH) and light (VL) chain amplicons were obtained via two rounds of PCR. All PCR reactions were performed in a nuclease-free water (DEPC) in a total volume of 25 pL/well. Briefly, 4 pL of cDNA were used for the first round of PCR (PCRI). PCRI-master mix contained 10 pM of VH and 10 pM VL primer-mix ,10mM dNTP mix, 0,125 pL of Kapa Long Range Polymerase (Sigma), 1,5 pL MgC12 and 5 pL of 5x Kapa Long Range Buffer. PCRI reaction was performed at 95°/3’, 5 cycles at 95°C/30”, 57°C/30”, 72°C/30” and 30 cycles at 95°C/30”, 60°C/30”, 72°C/30” and a final extension of 72 2’. All nested PCR reactions (PCRII) were performed using 3,5 pL of unpurified PCRI product using the same cycle conditions. PCRII products were then purified by Millipore MultiScreen® PCRp96 plate according to manufacture instructions. Samples were eluted with 30 pi nuclease-free water (DEPC) into 96-well plates and quantify by Qubit Fluorometric Quantitation assay (Invitrogen). Characterization of SARS-CoV-2 RBD-Antibodies binding by Flow cytometry
Flow cytometry analysis was performed to define antibodies interaction with S-protein- receptor-binding domain (RBD). Briefly, APEX™ Antibody Labeling Kits (Invitrogen) was used to conjugate 20 pg of selected antibodies to Alexa fluor 647, according to the manufacturer instructions. Then, 1 mg of magnetic bead (Dynabeads™ His-Tag, Invitrogen) were coated with 70 pg of histidine tagged RBD. To assess the ability of each antibody to bind the RBD domain, 20 pg/mL of labelled antibody were incubated with 40 pg/mL of beads-bound RBD for 1 hour on ice. Then, samples were washed with 200 pL of Phosphate- buffered saline (PBS), resuspended in 150 pL of PBS and assessed with a FACSCanto II flow cytometer (Becton Dickinson). Results were analyzed by FlowJo (version 10).
Flow Cytometry-Based S-protein Competition assay
Antibodies specificity to bind SARS-CoV-2 S-protein and their possible competition was analysed performing a Flow cytometer-based assay. To this aim, 200 pg of stabilized histidine tagged S-protein were coated with 1 mg of magnetic beads (Dynabeads™ His-Tag, invitrogen). Then, 20 pg of each antibody was labelled with Alexa fluor 647 working with the APEX™ Antibody Labeling Kits (invitrogen). To test competitive binding profiles of the antibody panel selected, beads-bound S-protein (40 pg/mL) were pre-incubated with unlabeled antibodies (40 pg/mL) for 1 hour on ice. Then, Beads-antibody complex was washed with Phosphate-buffered saline (PBS) and incubated with labelled antibodies (20 pg/mL) for 1 hour on ice. After incubation, the mix Beads-antibodies were washed, resuspended in 150 pL of PBS and read using FACScantoII flow cytometer (Becton Dickinson). Beads-bound and non-bound S-protein incubated with labelled antibodies were used as positive and negative control, respectively. Population gating and analysis was carried out using FlowJo (version 10).
Antigen-specific FCGR binding
Fluorescently coded microspheres were used to profile the ability of selected antibodies to interact with Fc receptors (Boudreau et ak, 2020). The antigen of interest (SARS -CoV-2 S- protein RBD) was covalently coupled to different bead sets via primary amine conjugation. The beads were incubated with diluted antibody (diluted in PBS), allowing “on bead” affinity purification of antigen-specific antibodies. The bound antibodies were subsequently probed with tetramerized recombinant human FCyR2A and FcRN and analyzed using Luminex. The data is reported as the median fluorescence intensity of PE for a specific bead channel.
Antibody-dependent neutrophil phagocytosis
Antibody-dependent neutrophil phagocytosis (ADNP) assesses the ability of antibodies to induce the phagocytosis of antigen-coated targets by primary neutrophils. The assay was performed as previously described (Karsten et al., 2019, Boudreau et al., 2020). Briefly, fluorescent streptavidin-conjugated polystyrene beads were coupled to biotinylated SARS- CoV-2 Spike trimer. Diluted antibody (diluted in PBS) was added, and unbound antibodies were washed away. The antibody:bead complexes are added to primary neutrophils isolated from healthy blood donors using negative selection (StemCell EasySep Direct Human Neutrophil Isolation Kit), and phagocytosis was allowed to proceed for 1 hour. The cells were then washed and fixed, and the extent of phagocytosis was measured by flow cytometry. The data is reported as a phagocytic score, which takes into account the proportion of effector cells that phagocytosed and the degree of phagocytosis. Each sample is run in biological duplicate using neutrophils isolated from two distinct donors. The monoclonal antibodies were tested for ADNP activity at a range of 30 pg/mL to 137.17 ng/ml.
Antibody-dependent NK cell activation
Antibody-dependent NK cell activation (ADNKA) assesses antigen-specific antibody- mediated NK cell activation against protein-coated plates. This assay was performed as previously described (Boudreau et al., 2020). Stabilized SARS-CoV-2 Spike trimer was used to coat ELISA plates, which were then washed and blocked. Diluted antibody (diluted in PBS) was added to the antigen coated plates, and unbound antibodies were washed away. NK cells, purified from healthy blood donor leukopaks using commercially available negative selection kits (StemCell EasySep Human NK Cell Isolation Kit) were added, and the levels of IFN-g was measured after 5 hours using flow cytometry. The data is reported as the percent of cells positive for IFN-g. Each sample is tested with at least two different NK cell donors, with all samples tested with each donor. The monoclonal antibodies were tested for ADNKA activity at a range of 20 pg/mL to 9.1449 ng/mL. Affinity evaluation of SARS-CoV-2 neutralizing antibodies
Anti -Human IgG Polyclonal Antibody (Southern Biotech 2040-01) was immobilized via amine group on two flow cells of a CM5 sensor chip. For the immobilization, anti-human IgG Ab diluted in lOmM Na acetate pH 5.0 at the concentration of 25 pg/ml was injected for 360 sec over the dextran matrix, which had been previously activated with a mixture of 0.1M 1 -ethyl-3 (3 -dimethylaminopropyl)-carbodiimide (EDC) and 0.4M N-hydroxyl succinimide (NHS) for 420 sec. After injection of the antibody, Ethanolamine 1M was injected to neutralize activated group. 10 mΐ/min flow rate was used during the whole procedure. Anti-SPIKE protein human mAbs were diluted in HBS-EP+ (Hepes 10 mM, NaCl 150 mM, EDTA 3.4 mM, 0.05% p20, pH 7.4) and injected for 120 sec at 10 pl/min flow rate over one of the two flow cells containing the immobilized Anti-Human IgG Antibody, while running buffer (HBS-EP+) was injected over the other flow cell to be taken as blank. Dilution of each mAb was adjusted in order to have comparable levels of RU for each capture mAb. Following the capture of each mAb by the immobilized anti-human IgG antibody, different concentrations of SPIKE protein (20 pg/ml, 10 pg/ml, 5 pg/ml, 2.5 pg/ml and 1 pg/ml in HBS-EP+) were injected over both the blank flow cell and the flow cell containing the captured mAb for 180 sec at a flow rate of 80 pl/min. Dissociation was followed for 800 sec, regeneration was achieved with a pulse (60 sec) of Glycine pH 1.5. Kinetic rates and affinity constant of SPIKE protein binding to each mAb were calculated applying a 1 : 1 binding as fitting model using the Bia T200 evaluation software 3.1.
Autoreactivity screening test on HEp-2 Cells
The NOVA Lite HEp-2 ANA Kit (Inova Diagnostics) was used in accordance to the manufacturer’s instructions to test antibodies the autoreactivity of selected antibodies which were tested at a concentration of 100 pg/mL. Kit positive and negative controls were used at three different dilutions (1:1 - 1:10 - 1:100). Images were acquired using a DMI3000 B microscope (Leica) and an exposure time of 300 ms, channel intensity of 2000 and a gamma of 2.
Negative-stain electron microscopy
Complexes were formed by incubating SARS-2 CoV-GSAS-6P-Mut7 and respective fabs at a 1:3 (trimer to fab) molar ratio for 30 minutes at room temperature. After diluting to 0.03 mg/ml in IX TBS pH 7.4, the samples were deposited on plasma-cleaned copper mesh grids and stained with 2% uranyl formate for 55 seconds. Automated data collection was made possible through the Leginon software (Suloway et al., 2005) and a FEI Tecnai Spirit (120keV, 56,000x mag) paired with a FEI Eagle (4k by 4k) CCD camera. Other details include a defocus value of -1.5 pm, a pixel size of 2.06 A per pixel, and a dose of 25 e-/A2. Raw micrographs were stored in the Appion database (Lander et al., 2009), particles were picked with DoGPicker (Voss et al., 2009), and 2D and 3D classification and refinements were performed in RELION 3.0 (Scheres, 2012). Map segmentation and model docking was done in UCSF Chimera (Pettersen et al., 2004).
Cryo-Electron Microscopy
The SARS-2 CoV-GSAS-6P-Mut7 and Fab J08 complex was formed by mixing trimer to fab at a 1 :3 molar ratio for 30 minutes at room temperature. The complex was briefly mixed with fluorinated octyl-maltoside (final concentration 0.02% w/v) and deposited on Quantifoil Au 1.2/1.3-300 mesh grids that had been plasma cleaned for 7 seconds. Grid freezing was facilitated by a Vitrobot Mark IV set to 4*C, 100% humidity, 3 s blot time, 6 s wait time, and blot force 0. The Leginon software was used once again for data collection automation on a FEI Talos Arctica (200 kEV) paired with a Gatan K2 (4k x 4k) camera.
Fill out remaining info when map and model are done.
Biological characteristics of the specific sequenced antibodies
Biological characteristics of the antibody herein identified as MAD0004J08
SUMMARY:
>MAD0004J08 shows a 100% inhibitory concentration (ICIOO) of 7.2 ng/mL when tested for an in vitro neutralization assay against the authentic SARS-CoV-2 virus (2019-nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
>MAD0004J08 shows specific binding to the SARS-CoV-2 Spike protein SI domain. >MAD0004J08 shows 53% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay. >MAD0004J08 heavy chain is composed of the rearrangement of IGHV1-69 and IGHJ4 immunoglobulin genes. The MAD0004J08 heavy chain shows less than 4% mutation rate with respect to germline genes.
>MAD0004J08 light chain is composed of the rearrangement of IGKV3-11 and IGKJ4 immunoglobulin genes. The MAD0004J08 light chain shows less than 2% mutation rate with respect to germline genes.
>MAD0004J08 heavy chain variable region has been successfully cloned and expressed into a mutant IgGl heavy chain constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0100I14 SUMMARY:
>MAD0100I14 shows a 100% inhibitory concentration (ICIOO) of 23.7 ng/mL when tested for an in vitro neutralization assay against the authentic SARS-CoV-2 virus (2019-nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
>MAD0100I14 shows specific binding to the SARS-CoV-2 Spike protein SI domain. >MAD0100I14 shows 98% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
>MAD0100I14 heavy chain is composed of the rearrangement of IGHV1-58 and IGHJ3 immunoglobulin genes. The MAD0100I14 heavy chain shows less than 4% mutation rate with respect to germline genes.
>MAD0100I14 light chain is composed of the rearrangement of IGKV3-20 and IGKJ1 immunoglobulin genes. The MAD0100I14 light chain shows less than 2% mutation rate with respect to germline genes.
>MAD0100I14 heavy chain variable region has been successfully cloned and expressed into a mutant IgGl heavy chain constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0102F05 SUMMARY:
>MAD0102F05 shows a 100% inhibitory concentration (ICIOO) of 8.1 ng/mL when tested for an in vitro neutralization assay against the authentic SARS-CoV-2 virus (2019-nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
>MAD0102F05 shows specific binding to the SARS-CoV-2 Spike protein SI domain. >MAD0102F05 shows 89% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
>MAD0102F05 heavy chain is composed of the rearrangement of IGHV3-53 and IGHJ6 immunoglobulin genes. The MAD0102F05 heavy chain shows less than 3% mutation rate with respect to germline genes.
>MAD0102F05 light chain is composed of the rearrangement of IGKV1-17 and IGKJ1 immunoglobulin genes. The MAD0102F05 light chain shows less than 5% mutation rate with respect to germline genes.
>MAD0102F05 heavy chain variable region has been successfully cloned and expressed into a mutant IgGl heavy chain constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ak, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ak, 2016; M428L/N434S as in Zalevsky et ak, 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0041G12 SUMMARY:
> MAD0041G12 shows a 100% inhibitory concentration (ICIOO) of 23.62 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0041G12 shows specific binding to the SARS-CoV-2 Spike protein S-l domain. > MAD0041G12 shows 100% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
> MAD0041G12 heavy chain is composed of the rearrangement of IGHV1-69 and IGHJ4 immunoglobulin genes. The MAD0041G12 heavy chain shows 95.59% identity to the germline genes.
> MAD0041G12 light chain is composed of the rearrangement of IGKV3-15 and IGKJ4 immunoglobulin genes.
> MAD0041G12 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ah, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ah, 2016; M428L/N434S as in Zalevsky et ah, 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0041I21 SUMMARY:
> MAD0041I21 shows a 100% inhibitory concentration (ICIOO) of 48.25 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0041I21 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
> MAD0041I21 heavy chain is composed of the rearrangement of IGHV3-30 and IGHJ4 immunoglobulin genes. The MAD0041I21 heavy chain shows 98.28% identity to the germline genes.
> MAD0041I21 light chain is composed of the rearrangement of IGKV1-9 and IGKJ4 immunoglobulin genes.
> MAD0041I21 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ah, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ah, 2016; M428L/N434S as in Zalevsky et ah, 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0041K22 SUMMARY:
> MAD0041K22 shows a 100% inhibitory concentration (ICIOO) of 48.42 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0041K22 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
> MAD0041K22 shows 63% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
> MAD0041K22 heavy chain is composed of the rearrangement of IGHV3-30 and IGHJ6 immunoglobulin genes. The MAD0041K22 heavy chain shows 97.30% identity to the germline genes.
> MAD0041K22 light chain is composed of the rearrangement of IGKV3-20 and IGKJ4 immunoglobulin genes.
> MAD0041K22 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ah, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ah, 2016; M428L/N434S as in Zalevsky et ah, 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0041M02 SUMMARY:
> MAD0041M02 shows a 100% inhibitory concentration (ICIOO) of 72.71 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0041M02 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
> MAD0041M02 shows 31% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay. > MAD0041M02 heavy chain is composed of the rearrangement of IGHV3-30 and IGHJ6 immunoglobulin genes. The MAD0041M02 heavy chain shows 96.97% identity to the germline genes.
> MAD0041M02 light chain is composed of the rearrangement of IGKV2-40 and IGKJ1 immunoglobulin genes.
> MAD0041M02 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0100F10 SUMMARY:
> MAD0100F10 shows a 100% inhibitory concentration (ICIOO) of 234.77 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0100F10 shows 45% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
> MAD0100F10 heavy chain is composed of the rearrangement of IGHV1-24 and IGHJ6 immunoglobulin genes. The MAD0100F10 heavy chain shows 96.61% identity to the germline genes.
> MAD0100F10 light chain is composed of the rearrangement of IGKV2-24 and IGKJ2 immunoglobulin genes.
> MAD0100F10 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein. Biological characteristics of the antibody herein identified as MAD0100L19 SUMMARY:
> MAD0100L19 shows a 100% inhibitory concentration (ICIOO) of 1473.67 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0100L19 shows specific binding to the SARS-CoV-2 Spike protein S-2 domain.
> MAD0100L19 shows 15% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
> MAD0100L19 heavy chain is composed of the rearrangement of IGHV3-11 and IGHJ5 immunoglobulin genes. The MAD0100L19 heavy chain shows 96.26% identity to the germline genes.
> MAD0100L19 light chain is composed of the rearrangement of IGKV1-33 and IGKJ5 immunoglobulin genes.
> MAD0100L19 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ah, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ah, 2016; M428L/N434S as in Zalevsky et ah, 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0101H20 SUMMARY:
> MAD0101H20 shows a 100% inhibitory concentration (ICIOO) of 93.11 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0101H20 heavy chain is composed of the rearrangement of IGHV1-69D and IGHJ2 immunoglobulin genes. The MAD0101H20 heavy chain shows 97.30% identity to the germline genes.
> MAD0101H20 light chain is composed of the rearrangement of IGKV3-11 and IGKJ1 immunoglobulin genes. > MAD0101H20 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0102F20 SUMMARY:
> MAD0102F20 shows a 100% inhibitory concentration (ICIOO) of 450.17 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0102F20 shows 21% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
> MAD0102F20 heavy chain is composed of the rearrangement of IGHV1-24 and IGHJ5 immunoglobulin genes. The MAD0102F20 heavy chain shows 96.93% identity to the germline genes.
> MAD0102F20 light chain is composed of the rearrangement of IGKV3-15 and IGKJ2 immunoglobulin genes.
> MAD0102F20 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0102F22 SUMMARY: > MAD0102F22 shows a 100% inhibitory concentration (ICIOO) of 1531.49 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0102F22 shows specific binding to the SARS-CoV-2 Spike protein S-2 domain.
> MAD0102F22 heavy chain is composed of the rearrangement of IGHV4-59 and IGHJ2 immunoglobulin genes. The MAD0102F22 heavy chain shows 96.58% identity to the germline genes.
> MAD0102F22 light chain is composed of the rearrangement of IGKV3-20 and IGKJ2 immunoglobulin genes.
> MAD0102F22 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ak, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ak, 2016; M428L/N434S as in Zalevsky et ak, 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0102G04 SUMMARY:
> MAD0102G04 shows a 100% inhibitory concentration (ICIOO) of 2889.47 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0102G04 shows specific binding to the SARS-CoV-2 Spike protein S-2 domain.
> MAD0102G04 shows 51% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
> MAD0102G04 heavy chain is composed of the rearrangement of IGHV1-69D and IGHJ3 immunoglobulin genes. The MAD0102G04 heavy chain shows 96.52% identity to the germline genes.
> MAD0102G04 light chain is composed of the rearrangement of IGKV1-27 and IGKJ3 immunoglobulin genes.
> MAD0102G04 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0008C14 SUMMARY:
> MAD0008C14 shows a 100% inhibitory concentration (ICIOO) of 56.19 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0008C14 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
> MAD0008C14 heavy chain is composed of the rearrangement of IGHV3-53 and IGHJ6 immunoglobulin genes. The MAD0008C14 heavy chain shows 96.64% identity to the germline genes.
> MAD0008C14 light chain is composed of the rearrangement of IGKV1-9 and IGKJ5 immunoglobulin genes.
> MAD0008C14 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0008D14 SUMMARY:
> MAD0008D14 shows a 100% inhibitory concentration (ICIOO) of 57.25 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0008D14 shows specific binding to the SARS-CoV-2 Spike protein S-l domain. > MAD0008D14 heavy chain is composed of the rearrangement of IGHV3-30 and IGHJ4 immunoglobulin genes. The MAD0008D14 heavy chain shows 96.61% identity to the germline genes.
> MAD0008D14 light chain is composed of the rearrangement of IGKV1-39 and IGKJ1 immunoglobulin genes.
> MAD0008D14 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0008B07 SUMMARY:
> MAD0008B07 shows a 100% inhibitory concentration (ICIOO) of 24.71 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0008B07 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
> MAD0008B07 shows 3% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
> MAD0008B07 heavy chain is composed of the rearrangement of IGHV1-46 and IGHJ4 immunoglobulin genes. The MAD0008B07 heavy chain shows 97.59% identity to the germline genes.
> MAD0008B07 light chain is composed of the rearrangement of IGKV1-16 and IGKJ5 immunoglobulin genes.
> MAD0008B07 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0008D12 SUMMARY:
> MAD0008D12 shows a 100% inhibitory concentration (ICIOO) of 92.94 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0008D12 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
> MAD0008D12 heavy chain is composed of the rearrangement of IGHV3-53 and IGHJ6 immunoglobulin genes. The MAD0008D12 heavy chain shows 95.95% identity to the germline genes.
> MAD0008D12 light chain is composed of the rearrangement of IGKV1-9 and IGKJ5 immunoglobulin genes.
> MAD0008D12 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et ah, 2001; Beltramello et ah, 2010; P329G as in Schlothauer et ah, 2016; M428L/N434S as in Zalevsky et ah, 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0102I15 SUMMARY:
> MAD0102I15 shows a 100% inhibitory concentration (ICIOO) of 123.18 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0102I15 shows 46% inhibition of the interaction between the human ACE2 receptor and the viral Spike protein as measured by the NOB assay.
> MAD0102I15 heavy chain is composed of the rearrangement of IGHV1-24 and IGHJ4 immunoglobulin genes. The MAD0102I15 heavy chain shows 96.93% identity to the germline genes. > MAD0102I15 light chain is composed of the rearrangement of IGKV1-9 and IGKJ2 immunoglobulin genes.
> MAD0102I15 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
Biological characteristics of the antibody herein identified as MAD0103J13 SUMMARY:
> MAD0103J13 shows a 100% inhibitory concentration (ICIOO) of 444.26 ng/mL when tested for an in vitro neutralisation assay against the authentic SARS-CoV-2 virus (2019- nCoV strain 2019-nCov/Italy-INMIl) at 100TCID50 viral dose.
> MAD0103J13 shows specific binding to the SARS-CoV-2 Spike protein S-l domain.
> MAD0103J13 heavy chain is composed of the rearrangement of IGHV1-69D and IGHJ6 immunoglobulin genes. The MAD0103J13 heavy chain shows 99.32% identity to the germline genes.
> MAD0103J13 light chain is composed of the rearrangement of IGKV3-11 and IGKJ3 immunoglobulin genes.
