WO2024101762A1 - Anticorps pour la détection du sars-cov-1, et biocapteur d'immunoaffinité l'utilisant - Google Patents

Anticorps pour la détection du sars-cov-1, et biocapteur d'immunoaffinité l'utilisant Download PDF

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WO2024101762A1
WO2024101762A1 PCT/KR2023/017217 KR2023017217W WO2024101762A1 WO 2024101762 A1 WO2024101762 A1 WO 2024101762A1 KR 2023017217 W KR2023017217 W KR 2023017217W WO 2024101762 A1 WO2024101762 A1 WO 2024101762A1
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antibody
seq
antigen
cov
binding fragment
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변재철
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연세대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present invention relates to an antibody for detecting SARS-CoV-1 and an immunoaffinity biosensor using the same. More specifically, it is derived from an Fv library expressed on the outer membrane of Escherichia coli ( E. coli ) and is used to detect SARS-CoV-1 SP. It relates to antibodies for detecting SARS-CoV-1 selected through screening of Fv antibodies that specifically bind to and immunoaffinity biosensors using the same.
  • CoVs Human coronaviruses
  • CoV strains OC43, HKU1, NL63, and CoV-229E are known to be one of the most common causative agents of seasonal colds and pneumonia (Jain et al., 2015).
  • severe acute respiratory syndrome (SARS) like MERS (Middle East Respiratory Syndrome) and COVID-19, has been reported to be caused by beta-CoV (Belouzard et al., 2012).
  • SARS-related coronavirus (SARS-CoV-1) infection occurs when the SARS-CoV-1 spike protein (SP) binds to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. reported (Lan et al., 2020).
  • SP SARS-CoV-1 spike protein
  • ACE2 angiotensin-converting enzyme 2
  • the receptor binding domain (RBD) of the homotrimeric spike glycoprotein is known to be the core region of the interaction between SP and ACE2, and the binding affinity of SP for ACE2 is known to be in the range of ⁇ 31-100nM ( Lan et al., 2020).
  • RBD receptor binding domain
  • Non-patent Document 1 Jain, S., Self, W.H., Wunderink, R.G., Fakhran, S., Balk, R., Bramley, A.M., Reed, C., Grijalva, C.G., Anderson, E.J., Courtney, D.M., 2015. Community-acquired pneumonia requiring hospitalization among US adults. New Engl. J. Med. 373(5), 415-427.
  • Non-patent Document 2 Belouzard, S., Millet, J.K., Licitra, B.N., Whittaker, G.R., 2012. Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 4(6), 1011-1033.
  • Non-patent Document 3 Lan, J., Ge, J., Yu, J., Shan, S., Zhou, H., Fan, S., Zhang, Q., Shi, X., Wang, Q. , Zhang, L., Wang, X., 2020. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581(7807), 215-220.
  • Non-patent Document 4 He, Y., Li, J., Du, L., Yan, X., Hu, G., Zhou, Y., Jiang, S., 2006. Identification and characterization of novel neutralizing epitopes in the receptor-binding domain of SARS-CoV spike protein: revealing the critical antigenic determinants in inactivated SARS-CoV vaccine. Vaccine 24(26), 5498-5508.
  • the present inventors were able to detect SARS-CoV-1 at an excellent level by screening Fv antibodies derived from an Fv library expressed in the outer membrane of Escherichia coli ( E. coli ) and specifically binding to SARS-CoV-1 SP.
  • the present invention was completed by selecting antibodies present.
  • the present invention is an antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-1, wherein the antibody contains CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4.
  • the antibody contains CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4.
  • the antibody or antigen-binding fragment thereof of the present invention includes a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 4; and a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 7.
  • the antibody or antigen-binding fragment thereof of the present invention includes a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 13; and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 14.
  • the antibody or antigen-binding fragment thereof of the present invention includes a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 14.
  • the antibody or antigen-binding fragment thereof of the present invention is technically characterized in that it is in the form of a single domain antibody.
  • the antibody or antigen-binding fragment thereof of the present invention is technically characterized in that it is used for detecting SARS-CoV-1.
  • the present invention makes medical diagnosis of SARS-CoV-1 feasible by using an SPR biosensor and impedance spectroscopy utilizing immobilized Fv-variants obtained from two clones screened for SARS-CoV-1 SP.
