Patent Application
20240043508
The present disclosure was carried out under project identifier number 1711120289 and project number NRF-2020M3A912107093, supported by the Ministry of Science and ICT of the Republic of Korea. The research management agency for the project is the National Research Foundation of Korea. The name of the research project is “Bio-Medical Technology Development Project”, and the title of the research task is “Development of novel bispecific antibodies effectively neutralizing SARS-CoV2 for clinical trials”. The institution conducting the research is the Industry-Academic Cooperation Foundation of Kookmin University, and the research period spans from Jul. 1, 2020, to Dec. 31, 2022.
The present disclosure relates to an antibody binding specifically to SARS-CoV-2 S protein or an antigen binding fragment thereof, and a use thereof. More specifically, the present disclosure concerns an anti-SARS-CoV-2 S protein-specific antibody and an antigen binding fragment thereof, and a use thereof for diagnosing or treating SARS-CoV-2 infection.
Also, the present disclosure relates to a diagnostic composition or kit comprising an antibody binding specifically to an RBD of SARS-CoV-2 S protein. More specifically, the present disclosure is concerned with a diagnostic composition comprising an antibody binding specifically to an RBD of SARS-CoV-2 S protein, or a kit suitable for sandwich ELISA using same.
In addition, the present disclosure is concerned with a bispecific antibody binding specifically to SARS-CoV-2.
The COVID-19 infectious disease caused by the SARS-CoV-2 virus erupted in late December 2019, infecting over 100 million people worldwide and leading to the deaths of over 2 million in just a year. The spike protein (S protein), located on the surface of SARS-CoV-2, binds to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of host cells, serving as the critical marker inducing infection. Accordingly, the receptor binding region (RBD) of the spike protein is a primary target in the development of therapeutics to counteract infections caused by SARS-CoV-2. Antibody-based treatments are recognized for their efficacy due to their high affinity and specificity towards their targets. Although various vaccines and therapeutic methods have been developed, the rapid emergence of SARS-CoV-2 variants continues to pose a persistent challenge in controlling COVID-19. Therefore, there is an urgent need for the development of new treatments for COVID-19. Moreover, many researchers are exerting significant efforts to develop fast and efficient methods for diagnosing SARS-CoV-2 infections.
Leading to the present disclosure, thorough and intensive research conducted by the present inventors with the aim of developing a pharmaceutical composition for prevention or treatment of COVID-19 constructed a novel anti-SARS-CoV-2 S protein-specific antibody or an antigen binding fragment thereof and resulted in the finding that the antibody or the antigen binding fragment has high affinity for S protein.
Accordingly, an aspect of the present disclosure is to provide an antibody binding specifically to SARS-CoV-2 S protein or an antigen binding fragment thereof.
Another aspect of the present disclosure is to provide a pharmaceutical composition comprising the antibody or the antigen binding fragment thereof, and a pharmaceutically acceptable carrier for prevention or treatment of SARS-CoV-2 infectious diseases.
A further aspect of the present disclosure is to provide a composition or kit for detecting SARS-CoV-2; or a composition or kit for diagnosing COVID-19, each comprising the antibody or the antigen binding fragment thereof.
Still another aspect of the present disclosure is to provide a bispecific antibody to SARS-CoV-2.
An additional further aspect of the present disclosure is to provide a pharmaceutical composition comprising a bispecific antibody to SARS-CoV-2 for the treatment of SARS-CoV-2 infections.
According to an aspect thereof, the present disclosure provides an antibody binding specifically to SARS-CoV-2 S protein or an antigen binding fragment thereof, selected from the group consisting of
In an embodiment of the present disclosure, the antibodies or the antigen binding fragments thereof selected from (i) to (v) are derived from the clones RG6, RB4, RB6, RD3, and RD10 selected from the working examples of the present disclosure, respectively.
In an embodiment of the present disclosure, the antibody or antigen binding fragment thererof (i) includes the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8; the antibody or antigen binding fragment thereof (ii) includes the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 17; the antibody or antigen binding fragment thereof (iii) includes the heavy chain variable region of SEQ ID NO: 25 and the light chain variable region of SEQ ID NO: 26; the antibody or antigen binding fragment thereof (iv) includes the heavy chain variable region of SEQ ID NO: 34 and the light chain variable region of SEQ ID NO: 35; and the antibody or antigen binding fragment thereof (v) includes the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 44, but with no limitations thereto.
In an embodiment of the present disclosure, the antibody or antigen binding fragment thereof (i) includes the amino acid sequence of SEQ ID NO: 9, the antibody or antigen binding fragment thereof (ii) includes the amino acid sequence of SEQ ID NO: 18, the antibody or antigen binding fragment thereof (iii) includes the amino acid sequence of SEQ ID NO: 27, the antibody or antigen binding fragment thereof (iv) includes the amino acid sequence of SEQ ID NO: 36, and the antibody or antigen binding fragment thereof (v) includes the amino acid sequence of SEQ ID NO: 45, but with no limitations thereto.
In an embodiment of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof according to the present disclosure binds to a receptor binding domain (RBD) of SARS-CoV-2 S protein (spike protein).
In an embodiment of the present disclosure, the RBD of SARS-CoV-2 S protein includes the amino acid sequence of SEQ ID NO: 51.
In an embodiment of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof according to the present disclosure inhibits binding of the RBD of SARS-CoV-2 S protein to human ACE2 (angiotensin converting enzyme 2).
In an embodiment of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or antigen binding fragment thereof the present invention binds to S1 domain of SARS-CoV-2 S protein.
In an embodiment of the present disclosure, the S1 domain of SARS-CoV-2 S protein includes the amino acid sequence of SEQ ID NO: 52.
In an embodiment of the present disclosure, the S2 domain of SARS-CoV-2 S protein includes the amino acid sequence of SEQ ID NO: 53.
In an embodiment of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or antigen binding fragment thereof the present disclosure binds to the full-length spike protein of SARS-CoV-2 S protein.
In an embodiment of the present disclosure, the full-length spike protein of SARS-CoV-2 includes the amino acid sequence of SEQ ID NO: 54.
In an embodiment of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or antigen binding fragment thereof the present disclosure binds specifically to a mutant virus having a mutation in SARS-CoV-2 S protein.
In an embodiment of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or antigen binding fragment binds specifically to a mutant virus having a mutation in an RBD region of SARS-CoV-2 S protein.
In a further embodiment of the present disclosure, the mutant virus, having a mutation in an RBD region of SARS-CoV-2 S protein, to which the anti-SARS-CoV-2 S protein-specific antibody or antigen binding fragment specifically binds, is a mutant virus that possesses mutation V431A at position 431, F342L at position 342, V367F at position 367, R408I at position 408, A435S at position 435, W436R at position 436, G476S at position 476, V483A at position 483, or N354D/D364Y at positions 354/364 in the RBD region, but with no limitations thereto.
In an embodiment of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof binds specifically to a mutant virus having a mutation in a site other than SARS-CoV-2 S protein.
In an embodiment of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof binds specifically to the S protein of SARS-CoV.
In some particular embodiments of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or antigen binding fragment binds specifically to an RBD region of S protein in SARS-CoV.
In some particular embodiments of the present disclosure, the RBD region of S protein includes the amino acid sequence of SEQ ID NO: 55, but with no limitations thereto.
The antibodies of the present disclosure can be produced using various phage display methods known in the art, as disclosed in [Brinkman et al., 1995, J. Immunol. Methods, 182:41-50]; [Ames et al., 1995, J. Immunol. Methods, 184, 177-186]; [Kettleborough et al. 1994, Eur. J. Immunol, 24, 952-958]; [Persic et al., 1997, Gene, 187, 9-18]; and [Burton et al., 1994, Adv. Immunol., 57, 191-280]; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; and WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.
As used herein, the term “antibody” refers to an antibody specific for SARS-CoV S protein and is intended to encompass an antigen-binding fragment thereof the antibody molecule as well as an intact antibody form.
An intact antibody consists of two full-length light chains and two full-length heavy chain, with disulfide linkages therebetween. Heavy chain constant regions are classified into gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with the subclassification of the gamma type into gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), and gamma 4 (γ4), and the alpha type into alpha 1 (α1) and alpha 2 (α2). Antibodies can be further classified by kappa (κ) and lambda (λ) types for light chain constant regions (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co. Philadelphia, Pa. (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, Mass. (1984)).
As used herein, the term “antigen-binding fragment” means a fragment retaining the function of binding to an antigen and is intended to encompass Fab, F(ab′), F(ab′)2, chemically linked F(ab′)2, Fv, and so on. Of the antibody fragments, Fab has one antigen-binding site which takes on the structure composed of light chain and heavy chain variable regions, a light chain constant region, and the first constant region (CH1 region) of the heavy chain. Fab′ is different from Fab in that the former has a hinge region containing at least one cysteine residue at the C terminus of the heavy chain CH1 region. An F(ab′)2 antibody is produced in such a way that a cysteine residue of the hinge region of Fab′ forms disulfide bonding. Fv is a minimum antibody fragment having only a heavy chain variable region and a light chain variable region. A recombination technology for producing an Fv fragment is disclosed in the International Patent Publication filed under the patent cooperation treaty (PCT) WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086, and WO 88/09344. In case of two-chain Fv, a heavy chain variable region and a light chain variable region are linked to each other by means of non-covalent bonding while single-chain Fv consists of a heavy chain variable region and a single chain variable region which are linked to each other by means of covalent bonding generally via a peptide linker, or directly linked to each other at C-terminus, and thus may form a structure like a dimer, as shown in the two-chain Fv. Such antibody fragments may be obtained by using protease (for example, Fab may be obtained by performing restriction digestion of a whole antibody with papain, while F(ab′)2 fragment may be obtained by doing so with pepsin), and may be prepared by means of a gene recombination technology.
The antibody in the present disclosure is particularly in the form of scFv or in an intact form. In addition, the heavy chain constant region may be any one isotype selected from gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε). Preferably, the constant region may be a gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), or gamma 4 (IgG4) isotype, with most preference for gamma 4 (IgG4) isotype. The light chain constant region may be a kappa or lambda isotype, with preference for kappa isotype.
In an embodiment of the present disclosure, the antibody of the present disclosure may be in the form of scFv or IgG4 comprising a kappa light chain or a gamma 4 heavy chain. In another embodiment of the present disclosure, a particular antibody of the present disclosure may be in the form of scFv or IgG1 comprising a kappa light chain and a gamma 1 heavy chain.
The term “heavy chain”, as used herein, refers to a full-length heavy chain comprising: a variable region VH, which comprises amino acid sequences having enough variable region sequences to allow the specificity to an antigen; and the three constant regions, CH1, CH2, and CH3, and to any fragment thereof. As used herein, the term “light chain” refers to a full-length light chain comprising: a variable region VL, which comprises amino acid sequences having enough variable region sequences to allow the specificity to an antigen; and a constant region, CL and to any fragment thereof.
The term “CDR” (complementarity determining region), as used herein, refers to an amino acid sequence in a hypervariable region of an immunoglobulin heavy chain or light chain (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). Three CDRs exist in each of the heavy chain (CDR-H1, CDR-H2, and CDR-H3) and the light chain (CDR-L1, CDR-L2, and CDR-L3). CDRs provide contact residues which play an important role in binding the antibody to an antigen or an epitope.
Herein, the antibody or antigen-binding fragment thereof encompasses not only full-length or intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab′, F(ab′)2, Fab3, Fv, and variants thereof), fusion proteins containing one or more antibody portions, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibodies (scFv), scFv-Fc, bispecific antibodies, multi-specific antibodies, and any other modified configuration of the immunoglobulin molecule that contains an antigen recognition site of the required specificity, comprising glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Concrete examples of the modified antibodies and antigen-binding fragments thereof include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable region immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies, and triomAbs, but with no limitations to the listing of such antibodies and antigen-binding fragments thereof.