> MAD0103J13 variable region has been successfully cloned and expressed into a mutant IgGl constant region backbone, which contains the following three groups of mutations: L234A/L235A as in Hezareh et al., 2001; Beltramello et al., 2010; P329G as in Schlothauer et al., 2016; M428L/N434S as in Zalevsky et al., 2010. The L234A/L235A and P329G mutations reduce the risk of antibody dependent enhancement of disease (ADE), whereas the M428L/N434S mutations increase the antibody half-life. This mutant version is referred to as "the mutant" herein.
FURTHER RESULTS In the present specification the antibodies MAD0004J08, MAD0100I14, MAD0102F05, MAD0041G12, MAD0041I21, MAD0041K22, MAD0041M02, MAD0100F10,
MAD0100L19, MAD0101H20, MAD0102F20, MAD0102F22, MAD0102G04,
MAD0008C14, MAD0008D14, MAD0008B07, MAD0008D12, MAD0102I15,
MAD0103J13 are also respectively abbreviated with J08, 114, F05, G12, 121, K22, M02,
F10, L19, H20, F20, F22, G04, C14, D14, B07, D12, 115, J13.
SARS-CoV-2 neutralizing antibodies (nAbs) can be classified in four groups into four groups
Based on the first round of screening, 14 nAbs were selected for further characterization. All nAbs were able to bind the SARS-CoV-2 S-protein in its trimeric conformation. The mAbs named J08, 114, F05, G12, C14, B07, 121, J13 and D14 were also able to specifically bind the SI domain. The nAbs named H20, 115, F10 and F20 were not able to bind single SI or S2 domains but only the S-protein in its trimeric state, while the nAb LI 9 bound only the S2 subunit. Among the group of SI specific nAbs only J08, 114, F05, G12, C14 and B07 were able to bind the SI receptor binding domain (RBD) and to strongly inhibit the interaction between the S-protein and Vero E6 receptors. On the other hand, 121, J13 and D14, despite showing SI binding specificity, did not show any binding to the RBD and NoB activity. Based on this description four different groups of nAbs against SARS-CoV-2 were identified. The first group (Group I) is composed by Sl-RBD specific nAbs (J08, 114, F05, G12 and Cl 4) and showed extremely high neutralization potency against both the WT and D614G live viruses ranging from 3,91 to 157,49 ng/mL. The second group (Group II) is composed by SI nAbs that did not bind the RBD (B07, 121, J13 and D14). These antibodies also showed good neutralization potency ranging from 49,61 to 500 ng/mL but inferior to Sl-RBD directed nAbs. Antibodies belonging to Group I and II showed picomolar affinity to the S-protein with a KD ranging from 0.78 to 6.30 E-10M. The third group (Group III) is composed by antibodies able to bind the S-protein only in its whole trimeric conformation (H20, 115, F10 and F20). Antibodies belonging to this group showed lower affinity to the S- protein (KD 7.57 E-8M - 6.40 E-9M) compared to Group I and II nAbs and medium neutralization potencies ranging from 155,02 to 492,16 ng/mL The fourth and final group (Group IV) composed only by L19 nAb and recognized exclusively the S2 domain of the S- protein showing the lowest neutralization potency with 19,8 pg/mL and 12,5 pg/mL for the authentic WT and D614G strain respectively.
All the antibodies described above have been also tested for their ability to cross-neutralize other human coronavirus strains. nAbs were tested against pseudoviruses expressing on the surface the SARS-CoV-2, SARS-CoV-2 D614G, SARS and Middle East Respiratory Syndrome (MERS) spike protein. Neutralization activity was shown against SARS-CoV-2 and D614G pseudoviruses confirming previous data. None of the antibodies herein reported were able to cross-neutralize other coronavirus species.
Different pathogen vulnerability regions identified on the S-protein
The fourteen selected nAbs were further characterized by a competition assay that allowed us to speculate on the regions recognized by these antibodies. Briefly, beads were coated with SARS-CoV-2 trimeric S-protein and incubated with a primary unlabeled antibody in order to saturate the binding site on the antigen surface. Following the first incubation step a secondary Alexa-647 labeled antibody was incubated with the antigen/unlabeled-mAb complex. If the secondary labeled-antibody did not recognize the same epitope of the primary unlabeled-mAb a fluorescent signal was detected when tested by flowcytometry. Through this assay, we observed that all Group I nAbs were competing among themselves for binding on the S-protein RBD, indicating that these antibodies possibly clash against each other and recognize a similar epitope region. All Group II nAbs, showed a different competition profiles and competed with Group II and Group III nAbs. These results confirm that Group III antibodies can recognize various regions on the S-protein surface as they compete with themselves as well as with antibodies belonging to the Group II. Interestingly nAbs belonging to the Group II compete also with the RBD-directed antibody B07 suggesting that this latter nAb may have a different orientation of binding compared to other nAbs included in the Group I. Finally, the Group IV nAb L19 did not compete with any of the other groups identified in this study suggesting that this class of nAbs recognize a distant epitope region with Group I - II and III nAbs.
Genetic characterization of SARS-CoV-2 nAbs
The 14 selected nAbs, were genetically characterized and their IGHV and IGKV genes compared with publicly available SARS-CoV-2 neutralizing antibody sequences. We observed that the majority of nAbs used one of the most predominant heavy chain gene for SARS-CoV-2 nAbs (IGHV1-69) as well as one of the least representative heavy chain V gene (IGHV1-24). Tested antibodies also showed to use the most common germline observed for SARS-CoV-2 nAbs which is the IGHV3-53 (Yuan et al., 2020). Interestingly, while IGHV1-69 and IGHV1-24 accommodate IGHJ diversity, nAbs belonging to the IGHV3-53 gene family showed to strictly use the IGHJ6 gene. The heavy chain V gene somatic hypermutation level and complementary determining region 3 (H-CDR3) length were also evaluated. Our selected nAbs showed low level of somatic mutations when compared to the inferred germlines with sequence identities ranging from 95,6 to 99,3% confirming what was observed in previous publications (Pinto et al., 2020, Zost et al., 2020b, Rogers et al., 2020b, Griffin et al., 2020). The H-CDR3 length spanned from 7 to 21 amino acids (aa) with the majority of the antibodies (N=6; 42%) showing a length of 14 to 16 aa which is slightly bigger to what previously observed. As for the light chain, all of our selected nAbs used the k-chain and the majority of them showed to use the common genes IGKV1- 9 and IGKV3-11 (N=6; 42%). The level of IGKV somatic hypermutation was extremely low for light chains as well showing a percentage of sequence identities ranging from 94,3 to 98,9% . The light chain CDR3 (L-CDR3) length were ranging from 5 to 10 aa which is in line with what previously observed for SARS-CoV-2 nAbs. When nAbs paired heavy and light chain gene analysis was performed, we observed that IGHV1-69 derived nAbs rearrange exclusively with IGKV3 gene family while IGHV1-24 derived nAbs accommodate light chain diversity. Interestingly some of our candidates showed unique heavy and light chain pairing when compared to the public SARS-CoV-2 nAb repertoire. Particularly, five different heavy and light chain rearrangements not previously described for nAbs against SARS-CoV-2 were identified. These include the IGHV1-24; IGKV1-9, IGH V 1 -24 ; IGKV 3-15, IGHV1-46;IGKV1-16, IGHV3-30;IGKVl-9, IGHV3-53;IGKV1- 17.
Fc-engineering of candidate nAbs to abrogate Fc-binding receptors and extend half- life
Antibody-dependent enhancement (ADE) disease, has been previously shown to be a potential clinical risk following coronaviruses infection (Lee et al., 2020). Therefore, to optimize the suitability for clinical development and reduce the risk of ADE, five different point mutations were introduced in the constant region (Fc) of the three most potent nAbs (J08, 114 and F05) which were renamed J08-MUT, I14-MUT and F05-MUT. The first two point mutations (M428L/N434S) were introduced to enhance antibody half-life and to increase tissue distribution and persistence (Zalevsky et al., 2010, Gaudinski et ak, 2018, Pegu et ak, 2017). The remaining three point mutations (L234A/L235A/ P329G) were introduced to reduce antibody dependent functions such as binding to FCyRs and cell-based activities (Schlothauer et ak, 2016).
To confirm the lack of FOyR binding as well as the extended half-life, a bead based Luminex assay was performed. Briefly the beads were coated with SARS-CoV-2 S-protein receptor binding domain (RBD). Antibodies were tested at eight-point dilutions and the binding was detected with FCyR2A and FcRn (Neonatal Fc receptor) at pH6.2 and 7.4. The FOyR2A was selected as it is predominantly expressed on the surface of phagocytic cells (such as monocytes, macrophages and neutrophils) and are associated with phagocytosis of immune complexes and antibody opsonized targets (Ackerman et ak, 2013). On the other hand, FcRn, which is highly expressed on endothelial cells and circulating monocytes, was selected as it is responsible for the recycling and serum half-life of IgG in the circulation (Mackness et ak, 2019). This latter receptor was shown to possess a tighter binding at lower pH (eg. pH 6.2) compared to physiological pHs (eg. pH 7.4) (Booth et ak, 2018). The results shown demonstrate that the binding to the FOyR2A was completely abrogated for the mutated version of candidate nAbs (J08-MUT, I14-MUT and F05-MUT) compared to their respective wild type (WT) versions (J08, 114 and F05) (Fig. S9A) and controls (CR3022 and unrelated protein). Furthermore, Fc-engineered antibodies showed an increased binding activity to the FcRn at both pH 6.2 and 7.4 compared to their wild type counterpart. Finally, to evaluate the lack of Fc-mediated cellular activities by our three candidate nAbs, the antibody-dependent neutrophil phagocytosis (ADNP) and antibody-dependent NK cell activation (ADNK) were evaluated (Butler et ak, 2019, Ackerman et ak, 2016, Karsten et ak, 2019, Boudreau et ak, 2020). For the ADNP assay primary human neutrophils were used to detect the antibody binding to SARS-CoV-2 S-protein RBD coated beads, while ADNK activity was evaluated by using primary human NK cells and detecting the release of the proinflammatory cytokine IFN-g. Complete abrogation of both ADNP and ADNK was observed for all three Fc-engineered candidate nAbs compared to their WT versions and control antibody (CR3022) confirming the lack of Fc-mediated cellular activities. Potency and autoreactivity evaluation of Fc-engineered candidates
The three engineered antibodies were tested to confirm their binding specificity, NoB ability and neutralization potency against both the WT (SARS-CoV-2/INMIl-Isolate/2020/Italy: MT066156) and the widespread SARS-CoV-2 D614G mutated strain (SARS-CoV- 2/human/ITA/INMI4/2020, clade GR, D614G (S): MT527178) to evaluate their cross neutralization ability. The three engineered nAbs maintained their Sl-doamin binding specificity and an extremely high NoB ability showing an half maximal effective concentration (EC50) of 78, 6, 15,6 and 68,5 ng/mL for J08, 114 and F05 respectively. Furthermore, the three engineered candidate nAbs showed an extremely high neutralization potency with J08 and F05 able to neutralize both strains with an ICIOO inferior to 10 ng/mL (both at 3,91 ng/mL for the WT and the D616G strains). Since it has been reported that SARS-CoV-2 elicited antibodies can cross-react with human tissues, cytokines, phospholipids and phospholipid-binding proteins (Zuo et al., 2020, Bastard et al., 2020, Kreer et al., 2020), the three candidate mAbs in both their WT ant MUT versions were tested through an indirect immunofluorescent assay against human epithelial type 2 (HEp-2) cells which expose clinically relevant proteins to detect autoantibody activities. The positive control presents a different range of detectable signals based on its initial dilution steps (from bright-green at 1:1 to very dim-green at 1:100). Among all samples tested, only F05-MUT showed moderate level of autoreactivity to human cells while the other antibodies tested showed complete absence of signal.
Structural analyses for candidate nAbs
Single particle negative stain electron microscopy (nsEM) was used to visualize a stabilized SARS-2-CoV-6P-Mut7 spike protein in complex with three separate Fabs: J08, 114 and F05. This recombinant, soluble spike protein primarily exhibits 3 RBD’s “down” but can switch to RBD “up” conformation with antibody bound. Inspection of the 2D class averages revealed a mixed stoichiometry of unbound spike protein, 1 fab bound, and 2 fab bound classes, which allowed for 3D refinements of each. The three different Fabs bind to the RBD in the “up” conformation, although at different angles and rotations, likely due to the flexibility of the RBD. Model docking of PDB 7BYR (one RBD “up” bound to antibody) shows that the fabs overlap with the receptor binding motif (RBM), and therefore are positioned to sterically block receptor hACE2 engagement. To determine the epitope, heavy chain (HC) and light chain (LC) sequences of Fabs J08, 114, and F05 were used to create synthetic models for docking into the nsEM maps. Based on the docking, a loop containing residues 477 to 489 (STPCNGVEGFNCY) appeared to be involved in binding-specifically, with residue F486 extending into a cavity that is in the middle of the HC and FC of each antibody. To investigate the epitope in greater detail, we performed single particle cryo-EM with SARS-2-CoV-6P-Mut7 spike protein in complex with Fab J08, resulting in about 3 A resolution reconstruction. Sorting of the particles in 3D showed similar results as nsEM, where we observed spike proteins with one or two fabs bound.
Table 3. Characteristics of selected neutralizing antibodies. The table summarizes the binding specificity, affinity, NoB and neutralization features for the fourteen neutralizing antibodies selected in this study.
Table 4. Summary table with the results for the first three antibodies
Table 5. Genetic analyses of fourteen selected SARS-CoV-2 nAbs. The table describes the heavy and light chain V-J gene usage, heacy complementary determining region 3 (H- CDR3) length and percentage of nucleotide germline identity for all the fourteen antibodies characterized in this study. Neutralization activity of selected antibodies against SARS-CoV-2 E484K escape mutant
To further assess the functional activity of identified human monoclonal antibodies, we performed a micro-neutralization assay against an internally generated escape mutant (SARS-CoV-2 PT188-EM) showing an amino acidic deletion (F141) and 11 amino acidic insertion in the N-terminal domain (NTD) of the SARS-CoV-2 S-protein as well as a point mutation in the RBD (E484K). Interestingly, we observed that no SI or S-protein directed antibodies were able to neutralize the SARS-CoV-2 PT188-EM and only four (J08, 114, C14 and B07) out of six (66.7%) Sl-RBD directed antibodies maintained their neutralization activity (Fig. 14).
Prophylactic in vivo experiments with the monoclonal antibody J08-MUT
Process: Six- to eight-month-old female Syrian hamsters were purchased from Charles River Laboratories and housed in microisolator units, allowed free access to food and water and cared for under U.S. Department of Agriculture (USD A) guidelines for laboratory animals. For the passive transfer prophylactic experiments, the day prior to SARS-CoV-2 infection six hamsters per group were intraperitoneally administered with 500 pL of a 4, 1 or 0.25 mg/kg dose of J08-MUT mAh. Another two groups (n=6/each) were administered with 500 pL of 4 mg/kg of the anti -influenza virus #1664 mAh (manuscript in preparation) or PBS only to serve as human IgGl isotype and mock control groups, respectively. The day after, hamsters were anesthetized using 5% isoflurane, and inoculated with 5x105 PFU of SARS- CoV-2 (2019-nCoV/USA-WAl/2020) via the intranasal route, in a final volume of 100 pL. Baseline body weights were measured before infection as well as monitored daily for 7 days post infection. All experiments with the hamsters were performed in accordance with the NRC Guide for Care and Use of Laboratory Animals, the Animal Welfare act, and the CDC/NIH Biosafety and Microbiological and Biomedical Laboratories as well as the guidelines set by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia who also approved the animal experimental protocol. All animal studies infection with SARS-CoV-2 were conducted in the Animal Health Research Center (AHRC) Biosafety Level 3 (BSL-3) laboratories of the University of Georgia.
Results: J08-MUT provides protection in golden Syrian hamster model of SARS-CoV-2 infection. The golden Syrian hamster model has been widely used to assess monoclonal antibody prophylactic and therapeutic activities against SARS-CoV-2 infection. This model has shown to manifest severe forms of SARS-CoV-2 infection mimicking more closely the clinical disease observed in humans (Baum et al., 2020, Imai et al., 2020, Rogers et al., 2020b, Sia et al., 2020). We designed a prophylactic study in golden Syrian hamster to evaluate the efficacy of our monoclonal antibody named J08-MUT in preventing SARS- CoV-2 infection. For this study 30 hamsters were divided into 5 arms (six animals each). The monoclonal antibody J08-MUT was administered at three different concentrations (4 - 1 - 0.25 mg/kg) via intraperitoneal injection. Placebo and IgGl isotype control groups were included in the study which received a saline solution and an anti-influenza antibody at the concentration of 4 mg/kg respectively. The J08-MUT 4 mg/kg group and the 1 and 0.25 mg/kg groups were tested in two independent experiments. The IgGl isotype control group was tested in parallel with the J08-MUT 4 mg/kg group while the placebo is an average of the two experiments. Twenty-four hours post-administration of the antibody or saline solution, animals were challenged with 100 pL of SARS-CoV-2 solution (5x105 PFU) via intranasal distillation. Three hamsters per group were sacrificed at three days post-infection while the remaining animals were culled at day 8. Body weight changes were daily evaluated throughout the study and reported in Fig.lOA. We observed that MAD000J08 significantly reduced weight loss at all concentrations tested in this study in a dose-response fashion compared to both the placebo and the IgGl isotype control groups. When J08-MUT was administered at 4 mg/kg we observed complete protection from SARS-CoV-2 infection and only a minimal weight loss was noticed one day post viral challenge. All animals quickly recovered at day 3 and hamsters reached their initial weight. From day 4 on hamsters gained weight increasing up to 5% from their initial body weight. A slightly bigger body weight loss was observed lday post infection in hamsters that received J08-MUT at 1 and 0.25 mg/kg. Anyway, hamsters in these groups completely recovered their initial body weight at day 6 and 8 for the 1 and 0.25 mg/kg dosage respectively. Hamsters in the control groups did not recover their initial body weight and at day 8 still show around 5% of weight loss.
Therapeutic in vivo experiments with the monoclonal antibody MAD0004J08 Process: Six- to eight-month-old female Syrian hamsters were purchased from Charles River Laboratories and housed in microisolator units, allowed free access to food and water and cared for under U.S. Department of Agriculture (USD A) guidelines for laboratory animals. For the passive transfer prophylactic experiments, the day after to SARS-CoV-2 infection six hamsters per group were intraperitoneally administered with 500 pL of a 4 mg/kg dose of MAD0004J08 mAh. Another two groups (n=6/each) were administered with 500 pL of 4 mg/kg of the anti -influenza virus #1664 mAh (manuscript in preparation) or PBS only to serve as human IgGl isotype and mock control groups, respectively. The day after, hamsters were anesthetized using 5% isoflurane, and inoculated with 5xl05 PFU of SARS-CoV-2 (2019-nCoV/USA-WAl/2020) via the intranasal route, in a final volume of 100 pL. Baseline body weights were measured before infection as well as monitored daily for 12 days post infection. All experiments with the hamsters were performed in accordance with the NRC Guide for Care and Use of Laboratory Animals, the Animal Welfare act, and the CDC/NIH Biosafety and Microbiological and Biomedical Laboratories as well as the guidelines set by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia who also approved the animal experimental protocol. All animal studies infection with SARS-CoV-2 were conducted in the Animal Health Research Center (AHRC) Biosafety Level 3 (BSL-3) laboratories of the University of Georgia.
Results:
MAD0004J08 was able to treat SARS-CoV-2 infection in golden Syrian hamster when administered at 4 mg/kg. We designed a therapeutic study in golden Syrian hamster to evaluate the efficacy of our monoclonal antibody named MAD0004J08 in preventing SARS- CoV-2 infection. For this study 20 hamsters were divided into 3 arms (six animals each). The monoclonal antibody MAD0004J08 was administered at one concentration (4 mg/kg) via intraperitoneal injection. Placebo and IgGl isotype control groups were included in the study which received a saline solution and an anti-influenza antibody at the concentration of 4 mg/kg respectively. Twenty-four hours prior-administration of the antibody or saline solution, animals were challenged with 100 pL of SARS-CoV-2 solution (5xl05 PFU) via intranasal distillation. Three hamsters per group were sacrificed at three days post-infection while the remaining animals were culled at day 12. Body weight changes were daily evaluated throughout the study and reported in Fig.13 A. We observed that MAD000J08 significantly reduced weight loss at tested concentration compared to both the placebo and the IgGl isotype control groups. These results, analyzed through a Two-Way ANOVA, allowed us to plan a second therapeutic experiment to evaluate a dose-response range and the therapeutic efficacy of MAD0004J08 12h post-infection.
The SARS-CoV-2 golden Syrian hamster infection model was previously shown to be a suitable small animal model to support the development of prophylactic and therapeutic tools against Covid-19. Indeed, several characteristics shared between SARS-CoV-2 infected human and hamster lungs, such as severe, bilateral, peripherally distributed, multilobular ground glass opacity, and regions of lung consolidation (DOI:10.1038/s41586-020-2342-5; DOI: 10.1073/pnas.2009799117). Based on the observed efficacy, where 0.25 pg/mL (i.e. 50 pg/animal) of J08-MUT was sufficient to prevent SARS-CoV-2 infection, and the average size of a golden Syrian hamster (approx.150 - 200 g), it’s possible to estimate that 250 pg/Kg will be an adequate dosage for protection and treatment of SARS-CoV-2 infection. If we evaluate 70 kg as average size of an adult human, we can estimate that 17.5 mg would be already sufficient to mediate protection or for treatment of the infection. Therefore, a proposed dosage of 100 mg or 400 mg for clinical studies will result in 6 and 22 times respectively more antibody than the minimum dosage needed. Example of Composition of MAD0004J08 drug product: The drug product MAD0004J08 consists of 2.5 ml of a sterile filtered solution containing 40 mg/mL of human monoclonal antibody (humAb) in single-use glass vials. The composition of the drug product is provided in Table 1. The solution is filled in 2R-3 mL glass vials and closed with a 13 mm flurotec rubber stopper. The drug product for early clinical trials was manufactured by Istituto Biochimico Italiani Lorenzini according to cGMP standards.