  • Figure 1a illustrates the screening and application procedure for Fv-variants used for SARS-CoV-1 detection.
  • Figure 1b illustrates the preparation process of an Fv-library with random CDR3 regions using site-directed mutagenesis.
  • Figure 1c shows the expression process of the Fv-library in the outer membrane of E. coli using a surface expression vector.
  • Figure 1d shows SDS-PAGE results (lane 1: Fv-library containing surface-expressed Fv-variant, lane 2: mutant strain (CDR1 and CDR2) containing surface-expressed Fv, lane 3: intact E. coli )
  • Figure 2a shows the results of flow cytometry after processing fluorescently labeled SP with the Fv library (from the left, Fv-library + SP, mutant strain + SP, intact E. coli + SP).
  • Figure 2b shows the screening procedure for target clones using magnetic beads attached with immobilized SP.
  • Figure 2c shows flow cytometry results and CDR3 sequencing results of each clone.
  • Figure 2d is a comparison of the binding affinities of the screened clones for SP and labeled fluorescent dye (fluorescent dye).
  • Figure 3a shows the results of flow cytometry after treating the screened clones (clone number 4, clone number 18 from the left) with fluorescently labeled SP compared to mutant strains with CDR1 and CDR2.
  • Figure 3b shows the measurement results of the binding constant (K D ) using quantitative binding analysis with flow cytometry.
  • Figure 4a shows the expression procedure for selected Fv-variants.
  • Figure 4c shows the binding constant (K D ) measurement results of SARS-CoV-1 SP to immobilized Fv-variants using the SPR biosensor.
  • Figure 5a shows the analysis process of SPR biosensor and impedance spectroscopy using immobilized Fv-variants.
  • Figure 5b shows the detection results of SARS-CoV-1 using the SPR biosensor with two types of immobilized Fv-variants in two screened clones (Anti-SP1 and Anti-SP2).
  • Figure 5c Detection results of SARS-CoV-1 using impedance spectroscopy using two types of immobilized Fv-variants in two screened clones (Anti-SP1 and Anti-SP2).
  • antibody used herein is the most comprehensive description and is meant to encompass all types of antibodies known to date. That is, antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, as long as they exhibit antigen-binding activity. Covers a variety of antibody structures.
  • antibody includes conventional four-chain antibodies, single domain antibodies, and antigen-binding fragments thereof.
  • the antibody according to the present invention may be in the form of a single domain antibody (sdAb).
  • Single domain antibodies include a heavy chain variable domain from a heavy chain-only antibody (e.g., VHH in Camelidae (the variable domain of the heavy chain of a heavy chain antibody)), a light chain derived from a conventional 4-chain antibody, and a single domain (e.g., VH or VL). ), humanized heavy chain-only antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. It may include without limitation.
  • the sdAb may be derived from any species, including but not limited to mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. It may also include naturally occurring single domain antibodies from species other than Camelidae.
  • single domain antibodies are derived from naturally occurring single domain antigen binding molecules known as heavy chain antibodies that lack a light chain. Such single domain molecules are disclosed, for example, in WO 94/04678 and Hamers-Casterman, et al., (1993) Nature 363:446-448.
  • VHH variable domains derived from heavy chain molecules that naturally lack a light chain are known herein as VHH, distinguishing them from the conventional VH of four-chain immunoglobulins.
  • VHH molecules may be derived from antibodies produced in camelid species, such as camelids, llamas, vicu ⁇ as, dromedaries, alpacas, and guanacos.
  • Species other than Camelidae can produce heavy chain molecules that naturally lack the light chain, and such VHHs are within the scope of this application.
  • the single domain antibody according to the present invention may be a chimeric antibody.
  • Specific chimeric antibodies include, for example, patents US4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984).
  • a chimeric antibody can comprise a non-human variable region (e.g., a variable region derived from a camelid species, such as a llama) and a human constant region.
  • chimeric antibodies can be humanized. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences.
  • a humanized antibody will optionally also comprise at least one portion of a human constant region.
  • some FR residues in a humanized antibody are cloned from the corresponding non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity. is replaced with a residue.