The term “framework” or “FR”, as used herein, refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL/Vk):
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain responsible for binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively.
The term “specifically binds” or similar expressions mean that an antibody or an antigen-binding fragment thereof, or another construct such as an scFv, forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10−6 M or less (e.g., 9×10−7 M, 8×10−7 M, 7×10−7 M, 6×10−7 M, 5×10−7 M, 4×10−7 M, 3×10−7 M, 2×10−7 M, or 1×10−7 M), preferably 1×10−7 M or less (e.g., 9×10−8 M, 8×10−8 M, 7×10−8 M, 6×10−8 M, 5×10−8 M, 4×10−8 M, 3×10−8 M, 2×10−8 M, or 1×10−8 M), and more preferably 1×10−8 M or less (e.g., 9×10−9 M, 8×10−9 M, 7×10−9 M, 6×10−9 M, 5×10−9 M, 4×10−9 M, 3×10−9 M, 2×10−9 M, or 1×10−9 M) (a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
As used herein, the term “affinity” refers to total strength of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless specified otherwise, “binding affinity”, as used herein, refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD). Affinity can be measured by common methods known in the art, comprising those disclosed herein.
As used herein, the term “human antibody” refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition for a human antibody specifically excludes a humanized antibody, which includes non-human antigen-binding residues.
The term “chimeric” antibody, as used herein, refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
As used herein, the term “humanized antibody” refers to a chimeric immunoglobulin which includes the minimal sequence derived from non-human immunoglobulin of non-human (e.g., mouse) antibodies, an immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequences of the antibody). In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit having desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further improve and optimize antibody performance. In general, the humanized antibody will include substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to CDR regions of a non-human immunoglobulin and all or substantially all of the FR regions have sequences of FR regions of a human immunoglobulin sequence. The humanized antibody includes at least a portion of an immunoglobulin constant region (Fc region), typically a constant region (Fc region) of a human immunoglobulin.
The term “substantial identity” or “substantially identical,” as used herein in the context of the variants, indicates that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 90% sequence identity and more preferably about 95%, 98%, or 99% sequence identity. Preferably, residue positions which 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 or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution.
Such amino acid variations may be provided on the basis of a relative similarity of amino acid side chains, e.g., hydrophobicity, hydrophilicity, charge, and size. By the analysis for size, shape, and type of the amino acid side chains, it is clear that all of arginine, lysine, and histidine residues are those having positive charge; alanine, glycine, and serine have a similar size; phenylalanine, tryptophan, and tyrosine have a similar shape. Accordingly, based on these considerable factors, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan, and tyrosine may be considered to be functional equivalents biologically.
In the introduction of variations, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of hydrophobicity and charge characteristics thereof: isoleucine (+4.5); valine (+4.2): leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5): aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
The hydrophobic amino acid indexes are very important in assigning interactive biological functions of proteins. It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index and still result in similar biological activity. In cases where a variation is introduced with reference to the hydrophobic indexes, the substitution is made between amino acids having a difference in the hydrophobic index within preferably ±2, more preferably ±1, and still more preferably ±0.5.
Meanwhile, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in U.S. Pat. No. 4,554,101, each amino acid residue has been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).
In cases where variations are introduced with reference to the hydrophilicity values, substitutions may be made between amino acids that exhibit a hydrophilicity value difference of preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.
Amino acid exchanges in proteins which do not entirely alter activity of a molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most common occurring exchanges are exchanges between amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
In an embodiment of the present disclosure, the antibody or the antigen binding fragment thereof according to the present disclosure, which binds specifically to SARS-CoV-2 S protein, are expressed as RG6, RB4, RB6, RD3, or RD10.
In an embodiment of the present disclosure, the antibody or antigen binding fragment has an equilibrium dissociation constant KD of 7.2×10−10 M or less for the RBD of the S protein.
In another embodiment of the present disclosure, the antibody or antigen binding fragment has an equilibrium dissociation constant KD of 3.2×10−9 M for the S1 antigen of the S protein.
In an embodiment of the present disclosure, RG6, RB4, RB6, RD3, and RD10, which are the antibody or the antigen binding fragment thereof binding specifically to SARS-CoV-2 S protein, binds specifically to RBD of S protein in SARS-CoV as well as to the RBD antigen of S protein in SARS-CoV-2.
The anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof according to the present disclosure may include a little change in the amino acid sequence, i.e., a modification that has little effect on the tertiary structure and function of the antibody. Therefore, although not identical to the aforementioned sequence, an anti-SARS-CoV-2 S protein-specific antibody or an antigen binding fragment thereof in accordance with some embodiments may at least 100%, 93%, 95%, 96%, 97%, or 98% similarity.
In an embodiment of the present disclosure, the anti-SARS-CoV-2 S antibody or the antigen binding fragment thereof according to the present disclosure includes, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of the antibodies, each comprising a heavy chain variable region and light chain variable region comprising CDRs of the sequences described above. In another embodiment of the present disclosure, the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof is an anti-SARS-CoV-2 S protein scFv.
In a concrete embodiment of the present disclosure, the heavy chain variable region and light chain variable region included in the antibody or the antigen binding fragment thereof are linked to each other via a linker such as Gly-Ser)n, (Gly2-Ser)n, (Gly3-Ser)n, or (Gly4-Ser)n. Here, n is an integer of 1 to 6 and specifically 3 or 4, but with no limitations thereto. The light chain variable region and heavy chain variable region in scFv may be arranged as follows: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.
Another aspect of the present disclosure provides a nucleic acid molecule encoding the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof.
As used herein, the term “nucleic acid molecule” is intended to comprehensively encompass RNA molecules as well as DNA (gDNA and cDNA), and nucleotides, which account for a basic unit of nucleic acid molecules, include not only natural nucleotides, but also analogues having modified sugar or base moieties (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584(1990)).
It would be obvious to a person skilled in the art that the nucleotide sequence encoding the antibody or the antigen binding fragment thereof according to the present disclosure is any nucleotide coding for the amino acid sequence constituting the antibody or the antigen binding fragment thereof and is not limited to any particular nucleotide sequence.
The reason is that even if the nucleotide sequence undergoes mutation, the expression of the mutated nucleotide sequence into a protein may not cause a change in the protein sequence. This is called the degeneracy of codons. Therefore, the nucleotide sequence includes nucleotide sequences containing functionally equivalent codons, codons encoding the same amino acids (e.g., due to the degeneracy of codons, the number of codons for arginine or serine being six), or codons containing biologically equivalent amino acids.
Considering biologically equivalent variations described in the foregoing, the nucleic acid molecule coding for the amino acid sequence accounting for the antibody or antigen-binding fragment thereof the present disclosure is construed to encompass sequences having substantial identity to them. Sequences having the substantial identity show at least 60% (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), particularly at least 70% (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), more particularly 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), even more particularly at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), most particularly at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) similarity to the nucleic acid molecule of this disclosure, as measured by using one of the sequence comparison algorithms for the sequences of the present disclosure aligned to any sequence, with maximum correspondence therebetween. With respect to % similarity, all integers from 60% to 100% and minor numbers existing therebetween fall within the scope of the present disclosure.
Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are disclosed in: Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins and Sharp, CABIOS 5:151-3(1989); Corpet et al., Nuc. Acids Res. 16:10881-90(1988); Huang et al., Comp. Appl. BioSci. 8:155-65(1992) and Pearson et al., Meth. Mol. Biol. 24:307-31(1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10(1990)) is accessible from the NBCI (National Center for Biotechnology Information) and on the Internet and may be used in connection with sequence analysis programs such as blastp, blastn, blastx, tblastn and tblastx. BLAST may be accessed through the BLAST webpage of the NCBI's website. The method for comparing sequence homology using such a program is available from the BLAST help page of the NCBI's website.
According to some particular embodiments of the present disclosure, polypeptides of the antibodies to SARS-CoV-2 S protein or antigen binding fragments thereof, e.g., polypeptides of heavy and light chains, e.g., heavy chain CDRs, light chain CDRs, heavy chain variable regions, light chain variable regions, etc., and nucleotide sequences coding therefor are given in the annexed sequence listings.
Provided according to another aspect of the present disclosure is a recombinant vector carrying the nucleic acid molecule coding for the anti-SARS-CoV-2 S protein-specific antibody or antigen binding fragment.
As used herein, the term “vector” refers to a means for expressing a target gene in a host cell and is intended to encompass a variety of vectors comprising: plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.
According to an embodiment of the present disclosure, the nucleic acid molecule coding for the heavy chain variable region and the nucleic acid molecule coding for the heavy chain variable region are operatively linked to a promoter in the vector of the present disclosure.
As used herein, “operatively linked” means that an expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulatory factors) and a nucleic acid of interest are linked so that the transcription and/or translation of the nucleic acid of interest can be governed by the control sequence.
The recombinant vector system of the present disclosure can be constructed by various methods known in the art. For concrete methods, reference may be made to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated in its entirety herein by reference.
The vector of the present disclosure may be typically constructed as a vector for cloning or expression. In addition, the vector of the present disclosure may be constructed with a prokaryotic cell or an eukaryotic cell serving as a host.
For example, when the vector of the present disclosure is an expression vector, with a eukaryotic cell serving as a host, advantage is taken of a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)), with a polyadenylated sequence commonly employed as a transcription termination sequence in the vector.
The vector of the present disclosure may be fused with the other sequences to facilitate the purification of the antibody expressed therefrom. Examples of the fusion sequence include glutathione 5-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6×His (hexahistidine; Qiagen, USA).
Since the protein expressed by the vector of the present disclosure is an antibody, the expressed antibodies can be easily purified through protein A column or the like even without additional sequences for purification.
Meanwhile, the expression vector of the present disclosure includes, as a selective marker, an antibiotic agent-resistant gene that is ordinarily used in the art, examples of which include resistant genes against ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.
Optionally, the vector may additionally carry a gene encoding a reporter molecule (e.g., luciferase and glucuronidase).
According to an embodiment of the present disclosure, the expression vector is a recombinant vector for host cell expression, into which a nucleotide sequence encoding the anti-SARS-CoV-2 S antibody or antigen binding fragment thereof is inserted, wherein the vector carries: a promoter, which is operatively linked to the nucleotide sequence and forms an RNA molecule in host cells; and a poly A signal sequence, which acts on the host cells to cause polyadenylation of the 3′-terminus of the RNA molecule.
According to another aspect thereof, the present disclosure provides an isolated host cell transformed with the recombinant vector.
So long as it is capable of performing continuous cloning and expression while stabilizing the vector of the present disclosure, any host cell known in the art may be used and, for instance, examples of eukaryotic host cells suitable for the vector include monkey kidney cells 7 (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK-293, but are not limited thereto.
As used herein, the term “transformed”, “transduced”, or “transfected” refers to pertaining to the delivery or introduction of a foreign nucleic acid into a host cell. The “transformed”, “transduced”, or “transfected” cells are cells into which a foreign nucleic acid is transformed, transduced, or transfected. Within the scope of the transformed, transduced, or transfected cells, the cells themselves and progeny cells thereof obtained through passages fall.
When host cells are eukaryotic cells, the vector may be injected into the host cells by microinjection (Capecchi, M. R., Cell, 22:479(1980)), calcium phosphate precipitation (Graham, F. L. et al., Virology, 52:456(1973)), electroporation (Neumann, E. et al., EMBO J., 1:841(1982)), liposome-mediated transfection (Wong, T. K. et al., Gene, 10:87(1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190(1985)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572(1990)), and the like.
