WO2022198314A1

WO2022198314A1 - Antibodies binding to o-linked carbohydrates on sars-cov-2 spike protein and uses thereof

Info

Publication number: WO2022198314A1
Application number: PCT/CA2022/050426
Authority: WO
Inventors: Serge Moffett; Ba Vuong LE
Original assignee: Koranex Capital
Priority date: 2021-03-23
Filing date: 2022-03-22
Publication date: 2022-09-29

Abstract

A protein complex comprising a SARS-CoV-2 S protein or fragment thereof, expressing one or more carbohydrate antigens, bound to a recombinant ligand having binding specificity for said carbohydrate antigen, is described herein. The carbohydrate antigen may be an O-linked carbohydrate antigen, such as unsialylated Thomsen-Friedenreich (TF) antigen, sialylated TF antigen, unsialylated Tn antigen, sialylated Tn antigen, or any combination thereof. The ligand may be a recombinant antibody or an antigen-binding fragment thereof. Also described herein is the use of such a recombinant ligand for binding to a SARS-CoV-2 S protein or fragment thereof, for treating or reducing the risk of SARS-CoV-2 viral infection in a subject.

Description

ANTI-CARBOHYDRATE ANTIGEN LIGANDS FOR INHIBITING BINDING OF SARS-CoV-2

SPIKE PROTEIN TO ACE2 RECEPTOR

The present description relates to the use of anti-carbohydrate antigen ligands for binding to coronavirus spike proteins. More specifically, the present description relates to the use of anti -carbohydrate antigen ligands, such as recombinant antibodies and antigen-binding fragments thereof, as potential neutralizing agents to inhibit the binding of coronavirus spike proteins to the ACE2 receptor.

The present description refers to a plurality of documents, the contents of which are herein incorporated by reference in their entirety.

BACKGROUND

SARS-CoV-2, the causative agent of the COVID-19 pandemic that began in late 2019, represents an ongoing threat to global human health that has also crippled global economies. Initial vaccine and therapeutic development efforts have largely focused on protein antigens and epitopes present on the spike (S) glycoprotein, which mediates cell entry and membrane fusion of SARS-CoV-2 into host cells. However, global health experts have strongly recommended that scientists explore different strategies in parallel for developing therapeutic interventions against SARS-CoV-2 to mitigate against potential failures or complications that may arise for a single strategy. Furthermore, novel variants of SARS-CoV-2 have been shown to be more resistant to neutralizing antibodies directed at protein antigens/epitopes within the S protein, raising concerns that the virus may be evolving in a manner that renders less effective vaccines and therapeutics directed at non-conserved regions of the S protein. Thus, there remains a need for developing therapeutic tools against SARS-CoV-2 in parallel to those focused on the protein antigens present on the S protein of SARS-CoV-2.

SUMMARY

In a first aspect, described herein is a protein complex comprising a SARS-CoV-2 S protein or fragment thereof, expressing one or more carbohydrate antigens, bound to a recombinant ligand having binding specificity for said carbohydrate antigen. In embodiments, the ligand comprises or consists of a recombinant antibody or an antigen-binding fragment thereof (e.g., single chain variable fragment (scFv), Fab, Fab’, F(ab’)2, minibody, diabody, triabody, or tetrabody). In embodiments, the carbohydrate antigen is an O-linked carbohydrate antigen, such as unsialylated Thomsen-Friedenreich (TF) antigen, sialylated TF antigen, unsialylated Tn antigen, sialylated Tn antigen, or any combination thereof.

In a further aspect, described herein is the use of a ligand as defined herein for binding to a SARS- CoV-2 S protein or fragment thereof, expressing the one or more carbohydrate antigens. In a further aspect, described herein is a method of treating or reducing the risk of SARS-CoV-2 viral infection in a subject, the method comprising administering to the subject one or more ligands as defined herein.

General Definitions

Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.

The term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

Fig. 1 shows examples of typical chemical structures of therapeutically relevant carbohydrate antigen.

Fig. 2 shows the relative abundance of all O-glycosylated forms of peptide VQPTESIVR on recombinant S 1 protein of SARS-CoV-2, analyzed by high-resolution LC-MS/MS on proteins over 75 kDa. Fig. 3 shows the relative abundance of all O-glycosylated forms of peptide VQPTESIVR on recombinant S 1 protein of SARS-CoV-2, analyzed by high-resolution LC-MS/MS on proteins between 75- 100 kDa.