  • SARS-CoV-1 severe acute respiratory syndrome coronavirus
  • SARS severe acute respiratory syndrome coronavirus
  • SP spike protein
  • Variable region fragment refers to the antigen-binding region of immunoglobulin G, which consists of three CDR regions (CDR1, CDR2, and CDR3) and a framework region (FR) between the CDR regions. It is a minimal antibody fragment consisting of only a heavy chain variable region and/or a light chain variable region.
  • the recombinant technique for producing Fv is described in international patent application WO88/10649, etc.
  • CDR complementarity determining region
  • phage display is a technique for displaying a variant polypeptide as a fusion protein with at least a portion of the coat protein on the surface of a phage, for example, a fibrous phage particle.
  • the utility of phage display lies in the fact that it can quickly and efficiently classify sequences that bind to target antigens with high affinity by targeting large libraries of randomized protein variants.
  • Expression of peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties. In particular, it is important to generate diverse libraries of antibodies or antigen-binding proteins in high-affinity antibody isolation.
  • CDR3 regions have been shown to often participate in antigen binding. Since the CDR3 region on the heavy chain varies considerably in size, sequence, and structural conformation, various libraries can be prepared using it.
  • Nucleic acid is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base sites. .
  • the sequences of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. The modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
  • amino acid sequence for the antibody or antigen-binding fragment thereof of the present invention or the nucleic acid encoding it is interpreted to also include a sequence showing substantial identity with the sequence shown in SEQ ID NO:
  • the above-mentioned substantial identity is at least 90% when the sequence of the present invention and any other sequence are aligned to the maximum extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a sequence showing homology, most preferably at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, and at least 99% homology.
  • the antibody or antigen-binding fragment thereof of the present invention has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the specified sequence or the entire sequence described in the specification. , may have 99% or more homology.
  • homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence homology of a nucleic acid or protein of the invention.
  • Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • vector refers to a means for expressing a gene of interest in a host cell, including a plasmid vector; cosmid vector; Includes viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated viral vectors.
  • the nucleic acid encoding the antibody is operably linked to a promoter.
  • “Operably linked” means a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence is linked to the other nucleic acid. It regulates the transcription and/or translation of the sequence.
  • a nucleic acid expression control sequence e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites
  • a strong promoter capable of advancing transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL*?* promoter, pR°C promoter, rac5 promoter, amp promoter, recA promoter
  • a ribosome binding site for initiation of translation e.g., SP6 promoter, trp promoter, and T7 promoter, etc.
  • promoters derived from the genome of mammalian cells e.g., metallothioneine promoter, actin promoter, human heroglobin promoter, and human muscle creatine promoter
  • mammalian viruses Promoters derived from e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, mol Promoters of Ronnie virus (e.g., the promoter of Epstein-Barr virus (EBV) and the promoter of Rouss sarcoma virus (RSV)) can be used and generally have a polyadenylation sequence as the transcription termination sequence.
  • adenovirus late promoter vaccinia virus 7.5K promoter
  • SV40 promoter cytomegalovirus (CMV) promoter
  • tk promoter of HSV e.
  • the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom.
  • Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).
  • the vector contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. There is a resistance gene.
  • the present invention relates to cells transformed with the above-mentioned vectors.
  • the cells used to produce the antibodies of the present invention may be, but are not limited to, prokaryotic, yeast, or higher eukaryotic cells.
  • Bacillus strains such as Escherichia coli , bacillus subtilis and Bacillus thuringiensis , and prokaryotic host cells such as Streptomyces , Pseudomonas , Proteus mirabilis and Staphylococcus can be used.
  • the present invention includes the steps of: (a) culturing cells; and (b) recovering the antibody or antigen-binding fragment thereof from the culture medium and/or the cells.
  • the cells can be cultured in various media. Any commercially available medium can be used as a culture medium without limitation. All other necessary supplements known to those skilled in the art may also be included in suitable concentrations. Culture conditions, such as temperature, pH, etc., are already used with host cells selected for expression and will be apparent to those skilled in the art.
  • transformation means introducing a vector containing a nucleic acid encoding a target protein into a host cell so that the protein encoded by the nucleic acid can be expressed within the host cell.