Herein, the recombinant vector injected into the host cells can express the recombined polypeptide or polypeptide complex in the host cells, and in such a case, a large amount of polypeptides or polypeptide complexes are obtained. For example, when the vector contains a lac promoter, gene expression can be induced by treatment of host cells with IPTG.
The culturing is usually carried out under aerobic conditions by, for example, a shaking culture or a rotation by a rotator. The culturing temperature is preferably in a range of 10-40° C., and the culturing time is generally for 5 hours to 7 days. The pH of the medium is preferably maintained at 3.0-9.0 during culturing. The pH of the medium can be adjusted with an inorganic or organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like. For maintenance and expression of recombinant vectors, if necessary, antibiotics, such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline, may be added during culturing. When host cells transformed by a recombinant expression vector having an inducible promoter is cultured, an inducer suitable for a medium may be added if necessary. For example, isopropyl-6-D-thiogalactopyranoside (IPTG) may be added when the expression vector contains a lac promoter, and indoleacrylic acid may be added when the expression vector contains a trp promoter.
Provided according to another aspect of the present disclosure is a polypeptide complex in which i) the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof and ii) and an additional polypeptide are linked to each other.
The ii) additional polypeptide is the aforementioned antibody or antigen binding fragment, or a “target binding polypeptide” or a “polypeptide of target” rather than an antibody or an antigen binding fragment.
The antibody or antigen binding fragment or the target binding polypeptide or the polypeptide of target as ii) the additional polypeptide may bind to the same antigen as for i) the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof or to a different antigen from that therefor. When i) the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof targets the same antigen as for ii) the additional polypeptide, the antigen preferably has different epitopes.
As used herein, the term “target-binding polypeptide” refers to a non-immunoglobulin polypeptide molecule which exhibits binding affinity for a target antigen or a hapten, like an antibody, but is structurally relevant to an antibody. The target-binding polypeptides, also called antibody-like molecules or antibody mimetics, generally have a molecular weight of 3-20 kDa, unlike antibodies, which have a molecular weight of about 150 kDa. Examples of the target-binding polypeptide include, but are not limited to, an affibody derived from Z-domain of protein A, an affilin derived from gamma-B crystallin or ubiquitin, affimer derived from cystatin, an affitin derived from Sac7d of Sulfolobus acidocaldarius, an alphabody derived from triple helix coiled coil, an anticalin derived from lipocalin, an avimer derived from a cell membrane receptor domain, DARPin derived from an ankyrin repeat motif, Fynomer derived from the SH3 domain of Fyn, a Kunits domain peptide derived from the Kunits domain of protease inhibitor, a monobody derived from the 10th type III domain of fibronectin, and nanoCLAMP derived from carbohydrate binding module 32-2 of NagH in Clostridium perfringens. Through various screening methods known in the art, such as phage display, ribosome display, etc., the target-binding polypeptide may be engineered to have binding affinity for any target antigen or hapten.
In an embodiment of the present disclosure, the target binding polypeptide may be a polypeptide derived from a host cell toward which SARS-CoV-2, the target of the present disclosure, is directed. By way of example, when the target binding polypeptide is an ACE2 receptor in a host cell, the ACE receptor linked to the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof is associated with SARS-CoV-2, followed by the neutralization of the SARS-CoV-2, with the consequent prevention of SARS-CoV-2 from entry into host cells.
As used herein, the term “polypeptide of target” refers to a polypeptide derived from SARS-CoV-2, the target of the present disclosure, which binds to a different polypeptide as a constituent of SARS-CoV-2. The polypeptide of target is associated with a different polypeptide that is a constituent of SARS-CoV-2, thereby preventing the entry of SARS-CoV-2 into host cells. The polypeptide of target may be a polypeptide that SARS-CoV-2 employs in invading host cells and may be specifically a polypeptide as a constituent of the spike protein, but with no limitations thereto.
The polypeptide complex according to an aspect of the present disclosure has a multimeric form in which individual antibodies or antigen binding fragments and monomers of the polypeptide are linked to each other. The polypeptide complexes of the present invention are linked to each other via a covalent linkage. According to an embodiment of the present disclosure, the polypeptide complex may be implemented in the form of a fused protein or a conjugate.
According to an embodiment of the present invention, the polypeptide complex may be implemented in the form of a fused protein or a conjugate. Therefore, the antibody or the antigen binding fragment thereof may be linked by means of chemical conjugation (using known organic chemistry methods) or by any other means (for example, via the expression of the complex as a fusion protein, or either directly, or indirectly via a linker (e.g., an amino acid linker)).
According to some particular embodiments of the present disclosure, individual polypeptides constituting the polypeptide complex are connected via at least one linker. The linker may be composed of the amino acid sequence represented by the general formula (GnSm)p or (SmGn)p:
In another particular embodiment of the present disclosure, the linker is (GnSm)p or (SmGn)p wherein n=1 to 5 or m=0 to 5. In a more particular embodiment, n=4 and m=1. In a further more particular embodiment, the linker is (GGGGS) 3. In another embodiment, the linker is GGGGS. In a further embodiment, the linker is VDGS. In a still further embodiment, the linker is ASGS.
In an embodiment of the present disclosure, the polypeptide complex may be a multispecific complex directing toward two or more targets.
In some particular embodiments of the present disclosure, the polypeptide complex of the present disclosure may include two or more antibodies or antigen binding fragments thereof selected from the following (i) to (v), but with no limitations thereto:
In some particular embodiments of the present disclosure, the polypeptide complex comprising at least two antibodies or antigen binding fragments thereof selected from the group consisting of (i) to (v) set forth above may be expressed into a fusion protein as the at least two antibodies or antigen binding fragments selected from (i) to (v) are linked to each other via an amino acid linker or may be prepared by chemical conjugation into a conjugate.
Another aspect of the present disclosure provides a nucleic acid molecule encoding the polypeptide complex. The common contents between the nucleic acid according to an aspect of the present disclosure and the anti-SARS-CoV-2 S protein-specific antibody or antigen binding fragment thereof according to an aspect of the present disclosure are omitted in order to avoid undue redundancy leading to the complexity of the description.
Another aspect of the present disclosure provides a pharmaceutical composition for prevention or treatment of SARS-CoV-2 infectious diseases, the composition comprising the antibody binding specifically to SARS-CoV-2 S protein or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier.
In an embodiment of the present disclosure, the SARS-CoV-2 causing SARS-CoV-2 infectious diseases is a mutant virus having a mutation in the S protein thereof.
In some particular embodiments of the present disclosure, the mutant virus has a mutation in an RBD region of the S protein.
In some more particular embodiments of the present disclosure, the mutant virus having a mutation in the RBD region of S protein may include the mutation V431A at position 431, F342L at position 342, V367F at position 367, R408I at position 408, A435S at position 435, W436R at position 436, G476S at position 476, V483A at position 483, or N354D/D364Y at positions 354 and 364 in the RBD region of S protein, but with no limitations thereto.
In some particular embodiments of the present disclosure, the monoclonal antibody of the present disclosure has the efficacy of reducing clinical severity for SARS-CoV-2 virus.
As will be elucidated, the monoclonal antibody of the present disclosure exhibits higher efficacy of reducing clinical severity of SARS-CoV-2 infectious diseases when being in the form of IgG4 than IgG1.
Also, another aspect of the present disclosure provides a pharmaceutical composition comprising the antibody binding specifically to SARS-CoV-2 S protein or the antigen binding fragment thereof for prevention or treatment of SARS-CoV infectious disease. The antibody binding specifically to SARS-CoV-2 S protein or the antigen binding fragment thereof according to the present disclosure binds specifically to the S protein of SARS-CoV as well as that of SARS-CoV-2 and thus can be advantageously used for preventing or treating SARS-CoV infectious diseases.
The pharmaceutical composition of the present disclosure employs the anti-SARS-CoV-2 S protein-specific antibody or antigen binding fragment thereof according to the present disclosure, and the common contents therebetween are omitted in order to avoid undue complexity of the description.
So long as it is typically used for formulation, any pharmaceutically acceptable carrier may be contained in the pharmaceutical composition of the present disclosure. Examples of the pharmaceutically acceptable carrier include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition of the present disclosure may further include a lubricant, a wetting agent, a sweetener, a flavorant, an emulsifier, a suspending agent, a preservative, and the like in addition to the above ingredients. With regard to suitable pharmaceutically acceptable carriers and preparations, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995).
The pharmaceutical composition of the present disclosure may be administered orally or parenterally, for example, by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.
Appropriate doses of the pharmaceutical composition of the present invention vary depending on various factors comprising a formulating method, a manner of administration, patient's age, body weight, sex, and morbidity, food, a time of administration, a route of administration, an excretion rate, and response sensitivity. An ordinarily skilled practitioner can easily determine and prescribe an effective dose for desired treatment or prevention. According to a preferable embodiment of the present disclosure, the daily dose of the pharmaceutical composition of the present disclosure is 0.0001-100 mg/kg. As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat the above-described diseases.
As used herein, the term “prevention” refers to a prophylactic or protective treatment of a disease or a disease condition. As used herein, the term “treatment” refers to a reduction, suppression, amelioration, or eradication of a disease condition.
The pharmaceutical composition of the present disclosure may be formulated into a unit dosage form or may be introduced into a multi-dose container by using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily implemented by a person having an ordinary skill in the art to which the present invention belongs. Here, the formulation may be in the form of a solution in an oily or aqueous medium, a suspension, an emulsion, an extract, a pulvis, a suppository, a powder, granules, a tablet, or a capsule, and may further contain a dispersant or a stabilizer.
According to an aspect thereof, the present disclosure provides a pharmaceutical composition for prevention or treatment of SARS-CoV-2 infectious diseases, the composition comprising: a polypeptide complex in which i) the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof and ii) an additional polypeptide are linked to each other; and a pharmaceutically acceptable carrier.
In an embodiment of the present disclosure, the additional polypeptide may be an anti-SARS-CoV-2 antibody or an antigen binding fragment thereof, or a target-binding, non-antibody polypeptide specifically binding to SARS-CoV-2.
The anti-SASRS-CoV-2 antibody or the antigen binding fragment thereof may be the aforementioned antibody or antigen binding fragment thereof according to the present disclosure.
Therefore, the polypeptide complex may include two or more of the aforementioned antibodies or antigen binding fragments thereof according to present disclosure, but is not limited thereto.
Since the pharmaceutical composition of the present disclosure employs the polypeptide complex of the present disclosure as an active ingredient, common contents therebetween are omitted to avoid undue complexity of the description.
According to another aspect thereof, the present disclosure provides a composition for detecting SARS-CoV-2 viruses or a composition for diagnosing SARS-CoV-2 infectious diseases (COVID-19), each comprising the antibody binding specifically to SARS-CoV-2 S protein or the antigen binding fragment thereof.
According to a further aspect thereof, the present disclosure provides a composition for detecting SARS-CoV-2 viruses or a composition for diagnosing SARS-CoV-2 infectious diseases (COVID-19), each comprising the antibody binding specifically to SARS-CoV-2 S protein or the antigen binding fragment thereof. Specifically binding to the S protein of SARS-CoV as well as SARS CoV-2, the antibody binding specifically to SARS-CoV-2 S protein or the antigen binding fragment thereof can also be advantageously used for preventing or treating SARS-CoV infectious diseases.