Fig. 4 shows the reactivity by ELISA of anti-TF (JAA-F11 IgG and SPM320 IgM) and anti-Tn (Tn218 IgM) monoclonal antibodies to culture supernatant from mammalian cells transfected with either SARS-CoV-2 SI protein or full-length S protein. Results are shown as fold increases over the same monoclonal antibodies exposed to culture supernatant from corresponding mammalian cells transfected with empty vector.

Fig. 5 shows the reactivity of a panel of lectins to culture supernatant from mammalian cells transfected with SARS-CoV-2 SI protein or full-length S protein. Results are shown as fold increases over the same monoclonal antibodies exposed to culture supernatant from corresponding mammalian cells transfected with empty vector.

Fig. 6A and 6B show the ability of diluted crude immune sera from mice previously immunized with TF-dTT (A1 and A2), but not a commercially available monoclonal anti-TF antibody (JAA-F11 mAh), to inhibit the binding of SARS-CoV-2 SI to the hACE2 receptor as evaluated by ELISA. Two different concentrations of hACE2 receptor were employed (15.625: Fig. 6A; and 31.25 ng/mL, Fig. 6B). Included as a control was a rabbit polyclonal antibody raised against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (anti-Sl-RBD pAb). Error bars represent ± S.E.M (n = 6-10).

Fig. 7A and 7B show the ability of a single-chain variable fragment (scFv) recombinant anti-TF antibody to inhibit the binding of SARS-CoV-2 SI to the hACE2 receptor in a dose-dependent manner. Two different types of recombinant SI proteins were employed in the ELISA assays (biotinylated SI, Fig.7A; and non-biotinylated SI, Fig. 7B). Rabbit polyclonal antibody raised against the RBD of the SARS- CoV-2 spike protein (anti-Sl-RBD pAb) was included as a positive control. Error bars represent ± S.E.M (n = 4).

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form created March 22, 2022 having a size of about 12 Kb. The computer readable form is incorporated herein by reference.

DETAILED DESCRIPTION

In some aspects, the present description relates to the discovery that certain O-linked carbohydrate antigens found on recombinantly-expressed SARS-CoV-2 spike (S) protein and/or the SI fragment thereof are accessible to binding by antibodies and other ligands having specificity of such carbohydrate antigens. In some embodiments, binding of recombinant antibodies and other ligands to the O-linked carbohydrate antigens may inhibit the ability of the spike protein to bind to the ACE2 receptor of host cells, thereby precluding spike protein-mediated entry of the SARS-CoV-2 virion into the host cells.

In some aspects, described herein is a protein complex comprising a SARS-CoV-2 S protein or fragment thereof, expressing one or more carbohydrate antigens, bound to a recombinant ligand having binding specificity for said carbohydrate antigen. In some embodiments, the one or more carbohydrate antigens may comprise an O-linked carbohydrate antigen. In some embodiments, the carbohydrate antigens may comprise or consist of unsialylated Thomsen-Friedenreich (TF) antigen, sialylated TF antigen, unsialylated Tn antigen, sialylated Tn antigen, or any combination thereof. The structures of some of these therapeutically relevant carbohydrate antigens is shown in Fig. 1.

In some embodiments, the carbohydrate antigens comprise a monosialylated TF antigen such as (2,3)-S-TF, and/or disialylated TF antigen such as disialyl core 1. These carbohydrate antigens were detected on recombinantly-expressed SARS-CoV-2 spike (S) protein and/or the SI fragment thereof at positions corresponding to positions 4 and/or 6 of the peptide fragment VQPTESIVRby quantitative high resolution mass spectrometry (Example 2; Figs. 2 and 3). Furthermore, the results shown in Example 3 and Figs. 4 and 5 demonstrate that at least some of these carbohydrate antigens are available to ligand binding (e.g., with lectins and/or antibodies), and the results shown in Examples 4, 5 and Figs. 6 and 7 demonstrate that anti-TF ligands have the ability to inhibit binding of the spike protein to the hACE2 receptor. These results were unforeseeable, given the multiple reports that the SARS-CoV-2 S protein being excessively shielded largely by N-linked glycans and that O-linked glycans (if detected, as reports are conflicting) represent a minor component to the overall glycosylation profile of the S protein of SARS- CoV-2 that may not be accessible for ligand binding in the context of a pathogenic virion particle (W atanabe et ah, 2020; Shajahan et ah, 2020; Grant et al, 2020).