Table 1 Composition of MAD0004J08 DP C) 20m M Phosphate buffered solution pH 7.0
No excipients are added to the DS solution to obtain the final formulation. All the ingredients listed in the previous table come from the DS solution.
Further properties of the Antibody MAD0004J08 (Abbreviated also as J08)
MATERIAL & METHODS
SARS-CoV-2 authentic virus neutralization assay
All SARS-CoV-2 authentic virus neutralization assays were performed in the biosafety level 3 (BSL3) laboratories at Toscana Life Sciences in Siena (Italy), Vismederi Sri, Siena (Italy) and Institute Pasteur, Paris (France). BSL3 laboratories are approved by a Certified Biosafety Professional and are inspected every year by local authorities. Two different approaches were used to evaluate the neutralization activity of J08 against SARS-CoV-2 and emerging variants and evaluate the breath of neutralization of this antibody. The first method is a cytopathic effect (CPE)-based neutralization assay described by Andreano and colleagues (Andreano et al. Cell 2021 , 184:1821-1835) while the second method is a S-fuse neutralization assay previously described by Planas et al. Nature Medicine 2021 , 27:917-9242. Briefly, the CPE-based neutralization assay sees the co-incubation of J08 with a SARS-CoV-2 viral solution containing 100 TCID50 of virus and after 1 hour incubation at 37°C, 5% CO2. The mixture was then added to the wells of a 96-well plate containing a sub-confluent Vero E6 cell monolayer. Plates were incubated for 3 days at 37°C in a humidified environment with 5% CO2, then examined for CPE by means of an inverted optical microscope. J08 was tested at a starting concentration of 1 pg/rnL and diluted step 1 :2 for twelve points. As for the S-fuse neutralization assay, U20S-ACE2 GFP1-10 orGFP 11 cells, also termed S-Fuse cells, become GFP+ cells when they are productively infected with SARS-CoV-2. The virus was incubated with J08 at a starting concentration of 5 pg/rnL and diluted step 1 :5 for eight points. Then, 18 h later, cells were fixed with 2% paraformaldehyde, washed and stained with Hoechst (1 :1 ,000 dilution; Invitrogen). Images were acquired with an Opera Phenix high-content confocal microscope (PerkinElmer). The percentage of neutralization was calculated using the number of syncytia as the value with the following formula: 100 c (1 - (value with monoclonal antibody - value in ‘noninfected’)/(value in ‘no monoclonal antibody’ - value in ‘noninfected’)). Using the CPE-based assay, the following SARS- CoV-2 viruses were tested: wild type (WT), B.1.1.7 (Alpha; UK), B.1.1.248 (Beta; BZ), B.1.351 (Gamma; SA) and B.1.617 (Delta; IN). By using the S-fuse neutralization assay the following SARS- CoV-2 viruses were tested: D614G, B.1.1.7 (Alpha; UK), B.1.1.248 (Beta; BZ), B.1.351 (Gamma; SA) and B.1.617 (Delta; IN).
Viral escape assay using authentic SARS-CoV-2
All SARS-CoV-2 authentic virus procedures were performed in the biosafety level 3 (BSL3) laboratories at Toscana Life Sciences in Siena (Italy) and Vismederi Sri, Siena (Italy). BSL3 laboratories are approved by a Certified Biosafety Professional and are inspected every year by local authorities. To detect neutralization-resistant SARS-CoV-2 escape variants, a standard concentration of the virus was sequentially passaged in cell cultures in the presence of serially diluted samples containing SARS-CoV-2-specific antibodies. This experimental procedure was previously described by Andreano et al SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma. bioRxiv. Briefly, 12 serial 2-fold dilutions of J08 at a starting concentration of 10 pg/mL were prepared in complete DMEM 2% FBS and added to the wells of one 24-well plate. Virus solution containing 105 TCID50 of authentic SARS-CoV-2 was dispensed in each antibody- containing well and the plates were incubated for 1 hour at 37°C, 5% C02. The mixture was then added to the wells of a 24-well plate containing a sub-confluent Vero E6 cell monolayer. Plates were incubated for 5-7 days at 37°C, 5% C02 and examined for the presence of CPE using an inverted optical microscope. A virus-only control and a cell-only control were included in each plate to assist in distinguishing absence or presence of CPE. At each virus passage, the content of the well corresponding to the lowest sample dilution that showed complete CPE was diluted 1 :100 and transferred to the antibody-containing wells of the pre-dilution 24-well plate prepared for the subsequent virus passage. At each passage, both the virus pressured with J08 and the virus-only control were harvested, propagated in 25cm2 flasks and aliquoted at -80°C to be used for RNA extraction, RT-PCR and sequencing.
Cell stimulation and pro-inflammatory cytokines detection hPBMC were pre-incubated for 1 hour at 37°C with live SARS-CoV-2 at multiplicity of infection (MOI)=0.1 and then cultured at 2x106 cells/ml in RPMI 1640 in presence of P/S (100 U/mL), L- glutamine (2mM) and 10% FBS for 24 or 48 hours. Cells were treated with R848, a specific TLR7/8 agonist (5mM, Invivogen) as positive control, or with CpG 2395 (3pg/mL, Invivogen) a specific TLR9 agonist as positive control. After 24 or 48 hours, cell culture supernatants were harvested and treated for 30 minutes at 56°C, then stored at -80°C for later use. SARS-CoV2 inactivation was tested by back titration for each experiment. Release of IFN-a in the supernatant was measured by a specific ELISA kit (PBL assay science). Production of IL-6 in the supernatant was quantified by specific cytometric bead arrays (BD Biosciences) on a FACS Canto (BD Biosciences) and analyzed by FCAP array software (BD Biosciences).
RESULTS WITH J08
Neutralization activity of J08 against SARS-CoV-2 wild type and emerging variants
The rise of SARS-CoV-2 emerging variants, also defined as variants of concern (VoC), are endangering the development of monoclonal antibodies (mAbs) for the treatment and prevention of COVID-19. The variants most in the spotlight are B.1.1.7 (isolated in the United Kingdom), B.1.351 (isolated in South Africa), B.1.1.248 (isolated in Brazil) and B.1.617 (isolated in India) now re-named by the World Health Organization (WHO) as alpha, beta, gamma and delta variants respectively. These SARS-CoV-2 variants have also been listed as variants of concern (VoC). To evaluate the effectiveness of J08 against the abovementioned VoCs, a CPE-based microneutralization assay and a S-2 fuse neutralization assay were performed. The CPE-based assay showed that J08 was able to maintain its extremely high neutralization activity against all tested VoCs with a 100% inhibitory concentration (ICioo) of 3.9, 3.9, and 4.9 and 9.7 ng/mL for the WT SARS-CoV-2 virus, the alpha (UK), beta (BZ) and gamma (SA) VoC respectively (Figure 15A - D). A similar scenario was observed with the S-fusion neutralization assay, where J08 was able to neutralize all the VoCs tested. Given the extreme neutralization potency of J08 we were not able to define a 50% inhibitory concentration (IC50) against the D614G, alpha (UK) and gamma (IN) variants and therefore we marked it as <1 ng/mL. On the other hand, it was possible to define the neutralization potency of J08 against the BZ and SA variants which showed an IC50 of around 8 and 40 ng/mL respectively. These data show that J08 is a pan-SARS-CoV-2 variants of concern human monoclonal antibody and it is able to maintain an extreme neutralization potence against all the VoCs currently emerged (Figure 16). J08 is able to bind the RBD in all conformational states due to a unique footprint
To better understand the reasons underlying the ability of J08 to neutralize all SARS-CoV-2 VoCs, cryogenic electron microscopy (Cryo-EM) was applied and the epitope recognize by this antibody was unraveled. Structural analyses revealed that J08 was able to bind the receptor binding domain (RBD) of the spike protein both in its up and down state (Figure 17A). Specifically, J08 is able to bind the RBD in its down tight state (state 1) by using residues S30 and Y32 in the light chain complementary determining region 1 (CDRL1) which interact with the RBD residues S477 and N487 respectively, and residues R50 in the heavy chain CDR2 (CDRH2) and Y100 in the CDRH3 which interact with residues Y489 and K417 respectively (Figure 17B - C). When the RBD is in its down- loose state (state 2), J08 is able to interact with RBDs in two juxtaposed protomers. In this scenario, J08 uses the CDRH2 residues R50, L54, R56 and M58 to interact with F486, F490 and Q493 on the RBD-1 protomer, while it uses the framework 1 (FW1) residue G26, the CDRH1 residue Y32 and the CDRH3 residue A96 to interact with residues T500, P499 and N440 respectively on the RBD-2 protomer (Figure 17B and D). Finally, J08 interacts with the RBD in its up conformation (state 3), using the CDRH2 residues D55, L54 and R56 which make contact with Y489, Q493, V483 and E484 respectively (Figure 17B and E). In addition, J08 epitope was compared with other two antibodies, named S2E12 and CV07-250, that recognize a similar region on the RBD (Figure 18A). As shown in Figure 18B, despite these three antibodies recognize the same external loop on the top of the RBD, J08 shows the smallest footprint (dark blue on the RBD shown in gray) among them all (Figure 18B). Despite this small footprint, J08 epitope still overlaps with the footprint of the angiotensin converting- enzyme 2 (ACE2), therefore interfering with the RBD/ACE2 interaction which is the onset of viral entry into the host cells.
Evolution of SARS-CoV-2 J08 escape mutant
To understand which mutations on the RBD can impairthe binding of J08 and evade its neutralization activity, we pressured SARS-COV-2 in vitro by co-incubating J08 with original Wuhan. Two-fold dilutions of purified J08 ranging from 10 to 0.004 pg/mL were co-incubated with 105 TCID50 of SARS- CoV-2 in a 24-well plate. This viral titer was approximately three logs more than what is conventionally used in microneutralization assays. The J08/virus mixture was co-incubated for 5-8 days. Then, the first well showing cytopathic effect (CPE) was diluted 1 :100 and incubated again with serial dilutions of J08 (Figure 19). For 6 passages and 38 days J08 neutralized the virus with a titer of 78.1 ng/mL and did not show any sign of escape. Flowever, after 7 passages and 45 days, the neutralizing titer dropped by 2-fold and J08 showed an IC100 of 156.2 ng/mL. Sequence analyses revealed a mutation of the glutamic acid in position 484 (E484) on the S-protein RBD in 17% of the virions (Figure 19; Table 1). Following this initial breakthrough, in the subsequent passage (P8), this mutation was observed in 100% of the sequenced virions which also introduced a nine residues deletion in the NTD N2 loop (68IHVSGTNGT76) an additional 4-fold decrease in neutralization activity was observed reaching an overall neutralization titer of 625.0 ng/mL. A third mutation occurred after 9 passages and 59 days of J08/virus co-incubation (P9). This time, the glutamine in position 493 of the RBD was substituted with a histidine (Q493H). This mutation occurred in 74%% of sequenced virions and led to a 8-fold decrease in neutralization activity and J08 reached an IC100 of 5000 ng/mL (Figure 19; Table 1). The Q493H substitution was rapidly followed by a fourth and final change comprising a third mutation in the RBD were the asparagine in position 487 was substituted with a lysine (N487K). With the appearance of this latter mutation, J08 lost completely its neutralization activity.
The evaluation of the impact of spike protein mutations of J08 escape mutant To better understand the impact of the mutations appeared on the authentic SARS-CoV-2 virus J08 escape mutant, a lentiviral pseudotype platform was implemented. Different constructs of lentiviral pseudotype were produced to include in the spike protein of the original Wuhan virus the same mutations observed in the J08 escape mutant (E484D, E484D + Del 68-76 NTD, E484D + Del 68-76 NTD + Q493H, E484D + Del 68-76 NTD + Q493H + N487K), single mutations in the RBD and NTD (E484D, Q493H, N487K, Del 68-76 NTD), or their combination (E484D + Q493H). Initially we assessed the evolution of J08 escape mutant using the lentiviral pseudotype platform (Figure 18). Here J08 showed a 50% neutralization dose (ND50) against the WT-pseudotype of 21.6 ng/mL and a 2.2-fold decrease (ND5o=47.7 ng/mL) in neutralization activity was observed when the E484D substitution was introduced in the spike protein. In contrast with what observed with the evolution of the authentic virus, the introduction of the Del 68-76 NTD led only to an additional 1.1-fold decrease in the neutralization activity of J08 (ND5o=50.9 ng/mL). Anyway, when we introduced the Q493H substitution in the RBD (E484D + Del 68-76 NTD + Q493H) we observed a drastic loss of activity by J08 with the ND50 dropping by 11-fold (ND5o=562.0 ng/mL) compared to the previous mutation. Finally, when the N487K substitution was introduced in addition to the previous mutations (E484D + Del 68-76 NTD + Q493H + N487K) the pseudotype was able to escape from J08 neutralization. We then evaluated the effect of single RBD or NTD mutations on J08 (Figure 20). Interestingly, we observed that E484D, Del 68-76 NTD and Q493FI alone reduced only by 2.2- (ND5o=47.7 ng/mL), 1.3- (ND5O=28.0 ng/mL) and 1.9-fold (ND5o=42.7 ng/mL) respectively the neutralization activity of J08, while N487K alone showed to be sufficient to escape from J08. In addition, we wanted to investigate whether the Del 68-76 in the NTD, in addition to the RBD substitutions E484D and Q493H, was necessary to drastically reduce the neutralization activity of J08 as observed with the authentic SARS-CoV-2 virus. Therefore, a final pseudotype construct presenting only the two RBD mutations E484D and Q493H was produced and tested in neutralization against J08. Interestingly, we observed that the Del 68-76 in the NTD was not essential to reduce the neutralization activity of J08 as E484D + Q493H alone led to an 18-fold reduction of J08 neutralization potency (ND5o=392.0 ng/mL). Finally, we interrogated the global initiative on sharing all influenza data (GISAID) database (which includes over >1 ,4 M complete and high coverage SARS-CoV-2 genomes) to understand how common are the RBD mutations evolved by the escape mutant of J08 compared to the RBD mutations emerged in nature. We observed that all three mutations in the RBD are extremely rare as E484D was identified 25 times (0.0017%), Q493H 36 times (0.0025%) and N487K was seen only 1 time (0.00007%).
Fc-engineering J08 does not induce the production of pro-inflammatory cytokines Recent data (unpublished) showed that cells of the immune system from human peripheral blood mononuclear cells (hPBMC) can sense SARS-CoV-2 leading to activation of innate response and production of pro-inflammatory cytokines such as Interferon a (IFN-a) and Interleukin 6 (IL-6). Therefore, to better understand if the mutations carried by Fc-mutated J08 (J08-MUT) are able to abrogate cellular activation and consequent production of pro-inflammatory cytokines, J08, in its WT and MUT versions, was co-incubated with hPBMC in presence of SARS-CoV-2. To assess the production of pro-inflammatory cytokines from hPBMC, cells were co-incubated with the SARS-CoV- 2 virus in presence of J08-WT and MUT used at three different concentrations, the neutralization dose (N), 2-times more the neutralization dose (2N) or 10-times less the neutralization dose (N/10). CpG 2395 and R848 were used as positive and negative control respectively for toll-like receptor 9 (TLR9) activation and IFN-a production. As shown in Figure 21A, CpG 2395 was able to properly activate hPBMC and produce IFN-a while R848 did not stimulate the cells. In addition, J08-MUT did not induced the production of IFN-a in presence of SARS-CoV-2 while J08-WT produced higher level than the positive control in a dose-response fashion (Figure 21A). We also evaluated the production of IL-6 using the same methodology described above. Also, in this case, J08-MUT did not induce IL- 6 production which is even lower than the virus alone control (-). On the other hand, J08-WT induced up to 3-fold increase of IL-6 production compared to hPBMC/SARS-CoV-2 alone (Figure 21 B). These data show that J08-MUT does not induce cellular activation and pro-inflamamtory production in presence during SARS-CoV-2 infection.
Experimental results show the following main aspects of the antibody J08:
1. J08 is a pan-SARS-CoV-2 variant neutralizing human monoclonal antibody with an extreme neutralization potency against all variants of concern (UK, BZ, SA and IN).
2. J08 shows a unique modality of binding to the RBD as its small epitope footprint allows the binding to all RBD states (up and down).
3. Extremely rare mutations on the SARS-CoV-2 spike protein RBD are needed to evade J08 highlighting that our antibody targets an epitope rarely recognized by the polyclonal response elicited following COVID-19 infection.
4. Fc-mutated J08 (J08-MUT) does not induce the production of pro-inflammatory cytokines by human PBMC in presence of SARS-CoV-2.