In an embodiment thereof, the present disclosure provides a composition for diagnosis of SARS-CoV-2 infection (COVID-19) or for detection of a SARS-CoV-2 antigen, the composition comprising a pair of the following antibodies or antigen binding fragments thereof, each antibody or fragment binding specifically to a receptor binding domain (RBD) of SARS-CoV-2 S protein:
Provided according to an embodiment of the present disclosure is the composition for diagnosis of SARS-CoV-2 infection (COVID-19) or for detection of a SARS-CoV-2 antigen, wherein the antibody or the antigen binding fragment of (i) includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 35, and the antibody or the antigen binding fragment of (ii) includes a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.
In an embodiment of the present disclosure, the pair of antibodies or antigen binding fragments thereof in the composition is derived from the clones RD3(=K102.1) and RB6(K102.2) selected in the following working examples.
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (i) comprises the amino acid sequence of SEQ ID NO: 36 and the antibody or antigen binding fragment of (ii) comprises the amino acid sequence of SEQ ID NO: 27, but with no limitations thereto.
In an embodiment of the present disclosure, the pair of the RBD antibodies of anti-SARS-CoV-2 S protein or an antigen binding fragments in the composition binds specifically to the receptor binding domain (RBD) of SARS-CoV-2 S protein. Therefore, the pair of antibodies or antigen binding fragments in the composition can be used for detecting SARS-CoV-2 S protein or diagnosing SARS-CoV-2 infection (COVID-19).
In a particular embodiment of the present disclosure, the pair of antibodies or antigen binding fragments in the composition is characterized by targeting different epitopes in the RBD of SARS-CoV-2 S protein.
As used herein, the term “epitope” is a specific antigen determinant that is recognized and bound by a paratope of an antibody to diagnose or detect the target. Specifically, antibodies with different antigenic determinants to the RBD of the SARS-CoV-2 S protein were selected for diagnosing or detecting the target SARS-CoV-2 virus. The utilization of the pairs of antibodies enables a faster and more accurate test.
In an embodiment of the present disclosure, the pair of antibodies and antigen-binding fragments thereof in the composition of the present disclosure specifically binds to variant viruses with mutations generated in the S protein of SARS-CoV-2.
In an embodiment of the present invention, the pair of antibodies and antigen-binding fragments thereof in the composition specifically binds to variant viruses where mutations have occurred in the RBD region of the S protein of SARS-CoV-2. The amino acid sequence of the RBD region is represented by SEQ ID NO: 19.
In a particular embodiment of the present disclosure, the variant virus with a mutation generated in the RBD region of SARS-CoV-2 to which the pair of antibodies or antigen binding fragments thereof in the composition bind is a virus where the mutation on the RBD region is selected from the group consisting of mutations A435S at position 435, N354D at position 354, G476S at position 476, V483A at position 483, F342L at position 342, V341I at position 341, N501Y at position 501, and L452R/T478K at positions 452/478, but with no limitations thereto.
In a more particular embodiment, the variant virus with a mutation generated in the RBD region of SARS-CoV-2 is selected from, but not limited to, the group consisting of the following viruses:
The detecting composition or diagnostic composition of the present disclosure contains the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof according to the present disclosure as constituents and is designed to detect the same virus or diagnose the same disease as for the pharmaceutical composition of the present disclosure. The common contents therebetween are omitted in order to avoid undue complexity of the description.
According to another aspect thereof, the present disclosure provides a kit for detecting SARS-CoV-2 viruses or for diagnosing SARS-CoV-2 infectious disease (COVID-19), the kit comprising the composition for detecting SARS-CoV-2 viruses or the composition for diagnosing SARS-CoV-2 infectious disease (COVID-19).
According to a further other aspect thereof, the present disclosure provides a kit for detecting SARS-CoV viruses or for diagnosing SARS-CoV infectious disease, the kit comprising the composition for detecting SARS-CoV viruses or the composition for diagnosing SARS-CoV infectious disease.
In an embodiment thereof, the present disclosure provides a kit for diagnosis of SARS-CoV-2 infection (COVID-19) or for detection of a SARS-CoV-2 antigen, the kit comprising a pair of the following antibodies or antigen binding fragments thereof, each antibody or fragment binding specifically to a receptor binding domain (RBD) of SARS-CoV-2 S protein:
As used herein, “detection” refers to verifying the presence or absence of an infection related to a certain disease in subject or the presence or absence of a specific target in a sample by using the purpose of the substance or product of the present disclosure. Specifically, the term involves confirming and identifying the presence of the SARS-CoV-2 virus by using the composition or kit of the present disclosure.
In detail, the “detection” means determining the presence or absence of an antibody-antigen-antibody complex in the kit of the present disclosure using various labeling substances. The term “antibody-antigen-antibody complex”, as used herein, refers to a conjugate between a SARS-CoV-2 RBD antigen and an antibody pair of the present disclosure which are reacted with each other to determine the presence or absence of SARS-CoV-2 virus in a sample. It also encompasses a conjugate between SARS-CoV-2 virus itself and an antibody pair of the present disclosure. The formation of the antibody-antigen-antibody may be confirmed by a method selected from the group consisting of a colorimetric method, an electrochemical method, a fluorometric method, luminometry, a particle counting method, absorbance measurement, a spectrometric method, a Raman spectroscopic method, a surface plasmon resonance method, visual assessment, and a scintillation counting method, but with no limitations thereto.
As used herein, the term “limit of detection” (LOD) refers to the lowest signal or the lowest corresponding quantity to be determined from the signal, which can be observed with a sufficient degree of confidence, completely distinct from the null (0) quantity or concentration.
The term “sensitivity”, as used herein, refers to the ratio of changes in measured values in response to changes in the quantity or concentration of the analyte in the test sample. The greater the sensitivity (slope of the calibration curve) of the test method, the more easily subtle changes in the amount or concentration of the analyte can be detected.
As used herein, the term “sample” is intended to encompass a “biological sample” and a “non-biological sample”. The “biological sample” includes, but is not limited to, nasal swab, nasopharyngeal lavage fluid, bronchoalveolar lavage fluid, pleural effusion, sputum, tissues, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, and urine. The “non-biological samples” include environmental samples such as soil, water, air, food, etc., and other samples.
SARS-CoV-2 virus can be detected by reacting these samples, whether either manipulated or not, with a pair of antibodies or antigen-binding fragments thereof included in the composition of the present disclosure, or by using the kit of the present disclosure.
As used herein, the term “kit” refers to an assembly of means, such as a solid support plate or a test strip, for diagnosing the presence or absence of a target or for detecting a target. Thus, a kit consists of one or more different component compositions, solutions or devices suitable for diagnostic and analytical methods.
The kit can be fabricated using methods typically used in the art. Specifically, because the kit contains a pair of antibodies or antigen-binding fragments thereof, it inherently enables the diagnosis or detection of the SARS-CoV-2 virus through the antigen-antibody binding reaction. Therefore, the kit can be fabricated for suitable use in various immunoassays or immunostaining methods. For example, measurement can be conducted by a method such as using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, flow cytometry, immunohistochemical staining, fluorescence immunoassay, and enzyme-substrate coloring assay, with preference for ELISA and most preference for sandwich ELISA. With respect to the immunoassay or immunostaining assay, reference may be made to Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J. M. ed., Humana Press, N J, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, the disclosures of which are hereby incorporated by reference.
In addition, the kit includes tools and reagents typically used for immunological analysis in the art as well as the detection of SARS-CoV-2 virus of the present disclosure. Examples of the tools and reagents include, but are not limited to, suitable carriers, solubilizers, detergents, buffers, stabilizers, and the like. Suitable carriers include soluble carriers, for example, biologically acceptable buffers known in the art, e.g., PBS, or insoluble carriers, for example polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymers, such as latex containing magnetic fine particles plated with metal, paper, glass, metal, agarose and combinations thereof, but with no limitations thereto.
In a particular embodiment of the present disclosure, the kit may be a kit adapted to utilize sandwich ELISA wherein the antibody or antigen binding fragment of (i) is used as a capture antibody and the antibody or antigen binding fragment of (ii) is used as a detection antibody, or vice versa.
As used herein, the term “sandwich ELISA” refers to a technique to detect or quantitatively analyze a target by utilizing two types of antibodies binding specifically to the target. As stated in the foregoing, the present disclosure provides a composition containing a pair of antibodies or antigen binding fragments thereof targeting different respective epitopes on the RBD of SARS-CoV-2 S protein, wherein any one of the paired antibodies or antigen binding fragments serves as a capture antibody and another is used as a detection antibody, thereby forming the sandwich pattern of an antibody-antigen-antibody complex.
In the case where the present disclosure is carried out in a Sandwich ELISA mode, a particular embodiment of the present disclosure includes the steps of (i) coating a surface of a solid support with the capture antibody; (ii) reacting a capture antibody with a SARS-CoV-2 RBD antigen in a sample; (iii) reacting the antigen on the capture antibody-antigen complex of step (ii) with a detection antibody; (iv) further reacting the resulting product (capture antibody-antigen-detection antibody) of step (iii) with a signal-detecting antibody conjugated with a signal-generating label; and (v) measuring the signal generated from the label.
As used herein, the term “capture antibody” refers to an antibody that first reacts with a target substance while being immobilized onto a solid support which may be usable as a reaction vessel.
Suitable as the solid support is a hydrocarbon polymer (e.g., polystyrene and polypropylene), glass, metal, or gel, with the most preference for microtiter plates.
The term “detection antibody”, as used herein, refers to an antibody that can react with the target immobilized by the capture antibody to form the sandwich pattern of a capture antibody-target substance (antigen)-detection antibody complex. In addition, the detection antibody is an antibody that can be indirectly detected by a different labeled antibody and forms a complex in a sandwich pattern, thereby facilitating the detection of the target substance.
The detection of the complex in a sandwich form is carried out by further adding a “signal-detecting antibody” conjugated with a label generating a signal, reacting same with the detection antibody, and measuring the signal generated from the signal-detecting antibody. Therefore, the “signal-detecting antibody” refers to an antibody that can be directly detected through the label conjugated thereto. The label includes, but is not limited to, chemicals (e.g., biotin), enzymes (e.g., alkaline phosphatase), (3-galactosidase, HRP (horseradish peroxidase) and cytochrome P450, radioisotopes (e.g., C14, I125, P32, and S35), fluorophores (e.g., fluorescein), luminophores, chemiluminescents, and FRET (fluorescence resonance energy transfer). With respect to various labels and labeling methods, reference may be made to Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.
In detail, in an embodiment of the present disclosure, the label conjugated to the signal-detecting antibody includes enzymes for catalyzing chromogenic reactions, fluorescent reactions, luminescent reactions, or infrared reactions, but is not limited thereto. For example, the label may include p-galactosidase, HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, FITC (poly L-lysine fluorescein isothiocyanate), RITC (rhodamine-B-isothiocyanate), luciferase, and cytochrome P450, but is not limited thereto. A substrate for the enzyme conjugated to the signal-detecting antibody may be a chromogenic reaction substrate such as BCIP (5-bromo-4-chloro-3-indolyl-phosphate), NBT (nitro blue tetrazolium), naphthol-AS-B1-phosphate, and ECR (enhanced chemifluorescence reaction) when the enzyme is alkaline phosphatase and may include chloronaphthol, AEC (3-amino-9-ethylcarbazole), DAB (3,3′-o-diaminobenzidine), D-luciferin, lucigenin (bis-N-methylacridinium nitrate), luminol, resorufin benzyl ether, Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine; ADHP), HYP (p-phenylenediamine-HCl and pyrocatechol), TMB (3,3′,5,5′-Tetramethylbenzidine), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), O-phenylenediamine (OPD) and naphthol/pyronine, glucose oxidase, t-NBT (nitroblue tetrazolium), and m-PMS (phenzaine methosulfate) when the enzyme is HRP.