In some embodiments, the ligands described herein may be bound to O-linked carbohydrate antigens at positions T323, S325, and/or T678 of the SARS-CoV-2 spike protein. Residues T323 are S325 are predicted to be relatively close to the spike-ACE2 docking site. Furthermore, residue T678 (which is close to the furin cleavage site of the spike protein at R682) has been reported to be O-glycosylated by core- 1 and core-2 structures. In some embodiments, the ligands described herein may be bound to one or more of the nine O-linked glycopeptides on the spike protein identified in Sanda et al., 2021.

In some embodiments, the anti-carbohydrate ligands described herein may comprise or consist of a recombinant antibody or an antigen-binding fragment thereof. In some embodiments, the anti carbohydrate ligands described herein may comprise or consist of a single chain variable fragment (scFv), Fab, Fab’, F(ab’)2, minibody, diabody, triabody, or tetrabody. In some embodiments, the anti-carbohydrate ligands described herein may comprise one or more of the CDRs of the monoclonal antibody of JAA-F11 or its humanized form (hJAA-Fl 1). In some embodiments, the anti-carbohydrate ligands described herein may comprise a detectable or functional label.

In some embodiments, the protein complex described herein may be an in vitro protein complex. In some embodiments, the protein complex described herein may be an in vivo protein complex.

In some aspects, described herein is the use of ligand as defined herein for binding to a SARS- CoV-2 S protein or fragment thereof, expressing the one or more carbohydrate antigens.

In some aspects, described herein is a method of treating or reducing the risk of SARS-CoV-2 viral infection in a subject, the method comprising administering to the subject one or more ligands as defined herein. In some embodiments, the subject is an animal, a mammal, preferably a human.

EXAMPLES Example 1: Methods

All methods were performed as described in US 17/025,978 (US patent no. 10,973,910) unless otherwise indicated herein.

Generation of the dTT-TF immunogen: The immunogen tetanus toxoid conjugated to TF (dTT-TF) was generated by incubating 20 nmols of dTT with 1000 nmols of COOH-TF and 2000 nmols of N-(3- dimethylaminopropyl)-N’ -ethyl -carbodiimide (EDC, Sigma- Aldrich) and N-hydroxysuccinimide (NHS, Sigma-Aldrich) in 500 pL of PBS pH 8 for 2 hours at room temperature under gentle agitation. The dTT- TF conjugate was then buffer exchanged to PBS pH 7.4 using a centrifugal filtration device with a membrane cut-off of 10 kDa and aliquoted at 4 mg/mL and stored frozen at -20 °C. The successful conjugation of TF to dTT was validated by ELISA and Western blotting using the TF-binding lectin PNA conjugated to horseradish peroxidase (PNA-hrp), as described in Example 14 and Fig. 17 of US 17/025978. The analysis of the dTT-TF by MALDI-TOFF mass spectrometry revealed that dTT gained an average mass of 3352.8 Da upon conjugation, indicating a molar ratio of TF conjugation of about 6.5: 1 relative to dTT.

Generation of immune serums against TF glycoantigen: Female BALB/c mice were immunized by intramuscular injection with dTT-TF immunogen emulsified in an adjuvant over a 2-month period as described in Example 15 of US 17/025978. The serums of immunized mice from two groups, A1 and A2, were confirmed for the presence of anti-TF antibodies (see Example 15 and Figs. 18 and 19 of US 17/025978). SARS-CoV-2 Sl/hACE2 binding assay. Assay procedure was adapted from the method previously described by Byrnes et al., 2020, as well as the supplier’s instructions (Sino Biological US Inc, Wayne, PA, USA). All assays were performed in 96-well Nunc MaxiSorp™ flat-bottom plates (Thermo Fisher Scientific, Burlington, ON, Canada) and each sample was run in duplicate. First, plate was coated with 50 pU of 0.5 pg/mL NeutrAvidin™ (Thermo Fisher Scientific) mixed in PBS (Wisent Inc., Saint-Jean- Baptiste, QC, Canada) for 60 min at room temperature. Plates were then washed three times with PBS containing 0.05% Tween™ 20 (PBS-T) and were washed similarly for each of the following steps. Next,