TABLE
Table S1. SARS-CoV-2 escape mutant summary. The table shows the starting dilution, passages, viral titer and neutralization activity of J08 per each passage to generate the authentic virus SARS- CoV-2 escape mutant. mAb ID SARS-CoV-2 WT SARS-CoV-2 WT IC100 (ng/mL) IC50 (ng/mL) Reference
J08 3.9 Andreano et al. , Cell, 2021
REGN10987 20.4 Hansen et al., Science, 2020
REGN10933 74.9 Hansen et al., Science, 2020
COV2-2196 15.0 Zost et al., Nature, 2020
COV2-2130 107.0 Zost et al., Nature, 2020
BD-503 140.0 Cao et al., Cell, 2020
BD-508 290.0 Cao et al., Cell, 2020
BD-515 200.0 Cao et al., Cell, 2020
Sequence Listing in the description
Sequence of the antibody herein identified as MAD0004J08 AMINO ACID SEQUENCES OF MAD0004J08:
>SEQ ID NO: l CDR 1 of variable domain of Heavy chain of MAD0004J08 GGTFSSYT
>SEQ ID NO:2 CDR 2 of variable domain of Heavy chain of MAD0004J08 IIPILDRV
>SEQ ID NO:3 CDR 3 of variable domain of Heavy chain of MAD0004J08 C ARRAID SDT YVEQ SHFD YW
>SEQ ID NO:4 CDR 1 of variable domain of Light chain of MAD0004J08 QSVSSY
>SEQ ID NO:5 CDR 2 of variable domain of Light chain of MAD0004J08 this sequence is not included in the sequence listing because is less than 4 amino acid DAS (Asp-Ala-Ser)
>SEQ ID NO:6 CDR 3 of variable domain of Light chain of MAD0004J08 CQQPLTF
>SEQ ID NO:7 variable domain of Heavy chain of MAD0004J08
Q VQL VQSGAEVKKPGS S VKVSCKASGGTF S S YTISWVRQ APGQGLEWMGRIIPILD RVM Y AQKF QGR VTIT ADK S T S T A YMEL S SLRSEDT A V Y Y CARR AID SDT YVEQ SH FD YWGQGTL VT VS S AS
>SEQ ID NO:8 variable domain of Light chain of MAD0004J08
EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPSLLIYDASNRAT GIPARF SGSGSGTDFTLTIS SLEPEDF AVYYCQQPLTFGGGTKVEIKRT >SEQ ID NO: 9 Heavy chain of MAD0004J08
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISW
VRQAPGQGLEWMGRIIPILDRVMYAQKFQGRVTITADKSTSTAYMELSSLRSEDT
A VYYCARRAIDSDTYVEQSHFD YWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGT
A ALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S S V VT VP S S SLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQ V SLTCL VKGF YP SDI A VEWE SN GQPENN YKTTPP VLD SDGSFFL Y SKLT VDK SR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO: 10 Light chain of MAD0004J08
GWSCIILFLVATATGVHSEIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPSLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQPLTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GN S QE S VTEQD SKD STYSLSSTLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRG EC*
NUCLEIC ACID SEQUENCES OF MAD0004J08:
> SEQ ID NO: 11 MAD0004J08_heavy chain LeaderSequence
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
> SEQ ID NO: 12 MAD0004J08_heavy chain Variable domain
CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGT
GAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATACTATCAGCT
GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCT
ATCCTTGATAGAGTAATGTACGCACAGAAGTTCCAGGGCAGAGTCACGATTAC
CGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTG
AGGACACGGCCGTGTATTACTGTGCGAGACGGGCCATTGATTCCGACACCTAT
GTTGAACAATCCCACTTTGACTACTGGGGCCAGGGAACCCTTGTCACCGTCTCC
TCAGCCTCC
> SEQ ID NO: 13 MAD0004J08_heavy chain Constant domain
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
> SEQ ID NO: 14 MAD0004J08_heavy chain Complete
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATACTATCA
GCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCAT
CCCTATCCTTGATAGAGTAATGTACGCACAGAAGTTCCAGGGCAGAGTCACGA
TTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGA
TCTGAGGACACGGCCGTGTATTACTGTGCGAGACGGGCCATTGATTCCGACAC
CTATGTTGAACAATCCCACTTTGACTACTGGGGCCAGGGAACCCTTGTCACCGT
CTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA
GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC
CCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC
ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGAC
AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC
AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCC
TGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
T C A AC T GGT AC GT GGAC GGC GT GGAGGT GC AT A AT GC C A AG AC A A AGC C GC G
GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
AC C AGGAC T GGC T G A AT GGC A AGGAGT AC A AGT GC A AGGT C TCC A AC A A AGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCA
GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG
AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT
GCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGA
GCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
> SEQ ID NO: 15 MAD0004J08 light chain LeaderSequence
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
> SEQ ID NO: 16 MAD0004J08_light_chain Variable Domain
GAAATTGTGATGACGCAGTCTCCAGCCACTCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGT
ACCAACAGAAACCTGGCCAGGCTCCCAGCCTCCTCATCTATGATGCCTCCAAC
AGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCA
GCAGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACT
> SEQ ID NO: 17 MAD0004J08_light_chain ConstantRegion
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
> SEQ ID NO: 18 MAD0004J08_light_kappa_complete_seq
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAAATTGTGATGACGCAGTCTCCAGCCACTCTGTCTTTGTCTCCAGGGGAA
AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTG
GTACCAACAGAAACCTGGCCAGGCTCCCAGCCTCCTCATCTATGATGCCTCCA
AC AGGGCC ACTGGC ATCCC AGCC AGGTT C AGT GGC AGT GGGTCTGGGAC AGAC
TTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT
C AGC AGCCTCTC ACTTTCGGCGGAGGGACC AAGGTGGAGAT C A AACGAACTGT GGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGG
AACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT
ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT
GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT
CAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
Sequence of the antibody herein identified as MAD0100I14 AMINO ACID SEQUENCES OF MAD0100I14:
>SEQ ID NO: 19 CDR 1 of variable domain of Heavy chain of MAD0100I14 GFTFTSSA
>SEQ ID NO:20 CDR 2 of variable domain of Heavy chain of MAD0100I14 IVVGSGNT
>SEQ ID NO:21 CDR 3 of variable domain of Heavy chain of MAD0100I14 CAAPYCSSTTCHDGFDIW
>SEQ ID NO:22 CDR 1 of variable domain of Light chain of MAD0100I14 QSVSSSY
>SEQ ID NO:23 CDR 2 of variable domain of Light chain of MAD0100I14 this sequence is not included in the sequence listing because is less than 4 amino acid GAS (Gly-Ala-Ser)
>SEQ ID NO:24 CDR 3 of variable domain of Light chain of MAD0100I14 CQQYGRSPWTF
>SEQ ID NO:25 variable domain of Heavy chain of MAD0100I14
VQL VQ S GPE VKKP GT S VK V SCKASGFTFTSS AMQ W VRQ ARGQRLE WIGWI V V GS
GNTDYVQKFQGRVTITRDMSTSTAYMELSSLRSEDTAVYYCAAPYCSSTTCHDGF
DIWGQGTMVT VS S AS
>SEQ ID NO:26 variable domain of Light chain of MAD0100I14 EIVMTQSPGTLSLAPGERATLSCRASQSVSSSYLGWYQQKPGQAPRLLIYGASSRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGRSPWTFGQGTKVEIKRT >SEQ ID NO:27 Heavy chain of MAD0100I14
MGW S CIILFL V AT AT GVHS Q V QL V Q S GPE VKKPGT S VK V S CK AS GF TF T S S AMQ W VRQ ARGQRLEWIGWI V V GS GNTD Y V QKF Q GRVTITRDM S T S T A YMEL S SLRSEDT AVYYCAAPYCSSTTCUDGFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKD YFPEP VT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTC VVVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO:28 Light chain of MAD0100I14
GWSCIILFLVATATGVHSEIVMTQSPGTLSLAPGERATLSCRASQSVSSSYLGWYQ QKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQ Y GRS PWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTK SFNRGEC *
NUCLEIC ACID SEQUENCES OF MAD0100I14:
> SEQ ID NO:29 MADOl 00114_heavy chain LeaderSequence
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
> SEQ ID NO:30 MADOl 00114_heavy chain Variable domain
CAGGTGCAGCTGGTGCAGTCTGGGCCTGAGGTGAAGAAGCCTGGGACCTCAGT
GAAGGTCTCCTGCAAGGCTTCTGGATTCACCTTTACTAGCTCTGCTATGCAGTG
GGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGATGGATCGTCGTTG
GC AGTGGT AAC AC AGACT ACGT GC AGAAGTTCC AAGGAAGAGT C ACC ATT ACC
AGGGACATGTCCACAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCCG
AGGACACGGCCGTGTATTACTGTGCGGCACCATATTGTAGTAGTACCACCTGC
CATGATGGCTTTGATATTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGCC
TCC
> SEQ ID NO:31 MADOl 00114_heavy chain Constant domain
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
> SEQ ID NO:32 MAD0100I14_heavy chain Complete
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGGTGCAGTCTGGGCCTGAGGTGAAGAAGCCTGGGACCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGATTCACCTTTACTAGCTCTGCTATGCA
GT GGGT GC GAC AGGC TCGT GGAC A AC GCC TT GAGT GGAT AGGAT GGAT C GTCG
TTGGCAGTGGTAACACAGACTACGTGCAGAAGTTCCAAGGAAGAGTCACCATT
ACCAGGGACATGTCCACAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATC
CGAGGACACGGCCGTGTATTACTGTGCGGCACCATATTGTAGTAGTACCACCT
GCCATGATGGCTTTGATATTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA
GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC
CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC
CTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC
CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCC
CAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT
CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT
CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA
GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG
ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAC
AGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGC
CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA
GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG
GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
> SEQ ID NO:33 MAD0100I14 light chain LeaderSequence
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
> SEQ ID NO:34 MAD0100I14_light_chain Variable Domain
GAAATTGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGGCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGGCT
GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC
AGC AGGGCC ACTGGC ATCCC AGAC AGGTT C AGT GGC AGT GGGTCTGGGAC AG
ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT
GTCAGCAGTATGGTAGGTCACCGTGGACGTTCGGCCAAGGGACCAAGGTGGA
AATCAAACGAACT
> SEQ ID NO:35 MAD0100I14_light_chain ConstantRegion
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
> SEQ ID NO:36 MAD0100I14_light_kappa_complete_seq
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAAATTGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGGCTCCAGGGGA A AGAGC C AC CC T C TCC T GC AGGGC C AGT C AGAGT GTT AGC AGC AGC T AC TT AG
GCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCC AGC AGGGCC ACTGGC ATCCC AGAC AGGTT C AGT GGC AGT GGGTCTGGGAC
AGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA
C T GT C AGC AGT AT GGT AGGT C AC CGT GGAC GTTCGGC C A AGGGAC C A AGGT GG
AAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATG
AGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC
CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA
CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC
AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG
CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC
AGGGGAGAGTGTTAG
Sequence of the antibody herein identified as MAD0102F05 AMINO ACID SEQUENCES OF MAD0102F05:
>SEQ ID NO:37 CDR 1 of variable domain of Heavy chain of MAD0102F05 GFTVSINY
>SEQ ID NO:38 CDR 2 of variable domain of Heavy chain of MAD0102F05 IYSGGST
>SEQ ID NO:39 CDR 3 of variable domain of Heavy chain of MAD0102F05 CAAPLLWADSYYMDVW
>SEQ ID NO:40 CDR 1 of variable domain of Light chain of MAD0102F05 QDIRNN
>SEQ ID NO:41 CDR 2 of variable domain of Light chain of MAD0102F05 this sequence is not included in the sequence listing because is less than 4 amino acid AAS (Ala-Ala-Ser)
>SEQ ID NO:42 CDR 3 of variable domain of Light chain of MAD0102F05 CLQHN S YLWTF
>SEQ ID NO:43 variable domain of Heavy chain of MAD0102F05 EVQLVESGGGLVQPGGSLRLSCAASGFTVSINYMSWVRQAPGKGLEWVSVIYSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAPLLWADSYYMDV WGKGTTVTVSSAS
>SEQ ID NO:44 variable domain of Light chain of MAD0102F05 AIQMTQSPSSLSASVGDRVTITCRASQDIRNNLGWFQQKPGKAPKRLIYAASTLQR GVPSRFSGSGSGTEFTLTIS SLQPEDF ATYY CLQHN S YLWTF GQGTKVEIKRT >SEQ ID NO:45 Heavy chain of MAD0102F05
MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTVSINYMSW VRQ APGKGLEWV S VIYSGGSTYYADS VKGRFTISRDN SKNTL YLQMN SLRAEDTA VYYCAAPLLWADSYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT Y ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVT C VVVD V SHEDPEVKFNW YVDGVEVHNAKTKPREEQYNST YRVV S VLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVF S C S VMHE ALHNH YT QK SL SL SPGK *
>SEQ ID NO:46 Light chain of MAD0102F05
MGW SCIILFL VAT AT GVHS AIQMT Q SP S SLS AS VGDRVTIT CRASQDIRNNLGWF Q QKPGKAPKRLIYAASTLQRGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSY LWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTK SFNRGEC *
NUCLEIC ACID SEQUENCES OF MAD0102F05:
> SEQ ID NO:47 MAD0102F05_heavy chain LeaderSequence
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
> SEQ ID NO:48 MAD0102F05_heavy chain Variable domain
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTATCAACTACATGAGTTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCG
GTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATTCACCATCTCCAGA
GACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGA
CACGGCTGTGTATTACTGTGCGGCCCCCTTACTATGGGCCGACTCCTACTACAT
GGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGCCTCC
> SEQ ID NO:49 MAD0102F05_heavy chain Constant domain ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
> SEQ ID NO:50 MAD0102F05_heavy chain Complete
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTATCAACTACATGAG
TTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATA
GCGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATTCACCATCTCC
AGAGAC AATTCC AAGAAC ACGCTGT ATCTTC AAAT GAAC AGCCTGAGAGCCGA
GGAC ACGGCTGTGT ATT ACTGTGCGGCCCCCTTACTATGGGCCGACTCCTACTA
CATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCA
AGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGT
CTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC
TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCC
ACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC
AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
T GGT GG AC GT G AGC C AC G A AG AC C C T G AGGT C A AGT T C A AC T GGT AC GT GG AC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA
GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
CTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGT C AAAGGCTTCT ATCCC AGCGAC ATCGCCGT GGAGTGGGAGAGC AAT GGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAA
CGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA
AGAGCCTCTCCCTGTCCCCGGGTAAATGA
> SEQ ID NO:51 MAD0102F05 light chain LeaderSequence
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
> SEQ ID NO:52 MAD0102F05_light_chain Variable Domain
GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGA
GTCACCATCACTTGCCGGGCAAGTCAGGACATTAGAAATAATTTAGGCTGGTT
TCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCACTT
TACAACGTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTC
ACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTA
CAGCATAATAGTTACCTTTGGACATTCGGCCAAGGGACCAAGGTGGAAATCAA
ACGAACT
> SEQ ID NO:53 MAD0102F05_light_chain ConstantRegion
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
> SEQ ID NO:54 MAD0102F05_light_kappa_complete_seq
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGAAATAATTTAGGCTG
GTTTCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCA
CTTTACAACGTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAA
TTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGT
CTACAGCATAATAGTTACCTTTGGACATTCGGCCAAGGGACCAAGGTGGAAAT
CAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA
GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG
AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC
AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG
CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC
GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG
AGAGTGTTAG
Sequence of the antibody herein identified as MAD0041G12 AMINO ACID SEQUENCES OF MAD0041G12:
>SEQ ID NO: 55 CDR 1 of variable domain of Heavy chain of MAD0041G12 GGTFSSYT
>SEQ ID NO:56 CDR 2 of variable domain of Heavy chain of MAD0041G12 IIPMLGIA
>SEQ ID NO:57 CDR 3 of variable domain of Heavy chain of MAD0041G12 C ARGIV GATPGYFD YW
>SEQ ID NO:58 CDR 1 of variable domain of Light chain of MAD0041G12 QSVSSN
>SEQ ID NO:59 CDR 2 of variable domain of Light chain of MAD0041G12 this sequence is not included in the sequence listing because is less than 4 amino acid
GAS
>SEQ ID NO:60 CDR 3 of variable domain of Light chain of MAD0041G12 CQQYNNWLTF >SEQ ID NO:61 variable domain of Heavy chain of MAD0041G12 Q VQL VQSGAEVKKPGPS VKVSCKASGGTF S S YTINWVRQAPGQGLEWMGRIIPML GIAK Y AQKF QGR VTIT ADK S T S T A YMEL S SLRSEDT A V Y Y C ARGI V GATPGYFD Y WGQGTLVTVSSAS
>SEQ ID NO: 62 variable domain of Light chain of MAD0041G12 DIVMT Q SP AALS V SPGERATL SCRASQ S V S SNL AW Y QQKPGQ APRLLI Y GASTRAT GIP ARF SGSW SGTEFTLTIS SLQSEDLAVYYCQQYNNWLTF GGGTKVEIKRT >SEQ ID NO: 63 Heavy chain of MAD0041G12
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGPSVKVSCKASGGTFSSYTINW VRQAPGQGLEWMGRIIPMLGIAKYAQKFQGRVTITADKSTSTAYMELSSLRSEDT AVYY C ARGIV GATPGYFD YW GQGTL VT V S S ASTKGP S VFPL AP S SKST SGGT AAL GCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT Y ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVT C VVVD V SHEDPEVKFNW YVDGVEVHNAKTKPREEQYNST YRVV S VLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVF S C S VMHE ALHNH YT QK SL SL SPGK *
>SEQ ID NO: 64 Light chain of MAD0041G12
MGW SCIILFL VAT AT GVHSDIVMT Q SP AAL S V SPGERATL SCRASQ SV S SNL AW Y Q QKPGQAPRLLIYGASTRATGIPARFSGSWSGTEFTLTISSLQSEDLAVYYCQQYNN WLTF GGGTKVEIKRT VAAP SVFIFPP SDEQLKSGT AS VV CLLNNF YPREAK V QWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTK SFNRGEC *
NUCLEIC ACID SEQUENCES OF MAD0041G12:
>SEQ ID NO: 65 leader sequence of Heavy chain of MAD0041G12
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 66 variable domain of Heavy chain of MAD0041G12
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGCCCTCGGT
GAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATACTATCAACT
GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCT
ATGCTTGGTATAGCAAAGTACGCACAGAAGTTTCAGGGCAGAGTCACGATTAC CGCGGACAAATCCACGAGCACAGCCTACATGGAGTTGAGTAGTCTGAGATCTG
AGGACACGGCCGTCTATTACTGTGCGAGAGGTATAGTGGGAGCTACTCCGGGG
TACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCC
>SEQ ID NO: 67 constant domain of Heavy chain of MAD0041G12
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 68 complete sequence of Heavy chain of MAD0041G12
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGCCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATACTATCA
ACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCAT
CCCTATGCTTGGTATAGCAAAGTACGCACAGAAGTTTCAGGGCAGAGTCACGA
TTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGTTGAGTAGTCTGAGA
TCTGAGGACACGGCCGTCTATTACTGTGCGAGAGGTATAGTGGGAGCTACTCC
GGGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTC CACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 69 leader sequence of Light chain of MAD0041G12
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:70 variable domain of Light chain of MAD0041G12
GACATCGTGATGACCCAGTCTCCAGCCGCCCTGTCTGTGTCTCCCGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGT
ACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACC
AGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTTGGTCTGGGACAGAGTT
CACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTAGCAGTTTATTACTGTCA
GCAGTATAATAACTGGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC
GAACT
>SEQ ID NO:71 constant domain of Light chain of MAD0041G12 GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO: 72 complete sequence of Light chain of MAD0041G12
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGACATCGTGATGACCCAGTCTCCAGCCGCCCTGTCTGTGTCTCCCGGGGAA
AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTG
GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCA
CCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTTGGTCTGGGACAGAG
TTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTAGCAGTTTATTACTGT
CAGCAGTATAATAACTGGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAA
ACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT
GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA
GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG
AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC
CCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA
GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA
GTGTTAG
Sequence of the antibody herein identified as MAD0041I21 AMINO ACID SEQUENCES OF MAD0041I21 :
>SEQ ID NO: 73 CDR 1 of variable domain of Heavy chain of MAD0041I21 GFTFSSYG
>SEQ ID NO: 74 CDR 2 of variable domain of Heavy chain of MAD0041I21 ISYDGSNK
>SEQ ID NO: 75 CDR 3 of variable domain of Heavy chain of MAD0041I21 C ATGT YDFW SDNHYLDYW
>SEQ ID NO:76 CDR 1 of variable domain of Light chain of MAD0041I21 QGISSS >SEQ ID NO:77 CDR 2 of variable domain of Light chain of MAD0041I21 this sequence is not included in the sequence listing because is less than 4 amino acid
AAS
>SEQ ID NO:78 CDR 3 of variable domain of Light chain of MAD0041I21 CQQLNSYPSTTF
>SEQ ID NO:79 variable domain of Heavy chain of MAD0041I21 EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYD GSNKYY AD S VKGRFTISRDN SKNTL SLQMN SLRAADT AVYY C ATGTYDF W SDNH YLD YWGQGTL VT VS S AS
>SEQ ID NO: 80 variable domain of Light chain of MAD0041I21 AIQMTQSPSFLSASVGDRVTITCRASQGISSSLAWYQQKPGKAPKLLIYAASTLQSG VPSRFSGSGS GTEF TLTIS SLQPEDF AT Y Y C QQLN S YP S TTF GGGTK VEIKRT >SEQ ID NO: 81 Heavy chain of MAD0041I21
MGW S CIILFL V AT AT GVHSE V QL VE S GGGV V QPGRSLRL S C A AS GF TF S S Y GMHW VRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLSLQMNSLRAAD TAVYYCATGTYDFWSDNHYLD YWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGT A ALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S S V VT VP S S SLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQ V SLTCL VKGF YP SDI AVEWE SN GQPENN YKTTPP VLD SDGSFFL Y SKLT VDK SR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO: 82 Light chain of MAD0041I21
MGW SCIILFL VAT AT GVHS AIQMT Q SP SFL S AS VGDRVTIT CRASQGIS S SL AW YQQ KPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPS TTF GGGTK VEIKRT V AAPS VFIFPP SDEQLKSGT AS VV CLLNNF YPREAK V QWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC*
NUCLEIC ACID SEQUENCES OF MAD0041I21 :
>SEQ ID NO: 83 leader sequence of Heavy chain of MAD0041I21
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC >SEQ ID NO: 84 variable domain of Heavy chain of MAD0041I21
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCT
GAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTG
GGTCCGCC AGGCTCC AGGC AAGGGGCTGGAGT GGGT GGC AGTT AT ATC AT ATG
ATGGAAGTAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCC
AGAGACAATTCCAAGAACACGCTGTCTCTGCAAATGAACAGCCTGAGAGCTGC
GGACACGGCAGTGTATTACTGTGCGACGGGTACTTACGATTTTTGGAGTGACA
ATCACTACCTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTT
CC
>SEQ ID NO:85 constant domain of Heavy chain of MAD0041I21
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 86 complete sequence of Heavy chain of MAD0041I21
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCA
CTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAT
AT GAT GG A AGT A AT AAAT ATT AT GC AGACTCC GT GA AGGGCC GATT C AC CATC
TCCAGAGACAATTCCAAGAACACGCTGTCTCTGCAAATGAACAGCCTGAGAGC
TGCGGACACGGCAGTGTATTACTGTGCGACGGGTACTTACGATTTTTGGAGTG
ACAATCACTACCTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
GCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC
TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC
TGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGC
CCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCC
AGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTC
ACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC
CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC
ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA
CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG
CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCC
TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTC
CGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGC
AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 87 leader sequence of Light chain of MAD0041I21
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:88 variable domain of Light chain of MAD0041I21 GCCATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGA GTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTCTTTAGCCTGGTAT CAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTT GCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCA
CTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAAC
AGCTTAATAGTTACCCTTCGACCACTTTCGGCGGAGGGACCAAGGTGGAGATC
AAACGAACT
>SEQ ID NO: 89 constant domain of Light chain of MAD0041I21
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO: 90 complete sequence of Light chain of MAD0041I21
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGCCATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTCTTTAGCCTG
GTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCA
CTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAA
TTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGT
CAACAGCTTAATAGTTACCCTTCGACCACTTTCGGCGGAGGGACCAAGGTGGA
GATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC
C AGAGAGGCC AAAGT AC AGT GGAAGGT GGAT AACGCCCTCC AATCGGGT AAC
TCCC AGGAGAGT GT C AC AGAGC AGGAC AGC AAGGAC AGC ACCT AC AGCCTC A
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC
CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA
GGGGAGAGTGTTAG
Sequence of the antibody herein identified as MAD0041K22 AMINO ACID SEQUENCES OF MAD0041K22:
>SEQ ID NO:91 CDR 1 of variable domain of Heavy chain of MAD0041K22 GFTFSAYG
>SEQ ID NO: 92 CDR 2 of variable domain of Heavy chain of MAD0041K22 ISYGGSDK
>SEQ ID NO: 93 CDR 3 of variable domain of Heavy chain of MAD0041K22 CAKDQDDAYYF YYYMD VW
>SEQ ID NO:94 CDR 1 of variable domain of Light chain of MAD0041K22 QSVSSSY
>SEQ ID NO:95 CDR 2 of variable domain of Light chain of MAD0041K22 this sequence is not included in the sequence listing because is less than 4 amino acid
GAS
>SEQ ID NO:96 CDR 3 of variable domain of Light chain of MAD0041K22 CQQYGGPLTF
>SEQ ID NO:97 variable domain of Heavy chain of MAD0041K22 QVQLVQSGGGVVQPGRSLRLSCAASGFTFSAYGMHWVRQAPGKGLEWVAVISY GGSDK YYVD S VKGRFTISRDN SKNTL YLQMN SLRAEDT AM YY C AKDQDD AYYF YYYMD VWGKGT AVT VS S AS
>SEQ ID NO:98 variable domain of Light chain of MAD0041K22 DIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLVYGASSRA TGIPGRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGGPLTFGGGTKVEIKRT >SEQ ID NO:99 Heavy chain of MAD0041K22
MGWSCIILFLVATATGVHSQVQLVQSGGGVVQPGRSLRLSCAASGFTFSAYGMH WVRQAPGKGLEWVAVIS Y GGSDKYYVDS VKGRFTISRDN SKNTL YLQMN SLRAE DTAMYYCAKDQDD AYYF YYYMD VWGKGT AVTVSSASTKGPSVFPLAPSSKSTS GGT A ALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S S LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO: 100 Light chain of MAD0041K22
MGWSCIILFLVATATGVHSDIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY QQKPGQAPRLL VYGAS SRATGIPGRF SGSGSGTDFTLTISRLEPEDF AVYYCQQYG GPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTK SFNRGEC *
NUCLEIC ACID SEQUENCES OF MAD0041K22:
>SEQ ID NO: 101 leader sequence of Heavy chain of MAD0041K22
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 102 variable domain of Heavy chain of MAD0041K22
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCT
GAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGCCTATGGCATGCACTG
GGTCCGCC AGGCTCC AGGC AAGGGGCTGGAGT GGGT GGC AGTT AT ATC AT ATG
GTGGAAGTGATAAATACTATGTAGACTCCGTGAAGGGCCGATTCACCATCTCC
AGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGA
GGACACGGCTATGTATTACTGTGCGAAAGACCAGGACGACGCCTACTACTTCT
ACTACTACATGGACGTCTGGGGCAAAGGGACCGCGGTCACCGTCTCCTCAGCT
TCC
>SEQ ID NO: 103 constant domain of Heavy chain of MAD0041K22
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 104 complete sequence of Heavy chain of MAD0041K22
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
T C C C AGGT GC AGC T GGT GC AGT C T GGGGG AGGC GT GGT C C AGC C T GGG AGGT C
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGCCTATGGCATGCA
CTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAT
AT GGT GG A AGT GAT AAAT ACT AT GT AGACTCC GT GA AGGGCC GATT C AC CATC
TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGC
TGAGGACACGGCTATGTATTACTGTGCGAAAGACCAGGACGACGCCTACTACT
TCTACTACTACATGGACGTCTGGGGCAAAGGGACCGCGGTCACCGTCTCCTCA
GCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC
TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC
TGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGC
CCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCC
AGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTC
ACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC
CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC
ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA
CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG
CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCC
TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTC
CGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGC
AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 105 leader sequence of Light chain of MAD0041K22 ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 106 variable domain of Light chain of MAD0041K22
GACATCGTGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCT
GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCGTCTATGGTGCATCC
AGC AGGGCC ACTGGC ATCCC AGGC AGGTT C AGT GGC AGT GGGTCTGGGAC AG
ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT
GTCAGCAGTATGGTGGCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATC
AAACGAACT
>SEQ ID NO: 107 constant domain of Light chain of MAD0041K22
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO: 108 complete sequence of Light chain of MAD0041K22
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGACATCGTGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA
AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCGTCTATGGTGCAT
CCAGC AGGGCC ACTGGC ATCCC AGGCAGGTTCAGTGGCAGTGGGTCTGGGACA
GACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTAC
TGTCAGCAGTATGGTGGCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGAT
CAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA
GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG
AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC
AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG
CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG
AGAGTGTTAG
Sequence of the antibody herein identified as MAD0041M02 AMINO ACID SEQUENCES OF MAD0041M02:
>SEQ ID NO: 109 CDR 1 of variable domain of Heavy chain of MAD0041M02 GFTFSSYV
>SEQ ID NO: 110 CDR 2 of variable domain of Heavy chain of MAD0041M02 ISYGGSNK
>SEQ ID NO: 111 CDR 3 of variable domain of Heavy chain of MAD0041M02 CARDGGDIVVVPGASTSENYYYYYMDVW
>SEQ ID NO: 112 CDR 1 of variable domain of Light chain of MAD0041M02 QSLLDSDDGNTY
>SEQ ID NO: 113 CDR 2 of variable domain of Light chain of MAD0041M02 this sequence is not included in the sequence listing because is less than 4 amino acid TLS
>SEQ ID NO: 114 CDR 3 of variable domain of Light chain of MAD0041M02 CMQRIEFPRTF
>SEQ ID NO: 115 variable domain of Heavy chain of MAD0041M02
QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYVMHWVRQAPGKGLEWVAVISYG
GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVCYCARDGGDIVVVPG
ASTSENYYYYYMDVWGKGTTVTVSSAS
>SEQ ID NO: 116 variable domain of Light chain of MAD0041M02
DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWCLQKPGQSPQLLIYTL
S YRASGVPDRF SGSGSGTDFTLKISRVEAED VGVYY CMQRIEFPRTF GQGTKVEIK
RT
>SEQ ID NO: 117 Heavy chain of MAD0041M02
MGWSCIILFL VAT ATGVHSQ VQLQESGGGVVQPGRSLRLSC AASGFTF S S YVMHW VRQ APGKGLEW VAVIS Y GGSNKYY AD S VKGRFTISRDN SKNTL YLQMN SLRAED TAVCYCARDGGDIVVVPGASTSENYYYYYMDVWGKGTTVTVSSASTKGPSVFPL AP S SK S T S GGT A ALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S S VVT VP S S SLGTQT YICNVNHKP SNTK VDKRVEPKSCDKTHT CPPCP APELLGGP S V FLFPPKPKDTLMISRTPE VT C VVVD V SHEDPEVKFNW YVDGVEVHNAKTKPREEQ YNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALP APIEKTISKAKGQPREPQ VYT LPP SREEMTKN Q VSLT CL VKGF YP SDI A VEWESN GQPENN YKTTPP VLD SD GSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO: 118 Light chain ofMAD0041M02
MGW SCIILFL VAT AT GVHSDIVMTQTPL SLP VTPGEP ASISCRS SQ SLLD SDDGNT Y LDWCLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQRIEFPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK V Q WK VDN ALQ S GN S QE S VTEQD SKD S T Y SL S S TLTL SK AD YEKHK V Y ACE VTHQ GLSSPVTKSFNRGEC*
NUCLEIC ACID SEQUENCES OF MAD0041M02:
>SEQ ID NO: 119 leader sequence of Heavy chain of MAD0041M02
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 120 variable domain of Heavy chain of MAD0041M02
CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGTTATGCACT
GGGTCCGCC AGGCTCC AGGC AAGGGGCTGGAGT GGGT GGC AGTT AT ATC AT AT
GGTGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTC
CAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTG
AGGACACGGCTGTGTGTTACTGTGCGAGAGATGGCGGGGATATTGTAGTAGTA
CCAGGTGCTTCAACCTCGGAGAACTACTACTACTACTACATGGACGTCTGGGG
CAAAGGGACCACGGTCACCGTCTCCTCAGCCTCC
>SEQ ID NO: 121 constant domain of Heavy chain of MAD0041M02
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 122 complete sequence of Heavy chain of MAD0041M02
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
T C C C AGGT GC AGC T GC AGG AGT C GGGGGG AGGC GT GGT C C AGC C T GGG AGGT
CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGTTATGC
ACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCA
TATGGTGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCAT
CTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
C T G AGG AC AC GGC T GT GT GT TACT GT GC GAG AG AT GGC GGGG AT ATT GT AGT A
GTACCAGGTGCTTCAACCTCGGAGAACTACTACTACTACTACATGGACGTCTG
GGGCAAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGG
TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTC
AGGC GCCCTGACC AGC GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAG
GACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACC
CAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA
AGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG
TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GT GC AT A AT GC C A AGAC A A AGCC GCGGG AGGAGC AGT AC A AC AGC AC GT AC C
GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG
CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA
ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT
ATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC
ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT
CCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 123 leader sequence of Light chain of MAD0041M02
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 124 variable domain of Light chain of MAD0041M02 GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCG GCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGGATAGTGATGATGGAAA CACCTATTTGGACTGGTGCCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGA TCTATACGCTTTCCTATCGGGCCTCTGGAGTCCCAGACAGGTTCAGTGGCAGTG GGT C AGGC ACTGATTTC AC AC T A A A A ATC AGC AGGGT GGAGGC T GAGGAT GTT GGAGTTTATTACTGCATGCAACGTATAGAGTTTCCTAGGACGTTCGGCCAAGG GAC C A AGGT GGA A AT C A A AC GA ACT
>SEQ ID NO: 125 constant domain of Light chain of MAD0041M02
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO: 126 complete sequence of Light chain of MAD0041M02
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAG
CCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGGATAGTGATGATGGA
AACACCTATTTGGACTGGTGCCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCT
GATCTATACGCTTTCCTATCGGGCCTCTGGAGTCCCAGACAGGTTCAGTGGCAG TGGGTCAGGCACTGATTTCACACTAAAAATCAGCAGGGTGGAGGCTGAGGATG
TTGGAGTTTATTACTGCATGCAACGTATAGAGTTTCCTAGGACGTTCGGCCAAG
GGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CC AATCGGGT AACTCCC AGGAGAGTGT C AC AGAGC AGGAC AGC AAGGAC AGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
A AGAGC TT C A AC AGGGGAGAGT GTT AG
Sequence of the antibody herein identified as MADOIOOFIO
AMINO ACID SEQUENCES OF MADOIOOFIO:
>SEQ ID NO: 127 CDR 1 of variable domain of Heavy chain of MADOIOOFIO GYTLTELS
>SEQ ID NO: 128 CDR 2 of variable domain of Heavy chain of MADOIOOFIO FDPADAET
>SEQ ID NO: 129 CDR 3 of variable domain of Heavy chain of MADOIOOFIO C ATALPITMVRGVQ YYYY GMD VW
>SEQ ID NO: 130 CDR 1 of variable domain of Light chain of MADOIOOFIO QSLVHSDGNTY
>SEQ ID NO: 131 CDR 2 of variable domain of Light chain of MADOIOOFIO this sequence is not included in the sequence listing because is less than 4 amino acid KIS
>SEQ ID NO: 132 CDR 3 of variable domain of Light chain of MADOIOOFIO CMQVTQFPYTF
>SEQ ID NO: 133 variable domain of Heavy chain of MADOIOOFIO QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQTPGKGLEWMGGFDP AD AETIY AQKLQGRVTMTEDT STDT AYMEL S SLRSEDT AVYY C AT ALPITMVRGV Q YYYYGMD VWGPGTT VT VS S AS
>SEQ ID NO: 134 variable domain of Light chain of MADOIOOFIO DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISN RF S GVPDRF S GS GAGTDFTLKISRVE AED V GI Y Y CMQ VT QFP YTF GQGTKLEIKRT >SEQ ID NO:135 Heavy chain of MADOIOOFIO MGW S CIILFL V AT AT GVHS Q V QL V Q S GAE VKKPGAS VK V S CK V S GYTLTEL SMH WVRQTPGKGLEWMGGFDPADAETIYAQKLQGRVTMTEDTSTDTAYMELSSLRSE DT AVYY CAT ALPITMVRGV Q YYYY GMD VW GPGTT VT V S S ASTKGP S VFPL AP S S K S T S GGT AALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTKVDKRVEPKSCDKTHT CPPCP APELLGGP S VFLFP PKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS T YRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VYTLPP SREEMTKN Q VSLT CL VKGF YP SDI A VEWE SN GQPENNYKTTPP VLD SDGSFFL Y SK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO: 136 Light chain ofMADOlOOFlO
MGW SCIILFL VAT AT GVHSDIVMTQTPL S SP VTLGQP ASISCRS SQ SL VHSDGNT YL SWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGIYYCMQ VTQFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ WK VDN ALQ S GN S QE S VTEQD SKD S T Y SL S STLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC *
NUCLEIC ACID SEQUENCES OF MAD0100F10:
>SEQ ID NO: 137 leader sequence of Heavy chain of MADOIOOFIO
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 138 variable domain of Heavy chain of MADOIOOFIO
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTCTCCTGCAAGGTTTCCGGATACACTCTCACTGAATTATCCATGCACTG
GGTGCGACAGACTCCTGGAAAAGGACTTGAGTGGATGGGAGGTTTTGATCCTG
CAGATGCTGAAACAATCTACGCACAGAAGTTGCAGGGCAGAGTCACCATGACC
GAGGAC AC ATCT AC AGAC AC AGCCT AC AT GGAGCTGAGC AGCCTGAGATCTGA
GGACACCGCCGTATATTACTGTGCAACAGCCCTGCCTATTACTATGGTTCGGGG
AGTTCAATATTACTACTACGGTATGGACGTCTGGGGCCCAGGGACCACGGTCA
CCGTCTCCTCAGCCTCC
>SEQ ID NO: 139 constant domain of Heavy chain of MADOIOOFIO ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 140 complete sequence of Heavy chain of MAD0100F10
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGTTTCCGGATACACTCTCACTGAATTATCCATGCA
CTGGGTGCGACAGACTCCTGGAAAAGGACTTGAGTGGATGGGAGGTTTTGATC
C T GC AGAT GC T GA A AC A AT C T AC GC AC AG A AGTT GC AGGGC AGAGT C AC C ATG
ACCGAGGACACATCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAGATC
TGAGGACACCGCCGTATATTACTGTGCAACAGCCCTGCCTATTACTATGGTTCG
GGGAGTTCAATATTACTACTACGGTATGGACGTCTGGGGCCCAGGGACCACGG
TCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GT C AAGTTC AACTGGT ACGTGGACGGCGT GGAGGT GC AT AATGCC AAGAC AAA
GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAG
AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC
CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGAC
AAGAGC AGGT GGC AGC AGGGGAACGTCTTCTC AT GCTCCGTGATGC ATGAGGC
TCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 141 leader sequence of Light chain of MAD0100F10
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 142 variable domain of Light chain of MADOIOOFIO
GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAGCCG
GCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTACACAGTGATGGAAACAC
CTACTTGAGTTGGCTTCAGCAGAGGCCAGGCCAGCCTCCAAGACTCCTAATTT
ATAAGATTTCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGG
GC AGGGAC AGATTTC AC ACTGAAGAT C AGC AGAGT GGAAGCTGAGGAT GTCG
GGATTTATTACTGCATGCAAGTTACACAATTTCCGTACACTTTTGGCCAGGGGA
CCAAGCTGGAGATCAAACGAACT
>SEQ ID NO: 143 constant domain of Light chain of MADOIOOFIO
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO: 144 complete sequence of Light chain of MADOIOOFIO ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAG
CCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTACACAGTGATGGAAA
CACCTACTTGAGTTGGCTTCAGCAGAGGCCAGGCCAGCCTCCAAGACTCCTAA
TTTATAAGATTTCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTG
GGGC AGGGAC AGATTTC AC AC T GA AGATC AGC AGAGT GGA AGC T GAGGAT GT
CGGGATTTATTACTGCATGCAAGTTACACAATTTCCGTACACTTTTGGCCAGGG
GACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCC
GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA
TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC
AATCGGGT AACTCCCAGG AGAGT GTC AC AGAGC AGGAC AGC AAGGAC AGC AC
CTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC
AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA
GAGC TT C A AC AGGGGAGAGT GTT AG
Sequence of the antibody herein identified as MAD0100L19
AMINO ACID SEQUENCES OF MAD0100L19:
>SEQ ID NO: 145 CDR 1 of variable domain of Heavy chain of MAD0100L19 GFTFSDYY
>SEQ ID NO: 146 CDR 2 of variable domain of Heavy chain of MAD0100L19 ISSSGSNI
>SEQ ID NO: 147 CDR 3 of variable domain of Heavy chain of MAD0100L19 C ARGRLW GWFDPW
>SEQ ID NO: 148 CDR 1 of variable domain of Light chain of MAD0100L19 QDISNY
>SEQ ID NO: 149 CDR 2 of variable domain of Light chain of MAD0100L19 this sequence is not included in the sequence listing because is less than 4 amino acid DAS
>SEQ ID NO: 150 CDR 3 of variable domain of Light chain of MAD0100L19 CQQYDHLLITF
>SEQ ID NO: 151 variable domain of Heavy chain of MAD0100L19 EVQLVESGGGLVKPGGSLRLFCAASGFTFSDYYMSWFRQAPGKGLEWVSYISSSG SNIY Y AD S VKGRFT V SRDNAKN SL YLQMN SLRAEDT AVYFC ARGRLW GWFDPW GQGTL VT V S S AS
>SEQ ID NO: 152 variable domain of Light chain of MAD0100L19 DIVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLQT GVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDHLLITFGQGTRLEIKRT >SEQ ID NO: 153 Heavy chain ofMAD0100L19
MGW S CIILFL V AT AT GVHSE V QL VE S GGGL VKPGGSLRLFC A AS GFTF SD Y YMS W FRQ APGKGLEW V S YIS S SGSNIYY AD S VKGRFT V SRDNAKN SL YLQMN SLRAEDT AVYFCARGRLWGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGT Q T YICN VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTC VVVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV S VLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO: 154 Light chain ofMAD0100L19
MGW SCIILFL VAT AT GVHSDIVMT Q SP S SLS AS VGDRVTITCQ ASQDISNYLNW Y Q QKPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDHL LITF GQGTRLEIKRT V A AP S VFIFPP SDEQLK S GT AS V V CLLNNF YPRE AK V Q WK V DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC*
NUCLEIC ACID SEQUENCES OF MAD0100L19:
>SEQ ID NO: 155 leader sequence of Heavy chain of MAD0100L19
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 156 variable domain of Heavy chain of MAD0100L19
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCCGGAGGGTCCCT
GAGACTTTTCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTG
GTTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTA
GTGGTAGTAACATATACTATGCAGACTCTGTGAAGGGCCGATTCACCGTCTCC
AGGGACAACGCCAAGAACTCTCTGTATCTGCAAATGAACAGCCTGAGAGCCGA GGACACGGCCGTTTATTTCTGTGCGAGAGGAAGATTGTGGGGGTGGTTCGACC
CCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCC
>SEQ ID NO: 157 constant domain of Heavy chain of MAD0100L19
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 158 complete sequence of Heavy chain of MAD0100L19
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCCGGAGGGTC
CCTGAGACTTTTCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAG
C T GGTTC CGC C AGGC T C C AGGGA AGGGGC T GGAGT GGGTTT CAT AC ATT AGT A
GTAGTGGTAGTAACATATACTATGCAGACTCTGTGAAGGGCCGATTCACCGTC
TCCAGGGACAACGCCAAGAACTCTCTGTATCTGCAAATGAACAGCCTGAGAGC
CGAGGACACGGCCGTTTATTTCTGTGCGAGAGGAAGATTGTGGGGGTGGTTCG
ACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGC
CCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGC GGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGT
GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG
TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG
GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGT
GGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAAC
CCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG
GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT
GGAGGT GC AT A AT GC C A AGAC A A AGC CGCGGGAGGAGC AGT AC A AC AGC AC G
TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC
CCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC
C TC T AT AGC A AGC TC ACC GT GGAC A AGAGC AGGT GGC AGC AGGGGA ACGT C TT
CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC
TCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 159 leader sequence of Light chain of MAD0100L19
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 160 variable domain of Light chain of MAD0100L19
GACATCGTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGA
GTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTA
TCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATT
T GC AAAC AGGGGTCCC ATC AAGGTT C AGT GGAAGT GGATCTGGGAC AGATTTT
ACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAG
CAGTATGATCATCTCTTGATCACCTTCGGCCAAGGGACACGACTGGAGATTAA
ACGAACT
>SEQ ID NO: 161 constant domain of Light chain of MAD0100L19
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO: 162 complete sequence of Light chain of MAD0100L19
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGACATCGTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGT C ACC AT C ACTT GCC AGGCGAGTC AGGAC ATT AGC AACT ATTT AAATTG
GTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCA
ATTTGCAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGAT
TTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGT
CAGCAGTATGATCATCTCTTGATCACCTTCGGCCAAGGGACACGACTGGAGAT
TAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA
GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG
AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC
AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG
CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC
GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG
AGAGT GTT AG
Sequence of the antibody herein identified as MAD0101H20 AMINO ACID SEQUENCES OF MAD0101H20:
>SEQ ID NO: 163 CDR 1 of variable domain of Heavy chain of MAD0101H20 GGTFSSYA
>SEQ ID NO: 164 CDR 2 of variable domain of Heavy chain of MAD0101H20 ITPIFHTA
>SEQ ID NO: 165 CDR 3 of variable domain of Heavy chain of MAD0101H20 CARETGDQGVTAPFDLW
>SEQ ID NO: 166 CDR 1 of variable domain of Light chain of MAD0101H20 QSVSSY
>SEQ ID NO: 167 CDR 2 of variable domain of Light chain of MAD0101H20 this sequence is not included in the sequence listing because is less than 4 amino acid DAS
>SEQ ID NO: 168 CDR 3 of variable domain of Light chain of MAD0101H20 C QLRGNWPP WTF
>SEQ ID NO: 169 variable domain of Heavy chain of MAD0101H20 Q VQL VQSGAEVKKPGS SVKVSCKASGGTF S S YAISWVRQ APGQGLEWMGGITPIF HT ANY AQKF QGR VTIT ADE STS T VYMEL SSLS SEDT A V Y Y CARET GDQGVT APFD LWGRGTLVTVSSAS
>SEQ ID NO: 170 variable domain of Light chain of MAD0101H20 EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARF SGSGSGTDFTLTIS SLEPEDF A V Y Y C QLRGNWPP WTF GQGTK VEIKRT >SEQ ID NO: 171 Heavy chain ofMAD0101H20
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW VRQ APGQGLEWMGGITPIFHT ANY AQKFQGR VTIT ADESTSTVYMELSSLSSEDTA VYYCARETGDQGVTAPFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT Y ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVT C VVVD V SHEDPEVKFNW YVDGVEVHNAKTKPREEQYNST YRVV S VLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVF S C S VMHE ALHNH YT QK SL SL SPGK *
>SEQ ID NO: 172 Light chain of MAD0101H20
MGW SCIILFL VAT AT GVHSEIVMTQ SP ATLSL SPGERATL SCRASQ S V S S YL AW Y Q QKPGQ APRLLIYD ASNRATGIPARF SGSGSGTDFTLTIS SLEPEDF AVYYCQLRGNW PPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTK SFNRGEC *
NUCLEIC ACID SEQUENCES OF MAD0101H20:
>SEQ ID NO: 173 leader sequence of Heavy chain of MAD0101H20
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 174 variable domain of Heavy chain of MAD0101H20 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGT
GAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCT
GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCACCCC
TATCTTTCACACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTA
CCGCGGACGAATCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGCTCT
GAGGACACGGCCGTCTATTACTGTGCGAGGGAGACCGGCGACCAGGGAGTTA
CAGCCCCTTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGCCT
CC
>SEQ ID NO: 175 constant domain of Heavy chain of MAD0101H20
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 176 complete sequence of Heavy chain of MAD0101H20
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCA GCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCAC
CCCTATCTTTCACACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGA
TTACCGCGGACGAATCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGC
TCTGAGGACACGGCCGTCTATTACTGTGCGAGGGAGACCGGCGACCAGGGAGT
TACAGCCCCTTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGC
CTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC
TGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTG
TGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG
GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG
C A AC AC C A AGGT GGAC A AGAGAGTT GAGC CC A A AT C TTGT GAC A A A AC TC AC
ACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCT
CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA
CATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG
T ACGT GGACGGCGT GGAGGT GC AT AAT GCC AAGAC AAAGCCGCGGGAGGAGC
AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGC
CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA
GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC
GACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT
ACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 177 leader sequence of Light chain of MAD0101H20
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 178 variable domain of Light chain of MAD0101H20
GAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGT
ACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAAC
AGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCA
GCTGCGTGGCAACTGGCCTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAA
TCAAACGAACT
>SEQ ID NO: 179 constant domain of Light chain of MAD0101H20
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO: 180 complete sequence of Light chain of MAD0101H20
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAA
AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTG
GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCA
AC AGGGCC ACTGGC ATCCC AGCC AGGTT C AGT GGC AGT GGGTCTGGGAC AGAC
TTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT
CAGCTGCGTGGCAACTGGCCTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGA
AATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC
C AGAGAGGCC AAAGT AC AGT GGAAGGT GGAT AACGCCCTCC AATCGGGT AAC
TCCC AGGAGAGT GT C AC AGAGC AGGAC AGC AAGGAC AGC ACCT AC AGCCTC A
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC
CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA
GGGGAGAGTGTTAG
Sequence of the antibody herein identified as MAD0102F20 AMINO ACID SEQUENCES OF MAD0102F20:
>SEQ ID NO: 181 CDR 1 of variable domain of Heavy chain of MAD0102F20 GYSLIELS
>SEQ ID NO: 182 CDR 2 of variable domain of Heavy chain of MAD0102F20 FDPEDVET >SEQ ID NO: 183 CDR 3 of variable domain of Heavy chain of MAD0102F20 CATFFAVRGALNWFDSW
>SEQ ID NO: 184 CDR 1 of variable domain of Light chain of MAD0102F20 QSVSSN
>SEQ ID NO: 185 CDR 2 of variable domain of Light chain of MAD0102F20 this sequence is not included in the sequence listing because is less than 4 amino acid GAS
>SEQ ID NO: 186 CDR 3 of variable domain of Light chain of MAD0102F20 CQQYNNWPPLTF
>SEQ ID NO: 187 variable domain of Heavy chain of MAD0102F20 Q VQL VQSGAEVKKPGAS VKVSCKVSGY SLIELSMHWVRQ APGKGLEWMGGFDP EDVETIYAQNLQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATFFAVRGALN WFDSWGQGTLVTVSSAS
>SEQ ID NO: 188 variable domain of Light chain of MAD0102F20 EIVLTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRAT GLPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPLTFGQGTKLEIKRT >SEQ ID NO: 189 Heavy chain of MAD0102F20
MGW S CIILFL V AT AT GVHS Q V QL V Q S GAE VKKPGAS VK V S CK V S GY SLIEL SMHW VRQAPGKGLEWMGGFDPEDVETIYAQNLQGRVTMTEDTSTDTAYMELSSLRSED T A V Y Y C ATFF A VRGALNWFD S W GQGTL VT V S S AS TKGP S VFPL AP S SK S T S GGT A ALGCLVKD YFPEP VT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTC VVVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVS VLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO: 190 Light chain of MAD0102F20
MGWSCIILFLVATATGVHSEIVLTQSPATLSVSPGERATLSCRASQSVSSNLAWYQ QKPGQAPRLLIYGASTRATGLPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNN WPPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS P VTK SFNRGEC * NUCLEIC ACID SEQUENCES OF MAD0102F20:
>SEQ ID NO: 191 leader sequence of Heavy chain of MAD0102F20
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 192 variable domain of Heavy chain of MAD0102F20
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTCTCCTGCAAGGTTTCCGGATACAGCCTCATTGAATTATCCATGCACTG
GGTGCGACAGGCTCCTGGAAAAGGGCTTGAGTGGATGGGAGGGTTTGATCCTG
AAGATGTTGAAACAATCTACGCACAGAACCTCCAGGGCAGAGTCACCATGACC
GAGGAC AC ATCT AC AGAC AC AGCCT AC AT GGAGCT AAGC AGCCTGAGATCTGA
GGACACGGCCGTGTATTACTGTGCAACTTTTTTTGCGGTTCGGGGAGCTCTGAA
CTGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCC
>SEQ ID NO: 193 constant domain of Heavy chain of MAD0102F20
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA >SEQ ID NO: 194 complete sequence of Heavy chain of MAD0102F20
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGTTTCCGGATACAGCCTCATTGAATTATCCATGCA
CTGGGTGCGACAGGCTCCTGGAAAAGGGCTTGAGTGGATGGGAGGGTTTGATC
C T GA AGAT GTT GA A AC A AT C T AC GC AC AGA ACC T C C AGGGC AGAGT C AC C ATG
ACCGAGGACACATCTACAGACACAGCCTACATGGAGCTAAGCAGCCTGAGATC
TGAGGACACGGCCGTGTATTACTGTGCAACTTTTTTTGCGGTTCGGGGAGCTCT
GAACTGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCT
CCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTG
GGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTG
ACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGC
TGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTC
CAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCA
ACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACAC
ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTT
CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT
ACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGAC
CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC
GGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA
CGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 195 leader sequence of Light chain of MAD0102F20
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 196 variable domain of Light chain of MAD0102F20 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAG AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGT ACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCAACC AGGGC C AC T GGTCTCC C AGCC AGGTT C AGT GGC AGT GGGT C T GGGAC AGAGTT CACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCA GCAGTATAATAACTGGCCTCCGCTCACTTTTGGCCAGGGGACCAAGCTCGAGA TCAAACGAACT
>SEQ ID NO: 197 constant domain of Light chain of MAD0102F20
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO: 198 complete sequence of Light chain of MAD0102F20
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAA
AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTG
GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCAA
CCAGGGCCACTGGTCTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAG
TTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGT
CAGCAGTATAATAACTGGCCTCCGCTCACTTTTGGCCAGGGGACCAAGCTCGA
GATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC
C AGAGAGGCC AAAGT AC AGT GGAAGGT GGAT AACGCCCTCC AATCGGGT AAC
TCCC AGGAGAGT GT C AC AGAGC AGGAC AGC AAGGAC AGC ACCT AC AGCCTC A
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC
CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA
GGGGAGAGTGTTAG
Sequence of the antibody herein identified as MAD0102F22 AMINO ACID SEQUENCES OF MAD0102F22: >SEQ ID NO: 199 CDR 1 of variable domain of Heavy chain of MAD0102F22 GGSISNNY
>SEQ ID NO:200 CDR 2 of variable domain of Heavy chain of MAD0102F22 IFYSGST
>SEQ ID NO:201 CDR 3 of variable domain of Heavy chain of MAD0102F22 CARGSGWYLYWYFDLW
>SEQ ID NO:202 CDR 1 of variable domain of Light chain of MAD0102F22 QSISSSY
>SEQ ID NO:203 CDR 2 of variable domain of Light chain of MAD0102F22 this sequence is not included in the sequence listing because is less than 4 amino acid GAS
>SEQ ID NO:204 CDR 3 of variable domain of Light chain of MAD0102F22 CQQYGSSPQTF
>SEQ ID NO:205 variable domain of Heavy chain of MAD0102F22 QVQLQESGPGLVKPSETLSLTCTVSGGSISNNYWSWIRQPPGKGLEWIGHIFYSGST NYNP SLKSRVTIS VDT SKNQF SLKLRS VT AADT AVYY CARGSGWYLYWYFDLW G RGTLVTVSSAS
>SEQ ID NO:206 variable domain of Light chain of MAD0102F22 EIVMTQSPGTLSLSPGERATLSCRASQSISSSYLAWYQQKPGQAPRLLLYGASSRAT GIPDRF SGSGSGTDFTLTISRLEPEDF AVYYCQQ YGS SPQTFGQGTKLEIKRT >SEQ ID NO:207 Heavy chain of MAD0102F22
MGWSCIILFLVATATGVHSQVQLQESGPGLVKPSETLSLTCTVSGGSISNNYWSWI RQPPGKGLEWIGHIFYSGSTNYNPSLKSRVTISVDTSKNQFSLKLRSVTAADTAVY Y C ARGS GW YL YW YFDLW GRGTL VT V S S AS TKGP S VFPL AP S SK S T S GGT A ALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGT Q T YICN VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTC VVVD V SHEDPEVKFNWYVDGVLVHNAKTKPREEQYNSTYRVV S VLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO:208 Light chain of MAD0102F22 MGWSCIILFLVATATGVHSEIVMTQSPGTLSLSPGERATLSCRASQSISSSYLAWYQ
QKPGQAPRLLLYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSS
PQTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGEC*
NUCLEIC ACID SEQUENCES OF MAD0102F22:
>SEQ ID NO:209 leader sequence of Heavy chain of MAD0102F22
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:210 variable domain of Heavy chain of MAD0102F22
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCT
GTCCCTCACTTGCACTGTCTCTGGTGGCTCCATCAGTAATAACTACTGGAGTTG
GATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGCATATCTTTTACA
GTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATCTCAGTA
GACACGTCCAAGAATCAGTTCTCCCTGAAGCTGAGGTCTGTGACCGCTGCGGA
CACGGCCGTCTATTACTGTGCGAGAGGGAGTGGCTGGTACCTATACTGGTACT
TCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGCTTCC
>SEQ ID NO:211 constant domain of Heavy chain of MAD0102F22
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:212 complete sequence of Heavy chain of MAD0102F22
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACTTGCACTGTCTCTGGTGGCTCCATCAGTAATAACTACTGGAG
TTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGCATATCTTTT
ACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATCTCA
GTAGACACGTCCAAGAATCAGTTCTCCCTGAAGCTGAGGTCTGTGACCGCTGC
GGACACGGCCGTCTATTACTGTGCGAGAGGGAGTGGCTGGTACCTATACTGGT
ACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGCTTCCACCA
AGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGT
CTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC
TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA
GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCC
ACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC
AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
T GGT GG AC GT G AGC C AC G A AG AC C C T G AGGT C A AGT T C A AC T GGT AC GT GG AC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA
GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
CTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGT C AAAGGCTTCT ATCCC AGCGAC ATCGCCGT GGAGTGGGAGAGC AAT GGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAA CGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA
AGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:213 leader sequence of Light chain of MAD0102F22
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:214 variable domain of Light chain of MAD0102F22 GAAATTGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCAGCTACTTAGCCT GGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCCTCTATGGTGCATCC AGC AGGGCC ACTGGC ATCCC AGAC AGGTT C AGT GGC AGT GGGTCTGGGAC AG ACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACT GT C AGC AGT AT GGT AGCTC ACCTC AAACTTTT GGCC AGGGGACC AAGCTGGAG ATCAAACGAACT
>SEQ ID NO:215 constant domain of Light chain of MAD0102F22
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO:216 complete sequence of Light chain of MAD0102F22
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAAATTGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA
AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCAGCTACTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCCTCTATGGTGCAT
CCAGC AGGGCC ACTGGC ATCCC AGACAGGTTCAGTGGCAGTGGGTCTGGGACA
GACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTAC
TGTCAGCAGTATGGTAGCTCACCTCAAACTTTTGGCCAGGGGACCAAGCTGGA
GATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC
C AGAGAGGCC AAAGT AC AGT GGAAGGT GGAT AACGCCCTCC AATCGGGT AAC TCCC AGGAGAGT GT C AC AGAGC AGGAC AGC AAGGAC AGC ACCT AC AGCCTC A GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGTTAG
Sequence of the antibody herein identified as MAD0102G04 AMINO ACID SEQUENCES OF MAD0102G04:
>SEQ ID NO:217 CDR 1 of variable domain of Heavy chain of MAD0102G04 GGTFSNFA
>SEQ ID NO:218 CDR 2 of variable domain of Heavy chain of MAD0102G04 IIPIFGTA
>SEQ ID NO:219 CDR 3 of variable domain of Heavy chain of MAD0102G04 CRADIALQWASDIW
>SEQ ID NO:220 CDR 1 of variable domain of Light chain of MAD0102G04 QSVSSY >SEQ ID NO:221 CDR 2 of variable domain of Light chain of MAD0102G04 this sequence is not included in the sequence listing because is less than 4 amino acid DAS (asp-ala-ser)
>SEQ ID NO:222 CDR 3 of variable domain of Light chain of MAD0102G04 CQQRSNWPPLITF >SEQ ID NO:223 variable domain of Heavy chain of MAD0102G04 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNFAISWVRQAPGQGLEWMGGIIPIF GT ANY AQKF QGRVTLIADESTRT AYMEL S SLT SEDT AVYY CRADIALQW ASDIWG QGTMV S VS S AS
>SEQ ID NO:224 variable domain of Light chain of MAD0102G04 EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARF SGSGSGTDFTLTIS SLEPEDF AVYYCQQRSNWPPLITFGPGTKVDIKRT>SE Q ID NO:225 Heavy chain of MAD0102G04
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNFAISW
VRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTLIADESTRTAYMELSSLTSEDTA
VYYCRADIALQWASDIWGQGTMVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGT Q T YICN
VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTC VVVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV S VLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO:226 Light chain of MAD0102G04
MGW SCIILFL VAT AT GVHSEIVMTQ SP ATLSL SPGERATL SCRASQ S V S S YL AW Y Q QKPGQAPRLLIYD ASNRATGIPARF SGSGSGTDFTLTIS SLEPEDF AVYYCQQRSNW PPLITFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC
NUCLEIC ACID SEQUENCES OF MAD0102G04:
>SEQ ID NO:227 leader sequence of Heavy chain of MAD0102G04
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:228 variable domain of Heavy chain of MAD0102G04
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGT
GAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAACTTTGCTATCAGCTG
GGTGCGACAGGCCCCTGGACAGGGACTTGAGTGGATGGGAGGGATCATCCCTA
TCTTTGGAACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGCTTATC
GCGGACGAATCCACGAGGACAGCCTACATGGAACTGAGCAGCCTGACATCTG
AGGACACGGCCGTGTATTACTGCCGGGCCGATATTGCACTACAATGGGCTTCT
GATATCTGGGGCCAAGGGACAATGGTCAGCGTCTCTTCGGCTTCC
>SEQ ID NO:229 constant domain of Heavy chain of MAD0102G04
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:230 complete sequence of Heavy chain of MAD0102G04
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAACTTTGCTATCA
GCTGGGTGCGACAGGCCCCTGGACAGGGACTTGAGTGGATGGGAGGGATCAT
CCCTATCTTTGGAACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGC
TTATCGCGGACGAATCCACGAGGACAGCCTACATGGAACTGAGCAGCCTGACA
TCTGAGGACACGGCCGTGTATTACTGCCGGGCCGATATTGCACTACAATGGGC
TTCTGATATCTGGGGCCAAGGGACAATGGTCAGCGTCTCTTCGGCTTCCACCAA
GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTC
TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT
ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA
GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCC
ACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC
AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
T GGT GG AC GT G AGC C AC G A AG AC C C T G AGGT C A AGT T C A AC T GGT AC GT GG AC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA
GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
CTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGT C AAAGGCTTCT ATCCC AGCGAC ATCGCCGT GGAGTGGGAGAGC AAT GGGC AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAA
CGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA
AGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:231 leader sequence of Light chain of MAD0102G04
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:232 variable domain of Light chain of MAD0102G04
GAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGT
ACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAAC
AGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT
CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCA
GCAGCGTAGCAACTGGCCTCCCTTAATCACTTTCGGCCCTGGGACCAAAGTGG
ATATCAAACGAACT>SEQ ID NO:233 constant domain of Light chain of
MAD0102G04
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO:234 complete sequence of Light chain of MAD0102G04
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAA
AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTG
GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCA
AC AGGGCC ACTGGC ATCCC AGCC AGGTT C AGT GGC AGT GGGTCTGGGAC AGAC
TTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT
CAGCAGCGTAGCAACTGGCCTCCCTTAATCACTTTCGGCCCTGGGACCAAAGT
GGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTA
TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGT
AACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCC
TCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTA
CGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA
ACAGGGGAGAGTGTTAGSequence of the antibody herein identified as MAD0008C14
AMINO ACID SEQUENCES OF MAD0008C14:
>SEQ ID NO:235 CDR 1 of variable domain of Heavy chain of MAD0008C14 GLTVSSNY
>SEQ ID NO:236 CDR 2 of variable domain of Heavy chain of MAD0008C14 IYSGGST
>SEQ ID NO:237 CDR 3 of variable domain of Heavy chain of MAD0008C14 CVRDLYSYGMDVW
>SEQ ID NO:238 CDR 1 of variable domain of Light chain of MAD0008C14 QGISSY
>SEQ ID NO:239 CDR 2 of variable domain of Light chain of MAD0008C14 this sequence is not included in the sequence listing because is less than 4 amino acid AAS
>SEQ ID NO:240 CDR 3 of variable domain of Light chain of MAD0008C14 CQQVNSYPTF
>SEQ ID NO:241 variable domain of Heavy chain of MAD0008C14 EVQLVESGGGLVQPGGSLRLSCAASGLTVSSNYMSWVRQAPGKGLEWVSVIYSG GSTF Y AD S VKDRFTISRDN SKNTL YLQMN SLRAEDT AVYYC VRDL Y S Y GMD VW G QGTTVTVSSAS
>SEQ ID NO:242 variable domain of Light chain of MAD0008C14 AIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQVNSYPTFGQGTRLEIKRT >SEQ ID NO:243 Heavy chain of MAD0008C14
MGW S CIILFL V AT AT GVHSE V QL VE S GGGL V QPGGSLRL S C A AS GLT V S SNYMS W VRQ APGKGLEW V S VI YSGGSTFYAD S VKDRFTISRDN SKNTL YLQMN SLRAEDT A VYYCVRDLYSYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGT Q T YICN VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTC VVVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV S VLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO:244 Light chain of MAD0008C14
MGWSCIILFLVATATGVHSAIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQ
QKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQVNSY
PTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
KSFNRGEC*
NUCLEIC ACID SEQUENCES OF MAD0008C14:
>SEQ ID NO:245 leader sequence of Heavy chain of MAD0008C14
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:246 variable domain of Heavy chain of MAD0008C14
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGCCTCTGGACTCACCGTCAGTAGCAACTACATGAGCT
GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGC
GGTGGTAGCACATTCTACGCAGACTCCGTGAAGGACAGATTCACCATCTCCAG
AGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGG
ACACGGCTGTATATTACTGTGTGAGAGATCTCTACTCCTACGGTATGGACGTCT
GGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCC
>SEQ ID NO:247 constant domain of Heavy chain of MAD0008C14
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:248 complete sequence of Heavy chain of MAD0008C14
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCGTCAGTAGCAACTACATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTAT
AGCGGTGGTAGCACATTCTACGCAGACTCCGTGAAGGACAGATTCACCATCTC
CAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCG
AGGACACGGCTGTATATTACTGTGTGAGAGATCTCTACTCCTACGGTATGGAC
GTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG
CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGG
AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTC
CTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG
GCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG
GACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTG
CCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGG
ACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG
GAGGT GC AT A AT GC C A AGAC A A AGC CGC GGGAGG AGC AGT AC A AC AGC AC GT
ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC
CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGA
GAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT
CTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT
CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC
TCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:249 leader sequence of Light chain of MAD0008C14
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:250 variable domain of Light chain of MAD0008C14
GCCATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGA
GTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTAT
CAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTT
GCAAAGTGGGGTCCCCTCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCA
CTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAAC
AGGTTAATAGTTACCCCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGA
ACT
>SEQ ID NO:251 constant domain of Light chain of MAD0008C14
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO:252 complete sequence of Light chain of MAD0008C14
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGCCATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTG
GTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCA
CTTTGCAAAGTGGGGTCCCCTCAAGGTTCAGCGGCAGTGGATCTGGGACAGAA TTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGT
CAACAGGTTAATAGTTACCCCACCTTCGGCCAAGGGACACGACTGGAGATTAA
ACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT
GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA
GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG
AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC
CCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA
GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA
GTGTTAG
Sequence of the antibody herein identified as MAD0008D14 AMINO ACID SEQUENCES OF MAD0008D14:
>SEQ ID NO:253 CDR 1 of variable domain of Heavy chain of MAD0008D14 GFTFSSYA
>SEQ ID NO:254 CDR 2 of variable domain of Heavy chain of MAD0008D14 ISFDGSKK
>SEQ ID NO:255 CDR 3 of variable domain of Heavy chain of MAD0008D14 CAREGQWLNWAFDYW
>SEQ ID NO:256 CDR 1 of variable domain of Light chain of MAD0008D14 QSISSY
>SEQ ID NO:257 CDR 2 of variable domain of Light chain of MAD0008D14 this sequence is not included in the sequence listing because is less than 4 amino acid LAS
>SEQ ID NO:258 CDR 3 of variable domain of Light chain of MAD0008D14 CQQSYSTPTWTF
>SEQ ID NO:259 variable domain of Heavy chain of MAD0008D14 EVQLVESGGGVVQPGKSLRLSCADSGFTFSSYAMHWVRQAPGKGLEWVAVISFD GSKKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGQWLNWAF DYWGQGTLVTVSSAS
>SEQ ID NO:260 variable domain of Light chain of MAD0008D14 AIQMTQSPSSLSASVGDRVTITCRAGQSISSYLNWYQQKPGKAPKLLIYLASTLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPTWTFGQGTKVEIKRT >SEQ ID NO:261 Heavy chain of MAD0008D14 MGW S CIILFL V AT AT GVHSE V QL VE S GGGV V QPGK SLRL SCADS GF TF S S YAMHW VRQAPGKGLEWVAVISFDGSKKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYY C AREGQWLNW AFD YW GQGTL VT V S S ASTKGP S VFPL AP S SKST SGGT AAL GCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT Y ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVT C VVVD V SHEDPEVKFNW YVDGVEVHNAKTKPREEQYNST YRVV S VLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVF S C S VMHE ALHNH YT QK SL SL SPGK *
>SEQ ID NO:262 Light chain of MAD0008D14
MGW SCIILFL VAT AT GVHS AIQMT Q SP S SLS AS VGDRVTIT CRAGQ SIS S YLNW Y Q QKPGKAPKLLIYLASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP TWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTK SFNRGEC *
NUCLEIC ACID SEQUENCES OF MAD0008D14:
>SEQ ID NO:263 leader sequence of Heavy chain of MAD0008D14
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:264 variable domain of Heavy chain of MAD0008D14
G AGGT GC AGC T GGT GGAGT C T GGGGG AGGC GT GGT C C AGC C AGGG A AGT C C C
TGAGACTCTCCTGTGCAGACTCTGGATTCACCTTCAGTAGCTATGCTATGCACT
GGGTCCGCCAGGCTCC AGGC AAGGGGCTGG AGT GGGT GGC AGTT AT ATC ATTT
GATGGAAGTAAGAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTC
CAGAGACAATTCCAAGAACACGCTGTACCTGCAAATGAACAGCCTGAGAGCTG
AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCAGTGGCTAAATTGGGCC
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCC
>SEQ ID NO:265 constant domain of Heavy chain of MAD0008D14
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:266 complete sequence of Heavy chain of MAD0008D14
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
T C C GAGGT GC AGC T GGT GG AGT C T GGGGG AGGC GT GGT C C AGC C AGGG A AGT