In an embodiment of the present disclosure, the concentration of the capture antibody and the detection antibody in the kit may be 0.1 to 15 μg/ml and, more particularly, 0.1 to 12 μg/ml, 0.1 to 10 μg/ml, 0.1 to 8 μg/ml, 0.1 to 7 μg/ml, 0.1 to 6 μg/ml, 0.1 to 5 μg/ml, 0.1 to 4 μg/ml, 0.1 to 3 μg/ml, 0.1 to 2 μg/ml, 0.1 to 1 μg/ml, 0.5 to 10 μg/ml, 0.5 to 8 μg/ml, 0.5 to 7 μg/ml, 0.5 to 6 μg/ml, 0.5 to 5 μg/ml, 0.5 to 4 μg/ml, 0.5 to 3 μg/ml, 0.5 to 2 μg/ml, 0.5 to 1 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, or 10 μg/ml, but is not limited thereto.
In another embodiment of the present disclosure, the concentration of the capture antibody in the kit may be higher than that of the detection antibody.
In an embodiment of the present disclosure, the kit may contain the capture antibody at a concentration of 5 μg/ml and the detection antibody at a concentration of 1 μg/ml.
According to another aspect thereof, the present disclosure provides a method for detecting a SARS-CoV-2 antigen from a sample, using the kit for detecting a SARS-CoV-2 antigen according to the present disclosure.
Since the method for detecting a SARS-CoV-2 antigen from a sample according to an aspect of the present disclosure includes the kit for detecting a SARS-CoV-2 antigen according to another aspect of the present disclosure described above, the common content is omitted to avoid undue complexity of the description.
The antibody of the present disclosure may be used for in vivo or in vitro imaging. According to another aspect of the present disclosure, the present disclosure provides a composition for imaging, containing a conjugate in which the antibody of the present disclosure is conjugated to a label generating a detectable signal conjugated to the antibody.
The label capable of generating a detectable signal includes T1 contrast materials (e.g., Gd chelate compounds), T2 contrast materials (e.g., superparamagnetic materials (i. e., magnetite, Fe3O4, γ-Fe2O3, manganese ferrite, cobalt ferrite, and nickel ferrite)), radioactive isotopes (e.g., 11C, 15O, 13N, P32, S35, 44Sc, 45Ti, 118I, 136La, 198Tl, 200Tl, 205Bi, and 206Bi), fluorescent materials (fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3/Cy5), chemiluminescent materials, magnetic particles, mass labels, and dense electron particles, but are not limited thereto.
According to an aspect thereof, the present disclosure provides a bispecific antibody to SARS-CoV-2.
In an embodiment of the present disclosure, the bispecific antibody binds specifically to SARS-CoV-2 and includes:
In an embodiment of the present disclosure, the antibodies or antigen binding fragments in (a) and (b) are derived respectively from the monoclonal antibodies RD3 (=K102.1) and RB6 (=K102.2) selected in the working examples.
The bispecific antibody according to an aspect of the present disclosure is in the form of a dimer or a multimer in which individual antibody or antigen binding fragment constituents are linked to each other. The antibody or antigen binding fragment constituents are linked to each other via a covalent bond. In an embodiment of the present disclosure, the bispecific antibody may be embodied into a fused protein or conjugate form. Hence, the antibodies or antigen binding fragments thereof may be indirectly coupled through chemical conjugation (known as an organic chemical method) or other means (e.g., expressed as a fusion protein or directly or via a linker (i.e., amino acid linker)).
In an embodiment of the present disclosure, the heavy variable region of (a) includes the amino acid sequence of SEQ ID NO: 34.
In an embodiment of the present disclosure, the light variable region of (a) includes the amino acid sequence of SEQ ID NO: 35.
In an embodiment of the present disclosure, the heavy variable region of (b) includes the amino acid sequence of SEQ ID NO: 25.
In an embodiment of the present disclosure, the light variable region of (b) includes the amino acid sequence of SEQ ID NO: 26.
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (a) includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 65.
In the bispecific antibody according to an embodiment of the present disclosure, the antibody or antigen binding fragment of (a) includes a light chain comprising the amino acid sequence of SEQ ID NO: 66.
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (b) is an scFv comprising the amino acid sequence of SEQ ID NO: 27.
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (a) is linked to the C terminus of the heavy chain in the antibody or antigen binding fragment of (b) or the antibody or antigen binding fragment of (b) is linked to the C terminus of the heavy chain in the antibody or antigen binding fragment of (a).
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (b) in the bispecific antibody is linked to the C terminus of the heavy chain in the antibody or antigen binding fragment of (a), but with no limitations thereto.
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (a) is linked to the N- or C-terminus of the light chain in the antibody or antigen binding fragment of (b) or the antibody or antigen binding fragment of (b) is linked to the N- or C-terminus of the light chain in the antibody or antigen binding fragment of (a).
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (b) is linked to the N-terminus of the light chain in the antibody or antigen binding fragment of (a), but with no limitations thereto.
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (b) is linked to the C-terminus of the light chain in the antibody or antigen binding fragment of (a).
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (a) and the antibody or antigen binding fragment of (b) bind to respective different epitopes on the receptor binding domain (RBD) of SARS-CoV-2 Spike protein.
In an embodiment of the present disclosure, the bispecific antibody inhibits the binding of the RBD of SARS-CoV-2 Spike protein to angiotensin converting enzyme 2 (ACE2) in host cells.
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (a) and the antibody or antigen binding fragment of (b) are each in a form of IgG1 or IgG4, but with no limitations thereto.
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (a) is in a form of IgG1 or IgG4, but with no limitations thereto.
In an embodiment of the present disclosure, the antibody or antigen binding fragment of (a) and the antibody or antigen binding fragment of (b) are each in a form of IgG1 or IgG4 and the antibody or antigen binding fragment of (b) is in a form of scFv.
According to another aspect thereof, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence coding for the bispecific antibody that binds specifically to SARS-CoV-2.
According to another aspect thereof, the present disclosure provides a recombinant vector carrying the nucleic acid molecule.
According to another aspect thereof, the present disclosure provides an isolate host cell anchoring the recombinant vector therein.
According to another aspect thereof, the present disclosure provides a pharmaceutical composition containing the bispecific antibody and a pharmaceutically acceptable carrier for treatment of SARS-CoV-2 infection.
In an embodiment of the present disclosure, the SARS-CoV-2 is a variant having, on the amino acid sequence of RBD, a mutation selected from the group consisting of N354D/D364Y, V367F, W436R, R408I, G476S, V483A, V341I, F342L, A435S, and a combination thereof, but with no limitations thereto.
In an embodiment of the present disclosure, the SARS-CoV-2 is selected from the group consisting of a wild type, an alpha variant, a beta variant, a gamma variant, a delta variant, and a kappa variant, but with no limitations thereto.
With the ability to inhibit the infection of SARS-CoV2 by binding specifically to the S protein responsible for the entry of SARS-CoV-2 into host cells, the anti-SARS-CoV-2 S protein-specific antibody or the antigen binding fragment thereof according to the present disclosure can be advantageously used as a therapeutic agent for COVID-19 and as diagnostic agent and kit for COVID-19.
A better understanding of the present disclosure may be obtained through the following examples which are set forth to illustrate, but are not to be construed to limit, the present disclosure.
Throughout the description, the term “%” used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt) % for solid/solid, (wt/vol) % for solid/liquid, and (vol/vol) % for liquid/liquid.
For use in selecting antibodies binding specifically to SARS-CoV-2, the spike full-length protein of SARS-CoV-2 and its constituent proteins 51 and RBD protein antigens were purchased from Sino Biological. The configuration of 51 and RBD proteins in the spike protein is depicted in
As shown in
A certain amount of secured SARS-CoV-2 RBD antigen was conjugated to epoxy-conjugated Dynabead (Invitrogen, USA), and human antibodies specific for RBD were selected using the phage display technique. After five rounds of bio-panning, binding antibody clones were measured for titer and degree of enrichment by round through titration. Subsequently, human antibody clones that had excellent reactivity to the RBD antigen and were specific for the antigen were selected using individual phage ELISA. The DNA was isolated through miniprep and ten types of RBD-specific human antibodies with different CDR sequences were secured after base sequencing. The results of the phage ELISA are presented in
3-1. Conversion of SARS-CoV-2 RBD Antigen-Specific scFv Antibody into IgG (IgG 4 Type)
For heavy and light chains of the ten kinds of RBD domain-specific scFv antibodies selected, insert DNAs were obtained. They were subcloned into bicistronic vectors for production full-IgG (IgG4 type) antibodies. For use in producing IgG antibodies in Expi293 cells, the recombinant DNA of each of the ten different scFvs was isolated in a large amount with high purity by using Maxi-prep kit. The purity of DNA was examined by NanoDrop.
The 10 different IgG antibodies were transfected into 1 L or greater of Expi293 cells with the aid of the Expi293 system (Invitrogen) comprising ExpiFectamine for transient expression, followed by incubation for 7 days. In order to purify the antibodies, the cultures were centrifuged and the supernatants (media) were obtained by removing the cell pellets.
The selected antibodies were purified by affinity column chromatography using protein A sepharose beads. In addition, the RBD-specific IgG antibodies were analyzed for yield after mass production and final purification.
The analysis of the 10 different RBD-specific antibodies for productivity showed that the five antibodies RB4, RB6, RD3, RD10, and RG6 were produced at high yields of 50 mg/L or higher, with the productivity of 140 mg/L or higher given for RD3, RD10, and RG6 clones (
The five selected SARS-CoV-2 RBD-specific antibodies were loaded at the same content to a polyacrylamide gel. After SDS-PAGE, all the five antibodies were observed to have a purity of 95% or higher and a molecular weight of 50 kDa for heavy chain and 25 kDa for light chain, as analyzed by Coomassie Brilliant Blue staining (
In order to examine the reactivity of the five selected SARS-CoV-2 RBD-specific antibodies to SARS-CoV-2 RBD, S1 domain, and full-length spike protein antigens, 0.1 μg of each antigen was coated on 96-well high binding plates (Corning, USA) and ELISA was conducted for each antibody. The results are depicted in
As can be seen in
In order to examine the reactivity of the five selected SARS-CoV-2-specific antibodies to SARS-CoV RBD as well as SARS-CoV-2 RBD, 0.1 μg of each of purchased SARS-CoV-2 and SARS-CoV RBD was coated on 96-well high binding plates (Corning, USA) and ELISA was conducted for each antibody. The results are depicted in
As shown in
The five selected antibodies were measured for KD values for SARS-CoV-2 RBD and S1. In this regard, SARS-CoV-2 RBD and S1 purchased were each bound to 96-well high binding plates (Corning, USA), and absorbance (450 nm) was read while increasing the concentrations of the selected antibodies. The results are depicted in
As shown in
In relation to SARS-CoV-2, nine typical RBD mutant antigens (V431A, F342L, V367F, R408I, A435S, W436R, G476S, V483A, and N354D/D364Y) that were classified worldwide were purchased from Sino Biological. Each of the antigens was measured for purity and molecular weight by SDS-PAGE. As a result, the RBD mutants were measured to have a size of about 30 kDa and a purity of 90% or higher (
In addition, in order to examine the reactivity of the five selected antibodies to the nine SARS-CoV-2 RBD mutant antigens, ELISA was performed. As a result, all the five selected RBD-specific antibodies were found to bind to the nine RBD mutant protein antigens, too (
The neutralization ability of antibody to inhibit protein-protein interaction between hACE2 receptor and SARS-CoV-2 spike protein was investigated by ELISA using purified proteins. To this end, neutralization ability was analyzed using the Spike RBD (SARS-CoV-2): ACE2 inhibitor screening assay kit (BPS Bioscience, Cat. No. 79931).