50 pU of 0.5 pg/mU biotinylated recombinant SARS-CoV-2 spike SI protein (Bioss Antibodies, Woburn, MA, USA) mixed in PBS was added to each well and allowed to bind for 30 min at room temperature. In the case of recombinant SARS-CoV-2 spike Sl-His recombinant protein (Sino Biological US Inc.), 100 pU of 0.5 pg/mU antigen was added directly into wells without precoating step with NeutrAvidin and incubated overnight at 4 °C. After washing, the plate was blocked with 300 pU per well of 1% BSA (Bio Basic Inc, Markham, ON, Canada) mixed in PBS-T for 60 min at room temperature. Then the plate was washed and incubated with: 10 pg/mU of mouse anti-TF monoclonal antibody JAA-F11; mouse anti-TF recombinant antibody scFv fragment (clone JAA-F11; Cat No.: PSBW-164; Creative Biolabs, Shirley, NY, USA) at different concentrations of 0, 5, 10, 20, and 40 pg/mU; rabbit polyclonal antibody raised against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (anti-RBD pAb; Sino Biological US Inc.) diluted at 1:500 in PBS-T; or immune serums from mice Al or A2 diluted 1: 100 in PBS. The incubation was performed for 60 min at room temperature before 100 pU per well of 31.25 ng/mU of human ACE2 hFc tag recombinant protein (hACE2; Sino Biological US Inc.) was added. The washing step was carried out after incubation for 60 min at room temperature and followed by a color development using 1-Step Ultra TMB- ELISA (Thermo Fisher Scientific) (100 pL/well) for 15 min at room temperature. The reactions were quenched with 0.5 N sulfuric acid (100 pL/well) and absorbance was measured at 450 nm using Biotek Synergy 4 microplate reader (Winooski, VT, USA). The OD450 values were normalized to that of the negative control (vehicle alone) and expressed as percentage of binding between the SARS-CoV-2 SI protein and hACE2.

Example 2: Quantitative O-glycosylation profile of recombinant SARS-CoV-2 spike protein subunit

51 by high resolution Mass spectrometry

SI protein preparation

27 pL of recombinant SARS-CoV-2 SI subunit with C-terminal His-tag from serum-free cell culture supernatant of transfected HEK293 cells (RayBiotech, USA) in reducing sample buffer was separated by electrophoresis on a 10% SDS-PAGE. The gel was stained with Coomassie blue G-250. The section of lane corresponding to 75 kDa and higher was cut into 10 pieces under a clean bench and each piece cut further into 1 mm³ pieces.

Gel pieces preparation

Gel pieces were first washed with water for 5 min and destained twice with a destaining buffer (100 mM sodium thiosulfate, 30 mM potassium ferricyanide) for 15 min. An extra wash of 5 min was performed after destaining with an ammonium bicarbonate buffer (50 mM). Gel pieces were then dehydrated with acetonitrile (ACN). Protein cysteine disulfide groups were reduced by adding the reduction buffer (10 mM Dithiothreiol (DTT), 100 mM ammonium bicarbonate) for 30 min at 40°C. The generated free-sulfhydryl groups were then alkylated to S-carboxyamidomethyl by adding the alkylation buffer (55 mM iodoacetamide, 100 mM ammonium bicarbonate) for 20 min in the dark at 40 °C. Gel pieces were then dehydrated and washed at 40°C by adding acetonitrile for 5 min before discarding all of the reagents.

Proteolytic digestion and peptides extraction steps

Gel pieces were dried for 5 min at 40 °C and then re-hydrated at 4 °C for 40 min with a trypsin solution (6 ng/pL of trypsin [sequencing grade] from Promega, 25 mM ammonium bicarbonate). Protein digestion was performed at 58 °C for 1 h and stopped by adding 15 pL of 1% formic acid/2% acetonitrile. Supernatant was then transferred into a 96-well plate and peptides extraction was performed with two 30- min extraction steps at room temperature using the extraction buffer (1% formic acid/50% ACN). All peptide extracts were completely dried in a vacuum centrifuge.