  • Transformed nucleic acids include all of them, regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell.
  • the nucleic acid includes DNA and RNA encoding the target protein.
  • the nucleic acid may be introduced in any form as long as it can be introduced into a host cell and expressed.
  • the nucleic acid can be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for self-expression.
  • the expression cassette typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked to the nucleic acid.
  • the expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the nucleic acid may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell.
  • the antibody or antigen-binding fragment thereof can be recovered by, for example, centrifugation or ultrafiltration to remove impurities, and the resulting product can be purified using, for example, affinity chromatography. Additional other purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc.
  • the diagnostic kit of the present invention may include essential elements necessary to perform ELISA.
  • the ELISA kit includes an antibody specific for a target protein (e.g., an antibody for detecting SARS-CoV-1 or an antigen-binding fragment thereof according to the present invention).
  • Antibodies are antibodies that have high specificity and affinity for a marker protein and almost no cross-reactivity to other proteins, and may be monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. Additionally, ELISA kits may include antibodies specific for control proteins.
  • Other ELISA kits include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g. conjugated with antibodies) and their substrates or those capable of binding to antibodies. It may contain other substances, etc.
  • the amino acid sequence of the CDR3 region was randomized using site-directed mutagenesis (Xie et al., 2020).
  • the CDR3 region consists of 11 amino acids
  • the Fv-library plasmid was prepared using an anti-thyroid peroxidase (TPO) antibody as a template sequence, as shown in Figure 1b.
  • TPO anti-thyroid peroxidase
  • the prepared Fv-library plasmid was inserted into an expression (surface expression, autodisplay) vector, and the Fv-library was expressed in the outer membrane of the colon using surface expression technology as shown in Figure 1a.
  • the outer membrane Fv-library was reported to have an expression efficiency as high as 10 5 Fv/ E.
  • FIG. 1c and 1d show that the Fv-variants were surface expressed on the outer membrane of E. coli ; Mutant strains with Fv-variants possessing CDR1 and CDR2 regions were also prepared as control strains. Targeted Fv-variants can be screened by treatment with fluorescently labeled antigen followed by flow cytometric isolation; Target Fv-variant can be isolated using magnetic beads with immobilized antigen, as shown in Figure 2b. Screening of target Fv-variants showing high affinity for SARS-CoV-1 SP can be achieved without repeated biopanning process due to the high expression efficiency of surface expression technology.
  • target Fv-variants are surface-expressed against various antigens such as biotin, dopamine, fluorescein, rhodamine B, and monosodium urate crystal. was screened using the Fv- library (Jung et al., 2021c; Jung et al., 2021d; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b).
  • Fv-variants with high affinity for SARS-CoV-1 SP were screened from the Fv library, and the binding affinities of the two screened clones were analyzed using flow cytometry.
  • expressed Fv-variants obtained from two screened clones against SARS-CoV-1 SP (Anti-SP1 and Anti-SP2) were expressed as fusion proteins with green fluorescent protein (GFP), and SARS- Detection of CoV-1 was performed using impedance spectroscopy using immobilized Fv-variants obtained from two screened clones for SARS-CoV-1 SP and viral culture of SARS-CoV-1 using a SPR biosensor. It has been proven.
  • the antibody or antigen-binding fragment thereof according to the present invention can be used for various purposes for detecting the SARS-CoV-1 virus.
  • the antibody or antigen-binding fragment thereof according to the present invention may be used in a SARS-CoV-1 diagnostic kit or biosensor.
  • SARS-CoV-1 diagnostic kit or biosensor for specific application, please refer to Republic of Korea Patent Publication Nos. 10-2022-0124972 and 10-2022-0021791.
  • SARS-CoV-1 SP was supplied by Optolane Inc. (Seongnam, Korea).
  • Luria-Bertani (LB) medium was purchased from Duchefa (Haarlem, Netherlands).
  • M-280 tosyl-activated magnetic beads (diameter: 2.8 ⁇ m) were purchased from Invitrogen Co. (Carlsbad, CA, USA).
  • Bovine serum albumin was purchased from Sigma-Aldrich Korea (Seoul, Korea). Primers were custom synthesized by Bionics (Seoul, Korea). Klenow DNA polymerase was purchased from New England BioLabs (Ipswich, MA, USA).