In brief, SARS-CoV-2 RBD protein (Fc-tagged) was coated on 96-well plates included within the assay kit and incubated with the ligand human ACE2 (His-tagged; hACE2-His). Then, anti-His-HRP and HRP substrate were added before measuring chemiluminescence on ELISA reader to analyze binding affinity between RBD domain-ACE2. A schematic diagram for assaying direct interaction between hACE2-His and SARS-CoV-2 RBD is given in
As shown in
Using the established direct interaction assay, the selected antibodies were measured for ability to inhibit interaction between SARS-CoV-2 RBD and hACE2.
In brief, SARS-CoV-2 RBD protein (Fc-tagged) was coated on 96-well plates included within the assay kit and incubated with the ligand human ACE2-His alone and in combination with the selected antibodies (0.016, 0.08, 0.4, 2, 10, and 50 nM). Then, anti-His-HRP and HRP substrate were added before measuring chemiluminescence on ELISA reader to analyze the ability of the antibodies to inhibit interaction between SARS-CoV-2 RBD and hACE2. A schematic diagram for assaying direct interaction is given in
As shown in
In
The binding kinetics of antibodies (RD3 and RB6) to SARS-CoV-2 RBD were analyzed at 25° C. on an iMSPR-mini instrument (iCLUEBIO, Seongnam, Republic of Korea) using 10 mM HEPES pH 7.4, 700 mM NaCl, 2 mM CaCl2), 1 mM MnCl2, and 0.005% (v/v) Tween-20 as a running buffer. The recombinant SARS-CoV-2 RBD (wild-type, Alpha, Beta, Gamma, Delta, or Kappa) was covalently immobilized on the surface of a COOH—Au chip (iCLUEBIO) up to 500 response units through standard amine coupling. The monoclonal antibodies (8, 16, 32, 64, and 128 nM) were injected onto the surface of a sensor chip at a flow rate of 50 μL/min. Curve fitting and data analysis were performed using the iMSPR analysis software (Tracedrawer; iCLUEBIO). The results are summarized in Tables 1 and 2 and depicted in
As understood from the data of Tables 1 and 2, the antibodies RD3 and RB6 of the present disclosure had high binding affinity for various types of SARS-CoV-2 RBD.
Pseudotyped replication-deficient lentiviral particles carrying the SARS-CoV-2 S protein of the wild-type or B.1 (D614G) variant, and a firefly luciferase reporter gene were prepared using Lenti-X™ SARS-CoV-2 packaging mix (Takara Bio, Kusatsu, Japan). Briefly, the packaging mix was transiently transfected into Expi293™ cells with ExpiFectamine™ 293 reagent. After culturing for 72 hours, the supernatants containing the pseudotyped viruses were collected and centrifuged briefly (500×g for 10 min) to remove cellular debris. Virus titration was measured using Lenti-X GoStix™ Plus (Takara Bio) according to the manufacturer's instructions.
The pseudotyped replication-deficient Moloney murine leukemia virus (MLV) particles carrying the SARS-CoV-2 S protein of B.1.1.7 (alpha), B.1.617.2 (delta) or B.1.617 (kappa) variant, and a firefly luciferase reporter gene were obtained from eEnzyme (Gaithersburg, MD, USA).
To determine the neutralization activity of monoclonal antibodies on pseudotyped virus infection, 1×104 293T/hACE2 cells in 50 μL culture medium were seeded in 96-well tissue culture plates overnight. Serial dilutions of the antibodies were pre-incubated at room temperature for 10 minutes with 50 μL of pseudotyped virus [1×107 PFU/mL], and the mixture was subsequently incubated with the cells for 24 hours.
The firefly luciferase reporter gene expression (indicative of viral presence) was measured using ONE-Glo™ luciferase substrate (Promega, Madison, WI, USA). In brief, the culture medium was removed and incubated with 100 μl of ONE-Glo™ substrate. After 5 minutes, 70 μl supernatant was transferred to white flat-bottom 96-well assay plates (Corning; Lowell, MA, USA) and the luminescence signal was measured using the Synergy H1 microplate reader. The recorded relative luminescence units were normalized to those derived from cells infected with each SARS-CoV-2 pseudotyped virus in the absence of antibodies. Dose-response curves for IC50 values were determined using 4-parameter non-linear regression analysis (Graph Pad Prism 8.0).
Results are summarized in Table 3 and depicted in
All experiments for true wild-type SARS-CoV-2 viruses were performed in a Biosafety Level 3 laboratory facility. A dilution of 40 μl of the patient sample in medium was inoculated into 150,000 VERO E6 cells in a 6-well plate. After 72 hours of infection, the supernatant was collected, centrifuged, and stored at −80° C. After two consecutive passages, RNA samples were prepared from the supernatant, and NGS confirmed that the clinical isolate was wild-type.
All procedures for in vivo efficacy studies of monoclonal antibodies were approved by the Institutional Animal Care and Use Committee (IACUC) at KNOTUS (KNOTUS IACUC, Protocol Number: 22-KE-0076) and performed in a biosafety cabinet at the Biosafety Level 3 facilities. Female K18-hACE2 c57BL/6J mice 8-10 weeks old were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and housed in a specific pathogen-free condition and allowed to freely access foods and water. They were randomly assigned into experimental groups.
The mice (n=7/group) were anesthetized by isoflurane inhalation and intranasally inoculated with 104 PFU wild-type SARS-CoV-2 in 30 μL of PBS. After viral infection, the monoclonal antibody IgG1 or IgG4 was intravenously injected once (+3 h) at a dose of 30 mg/kg. After infection, the mice were monitored for weight change and the results are depicted in
In addition, clinical severity was scored according to the criteria of Table 4, below, and the results are depicted in
As shown in
Selection was made of SARS-CoV-2 RBD antigen-specific human antibodies from the human synthetic single-chain variable fragment (scFv) antibody library. As illustrated in
First, SARS-CoV-2 RBD antigen-specific scFv clones were selected through five rounds of bio-panning using magnetic beads Dynabeads M-270 epoxy, Invitrogen) coated with 4 μg of the recombinant SARS-CoV-2 RBD antigen.
Then, 96 clones were randomly selected from output colonies formed on plates and tested for their reactivity to the SARS-CoV-2 RBD antigen by phase ELISA to pick out human antibody clones that are highly reactive to and specific for the corresponding antigen. Results of the phase ELISA are depicted in
2.1. SARS-CoV-2 RBD Antigen-Specific scFv Antibody Conversion to IgG
Heavy and light chains of four types of the selected RBD-specific scFv antibodies were amplified by PCR to obtain respective insert DNAs which were then cloned into mammalian expression vector pcDNA3.1 for production of IgG antibodies. Each recombinant DNA of the four types of scFvs was overproduced with high purity in Expi293 cell using a Maxi-prep kit. The purity of DNA was measured using nanodrop 2000 spectrophotometer (Thermo Fisher Scientific).
Transient expression was performed using the Expi293 system (Invitrogen). In this regard, the four types of IgG antibodies were transfected into Expi293 cells with the ExpiFectamine 293 transfection kit (Gibco) and then incubated for five days. Following centrifugation of the cell culture, the cell pellet was removed. The antibodies were purified from the supernatant (medium).
The purification of the selected antibodies was conducted by affinity chromatography using protein A sepharose bead (Repligen, Waltha, MA, USA).
The four SARS-CoV-2 RBD-specific antibodies (K102.1, K102.2, K102.3, and K102.4) were loaded in the same amount onto a polyacrylamide gel and resolved by SDS-PAGE. From Coomassie Brilliant Blue staining, it was observed that each of the four antibodies had a final purity of 90% or higher and molecular weights of approximately 50 kDa for the heavy chain and 25 kDa for the light chain (
In addition, the binding affinity (KD) of the selected antibodies was observed to be 1.643×10−9 M for K102.1 and 2.465×10−9 M for K102.2 (
For use as a detection antibody in sandwich ELISA, the antibody K102.1 produced in Example 2 was conjugated with the marker HRP (horseradish peroxidase), using EZ-Link™ Plus Activated Peroxidase Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.
To select an antibody pair recognizing different epitopes of SARS-CoV-2 RBD from the selected SARS-CoV-2 RBD-specific antibody group, the four selected antibodies were subjected to competition ELISA with the HRP-conjugated antibody (K102.1-HRP).
The results are depicted in
Therefore, K102.1 and K102.2 were selected as a pair of antibodies that can be utilized for the development of Sandwich ELISA.
The selected antibody pair (K102.1 and K102.2) determined in Example 4 was evaluated for suitability for use in the sandwich ELISA of the present disclosure by verifying pairing therebetween. The sandwich ELISA was performed using K102.1 or K102.2 as the capture antibody and K102.1-HA or K102.2-HA as the detection antibody.
As shown in
Furthermore, SPR (Surface Plasmon Resonance)-based competition binding assay was performed to determine whether the selected antibody pair bind to distinct epitopes on the SARS-CoV-2 RBD. By measuring real-time binding of the selected antibodies to the SARS-CoV-2 RBD an iMSPR mini-instrument (icluebio, South Korea), the competition binding assay was used to evaluate whether K102.1 or K102.2-HA possess unique or overlapping binding sites.
In brief, 128 nM K102.1 in a HEPES buffered Steinberg's solution containing 0.005% (v/v) Tween-20. was injected onto the SARS-CoV-2 RBD surface (ca. 1,000 RU) at a flow rate of 50 μl/min for 240 seconds. Subsequently, 128 nM K102.2-HA was introduced under the same conditions onto the surface where K102.1 and SARS-CoV-2 RBD were bound. The resulting curves were obtained as sensorgrams using the iMSPR analysis software.
As shown in
A Sandwich ELISA method for detecting SARS-CoV-2 was developed using the SARS-CoV-2 specific monoclonal antibodies K102.1 (capture antibody) and K102.2 (detection antibody). The sandwich ELISA method of the present disclosure is schematically illustrated in
To determine the optimal conditions therefor, sandwich ELISA was carried out with increasing concentrations of either the capture antibody or the detection antibody.
As a result, it was confirmed that 5 μg/ml of the capture antibody (K102.1) and 1 μg/ml of the detection antibody (K102.2-HA) are optimal concentrations for sandwich ELISA method (
A calibration curve was derived to determine the limit of detection of SARS-CoV-2 RBD in the sandwich ELISA of the present disclosure. The reproducibility of the calibration curve was validated through six independent analyses, and the linear dynamic range of the derived calibration curve was determined to be between 0 ng/ml and 12 ng/ml (equivalent to 0 pM to 480 pM) for the SARS-CoV-2 RBD (
The limit of detection (LOD) for the sandwich ELISA of the present disclosure was derived by calculating the standard deviation (SD) and slope (S) of the calibration curve according to Equation 1. The determined LOD was found to be 0.55 ng/ml (equivalent to 22 pM).
Limit of Detection (LOD)=3×(standard deviation (SD)/slope (S) of calibration curve) [Equation 1]
In addition, the sensitivity was derived by calculating standard deviation (SD) and mean of the blank according to the following Equation 2. The sensitivity was determined to be 0.48 ng/ml (19.2 pM).
Sensitivity)=3×standard deviation (SD)+mean of blank [Equation 2]
Table 5 shows a comparison of the performance of the Sandwich ELISA of the present disclosure with two types of Sandwich ELISA kits previously developed and sold on the market.
From the data of Table 5, it is understood that the kit of the present disclosure with a sensitivity of 48 ng/ml (19.2 pM) is superior in terms of sensitivity to the previously approved and commercially available kits with respective sensitivities of 12 ng/ml (Eaglebio) and 0.58 ng/ml (Biozol).