LC-MS/MS analysis

Prior to LC-MS/MS, protein digests were re-solubilized under agitation for 15 min in 10 pL of 1% ACN / 0.5% formic acid. A 15 cm long, 75 pm i.d. Self-Pack PicoFrit™ fused silica capillary column (New Objective, Woburn, MA) was packed with C18 Jupiter™ (5 pm, 300 A) reverse-phase material (Phenomenex, Torrance, CA). This column was installed on the Easy-nLC™ II system (Proxeon Biosystems, Odense, Denmark) and coupled to the Orbitrap Fusion™ (Thermo-Fisher Scientific, Bremen, Germany) equipped with a Nanospray Flex™ Ion Source (Thermo-Fisher Scientific). The buffers used for chromatography were 0.2% formic acid (buffer A) and 100% acetonitrile/0.2% formic acid (buffer B). Peptides were eluted with a two-slope gradient at a flowrate of 250 nL/min. Solvent B first increased from 1 to 35% in 75 min and then from 35 to 86% in 15 min. Nanospray and S-lens voltages were set to 1.3-1.7 kV and 50 V, respectively. Capillary temperature was set to 225°C. Full scan MS survey spectra (360-1560 m/z) in profile mode were acquired in the Orbitrap with a resolution of 120 000 with a target value set at 8e5. A cycle time of 3 seconds was used for the data dependent MS/MS analysis, where the selected precursor ions were fragmented in the HCD (Higher-energy C-trap dissociation) collision cell and analyzed in the Orbitrap with the resolution set at 30 000, the target value at 7e4 and a normalized collision energy at 28 V. A subsequent MS/MS analysis using CID (Collision Induced Dissociation) was performed in the Orbitrap upon detection of oxonium ions. An inclusion list was also used for all know forms of peptide 320-328 of SARS-Cov-2. A second series of analysis was performed on these peptides by using a SIM (Single Ion Monitoring) and targeted MS2 method.

Data analysis

Protein database searches were performed with Mascot 2.6 (Matrix Science) against the Uniprot protein database (2017-04-11). The mass tolerances for precursor and fragment ions were set to 10 ppm and 0.6 Da, respectively. The enzyme specified was trypsin and one missed cleavage was allowed. Cysteine carbamidomethylation was specified as a fixed modification and methionine oxidation as variable modification. A second series of searches was performed against the SARS-CoV-2 sequence using Mascot 2.6 and Byos 3.8 (Protein Metrics). The enzyme specified was semi-trypsin and one missed cleavage was allowed. Cysteine carbamidomethylation was specified as a fixed modification. Methionine oxidation and all known O-glycosylated forms of peptide 320-328 were used as variable modifications.

Fig. 2 shows the quantitative O-glycosylation profile of peptide VQPTESIVR (SEQ ID NO: 1) from SARS-CoV-2 SI protein characterized by high-resolution LC-MS/MS of proteins over 75 kDa. Fig. 3 shows the same analysis done by high-resolution LC-MS/MS of proteins between 75-100 kDa. The symbols nomenclature for graphical representation of individual glycans are as follows (Varki et ah, 2015): yellow square = GalNAc, yellow circle = Gal, purple diamond = Neu5Ac (sialic acid). TF is formed by the di saccharide Gal-GalNAc. The figures show a different pattern of glycosylated-peptide with the predominant species being di-sialyl-TF, and the second most abundant being TF, amongst recombinant SI protein fragments containing O-linked glycosylation (only about 5-10% of the total SI peptides were O- glycosylated).

Example 3: Reactivity of anti-Tn and anti-TF ligands to recombinant SARS-CoV-2 SI and S proteins by ELISA and Western blot

Wells of a 96-well plate were coated for lh with 100pL PBS pH 7.4 containing 10 pL of serum- free culture supernatant of mammalian cells transfected with either empty vector or with DNA encoding either recombinant SARS-CoV-2 SI (SI subdomain of spike protein; RayBiotech, USA, Cat. No. 230- 20407) or S (full length spike protein; NRC, Canada) with a C-terminal His-tag. Wells were then washed with PBS-Tween 0.05% and blocked with PBS-T 0.05% + 1% BSA for 30 minutes. The wells were then washed with PBS-T 0.05% and further incubated for lh with the indicated anti-TF (JAA-F 11 IgG, 1 pg/mL; SPM320 IgM, Abnova, Cat. No. MAB13207, 0.2 mg/mL, 1: 100 dilution) or anti-Tn (Tn218 IgM, Abnova, Cat. No. MAB6198, 1: 100 dilution) antibodies in PBS-T 0.01% followed by incubating 30 minutes with their appropriate HRP-conjugated secondary antibodies (goat anti-mouse IgG-HRP or goat anti-mouse IgM-HRP; Jackson Immuno, 1/1000) in PBS-T. The binding of the ligands was revealed with the HRP colorimetric substrate ultra-TMB (Thermo). The reaction was stopped by adding an equivalent volume of 0.5M sulfuric acid and the optical density was measured at 450nm on a plate reader (Biotek). Results shown in Fig. 4 are the fold increase of the OD450 with supernatant from HEK cells expressing the recombinant S 1 or S proteins over the OD450 of the supernatant from HEK cells transfected with the empty vector, for each antibody. The fold increases shown represent the means of at least six separate experiments and the error bars represent standard error of the means. The experiment was also repeated using recombinant SI protein expressed from insect (Sf9) cells (Sl-His, SinoBiological, Cat, No. 40591-V08B1) and the results yielded mean fold increases of 3.32 ± 1.35 with JAA-F11, 5.50 ± 1.80 with SPM320, and 1.77 ± 0.64 with Tn218 (mean of three experiments ± SEM).