  • PCR purification mini kit was purchased from Favorgen (Pingtung, Taiwan).
  • Phusion high-fidelity DNA polymerase was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
  • the sfGFP-labeled Fv-variant fusion protein plasmid was custom synthesized by Cosmogenetech (Seoul, Korea).
  • NATtrol TM SARS-CoV-1 reagent cycle threshold: 25-28
  • NATtrol TM negative control reagent A-549 cells, 50,000 cells/mL
  • a single-stranded forward primer with a randomized sequence of CDR3 was combined with the corresponding reverse primer (22 bp) as shown in Figure 1(b). mixed with.
  • Primer sequences for the Fv library are summarized in Table 1.
  • N, R, K, Y, W, and V are substituted with the nucleotide corresponding to each letter listed in the table. For example, N is replaced with A, C, G, and T, and R is replaced with A and G.
  • annealing of primers (2 ⁇ L each) was performed by heating at 95°C for 5 minutes and then cooling to 36°C, and consisted of NEB buffer (4 ⁇ L) and DW (32 ⁇ L) in a total volume of 40 ⁇ L.
  • NEB buffer 4 ⁇ L
  • DW 32 ⁇ L
  • For the extension reaction add Klenow (exo-) DNA polymerase (3 ⁇ L), NEB buffer (16 ⁇ L), 10 mM dNTP (8 ⁇ L), and DW (133 ⁇ L) to make a total volume of 200 ⁇ L. did. Afterwards, the extension reaction was performed at 37°C for 15 minutes.
  • the PCP product double-stranded primer from the Fv library with randomized CDR3 region
  • the PCP product double-stranded primer from the Fv library with randomized CDR3 region
  • Fv-library plasmids containing randomized CDR3 sequences were prepared through PCR.
  • the Fv-library plasmid was prepared in a total volume of template plasmid (pST009, 150 ng), double-stranded Fv-library primer mixture (150 ng), HF buffer (10 ⁇ L), 10 mM dNTP (1 ⁇ L), and Phusion high-fidelity DNA polymerase (0.5 ⁇ L). It was synthesized using a 50 ⁇ L mixture.
  • PCR was performed according to the following steps: (1) initial denaturation (98 °C for 1 min), (2) denaturation (98 °C for 30 s), (3) annealing (68 °C for 1 min), ( 4) Elongation (5 minutes at 72°C), (5) repeat (2)-(4) 30 times, and (6) termination (10 minutes at 72°C). Finally, the template plasmid was digested with DpnI restriction enzyme (37°C for 16 hours). The cut Fv-library plasmid was filtered using a 100K Amicon filter (Millipore, Billerica, MA, USA). Surface expression of the Fv-library on the outer membrane of E.
  • coli BL21(DE3) was performed by transforming the Fv-library plasmid prepared as shown in Figure 1c into competent cells through electrophoresis.
  • Transformed E. coli cells were cultured in LB medium containing 50 ⁇ g/mL kanamycin at 37°C for 16 hours.
  • Cultured E. coli cells (100 ⁇ L) were incubated at 99% °C in LB medium (10 mL) containing mercaptoethanol ( ⁇ -mercaptoethanol, 8 ⁇ L), kanamycin (50 ⁇ g/mL), and ethylenediaminetetraacetic acid (5 ⁇ M). to an OD of 0.6 at 600 nm while shaking (150 rpm) at 37°C. Re-cultivated until reached.
  • Plasmids pJY003 and pJY004 encoding the open reading frames of Fv-variants with binding activity to SARS-CoV-1 SP, sfGFP and His-tag fusion proteins were custom synthesized by Cosmogenetech as shown in Figure 4A.
  • Fv-variants were expressed intracellularly in E. coli by transforming custom plasmids into competent cells. Transformed E. coli was cultured in 20mL high-salt LB medium containing 1mM IPTG and 30 ⁇ g/mL carbenicillin at 30°C for 16 hours. The E. coli pellet was collected by centrifugation (3,000 E.