CCCTGAGACTCTCCTGTGCAGACTCTGGATTCACCTTCAGTAGCTATGCTATGC
ACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCA
TTTGATGGAAGTAAGAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCAT
CTCCAGAGACAATTCCAAGAACACGCTGTACCTGCAAATGAACAGCCTGAGAG
CTGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGCAGTGGCTAAATTGG
GCCTTTGACTACTGGGGCC AGGG AACCCTGGTCACCGTCTCCTCAGCCTCC ACC
AAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGG
CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGG
TCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC
CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC
AGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACAC
CAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCC
CACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA
CGGCGT GGAGGT GC AT AAT GCC AAGAC A AAGCCGCGGGAGGAGC AGT AC AAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG
AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC
CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC
TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG
CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC
CTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG
AAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:267 leader sequence of Light chain of MAD0008D14
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:268 variable domain of Light chain of MAD0008D14
GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGA
GTCACCATCACTTGCCGGGCAGGTCAGAGCATTAGCAGCTATTTAAATTGGTA
TCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCTTGCATCCACTTT
GCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCA
CTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAC
AGAGTTACAGTACCCCCACGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATC
AAACGAACT
>SEQ ID NO:269 constant domain of Light chain of MAD0008D14
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO:270 complete sequence of Light chain of MAD0008D14 ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCACTTGCCGGGCAGGTCAGAGCATTAGCAGCTATTTAAATTG
GTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCTTGCATCCA
CTTTGC AAAGTGGGGTCCC ATC AAGGTT C AGT GGC AGT GGATCTGGGAC AGAT
TTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGT
CAACAGAGTTACAGTACCCCCACGTGGACGTTCGGCCAAGGGACCAAGGTGG
AAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATG
AGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC
CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA
CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC
AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG
CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC
AGGGGAGAGTGTTAG
Sequence of the antibody herein identified as MAD0008B07 AMINO ACID SEQUENCES OF MAD0008B07:
>SEQ ID NO:271 CDR 1 of variable domain of Heavy chain of MAD0008B07 GFTFTNYY
>SEQ ID NO:272 CDR 2 of variable domain of Heavy chain of MAD0008B07 INPSGGST
>SEQ ID NO:273 CDR 3 of variable domain of Heavy chain of MAD0008B07 CADLLLDYW
>SEQ ID NO:274 CDR 1 of variable domain of Light chain of MAD0008B07 QGINNY
>SEQ ID NO:275 CDR 2 of variable domain of Light chain of MAD0008B07 this sequence is not included in the sequence listing because is less than 4 amino acid AAS
>SEQ ID NO:276 CDR 3 of variable domain of Light chain of MAD0008B07 CQQYNSYPFTF
>SEQ ID NO:277 variable domain of Heavy chain of MAD0008B07 QVQLVQSGAEVKKPGASVKVSCKASGFTFTNYYIHWVRQAPGQGLEWMGIINPS
GGSTIYAQKFQGRVTMTRDTSTRTVYMELSSLRSEDTAVYYCADLLLDYWGQGT
LVTVSSAS
>SEQ ID NO:278 variable domain of Light chain of MAD0008B07 AIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWFQQKPGKAPKSLIYAASSLQS GVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPFTFGQGTRLEIKRT >SEQ ID NO:279 Heavy chain of MAD0008B07
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGFTFTNYYIHW VRQAPGQGLEWMGIINPSGGSTIYAQKFQGRVTMTRDTSTRTVYMELSSLRSEDT AVYYCADLLLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV S VLTVLHQDWLN GKEYKCK V SNK ALP APIEKTISK AKGQPREPQ VYTLPP SREEMTKNQ V SLT CLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNH YT QK SL SL SPGK *
>SEQ ID NO:280 Light chain of MAD0008B07
MGW SCIILFL VAT AT GVHS AIQMT Q SP S SLS AS VGDRVTIT CRASQGINNYL AWF Q QKPGKAPKSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSY PFTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC*
NUCLEIC ACID SEQUENCES OF MAD0008B07:
>SEQ ID NO:281 leader sequence of Heavy chain of MAD0008B07
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:282 variable domain of Heavy chain of MAD0008B07
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT
GAAGGTTTCCTGCAAAGCATCTGGATTCACCTTCACCAACTACTATATTCACTG
GGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTA
GT GGTGGT AGC AC A ATCT ACGC AC AGAAGTTCC AGGGC AGAGT C ACC AT GACC
AGGGACACGTCCACGCGCACAGTCTACATGGAACTGAGCAGCCTGAGATCTGA GGACACGGCCGTGTATTACTGTGCGGATCTACTCCTGGACTACTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTTCC
>SEQ ID NO:283 constant domain of Heavy chain of MAD0008B07
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:284 complete sequence of Heavy chain of MAD0008B07
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTTTCCTGCAAAGCATCTGGATTCACCTTCACCAACTACTATATTCA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACC
CTAGTGGTGGTAGCACAATCTACGCACAGAAGTTCCAGGGCAGAGTCACCATG
ACCAGGGACACGTCCACGCGCACAGTCTACATGGAACTGAGCAGCCTGAGATC
TGAGGACACGGCCGTGTATTACTGTGCGGATCTACTCCTGGACTACTGGGGCC
AGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCATCGGTCTTCC
CCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGC CTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGC
CCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA
CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCT
ACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC
CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
C G A AG AC C C T G AGGT C A AGTT C A AC T GGT AC GT GG AC GGC GT GG AGGT GC AT A
ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT
CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA
GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA
CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAA
GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG
TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCC
CCGGGTAAATGA
>SEQ ID NO:285 leader sequence of Light chain of MAD0008B07
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGGGTACAT
TCC
>SEQ ID NO:286 variable domain of Light chain of MAD0008B07
GCCATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAG
AGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAACAATTATTTAGCCTGGTT
TCAGCAGAAACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTT
T GC AAAGT GGGGTCCC AT C AAAGTTC AGCGGC AGTGGATCTGGGAC AGATTTC
ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAA
CAATATAATAGTTACCCCTTCACCTTCGGCCAAGGGACACGACTGGAGATTAA
ACGAACT
>SEQ ID NO:287 constant domain of Light chain of MAD0008B07
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO:288 complete sequence of Light chain of MAD0008B07
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGGGTACAT
TCCGCCATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAACAATTATTTAGCCTG
GTTTCAGCAGAAACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCA
GTTT GC AAAGT GGGGTCCC AT C AAAGTTC AGCGGC AGTGGATCTGGGAC AGAT
TTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGC
CAACAATATAATAGTTACCCCTTCACCTTCGGCCAAGGGACACGACTGGAGAT
TAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA
GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG
AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC
AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG
CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC
GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG
AGAGTGTTAG
Sequence of the antibody herein identified as MAD0008D12 AMINO ACID SEQUENCES OF MAD0008D12:
>SEQ ID NO:289 CDR 1 of variable domain of Heavy chain of MAD0008D12 GLIVSSNY
>SEQ ID NO:290 CDR 2 of variable domain of Heavy chain of MAD0008D12 IYSGGTT
>SEQ ID NO:291 CDR 3 of variable domain of Heavy chain of MAD0008D12 CARDLQYYGMDVW
>SEQ ID NO:292 CDR 1 of variable domain of Light chain of MAD0008D12 QGISSY
>SEQ ID NO:293 CDR 2 of variable domain of Light chain of MAD0008D12 this sequence is not included in the sequence listing because is less than 4 amino acid AAS
>SEQ ID NO:294 CDR 3 of variable domain of Light chain of MAD0008D12 CRHLN S YPPITF
>SEQ ID NO:295 variable domain of Heavy chain of MAD0008D12 EVQLVESGGGLIQPGGSLRLSC AASGLIV S SNYMSWVRQ APGKGLEWV SLIYSGG TT YY AD S VKGRFTISRDN SKNTL YLQMN SLRAEDT AVY Y C ARDLQ YY GMD VW G QGTTVTVSSAS
>SEQ ID NO:296 variable domain of Light chain of MAD0008D12 AIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQS GVP SRF SGSGSGT QFTLTIS SLQPEDF AT YY CRHLN S YPPITF GQGTRLEIKRT >SEQ ID NO:297 Heavy chain of MAD0008D12
MGW S CIILFL V AT AT GVHSE V QL VE S GGGLIQPGGSLRL S C A AS GLI V S SN YMS W V RQ APGKGLEWV SLIYSGGTT YY AD S VKGRFTISRDN SKNTL YLQMN SLRAEDT AV YYCARDLQYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICN V NHKP SNTK VDKRVEPK S CDKTHT CPPCP APELLGGP S VFLFPPKPKD TLMI SRTPE V T C VVVD V SHEDPEVKFNW YVDGVEVHNAKTKPREEQ YNSTYRVV S VLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO:298 Light chain of MAD0008D12
MGWSCIILFLVATATGVHSAIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQ
QKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCRHLNSY
PPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGEC*
NUCLEIC ACID SEQUENCES OF MAD0008D12:
>SEQ ID NO:299 leader sequence of Heavy chain of MAD0008D12
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 300 variable domain of Heavy chain of MAD0008D12 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGATCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGCCTCTGGGCTCATTGTCAGTAGTAACTACATGAGTTG
GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACTTATTTATAGTG
GAGGAACCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGA
GACAATTCCAAGAACACTCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGA
CACGGCCGTGTATTACTGTGCGAGAGATCTTCAATACTACGGTATGGACGTCT
GGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCC
>SEQ ID NO:301 constant domain of Heavy chain of MAD0008D12
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 302 complete sequence of Heavy chain of MAD0008D12
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGATCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGGCTCATTGTCAGTAGTAACTACATGAG
TTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACTTATTTATA GTGGAGGAACCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCC
AGAGACAATTCCAAGAACACTCTGTATCTTCAAATGAACAGCCTGAGAGCCGA
GGACACGGCCGTGTATTACTGTGCGAGAGATCTTCAATACTACGGTATGGACG
TCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGC
CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGA
ACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC
ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGA
CAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCC
CAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC
GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA
GGT GC AT AAT GCC AAGAC AAAGCCGCGGGAGGAGC AGT AC AAC AGC ACGT AC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA
TCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC
ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT
CCCTGTCCCCGGGTAAATGA
>SEQ ID NO: 303 leader sequence of Light chain of MAD0008D12
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO: 304 variable domain of Light chain of MAD0008D12
GCCATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGA
GTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTAT
CAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTT
GCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACACAATTCA
CTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCGAC ACCTTAATAGTTACCCCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATT
AAACGAACG
>SEQ ID NO: 305 constant domain of Light chain of MAD0008D12
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO: 306 complete sequence of Light chain of MAD0008D12
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGCCATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTG
GTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCA
CTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACACAA
TTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGT
CGACACCTTAATAGTTACCCCCCGATCACCTTCGGCCAAGGGACACGACTGGA
GATTAAACGAACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC
C AGAGAGGCC AAAGT AC AGT GGAAGGT GGAT AACGCCCTCC AATCGGGT AAC
TCCC AGGAGAGT GT C AC AGAGC AGGAC AGC AAGGAC AGC ACCT AC AGCCTC A
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC
CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA
GGGGAGAGTGTTAG
Sequence of the antibody herein identified as MAD0102I15 AMINO ACID SEQUENCES OF MAD0102I15:
>SEQ ID NO:307 CDR 1 of variable domain of Heavy chain of MAD0102I15 GYTLTELS
>SEQ ID NO:308 CDR 2 of variable domain of Heavy chain of MAD0102I15 FDPEDGET
>SEQ ID NO:309 CDR 3 of variable domain of Heavy chain of MAD0102I15 C TT V GFPD YYD S S V YLRHFD YW
>SEQ ID NO:310 CDR 1 of variable domain of Light chain of MAD0102I15 QGISSY
>SEQ ID NO:311 CDR 2 of variable domain of Light chain ofMAD0102I15 AAS (Ala-Ala-Ser)
>SEQ ID NO:312 CDR 3 of variable domain of Light chain of MAD0102I15 CQQLNSYPRTF
>SEQ ID NO:313 variable domain of Heavy chain of MAD0102I15 QVQLVQSGAEVKKPGASAKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDP EDGETIYAQRF QGRVTMTED ASTDTAYMELS SLRSEDTAVYYCTTVGFPD YYDS S VYLRHFDYWGQGTL VT VS S AS
>SEQ ID NO:314 variable domain of Light chain of MAD0102I15 AIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQS GVP SRF S GS GS GTEF TLTIS SLQPEDF AT Y Y C QQLN S YPRTF GQGTKLEIKRT >SEQ ID NO:315 Heavy chain ofMAD0102I15
MGW S CIILFL V AT AT GVHS Q V QL V Q S GAE VKKPGAS AK V S CK V S GYTLTEL SMH WVRQ APGKGLEWMGGFDPEDGETIY AQRFQGRVTMTED ASTDT AYMEL S SLRSE DT AVYYCTT VGFPD YYD S S VYLRHFDYWGQGTL VT VS S ASTKGP S VFPL AP SSKS T S GGT A ALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO:316 Light chain ofMAD0102I15
MGWSCIILFLVATATGVHSAIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQ
QKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSY
PRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGEC*
NUCLEIC ACID SEQUENCES OF MAD0102I15:
>SEQ ID NO:317 leader sequence of Heavy chain of MAD0102I15 ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:318 variable domain of Heavy chain of MAD0102I15
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGC
GAAGGTCTCCTGCAAGGTTTCCGGATACACCCTCACTGAATTATCCATGCACTG
GGTGCGACAGGCTCCTGGAAAAGGGCTTGAGTGGATGGGAGGTTTTGATCCTG
AAGAT GGT GAAACGATCT ACGC AC AGAGGTTCC AGGGC AGGGT C ACC AT GAC
CGAGGACGCATCTACAGACACAGCCTACATGGAGCTGAGTAGCCTGAGATCTG
AGGACACGGCCGTCTATTACTGTACAACGGTGGGATTTCCGGATTATTATGAT
AGTAGTGTTTATTTGCGGCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACC
GTCTCCTCAGCTTCC
>SEQ ID NO:319 constant domain of Heavy chain of MAD0102I15
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:320 complete sequence of Heavy chain of MAD0102I15 ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGCGAAGGTCTCCTGCAAGGTTTCCGGATACACCCTCACTGAATTATCCATGC
ACTGGGTGCGACAGGCTCCTGGAAAAGGGCTTGAGTGGATGGGAGGTTTTGAT
CCTGAAGATGGTGAAACGATCTACGCACAGAGGTTCCAGGGCAGGGTCACCAT
GACCGAGGACGCATCTACAGACACAGCCTACATGGAGCTGAGTAGCCTGAGAT
CTGAGGACACGGCCGTCTATTACTGTACAACGGTGGGATTTCCGGATTATTATG
ATAGTAGTGTTTATTTGCGGCACTTTGACTACTGGGGCCAGGGAACCCTGGTCA
CCGTCTCCTCAGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT
CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTAC
TTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT
GGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA
ATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCA
AGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC
T GC AC C AGGAC T GGCTGA AT GGC A AGGAGT AC A AGT GC A AGGT C TC C A AC A A
AGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAA
CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAA
GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:321 leader sequence of Light chain of MAD0102I15
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:322 variable domain of Light chain of MAD0102I15 GCCATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGA
GTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTAT
CAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTT
GCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCA
CTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAAC
AGCTTAATAGTTACCCCCGAACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
CGAACT
>SEQ ID NO:323 constant domain of Light chain of MAD0102I15
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO:324 complete sequence of Light chain of MAD0102I15
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGCCATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTG
GTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCA
CTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAA
TTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGT
CAACAGCTTAATAGTTACCCCCGAACTTTTGGCCAGGGGACCAAGCTGGAGAT
CAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA
GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG
AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCC
AGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG
CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC
GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG
AGAGTGTTAG
Sequence of the antibody herein identified as MAD0103J13 AMINO ACID SEQUENCES OF MAD0103J13: >SEQ ID NO:325 CDR 1 of variable domain of Heavy chain of MAD0103J13 GGTFSSYA
>SEQ ID NO:326 CDR 2 of variable domain of Heavy chain of MAD0103J13 LIPIFGTA
>SEQ ID NO:327 CDR 3 of variable domain of Heavy chain of MAD0103J13 CANFIGDGYNYEEDYMDVW
>SEQ ID NO:328 CDR 1 of variable domain of Light chain of MAD0103J13 QSVSSF
>SEQ ID NO:329 CDR 2 of variable domain of Light chain of MAD0103J13 DAS (Asp-Ala-Ser)
>SEQ ID NO:330 CDR 3 of variable domain of Light chain of MAD0103J13 CQQRSNWPPFTF
>SEQ ID NO:331 variable domain of Heavy chain of MAD0103J13 QVQLQESGAEVKKPGS SVKVSCKASGGTF S S YAINWVRQ APGQGLEWMGGLIPIF GTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCANFIGDGYNYEEDY MD VWGKGTT VT VS S AS
>SEQ ID NO:332 variable domain of Light chain of MAD0103J13 EIVMTQSPATLSLSPGERATLSCRASQSVSSFLAWYQQKPGQAPRLLIYDASNRAT GIPARF SGSGSGTDFTLTIS SLEPEDF AVYYCQQRSNWPPFTFGPGTKVDIKRT >SEQ ID NO:333 Heavy chain of MAD0103J13
MGWSCIILFL VAT ATGVHSQVQLQESGAEVKKPGS SVKVSCKASGGTF S SYAINW VRQ APGQGLEWMGGLIPIF GT ANY AQKF QGRVTIT ADEST ST AYMEL S SLRSEDT A VYYCANFIGDGYNYEEDYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKD YFPEP VT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTC VVVD V SHEDPEVKFNWYVDGVLVHNAKTKPREEQYNSTYRVVS VLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
>SEQ ID NO:334 Light chain of MAD0103J13
MGW S CIILFL V AT AT GVHSEIVMT Q SP ATL SL SPGER ATL S CR ASQ S V S SFL AW Y Q QKPGQAPRLLIYDASNRATGIPARF SGSGSGTDFTLTIS SLEPEDF AVYYCQQRSNW PPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTK SFNRGEC *
NUCLEIC ACID SEQUENCES OF MAD0103J13:
>SEQ ID NO:335 leader sequence of Heavy chain of MAD0103J13
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:336 variable domain of Heavy chain of MAD0103J13
C AGGTGC AGCTGC AGGAGTCGGGGGCTGAGGT GAAGAAGCCTGGGTCCTCGG
TGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAACT
GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGCTCATCCCT
ATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTAC
CGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTG
AGGACACGGCCGTGTATTACTGTGCGAATTTTATAGGGGATGGCTACAATTAC
GAGGAGGACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACTGTCTCCTC
AGCTTCC
>SEQ ID NO:337 constant domain of Heavy chain of MAD0103J13
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG
GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGA
CGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GT GGT GGT GGAC GT GAGC C AC GA AGACC C T GAGGT C A AGTT C A ACTGGT AC GT
GGAC GGCGT GGAGGT GC AT A AT GC C A AG AC A A AGC CGCGGGAGGAGC AGT AC
AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA
CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
>SEQ ID NO:338 complete sequence of Heavy chain of MAD0103J13
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCCAGGTGCAGCTGCAGGAGTCGGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCA
ACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGCTCATC
CCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGAT
TACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGAT
CTGAGGACACGGCCGTGTATTACTGTGCGAATTTTATAGGGGATGGCTACAAT
TACGAGGAGGACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACTGTCTC
CTCAGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAG
CACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCG
AACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC
TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAA
GCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAA
ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA
GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA
ACT GGT AC GT GG AC GGC GT GG AGGT GC AT A AT GC C A AG AC A A AGC C GC GGG A
GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT
CCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGT
CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT
GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT
GGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCA
GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA >SEQ ID NO:339 leader sequence of Light chain of MAD0103J13
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCC
>SEQ ID NO:340 variable domain of Light chain of MAD0103J13
GAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAG
AGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGCCTGGTA
CCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACA
GGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC
ACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAG
CAGCGTAGCAACTGGCCTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATAT
CAAACGAACT
>SEQ ID NO:341 constant domain of Light chain of MAD0103J13
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC
CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
>SEQ ID NO:342 complete sequence of Light chain of MAD0103J13
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACAT
TCCGAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAA
AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGCCTG
GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCA
AC AGGGCC ACTGGC ATCCC AGCC AGGTT C AGT GGC AGT GGGTCTGGGAC AGAC
TTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT
CAGCAGCGTAGCAACTGGCCTCCATTCACTTTCGGCCCTGGGACCAAAGTGGA
TATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC
C AGAGAGGCC AAAGT AC AGT GGAAGGT GGAT AACGCCCTCC AATCGGGT AAC
TCCC AGGAGAGT GT C AC AGAGC AGGAC AGC AAGGAC AGC ACCT AC AGCCTC A
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA
GGGGAGAGTGTTAG
>SEQ ID NO:343 epitope residues 477 to 489 in the RBD of the S-protein STPCNGVEGFNCY
>SEQ ID NO:344 surface glycoprotein [Severe acute respiratory syndrome coronavirus 2] QHD43416.1
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSN
VTWFHAIHV
SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNW IKVC
EFQFCNDPF
LGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYF
KIYSKHTPI
NLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQ
PRTFLLKYN
ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTES IVRFPNITNLCPFGEV FNATRFASV
YAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA
PGQTGKIAD
YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTE IYQAGSTPCN GVEGFNCYF
PLQSYGFQPTNGVGYQPYRW VLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGV
LTESNKKFL
PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVP
VAIHADQLT
PTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQS IIAYTMSLG
AENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCT
QLNRALTGI
AVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADA GFIKQYGDC
LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQ
MAYRFNGIG
VTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDW NQNAQALNTLVKQLSSNFG
AISSVLNDI
LSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVD
FCGKGYHLM
SFPQSAPHGW FLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYE
PQIITTDNT
FVSGNCDW IGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASW NIQK
EIDRLNEVA
KNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCG
SCCKFDEDD
SEPVLKGVKLHYT
Sequences of the S-protein Sequences of the surface glycoprotein of Severe acute respiratory syndrome coronavirus 2 are known in the art. Sequences of SI (“Spike rec bind”) and S2 (“Coronavirus S2 glycoprotein”) domains are in the GenBank with the ID QHD43416.1.