A similar ELISA experiment as above was performed with a panel of HRP-conjugated lectins using the serum -free culture supernatants of HEK293 cells transfected with empty vector, DNA encoding the SI protein, or DNA encoding the full-length S protein of SARS-CoV-2 (see above). The lectin panel included lectins from: Arachis hypogaea (PNA; Cat. No: H-2301-1), Vicia villosa (VVA; Cat. No: H-4601-1), Salvia sclarea (SSA; Cat. No: H-3501-1), Maackia amurensis (MAA; Cat. No: H-7801-1), Maclura pomifera (MPA; Cat. No: H-3901-1). All lectins were purchased from EY Laboratories (CA, USA; 1 mg/mL, HRP- conjugated) and used at 10 pg/mL in PBS-Tween 0.01%. Results shown in Fig. 5 are the fold increase of the OD450 with supernatant from HEK cells expressing the recombinant SI or S proteins over the OD450 of the supernatant from HEK cells transfected with the empty vector, for each lectin. The fold increases shown represent the means of at least seven separate experiments and the error bars represent standard error of the means. The experiment was also repeated using recombinant SI protein expressed from insect (Sf9) cells (Sl-His, SinoBiological, Cat, No. 40591-V08B1) and the results yielded mean fold increases of 22.67 ± 12.52 with PNA, 22.51 ± 9.71 with VVA, 3.97 ± 1.09 with SSA, 4.30 ± 1.23 with MAA, and 7.51 ± 3.64 with MPA.

For Western blotting, 2 pL of serum-free culture supernatant of HEK293 cells transfected with DNA encoding SARS-CoV-2 SI with C-terminal His-tag in reducing sample buffer was separated by SDS- PAGE on a 10% polyacrylamide gel. The proteins were then transferred to a PVDF membrane for analysis by Western blot with anti-glycan lectins, antibodies, and sera. The reactivity of the anti-glycans was compared to that of an anti -His tag antibody in corresponding conditions as a control. Bands detected with PNA, VVA, and the JAA-F11 mAb were similar to those detected with the control anti-His-Tag Ab, suggesting that they recognize the same SI protein (data not shown). The mouse A1 immune serum (Example 15 of US 17/025,978, now US patent no. 10,973,910) also detected SI strongly as compared to a pool of pre-immune serum.

Collectively, the results in this Example consistently show that the carbohydrate antigens Tn and TF are present on recombinant SARS-CoV-2 SI and S proteins produced by different expression systems, and that these antigens are accessible for binding by anti-Tn and anti-TF ligands.

Example 4: Polyclonal anti-TF antibodies inhibit SARS-CoV-2 SI to hACE2

The ability of anti-TF antibodies to inhibit the binding of SARS-CoV-2 SI to the hACE2 receptor was evaluated by ELISA as described in Example 1. Two different concentrations of hACE2 receptor were employed (15.625 and 31.25 ng/mL) and results are shown in the Table below and in Fig. 6A and 6B. Included as a control was a rabbit polyclonal antibody raised against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (anti-RBD pAb), the latter of which has been shown to possess neutralizing activity against SARS-CoV-2.