  • coli was sonicated using an ultrasonic reactor (Vibra cell VCX-130, Sonics, USA) and the lysate was centrifuged at 25,000
  • the purified protein was purified on a His-tag purification column (Roche, Bassel, Switzerland) using binding buffer containing 3M urea and 500mM imidazole, and dialyzed at 100rpm for 16 hours at 4°C to separate urea and imidazole. removed.
  • SPR measurements of expressed Fv-variants obtained from Anti-SP1 and Anti-SP2 were measured using an SPR biosensor from I-Cluebio (Seongnam, Korea). Assay constructs were prepared using expressed Fv-variant or SARS-CoV-1 SP immobilized on the gold surface of the SPR biosensor.
  • the SPR chip for SPR measurements was fabricated using a step sputter-coated with titanium (thickness: 2 nm) and gold (thickness: 48 nm) as an adhesive layer on BK-7 glass (11 °C cm 2 ).
  • Figures 4b and 4c show that the SPR chip was incubated with Fv-variant or SARS-CoV-1 SP (20 ⁇ g/mL each) at 4°C for 16 hours.
  • Impedance spectroscopy was performed using a three-electrode system from IVIUM Technologies (Eindhoven, Netherlands) and a commercial potentiostat.
  • a SiO 2 wafer was deposited with gold (thickness: 100 nm) and used as an electrode.
  • the expressed Fv-variant (20 ⁇ g/mL) was immobilized on the electrode for 2 hours.
  • Incubation for nonspecific blocking with BSA (1 mg/mL) was performed on the electrodes for 1 hour.
  • R ct parallel charge transfer resistance
  • C dl double-layer capacitance
  • R s series intermediate resistance
  • the Fv-library with three CDR regions was prepared as previously reported by randomizing the CDR3 region (Jung et al., 2021c; Jung et al., 2021d; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b). Additionally, a mutant strain with the same CDR1 and CDR2 regions as the Fv-library was also prepared. The sequences of CDR1 and 2 are listed in Table 2.
  • FIG. 2A is a dot plot of the Fv-library compared to a control strain (only CDR1 and CDR2) and E. coli intact for cleavage (no Fv expressed on the surface). It shows that a region of high fluorescence exceeds the cut-off value (indicated by a dotted line in the graph). Therefore, the highly fluorescent region of the Fv-library indicates that the CDR3 region of some Fv-variants can bind fluorescently labeled SP with high affinity.
  • Candidate Fv-variants with high affinity for SP were isolated using magnetic beads with immobilized SP.
  • Fv-variants with specific affinity for SP are It was selected for surface expression from the Fv-library in the outer membrane.
  • Figure 2(b) shows that SP is covalently immobilized on tosyl group-activated magnetic beads. Magnetic beads with immobilized SP were incubated with the Fv-library to screen E. coli with Fv-variants having binding affinity for SP. The magnetic beads bound to E. coli were separated using an external magnet and then cultured on an agar plate. These clones were further analyzed to determine (1) that the Fv-variants of the cultured clones had different genetic sequences for the CDR3 region when compared to the template sequence, and (2) that the Fv-variants had different genetic sequences for the SP using fluorescently labeled SP. It was confirmed that it had specific affinity.
  • Figure 2(c) shows that the base sequences for the randomized CDR3 region were analyzed, and six clones (clone numbers 1, 4, 7, 9, 18, and 20) were site-directed when compared to the template sequence. ) show that it was determined from mutagenesis to have binding affinity for fluorescently labeled SP with the appropriate nucleotide sequence.
  • the amino acid sequences of the six clones are listed in Table 3.
  • the remaining 14 clones had inappropriate nucleotide sequences: 2 clones had base deletions, 2 clones had repetitive sequences, and 10 clones had no binding activity or had identical nucleotide sequences to the template plasmid before site-directed mutation.
  • fluorescent dye fluorescein
  • six selected clones (clone numbers 1, 4, 7, 9, 18, and 20) with appropriate base sequences were combined with the fluorescently labeled SP and It was reacted with fluorescent dye. When the six selected clones were reacted with a fluorescent dye, significant fluorescent signals were observed for four clones (Clone No.
  • Figure 3A shows that the dot plots obtained for the two screened clones (Anti-SP1 and Anti-SP2) show much higher fluorescence signals when compared to the mutant strain. These results show that the two screened clones had Fv-variants with much higher affinity for SP.