Claims

1. A human monoclonal antibody or antigen-binding portion thereof that specifically binds to a region of human severe acute respiratory syndrome (SARS) Corona Virus 2 (SARS- CoV-2) Spike (S) protein.
2. The human monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein said region is i) in the S 1 domain of S ARS-CoV-2 S-protein; or (ii) in the S2 domain of SARS-CoV-2 S-protein; or (iii) in the SARS-CoV-2 S-protein trimer in its pre-fusion conformation; or (iv) in the SARS-CoV-2 S-protein trimer in its post-fusion conformation or (v) in the receptor binding domain (RBD) of SARS-CoV-2 S-protein or in a combination thereof.
3. The human monoclonal antibody or antigen-binding portion thereof according to claim 1 or 2, wherein said region is in the receptor binding domain (RBD) of SARS-CoV-2 S- protein, preferably wherein said human monoclonal antibody or antigen-binding portion thereof specifically binds the receptor binding domain (RBD) of the spike protein of SARS- CoV-2 both in its up and down state, more preferably with a footprint of less than 1000 A.
4. The human monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 3, wherein said antibody or antigen-binding portion thereof binds to an epitope comprising the residues from 477 to 489 in the receptor binding domain (RBD) of SARS-CoV-2 S-protein.
5. The human monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 4, wherein said epitope comprising the sequence SEQ ID NO:343 or a sequence at least 80% identical to SEQ ID NO: 343.
6. The human monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 5, wherein said antibody or antigen-binding portion thereof providing equal or more than 25%inhibition of the binding between the human ACE2 receptor and the viral Spike (S) protein preferably more than 80%, more preferably more than 95%, as measured by the NOB assay.
7. The human monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 6, wherein said antibody or antigen-binding portion thereof showing 100% inhibitory concentration (ICIOO) of less than 100 ng/ml, preferably less than 10 ng/ml, when tested in an in vitro neutralization assay against the SARS-CoV-2 vims.
8. The human monoclonal antibody or an antigen-binding portion thereof according to claim 7, wherein said antibody or antigen-binding portion is tested in an in vitro neutralization assay against the SARS-CoV-2 vims wild type, the SARS-CoV-2 vims mutant E484K and/or the SARS-CoV- 2 vims mutant D614G and/or the escape mutant SARS-CoV-2 PT188-EM.
9. The human monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 8, which specifically binds to human severe acute respiratory syndrome (SARS) Corona Vims (SARS-CoV-2) S-protein with an affinity constant (KD) of equal to or less than about 1000 pM, preferably with a KD of equal to or less than about 100 pM, more preferably equal to or less than 10 pM, as measured by surface plasmon resonance (SPR).
10. The human monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 9, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH and VL comprise the following complementarity-determining regions (CDRs):
-CDR1 of VH having SEQ ID NO: 1,
-CDR2 of VH having SEQ ID NO:2,
-CDR3 of VH having SEQ ID NO:3,
-CDR1 of VL having SEQ ID NO:4,
-CDR2 of VL having the sequence DAS (Asp-Ala-Ser) and -CDR3 of VL having SEQ ID NO:6; or
-CDR1 of VH having SEQ ID NO: 19,
-CDR2 of VH having SEQ ID NO:20,
-CDR3 of VH having SEQ ID NO:21,
-CDR1 of VL having SEQ ID NO:22, -CDR2 of VL having the sequence GAS (Gly-Ala-Ser) and -CDR3 of VL having SEQ ID NO:24; or
-CDR1 of VH having SEQ ID NO:37,
-CDR2 of VH having SEQ ID NO:38,
-CDR3 of VH having SEQ ID NO:39,
-CDR1 of VL having SEQ ID NO:40,
-CDR2 of VL having the sequence AAS (Ala-Ala-Ser) and -CDR3 of VL having SEQ ID NO:42. or
-CDR1 of VH having SEQ ID NO:55,
-CDR2 of VH having SEQ ID NO: 56,
-CDR3 of VH having SEQ ID NO: 57,
-CDR1 of VL having SEQ IDNO:58,
-CDR2 of VL having the sequence GAS (Gly-Ala-Ser) and -CDR3 of VL having SEQ ID NO:60 or
-CDR1 of VH having SEQ ID NO:73,
-CDR2 of VH having SEQ ID NO:74,
-CDR3 of VH having SEQ ID NO:75,
-CDR1 of VL having SEQ ID NO:76,
-CDR2 of VL having the sequence AAS (Ala-Ala-Ser) and -CDR3 of VL having SEQ ID NO:78 or-CDRl of VH having SEQ ID NO:91,
-CDR2 of VH having SEQ ID NO:92,
-CDR3 of VH having SEQ ID NO:93,
-CDR1 of VL having SEQ ID NO:94,
-CDR2 of VL having the sequence GAS (Gly-Ala-Ser) and -CDR3 of VL having SEQ ID NO:96 or
-CDR1 of VH having SEQ ID NO: 109,
-CDR2 of VH having SEQ ID NO: 110, -CDR3 of VH having SEQ ID NO: 111,
-CDR1 of VL having SEQ ID NO: 112,
-CDR2 of VL having the sequence TLS (Thr-Leu-Ser) and -CDR3 of VL having SEQ ID NO: 114 or
-CDR1 of VH having SEQ ID NO: 127,
-CDR2 of VH having SEQ ID NO: 128,
-CDR3 of VH having SEQ ID NO: 129,
-CDR1 of VL having SEQ ID NO: 130,
-CDR2 of VL having the sequence KIS (Lys-Iso-Ser) and -CDR3 of VL having SEQ ID NO: 132 or
-CDR1 of VH having SEQ ID NO: 145,
-CDR2 of VH having SEQ ID NO: 146,
-CDR3 of VH having SEQ ID NO: 147,
-CDR1 of VL having SEQ ID NO: 148,
-CDR2 of VL having the sequence DAS (asp-ala-ser) and -CDR3 of VL having SEQ ID NO: 150 or
-CDR1 of VH having SEQ ID NO: 163,
-CDR2 of VH having SEQ ID NO: 164,
-CDR3 of VH having SEQ ID NO: 165,
-CDR1 of VL having SEQ ID NO: 166,
-CDR2 of VL having the sequence DAS (asp-ala-ser) and -CDR3 of VL having SEQ ID NO: 168 or
-CDR1 of VH having SEQ ID NO: 181,
-CDR2 of VH having SEQ ID NO: 182,
-CDR3 of VH having SEQ ID NO: 183,
-CDR1 of VL having SEQ ID NO: 184,
-CDR2 of VL having the sequence GAS (gly-ala-ser) and -CDR3 of VL having SEQ ID NO: 186 or
-CDR1 of VH having SEQ ID NO: 199,
-CDR2 of VH having SEQ ID NO:200,
-CDR3 of VH having SEQ ID NO:201,
-CDR1 of VL having SEQ IDNO:202,
-CDR2 of VL having the sequence GAS (gly-ala-ser) and -CDR3 of VL having SEQ ID NO:204 or
-CDR1 of VH having SEQ ID NO:217,
-CDR2 of VH having SEQ ID NO:218,
-CDR3 of VH having SEQ ID NO:219,
-CDR1 of VL having SEQ IDNO:220,
-CDR2 of VL having the sequence DAS (asp-ala-ser) and -CDR3 of VL having SEQ ID NO:222 or
-CDR1 of VH having SEQ ID NO:235,
-CDR2 of VH having SEQ ID NO:236,
-CDR3 of VH having SEQ ID NO:237,
-CDR1 of VL having SEQ IDNO:238,
-CDR2 of VL having the sequence AAS (ala-ala-ser) and -CDR3 of VL having SEQ ID NO:240 or
-CDR1 of VH having SEQ ID NO:253,
-CDR2 of VH having SEQ ID NO:254,
-CDR3 of VH having SEQ ID NO:255,
-CDR1 of VL having SEQ IDNO:256,
-CDR2 of VL having the sequence LAS (leu-ala-ser) and -CDR3 of VL having SEQ ID NO:258 or
-CDR1 of VH having SEQ ID NO:271,
-CDR2 of VH having SEQ ID NO:272,
-CDR3 of VH having SEQ ID NO:273, -CDR1 of VL having SEQ IDNO:274,
-CDR2 of VL having the sequence AAS (ala-ala-ser) and -CDR3 of VL having SEQ ID NO:276 or
-CDR1 of VH having SEQ ID NO:289,
-CDR2 of VH having SEQ ID NO:290,
-CDR3 of VH having SEQ ID NO:291,
-CDR1 of VL having SEQ IDNO:292,
-CDR2 of VL having the sequence AAS (ala-ala-ser) and -CDR3 of VL having SEQ ID NO:294 or
-CDR1 of VH having SEQ ID NO:307,
-CDR2 of VH having SEQ ID NO:308,
-CDR3 of VH having SEQ ID NO:309,
-CDR1 of VL having SEQ IDNO:310,
-CDR2 of VL having the sequence AAS (ala-ala-ser) and -CDR3 of VL having SEQ IDNO:312 or
-CDR1 of VH having SEQ ID NO:325,
-CDR2 of VH having SEQ ID NO:326,
-CDR3 of VH having SEQ ID NO:327,
-CDR1 of VL having SEQ IDNO:328,
-CDR2 of VL having the sequence DAS (asp-ala-ser) and -CDR3 of VL having SEQ IDNO:330.
11. The human monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 10, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH having SEQ ID NO:7 and said VL having SEQ ID NO:8; or wherein said VH having SEQ ID NO:25 and said VL having SEQ ID NO:26; or wherein said VH having SEQ ID NO:43 and said VL having SEQ ID NO:44 or wherein said VH having SEQ ID NO:61 and said VL having SEQ ID NO:62 or wherein said VH having SEQ ID NO:79 and said VL having SEQ ID NO:80 or wherein said VH having SEQ ID NO:97 and said VL having SEQ ID NO:98 or wherein said VH having SEQ ID NO: 115 and said VL having SEQ ID NO: 116 or wherein said VH having SEQ ID NO: 133 and said VL having SEQ ID NO: 134 or wherein said VH having SEQ ID NO: 151 and said VL having SEQ ID NO: 152 or wherein said VH having SEQ ID NO: 169 and said VL having SEQ ID NO: 170 or wherein said VH having SEQ ID NO: 187 and said VL having SEQ ID NO: 188 or wherein said VH having SEQ ID NO:205 and said VL having SEQ ID NO:206 or wherein said VH having SEQ ID NO:223 and said VL having SEQ ID NO:224 or wherein said VH having SEQ ID NO:241 and said VL having SEQ ID NO:242 or wherein said VH having SEQ ID NO:259 and said VL having SEQ ID NO:260 or wherein said VH having SEQ ID NO:277 and said VL having SEQ ID NO:278 or wherein said VH having SEQ ID NO:295 and said VL having SEQ ID NO:296 or wherein said VH having SEQ ID NO:313 and said VL having SEQ ID NO:314 or wherein said VH having SEQ ID NO:331 and said VL having SEQ ID NO:332.
12. The human monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 11, wherein said VL and said VH is at least 85% identical in amino acid sequence, preferably at least 95%, more preferably at least 99% of
VH having SEQ ID NO:7 and said VL having SEQ ID NO:8; or
VH having SEQ ID NO:25 and said VL having SEQ ID NO:26; or
VH having SEQ ID NO:43 and said VL having SEQ ID NO:44 or
VH having SEQ ID NO:61 and said VL having SEQ ID NO: 62 or
VH having SEQ ID NO:79 and said VL having SEQ ID NO:80 or
VH having SEQ ID NO: 97 and said VL having SEQ ID NO: 98 or
VH having SEQ ID NO: 115 and said VL having SEQ ID NO: 116 or
VH having SEQ ID NO: 133 and said VL having SEQ ID NO: 134 or
VH having SEQ ID NO: 151 and said VL having SEQ ID NO: 152 or
VH having SEQ ID NO: 169 and said VL having SEQ ID NO: 170 or
VH having SEQ ID NO: 187 and said VL having SEQ ID NO: 188 or
VH having SEQ ID NO:205 and said VL having SEQ ID NO:206 or
VH having SEQ ID NO:223 and said VL having SEQ ID NO:224 or VH having SEQ ID NO:241 and said VL having SEQ ID NO:242 or VH having SEQ ID NO:259 and said VL having SEQ ID NO:260 or VH having SEQ ID NO:277 and said VL having SEQ ID NO:278 or VH having SEQ ID NO:295 and said VL having SEQ ID NO:296 or VH having SEQ ID NO:313 and said VL having SEQ ID NO:314 or VH having SEQ ID NO:331 and said VL having SEQ ID NO:332.
13. The human monoclonal antibody according to any one of the claims from 1 to 12, wherein the heavy chain of said antibody having SEQ ID NO:9 and the light chain of said antibody having SEQ ID NO: 10; or the heavy chain of said antibody having SEQ ID NO:27 and the light chain of said antibody having SEQ ID NO:28 ,or the heavy chain of said antibody having SEQ ID NO:45 and the light chain of said antibody having SEQ ID NO:46,or the heavy chain of said antibody having SEQ ID NO:63 and the light chain of said antibody having SEQ ID NO: 64, or the heavy chain of said antibody having SEQ ID NO:81 and the light chain of said antibody having SEQ ID NO:82, or the heavy chain of said antibody having SEQ ID NO:99 and the light chain of said antibody having SEQ ID NO: 100 ,or the heavy chain of said antibody having SEQ ID NO: 117 and the light chain of said antibody having SEQ ID NO: 118 ,or the heavy chain of said antibody having SEQ ID NO: 135 and the light chain of said antibody having SEQ ID NO: 136 ,or the heavy chain of said antibody having SEQ ID NO: 153 and the light chain of said antibody having SEQ ID NO: 154 ,or the heavy chain of said antibody having SEQ ID NO: 171 and the light chain of said antibody having SEQ ID NO: 172 ,or the heavy chain of said antibody having SEQ ID NO: 189 and the light chain of said antibody having SEQ ID NO: 190 ,or the heavy chain of said antibody having SEQ ID NO:207 and the light chain of said antibody having SEQ ID NO:208 ,or the heavy chain of said antibody having SEQ ID NO:225 and the light chain of said antibody having SEQ ID NO:226,or the heavy chain of said antibody having SEQ ID NO:243 and the light chain of said antibody having SEQ ID NO:244,OG the heavy chain of said antibody having SEQ ID NO:261 and the light chain of said antibody having SEQ ID NO:262,or the heavy chain of said antibody having SEQ ID NO:279 and the light chain of said antibody having SEQ ID NO:280 or the heavy chain of said antibody having SEQ ID NO:297 and the light chain of said antibody having SEQ ID NO:298, or the heavy chain of said antibody having SEQ ID NO:315 and the light chain of said antibody having SEQ ID NO:316. or the heavy chain of said antibody having SEQ ID NO:333 and the light chain of said antibody having SEQ ID NO:334.
14. The human monoclonal antibody according to any one of the claims from 1 to 13, wherein the IgGl constant region backbone contains one or more of the following mutations L234A, L235A, P329G, M428L, N434S.
15. The human monoclonal antibody according to any one of the claims from 1 to 14, wherein the IgGl constant region backbone contains the following mutations L234A, L235A, P329G, M428L, N434S.
16. A human monoclonal antibody or an antigen-binding portion thereof that compete for the binding to Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike (S) protein with any one of the antibody or antigen-binding portion according to any one of the claims 1 to 15.
17. The human monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16 for use in a prophylactic or therapeutic treatment of a virus infection or conditions or disorders resulting from such infection, in particular for use in the prevention and/or treatment of a coronavirus infection, in particular SARS-CoV-2.
18. The human monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16, for use in a prophylactic or therapeutic treatment of the SARS- CoV-2 infection or conditions or disorders resulting from such infection, in particular Coronavirus disease 2019 (COVID-19).
19. The human monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16, for use in a method for prophylactic or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, wherein said method comprising the step of administering to a patient between 0.025 to 5 mg/kg of said human monoclonal antibody or an antigen-binding portion,
20. The human monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16, for use in a method for prophylactic or therapeutic treatment of the SARS-CoV-2 infection or conditions or disorders resulting from such infection, wherein said method comprising the step of administering to a patient between 0.1 to 3 mg/kg of said human monoclonal antibody or an antigen-binding portion, preferably 0,25 mg/kg.
21 The human monoclonal antibody or an antigen-binding portion thereof for use according to any claims from 17 to 21wherein said human monoclonal antibody or an antigen-binding portion thereof is administered by intravenous, subcutaneous, intraperitoneal or intramuscular route.
22. The human monoclonal antibody or an antigen-binding portion thereof for use according to any one of the claims from 17 to 21, wherein said method comprising the step of administering to a patient said human monoclonal antibody or an antigen-binding portion once a day for at least 3 days.
23. A pharmaceutical composition comprising one or more human monoclonal antibody or antigen-binding portion thereof according to any one of the claims from 1 to 16 and a pharmaceutically acceptable carrier.
24. The pharmaceutical composition according to claim 23 comprising equal or less than 400 mg for unit dosage of said human monoclonal antibody or antigen-binding portion thereof.
25. The pharmaceutical composition according to claim 23 comprising equal or less than 100 mg for unit dosage of said human monoclonal antibody or antigen-binding portion thereof.
26. The pharmaceutical composition according to any one of the claims from 23 to 25 for intravenous, subcutaneous, intraperitoneal or intramuscular administration.
27. The composition according to any one of the claims from 23 to 26, for use in the prevention and/or treatment of a SARS-CoV-2 infection.
28. An isolated cell line that produces the antibody or antigen-binding portion thereof according to any one of the claims from 1 to 16.
29. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes the antibody or antigen-binding portion thereof according to any one of the claims from 1 to 16.
30. A vector comprising the nucleic acid molecule according to claim 29, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule.
31. The vector according to claim 30, wherein said vector is a selected from RNA vims vectors, DNA vims vectors, plasmid viral vectors, adenovims vectors, adenovims associated vims vectors, herpes vims vectors and retrovims vectors.
32. A composition, comprising an isolated nucleic acid molecule according to claim 29 or a vector according to claim 30 or 31 for use in the prevention and/or treatment of a SARS-CoV-2 infection.
33. The composition according to claim 32 wherein said nucleic acid molecule or said vector is formulated in a lipid nanoparticle.
34. A host cell comprising the vector according to claim 30 or the nucleic acid molecule according to claim 29.
35. A non-human transgenic animal or transgenic plant comprising the nucleic acid according to claim 29, wherein the non-human transgenic animal or transgenic plant expresses said nucleic acid.
36. Use of the human monoclonal antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16 in the diagnosis of the SARS-CoV-2 infection.
37. An in vitro method for revealing the presence of SARS-CoV-2 in a sample comprising the following steps: i) Contacting the antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16 with said sample; ii) Detecting the binding of said antibody or an antigen-binding portion thereof with the S-protein of the SARS-CoV-2.
38. An in vitro method for the diagnosis of the SARS-CoV-2 infection in a subject comprising the following steps: i) Contacting the antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16 with a biological sample of said subject; ii) Detecting the binding of said antibody or an antigen-binding portion thereof with the S-protein of the SARS-CoV-2.
39. The method in vitro according to claim 37 or 38, wherein said method is immunoassay selected from ELISA, RIA, flow cytometry, tissue immunohistochemistry, Western blot (immunoblot), immunoprecipitation or any other equivalent assays.
40. An in vitro method according to claims 37 to 39, wherein said antibody or an antigen-binding portion thereof is directly labelled with a detectable label or wherein said antibody or an antigen-binding portion thereof (the first antibody) is bound to a second labelled antibody or to another labelled molecule.
41. A diagnostic kit comprising as a specific reagent an antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16, said kit being intended in particular for use in a method for detecting or quantifying, in a biological sample from a patient, anti- coronavirus antibodies and/or the coronavirus S-protein, in particular anti-Sars-Cov-2 antibodies and/or Sars-Cov-2 S-protein.
42. Use of an antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16 for the design of a vaccine against a Coronavirus.
43. Use of an antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16 for the design of a vaccine against Sars-Cov-2.
44. A mimotope specifically directed against the idiotype of an antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16.
45. An anti -idiotype antibody that is specifically directed against the idiotype of an antibody or an antigen-binding portion thereof according to any one of the claims from 1 to 16.
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