The results in the Table above and in Fig. 6A and 6B show that a polyclonal antibody raised against the receptor binding domain (RBD) of the SARS-CoV-2 SI protein inhibited binding between the SI protein and hACE2, which was consistent with the neutralizing activity associated with this antibody reported by the manufacturer. Interestingly, diluted crude immune sera from mice immunized with TF-dTT (A1 and A2) exhibited up to 30% inhibition of Sl/hACE2 binding, but a commercially available monoclonal anti-TF antibody (JAA-F11) did not exhibit any such inhibition at the tested concentrations. These results suggest that immune sera from A1 and A2 mice contain antibody species that interfere with the spike-hACE2 binding, potentially either by direct steric hindrance and/or by inducing the spike protein to adopt a conformation that precludes hACE2 binding.

Example 5: Recombinant anti-TF scFv antibody inhibits SARS-CoV-2 SI to hACE2

The ability of a single-chain variable fragment (scFv) recombinant anti-TF antibody to inhibit the binding of SARS-CoV-2 SI to the hACE2 receptor was evaluated by ELISA as described in Example 1. Two different types of recombinant SI proteins were employed in the ELISA assays (biotinylated SI and non-biotinylated SI) and results are shown in the Table below and in Fig. 7A and 7B. Rabbit polyclonal antibody raised against the RBD of the SARS-CoV-2 spike protein (anti -RBD pAb) was included as a positive control.

The results in the Table above and in Fig. 7A and 7B show that a single-chain variable fragment (scFv) recombinant anti-TF antibody was able to inhibit the binding of SARS-CoV-2 SI to the hACE2 receptor in a dose-dependent manner, with the highest concentration tested (40 pg/mL) yielding results comparable to the inhibition observed with a polyclonal antibody raised against the RBD of the SARS- CoV-2 SI.

REFERENCES

Byrnes et al., “Competitive SARS-CoV-2 serology reveals most antibodies targeting the spike receptor binding domain compete for ACE2 binding”, mSphere (2020), 5: 200802-20.

Grant et al., “Analysis of the SARS-CoV-2 Spike Protein Glycan Shield: Implications for Immune Recognition”, Preprint. bioRxiv. 2020;2020.04.07.030445. Published 2020 May 1. doi: 10.1101/2020.04.07.030445

Sanda et al., “N- and O-Glycosylation of the SARS-CoV-2 Spike Protein”, Analytical Chemistry, (2021), 93, 4, 2003-2009.

Shajahan et al., “Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2”, Glycobiology, (2020), 1-8.

Varki et al., “Symbol Nomenclature for Graphical Representations of Glycans”, Glycobiology, (2015), 25(12): 1323-4.

Watanabe etak, “Site-specific glycan analysis of the SARS-CoV-2 spike . Science (2020), Published online 2020 May 4. doi: 10.1126/science. abb9983.

Claims

1. A protein complex comprising a SARS-CoV-2 S protein or fragment thereof, expressing one or more carbohydrate antigens, bound to a recombinant ligand having binding specificity for said carbohydrate antigen.

2. The protein complex of claim 1 , wherein the ligand comprises or consists of a recombinant antibody or an antigen-binding fragment thereof.

3. The protein complex of claim 1 or 2, wherein the recombinant antibody comprises or consists of a single chain variable fragment (scFv), Fab, Fab’, F(ab’)2, minibody, diabody, triabody, ortetrabody.

4. The protein complex of any one of claims 1 to 3, wherein the carbohydrate antigen is an O-linked carbohydrate antigen.

5. The protein complex of any one of claims 1 to 4, wherein the carbohydrate antigen comprises or consists of unsialylated Thomsen-Friedenreich (TF) antigen, sialylated TF antigen, unsialylated Tn antigen, sialylated Tn antigen, or any combination thereof.

6. The protein complex of any one of claims 1 to 5, wherein the ligand further comprises a detectable or functional label.

7. The protein complex of any one of claims 1 to 6, which is an in vitro protein complex.

8. The protein complex of any one of claims 1 to 6, which is an in vivo protein complex.

9. Use of the ligand as defined in any one of claims 1 to 6 for binding to a SARS-CoV-2 S protein or fragment thereof, expressing the one or more carbohydrate antigens.

10. A method of treating or reducing the risk of SARS-CoV-2 viral infection in a subject, the method comprising administering to the subject one or more ligands as defined in any one of claims 1 to 6.

11. Use of the ligand as defined in any one of claims 1 to 6 for the manufacture of a medicament for treating and/or preventing a SARS-CoV-2 viral infection in a subject; or for treating and/or preventing a SARS-CoV-2 viral infection in a subject.