  • K D binding constants
  • Figure 3b shows that a quantitatively increasing fluorescence signal was observed over the concentration range studied and that the signal observed for the mutant strain was maintained at baseline levels.
  • the K D value of Fv-variant the peak value was taken as the fluorescence signal.
  • Fv-variants containing three CDR regions (CDR1, CDR2, and CDR3) and the framework region between the CDR regions were expressed as fusion proteins of sfGFP, as shown in Figure 4(a).
  • SP was immobilized on the SPR chip
  • different concentrations of expressed Fv-variant were added in the range of 3.8-30.0 nM, as shown in Figure 4b.
  • Binding of expressed Fv-variants to SP was monitored using a SPR sensor, which associated and dissociated under continuous flow conditions.
  • the K D value of the Fv-variant immobilized on SARS-CoV-1 SP was also measured using the SPR biosensor.
  • the expressed Fv-variant was immobilized on the SPR biosensor, and various concentrations of SARS-CoV-1 SP ranging from 15.0 to 120.0 nM were added, as shown in Figure 4c.
  • the binding of SP to the expressed Fv-variant was monitored using a SPR sensor under the same continuous flow conditions, and the K D value of the expressed Fv-variant to SP was 41.7 ⁇ 41.7 ⁇ 4.0 for the Fv-variant expressed in Anti-SP1.
  • the expressed Fv-variant was used to detect SARS-CoV-1 using SPR biosensor and impedance spectroscopy.
  • the actual sample used was the SARS-CoV-1 NATtrol TM reagent from Zeptometrix (Buffalo, NY, USA), which has a similar composition to the medical sample.
  • the cycle threshold (Ct) values of NATtrol TM reagent averaged in the range of 26.5 for SARS-CoV-1 (Wei et al., 2021; Park et al., 2022).
  • the cutoff Ct value for SARS-CoV-1 diagnosis using RT-PCR has been reported to be 35 (Lau et al., 2003; Poon et al., 2003).
  • NATtrol TM reagent was serially diluted to prepare standard samples for diagnosis using SPR biosensor and impedance spectroscopy.
  • FIG. 5a shows the assay setup used for SARS-CoV-1 detection prepared by immobilizing the expressed Fv variant on the sensor surface of the SPR biosensor and the gold electrode of the impedance spectrometer.
  • impedance spectroscopy was performed after processing the sample under flow conditions and an impedance signal according to the amount of bound analyte recorded in the x-cut of the impedance spectrum after the washing step.
  • the X-cut value represents the charge transfer resistance (R ct ), which is the resistance required for charge transfer to the electrode.
  • the Fv library was prepared on the outer membrane. Fv-variants with specific affinity for SARS-CoV-1 SP were screened using magnetic beads immobilized with SP. Through screening of the Fv-library, two target clones (clone number 4 and clone number 18) with specific binding ability to SP were determined, and the screened clone was clone number 4 (CDR3 amino acid sequence: 1 GRTTG 5 NDRPD 11 Y) and "Anti-SP2" for clone number 18 (CDR3 amino acid sequence: 1 CLRQA 5 GTADD 11 V). The binding affinity of the two screened clones was analyzed using flow cytometry.
  • KD of immobilized Fv-variants against SARS-CoV-1 SP The values were also measured using an SPR biosensor.

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Abstract

La présente invention concerne un anticorps se liant spécifiquement au SARS-CoV-1, ou un fragment de liaison à l'antigène de celui-ci. L'anticorps est choisi dans le groupe constitué par : une région variable comprenant CDR1 de SEQ ID NO : 1, CDR2 de SEQ ID NO : 2, et CDR3 de SEQ ID NO : 4 ; et une région variable comprenant CDR1 de SEQ ID NO : 1, CDR2 de SEQ ID NO : 2, et CDR3 de SEQ ID NO : 7.
PCT/KR2023/017217 2022-11-11 2023-11-01 Anticorps pour la détection du sars-cov-1, et biocapteur d'immunoaffinité l'utilisant WO2024101762A1 (fr)

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KR20150115747A (ko) * 2013-01-24 2015-10-14 (재) 스크립스코리아항체연구원 단백질 조합 기반의 Fv 라이브러리 및 이의 제조 방법
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