To validate the Sandwich ELISA method developed using the selected antibody pair of the present disclosure, the coefficient of variation (CV) and recovery were measured by performing both intra- and inter-assays. Intra-assay precision was determined by measuring samples six times in triplicate within the same assay run. Inter-assay precision was determined by measuring a sample in triplicate in six separate assay runs.
The mean and standard deviation (SD) was calculated. The coefficient of variation (CV) was calculated according to the following equation 3:
CV(%)=(SD/mean)×100. [Equation 3]
Recovery was calculated according to the following equation 4:
Recovery (%)=Average measured concentration/expected concentration]×100 [Equation 4]
As shown in
Therefore, it was confirmed that the sandwich ELISA method of the present disclosure is a sensitive, accurate, and reliable technique for detecting SARS-CoV-2 RBD.
The optimized sandwich ELISA method of the present disclosure was evaluated for ability to detect SARS-CoV-2 RBD mutants.
Regarding SARS-CoV-2, representative RBD mutants categorized by countries worldwide were secured, comprising eight types: A435S (Finland), N354D (China), G476S and V483A (USA), F342L, V341I, and N501Y (UK), and L452R/T478K (India). The Sandwich ELISA was performed in the presence of increasing concentrations of these antigens to 32 pM, 96 pM, and 200 pM.
Briefly, 96-well high-binding microplates (Corning) were coated with the capture antibody K102.1 and then blocked using 3% (w/v) bovine serum albumin (BSA) in PBS for 2 hours at 37° C. Next, 100 μL of increasing concentrations of the RBDs of wild-type SARS-CoV-2 mutants (A435S, N354D, G476S, V483A, F342L, V341I, N501Y, and L452R/T478K) were added to each well, and the microplates were incubated for 3 hours at 37° C. The plates were washed thrice with 0.05% (v/v) PBST and incubated with the detection antibody HA-tagged K102.2 (K102.2-HA). Subsequently, the plates were washed thrice with 0.05% (v/v) PBST and incubated with HRP-conjugated anti-HA antibody to detect K102.2-HA. To this end, the plates were washed thrice and a TMB substrate solution (Thermo Fisher Scientific) was added to each well, followed by reaction with HRP for 15 minutes. The reaction was terminated by adding 1 M H2504. (100 100 μL/well). The absorbance of each sample was read at 450 nm on a microplate reader.
Consequently, as shown in
Four SARS-CoV-2 RBD-specific human scFvs with a complementarity determining region (CDR) sequence were isolated using phage-display technology from the human synthetic scFv library. To prevent Fab arm exchange that results in an unwanted heterogeneous mixture of antibodies by half molecule exchanged with endogenous IgG4, IgG4-based mAbs with S228P mutations [IgG4 (S228P)] were constructed. Among the antibodies, a noncompeting pair of mAbs, K102.1 and K102.2, which recognize independent epitopes of the SARS-CoV-2 RBD, was identified using a competition ELISA (
Based on these parental mAbs, three forms of IgG4 (5228P)-(scFv)2 bispecific antibody (bsAb), such as structures of K202.A (
Of the designed bispecific antibody forms, the two IgG4(S228P)-(scFv)2 bsAb forms K202.A and K202.B were produced with a purity of 90% or higher (
As can be seen in
To further confirm whether K202.B could recognize two independent epitopes of SARS-CoV-2 RBD, competition assays were performed using SPR. The results are depicted in
As shown in
2-1. Neutralizing Activity of bsAbs in hACE2-RBD Interaction
To assess the neutralizing activity of the bsAbs in hACE2-RBD interactions, ELISA-based neutralization assays were performed with microtiter plates to which the recombinant RBD proteins of wild-type SARS-CoV-2 and SARS-CoV-2 variants comprising alpha, beta, gamma, delta, and kappa variants. The microtiter plates were incubated with recombinant hACE2 in the presence or absence of parental mAb, an mAb cocktail containing parental mAb, or bsAbs. The results are depicted in
As is understood from data of
Furthermore, K202.B also exhibited a strong inhibitory effect on hACE2 binding to RBDs with N354D/D364Y, V367F, W436R, R408I, G476S, V483A, V341I, F342L, or A435S mutations (
2-2. Neutralizing Activity of bsAbs Against SARS-CoV-2 Pseudotyped Virus Infection
To assess the neutralizing ability of the bsAbs of the present disclosure against SARS-CoV-2 pseudotyped virus infection, SARS-CoV-2 pseudotyped virus neutralization assays were conducted using hACE2-overexpressing 293T stable cell line (293T/hACE2 cells) (
As shown in
In contrast, K202.B exhibited stronger inhibitory effects on the infection of almost all the tested pseudotyped viruses than parental mAbs or the mAb cocktail with the IC50 values of mostly subnanomolar or nanomolar concentrations. The inhibitory effect of the bispecific antibody K202.A was similar to or slightly lower than that of the bispecific antibody K202.B (
2-3. Neutralizing Activity of bsAbs in Live Virus Infection
Next, the effect of the bispecific antibody K202.B of the present disclosure on the antibody-dependent enhancement (ADE) was evaluated using permissive cells (293T/hACE2 cells) and Fc gamma receptor-bearing cells (293T, K562, and THP-1 cells). Changes in the pseudotyped virus infection rate of various SARS-CoV-2 wild-type and variants were monitored in the presence of K202.B. The results are depicted in
As can be seen in
To investigate the pharmacokinetics of the bispecific antibody K202.B, K202.B was intravenously injected at a dose of 5 mg/kg into ICR mice from which blood was then sampled at various times. Serum levels of K202.B were measured by ELISA. The results are depicted in
As shown in
Next, an analysis was made of in vivo efficacy of the bispecific antibody K202.B on wild-type SARS-CoV-2.
Briefly, wild-type SARS-CoV-2 viruses were intranasally administered to the K18-hACE2 transgenic (TG) mice. After 3 hours, the mice were intravenously injected with two doses (5 and 30 mg/kg) of K202.B, or a single dose (30 mg/kg) of K102.1. At 6 days post-infection, the mice were sacrificed and analyzed as illustrated in
As shown in
In addition, as shown in
In addition, lung samples from all mice sacrificed at 6 dpi were subjected to RT-qPCR to determine the relative expression of viral E and RNA-dependent RNA polymerase (RdRp) genes. The results are depicted in
As shown in
Furthermore, histopathological examination made of lungs from the infected mice at 6th dpi and the results are depicted in
As can be seen in Table 9, PBS- and K102.1-treated mice scored 1 or more due to significant pulmonary lesions. In contrast, a high proportion of the K202.6-treated group showed a score of 0 at both 5 and 30 mg/kg doses.
As shown in
To assay in vitro cytotoxicity of the bispecific antibody of the present disclosure, endothelial cells were measured for cell viability after treatment with 20 μg/ml K202.B or 36 μg/ml 5-fluorouracil. The results are given in
As shown in
Moreover, to confirm the effect of the bispecific antibody on endothelial activation, the cells were treated with inflammatory cytokine (hTNF-α) or bispecific antibody K202.B and measured for expression levels of cell adhesion molecules (ICAM-1 and VCAM-1). The results are depicted in
As shown in
To evaluate in vivo cytotoxicity of the bispecific antibody K202.B, K202.B was intravenously injected to mice and hepatic and renal toxicity were assayed using the sera.
The results are depicted in
As shown in
In vivo efficacy of bispecific K202.B against SARS-CoV-2 delta variant was analyzed. To evaluate the in vivo efficacy of K202.B against SARS-CoV-2 delta variant, K18-hACE2 TG mice was intranasally challenged with SARS-CoV-2 delta virus. After 3 hours, the mice received intravenous injections of a dose of 5 or 30 mg/kg of K202.B (
As shown in
In addition, as shown in
Lung samples from all mice sacrificed at 6 dpi were subjected to RT-qPCR to determine the relative expression of viral E and RNA-dependent RNA polymerase (RdRp) genes. As can be seen in
Furthermore, histopathological examination made of lungs from the infected mice at 6 dpi and the results are depicted in
As shown in
The cell lines 293T, K562, and THP-1 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). A purchase was made of CHOZN-GS cells (derived from CHO-K1 and adapted to serum-free and suspension conditions) from Merck (Merck, Whitehouse Station, NJ, USA), Expi293 cells from Thermo Fisher Scientific (Waltham, MA, USA), and human umbilical vein endothelial cells (HUVEC) from Lonza (Basel, Switzerland). 293T cells were cultured in DMEM (Thermo Fisher Scientific) whereas K562 and THP-1 cells were cultured in RPMI media (Thermo Fisher Scientific) media supplemented with 10% (v/v) FBS (Thermo Fisher Scientific) and 100 U/mL penicillin-streptomycin (Thermo Fisher Scientific) at 37° C. in 5% CO2. HUVECs were maintained in EGM-2 (Lonza). Expi293 cells were cultured in Expi293™ Expression Media in shaking incubators at 37° C., 125 rpm, and 8% CO2. CHOZN-GS cells were in EX-CELL Advanced CHO Fed-batch medium (Sigma-Aldrich, Burlington, MA, USA) in a microscale bioreactor Ambr® 15 (Sartorius, Gottingen, Germany) at 37° C.
The binding kinetics of antibodies to SARS-CoV-2 RBDs were analyzed at room temperature on an iMSPR-mini instrument (iCLUEBIO, Seongnam, Republic of Korea) using 10 mM HEPES pH 7.4, 700 mM NaCl, 2 mM CaCl2), 1 mM MnCl2, and 0.005% (v/v) Tween-20 as a running buffer. The recombinant SARS-CoV-2 RBDs (wild-type and variants comprising B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), and B.1.617 (kappa)) were covalently immobilized on the surface of a COOH—Au chip (iCLUEBIO) up to 500 response units through standard amine coupling. Antibodies (8, 16, 32, 64, and 128 nM, respectively) were injected onto the surface of a sensor chip at a flow rate of 50 μL/min. Kinetics evaluation data was obtained using a 1:1 binding model.
To evaluate the ability of K202.B to bind to different regions of the RBD, competition experiments were performed. After the immobilization of 5 nM WT-RBD-His on the surface of a COOH—Au chip, a high concentration (512 nM) of K102.1 or K102.2 antibody was added to saturate the corresponding binding sites on the RBD. Then, 128 nM K202.B was added. Conversely, following the addition of 512 nM K202.B to the surface of recombinant WT-RBD-His-immobilized sensor chip, 256 nM K102.1 or K102.2 was subsequently added. Curve fitting and data analysis were performed using the iMSPR analysis software (Tracedrawer; iCLUEBIO).
The ability of antibodies to inhibit the interaction of the SARS-CoV-2 RBD with hACE2 was investigated using ELISA. 50 ng of purified Fc-tagged hACE2 (hACE2-Fc) (R&D Systems, Minneapolis, MN, USA) was coated in each well of a 96-well plate and incubated for 2 hours at room temperature. After washing with immunobuffer (BPS Bioscience, San Diego, CA, USA), the plates were blocked with blocking buffer (BPS bioscience) for 1 hour at room temperature. Simultaneously, 25 nM of purified SARS-CoV-2 WT- or variant-RBD-His (alpha, beta, gamma, delta, and kappa) (Sino Biological) was pre-incubated in the presence or absence of mAb or bsAbs (0.006, 0.024, 0.097, 0.39, 1.56, 6.25, 25, and 100 nM) for 1 hour at room temperature.
To generate stable 293T/hACE2 cell lines, a pUNO1-hACE2 plasmid (InvivoGen, San Diego, CA, USA) was transfected into 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 48 hours of transfection, the cells were cultured in a medium containing 20 μg/mL blasticidin (InvivoGen) to select positive cell populations. The expression of hACE2 was determined using immunoblot and immunocytochemical analysis. For immunoblot analysis, 293T and 293T/hACE2 cell lines were lysed with SDS sample buffer, and subjected to immunoblot analysis using a polyclonal anti-hACE2 antibody (R&D Systems). The distribution of hACE2 in the cell membrane was studied through immunocytochemical analysis. Briefly, 293T and 293T/hACE2 cells were plated on Nunc® Lab-Tek® II chamber slides (Thermo Fisher Scientific) coated with poly-L-lysine (0.1 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA). After 24 hours, the cells were fixed with 4% formaldehyde for 10 min and washed twice with PBS.
The cells were blocked using PBS containing 1% (w/v) BSA and incubated with polyclonal anti-hACE2 antibody overnight at 4° C. After washing thrice with PBS, the cells were subsequently incubated with Alexa Fluor 488-labeled anti-goat secondary antibody (Invitrogen) at room temperature for 1 hour, then mounted with mounting solution (Dako North America, Carpinteria, CA, USA). The stained cells were imaged using confocal microscopy (LSM510; Carl Zeiss, Oberkochen, Germany).
Pseudotyped replication-deficient lentiviral particles carrying the SARS-CoV-2 spike (S) protein of the wild-type or D614G variant, and a firefly luciferase reporter gene were prepared using Lenti-X™ SARS-CoV-2 packaging mix according to the manufacturer's instruction (Takara Bio, Kusatsu, Japan). Briefly, the packaging mix was transiently transfected into Expi293™ cells with ExpiFectamine 293 reagent. After culturing for 72 hours, the supernatants containing the pseudotyped viruses were collected and centrifuged briefly (500×g for 10 min) to remove cellular debris. Virus titration was measured using Lenti-X GoStix Plus (Takara Bio) according to the manufacturer's instructions. The pseudotyped replication-deficient Moloney murine leukemia virus particles carrying the SARS-CoV-2 S protein of alpha, beta, gamma, delta, or kappa variants and a firefly luciferase reporter gene were obtained from eEnzyme (Gaithersburg, MD, USA).
To determine the neutralization activity of mAbs or bsAbs against pseudotyped virus infection, 1×10 4 293T/hACE2 cells in 50 μL culture medium were seeded in 96-well tissue culture plates overnight. Serial dilutions of the antibodies were pre-incubated at room temperature for 10 min with 50 μL of each pseudotyped virus (1×107 PFU/mL), and the mixture was subsequently incubated with the cells for 24 hours. The firefly luciferase reporter gene expression (which is indicative of viral presence) was measured using ONE-Glo luciferase substrate (Promega, Madison, WI, USA). Next, the culture medium was removed and incubated with 100 μL of ONE-Glo substrate. After 5 min, 70 μL supernatant was transferred to white flat-bottom 96-well assay plates (Corning) and the luminescence signal was measured using the Synergy H1 microplate reader. The recorded relative luminescence units were normalized to those derived from cells infected with each SARS-CoV-2 pseudotyped virus in the absence of antibodies. Dose-response curves for IC50 values were determined by nonlinear regression (GraphPad Prism 8.0 software).
For in vivo efficacy studies, 8-week-old female B6.Cg-Tg(K18-ACE2)2Prlmn/J (hACE2) mice (The Jackson Laboratory, CA, USA), were housed in a certified A/BSL3 facility (Korea Zoonosis Research Institute, lksan, Republic of Korea). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at KNOTUS (No. 22-KE-0076), and all experimental protocols requiring biosafety were approved by the Institutional Biosafety Committee of Jeonbuk National University (approval number: JBNU 2020-11-003-003) and performed in a biosafety cabinet at the BL3 and ABL3 facilities of Korea Zoonosis Research Institute at Jeonbuk National University.
The hACE2-transgenic (hACE2-TG) mice (n=7) was intranasally inoculated with 30 μL of wild-type or delta variant virus (1×10 4 PFU) under anesthesia. Three hours after infection, PBS, mAbs, or bsAbs were injected intravenously.
The mice were monitored daily for weight change and clinical severity based on the criteria as shown in the table 11.
The SARS-CoV-2 burden in lung tissues was determined via RT-qPCR.
Lung tissues were harvested from hACE2-TG mice 6 days after SARS-CoV-2 wild-type or delta variant infection, and total RNAs were extracted from the collected tissues using Wizol Reagent (Wizbiosolutions, Seongnam, Republic of Korea). The samples were subjected to RT-qPCR using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA).
Following the reverse transcription of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA), the reaction mixture (20 μL total) contained 2 μL of template cDNA, 10 μL of 2× Premix Ex Taq, 200 nM primer, and a probe (E gene: forward primer 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′ (SEQ ID NO: 67), reverse primer 5′-ATATTGCAGCAGTACGCACACA-3′ (SEQ ID NO: 68), probe 5′-FAM-ACACTAGCCATCCTTACTGCGC TTCG-BHQ1-3′ (SEQ ID NO: 69); RdRp gene: forward primer 5′-ATGAGCTTAGTCCTGTTG-3′ (SEQ ID NO: 70), reverse primer 5′-CTCCCTTTGTTGTGTTGT-3′ (SEQ ID NO: 71), probe 5′-HEX-AGATTGTCTTGTGCTGCCGGTA-BHQ1-3′ (SEQ ID NO: 72)). These reactions were denatured at 95° C. for 30 seconds, and then subjected to 45 cycles of 95° C. for 5 seconds and 60° C. for 20 seconds. After completion of the reaction cycles, the temperature was increased from 65 to 95° C. at a rate of 0.2° C./15 seconds and fluorescence was measured every 5 seconds to construct a melting curve. A control sample lacking template DNA was run with each assay. All measurements were performed in duplicate to ensure reproducibility. The authenticity of the amplified product was determined using melting curve analysis. All data were analyzed using Bio-Rad CFX Manager analysis software version 2.1 (Bio-Rad Laboratories). The viral burden was expressed by the copy number of viral RNA per nanogram of total RNA after calculating the absolute copy number of viral RNA in comparison with the standard cDNA template.
Excised mouse lung tissues were fixed with 4% (v/v) paraformaldehyde (PFA) in PBS and processed for paraffin embedding. The paraffin blocks were sliced into 3 μm-thick sections using a microtome (HistoCore MULTICUT R; Leica, Germany) and mounted on silane-coated glass slides (5116-20F; Muto, Tokyo, Japan). Hematoxylin and eosin, periodic acid—Schiff, and modified Masson's trichrome stains were used to identify histopathological changes in all the organs. The histopathology of the lung tissue was observed using light microscopy (Axio Scope A1; Carl Zeiss). Pathological scores were determined based on the percentage of inflammation area for each section in each group using the following scoring system: 0, no pathological change; 1, affected area (10%); 2, affected area (10-50%); 3, affected area (50%); an additional 0.5 point was added when pulmonary edema and/or alveolar hemorrhage was observed.
Fifty microliters of each SARS-CoV-2 pseudotyped virus (1×10 7 PFU/mL) was preincubated with different concentrations of K202.B (0.044, 0.138, 0.42, 1.24, 3.7, 11.1, 33.3, and 100 nM) in culture medium. After 30 minutes of incubation at room temperature, the mixture was added to 293T, 293T/hACE2, K562, or THP-1 cells (1×10 4 cells in a 96-well plate). The cells were cultured for 24 hours, and the luciferase activity of infected cells was measured as described in “pseudotyped virus neutralization assay”.
A total of 5×103 HUVECs were plated in 96-well plates and incubated in the presence or absence of 20 μg/mL K202.B or 36 μg/mL 5-fluorouracil for 24 hours at 37° C. Cell viability was determined using the Cell Counting Kit-8 (Sigma) according to the manufacturer's instructions. The final absorbance was measured at 450 nm using a spectrophotometer (BioTek).
The effect of K202.B on endothelial cell activation was determined by treating cells with 20 ng/ml of human tumor necrosis factor-α (hTNF-α; Millipore), 20 μg/ml of K202.B, or PBS for 24 hours and fixing with 4% (v/v) PFA. The cells were fixed with 4% (v/v) PFA in PBS and incubated with 10 μg/well of intercellular cell adhesion molecule-1 (ICAM-1; Abcam, Cambridge, MA, USA) or vascular cell adhesion molecule-1 (VCAM-1; Abcam) antibody for 1 hour at 25° C. Then, Alexa Fluor 647-conjugated anti-mouse IgG or anti-rabbit IgG (1:1000; Invitrogen) was incubated for 1 hour at 25° C. All samples were analyzed using flow cytometry with the aid of FlowJo software (TreeStar, Ashland, OR, USA).
In vivo toxicity and serum pharmacokinetic studies using animals were approved by the IACUC (Approval No. NCC-21-693) of the National Cancer Center, Republic of Korea. Eight-week-old female Institute of Cancer Research (ICR) mice (Orient Bio Inc., Seongnam, Republic of Korea) were intravenously injected with 5 or 30 mg/kg of K202.B (n=3 per group). At 4, 8, 24, 72, 120, 168, 264, 384, and 504 hours post-inoculation, blood samples (50 μL) were collected from each mouse and centrifuged at 5000×g for 20 min at 4° C. The serum was stored at −80° C. for evaluation of biochemical parameters. Serum levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatinine (CRE), and blood urea nitrogen (BUN) were measured using a Fuji Dri-Chem 3500 Biochemistry Analyzer (Fujifilm, Tokyo, Japan).
Serum levels of K202.B were determined using a human IgG ELISA kit (Abcam) according to the manufacturer's instructions. Optical density was measured using a Synergy H1 microplate reader, and values were compared to those from a concurrently analyzed standard curve.
Data were analyzed with GraphPad Prism 8.0 software using two-tailed Student's t-test for comparisons between two groups, and one-way analysis of variance (ANOVA) with Bonferroni's correction for multiple comparisons. All data represent the mean±standard deviation (S.D.). A P-value less than 0.05 was considered statistically significant (*P<0.05, **P<0.01, ***P<0.001).
This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing XML file entitled “000352uscoa_SequenceListing.XML”, file size 72.2 kilobytes, created on 25 Oct. 2023. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2021-0054590 | Apr 2021 | KR | national |
| 10-2021-0054596 | Apr 2021 | KR | national |
| 10-2021-0054600 | Apr 2021 | KR | national |
| 10-2021-0054603 | Apr 2021 | KR | national |
| 10-2021-0054606 | Apr 2021 | KR | national |
| 10-2021-0118656 | Sep 2021 | KR | national |
| 10-2022-0019199 | Feb 2022 | KR | national |
This application is a continuation of PCT Application No. PCT/KR2022/006054, filed on 27 Apr. 2022, PCT Application No. PCT/KR2022/006055, filed on 27 Apr. 2022 and PCT Application No. PCT/KR2022/010843, filed on 22 Jul. 2022, which claims benefit of Korean Patent Application Nos. 10-2021-0054590, 10-2021-0054596, 10-2021-0054600, 10-2021-0054603, and 10-2021-0054606, filed on 27 Apr. 2021, 10-2022-0019199, filed on 14 Feb. 2022 and 10-2021-0118656, filed on 6 Sep. 2021. The entire disclosures of the applications identified in this paragraph are incorporated herein by references.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/KR2022/010843 | Jul 2022 | US |
| Child | 18383595 | US | |
| Parent | PCT/KR2022/006054 | Apr 2022 | US |
| Child | PCT/KR2022/010843 | US | |
| Parent | PCT/KR2022/006055 | Apr 2022 | US |
| Child | PCT/KR2022/006054 | US |
