SARS-CoV-2 nucleocapsid antibody

Abstract

The present invention relates to monoclonal antibodies that bind to the nucleocapsid protein of the SARS-CoV-2 virus, nucleic acids encoding the antibodies, host cells that produce the antibodies, compositions and kits comprising the antibodies, and methods for detecting the SARS-CoV-2 virus in a sample comprising using the antibodies.

Description

[Technical Field]

[0001] The present invention relates to a monoclonal antibody that binds to the nucleocapsid protein of the SARS-CoV-2 virus, a nucleic acid encoding the antibody, a host cell that produces the antibody, a composition and kit comprising the antibody, and a method for detecting the SARS-CoV-2 virus in a sample, comprising using the antibody. [Background technology]

[0002] Background of the Invention Coronaviruses (CoVs) are large, enveloped, positive-sense single-stranded RNA viruses that can be further subdivided into alphaviruses, betaviruses, gammaviruses, and deltacornoviruses based on their serological and genotypic characteristics. Two betacoronaviruses, SARS-CoV-1 (Severe Acute Respiratory Syndrome Coronavirus) and MERS-CoV (Middle East Respiratory Syndrome Coronavirus), have caused two severe coronavirus epidemics in the past decade (SARS 2002 / 2003, MERS 2012). In December 2019, an outbreak of a novel infectious respiratory illness called Coronavirus Disease 2019 (COVID-19) occurred in China, and by March 2020 it had become a global pandemic. As of October 17, 2020, since December 31, 2019, 39,196,259 cases have been reported worldwide, with 1,101,298 confirmed deaths, affecting 235 countries or territories (Source: World Health Organization - https: / / www.who.int / emergencies / diseases / novel-coronavirus-2019). COVID-19 is caused by a novel coronavirus, namely Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infects the respiratory tract by binding to the host cell receptor ACE2 (angiotensin-converting enzyme 2), a receptor widely present in the lower respiratory tract. The surface spike (S) glycoprotein of SARS-CoV-2 mediates this interaction with the ACE2 receptor, driving membrane fusion and thus driving entry into host cells. Viral replication in host cells is driven by the SARS-CoV-2N (nucleocapsid) protein, a multifunctional RNA-binding protein that enters the host cell along with viral RNA, mediates viral replication, and processes the assembly and release of viral particles. The N protein is described as highly immunogenic and abundantly expressed during SARS-CoV-2 infection.

[0003] Common symptoms of COVID-19 include fever, cough, fatigue, shortness of breath, or difficulty breathing. These symptoms are relatively nonspecific and can be seen in a variety of other illnesses. Most COVID-19 patients have mild symptoms, but some patients develop pneumonia, acute respiratory distress syndrome, septic shock, and renal failure.

[0004] The burden of COVID-19 far exceeds that of other infectious diseases and threatens to overwhelm healthcare systems. Identifying areas of high disease burden is crucial to ensure the careful and effective allocation of emergency medical and public health resources. The risk of severe outcomes associated with COVID-19 appears to increase with age, frailty, and vascular comorbidities. This scenario is likely to increase hospitalizations, admissions to intensive care units, and readmissions. Because SARS-CoV-2 is a novel virus, there is a lack of experience in patient management, from diagnosis to treatment and vaccination.

[0005] The standard method for testing for SARS-CoV-2 infection is real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) of nasopharyngeal and oropharyngeal swab samples from patients. However, molecular testing is considerably slower and more expensive, and cannot provide the scale of testing needed to respond to the COVID-19 pandemic. Demand for PCR-based SARS-CoV-2 testing is high, and supply remains a problem as the pandemic continues.

[0006] Antibody tests, such as anti-nucleocapsid or anti-spike immunoassays, followed PCR testing in a laboratory setting to assess a patient's immunity. Antigen tests bridge the gap between molecular tests (PCR) and immunological tests (antibody tests).

[0007] Rapid antigen tests were developed in point-of-care settings to meet the high demand for testing and enable SARS-CoV-2 infection detection as quickly as possible. However, there are no antigen tests on the market for central laboratory settings that enable high-throughput testing and increase SARS-CoV-2 testing capacity worldwide. Given the ongoing pandemic and the increasing number of infected patients, and therefore the demand for testing, there is a high demand for cost-effective and high-throughput antigen tests in centralized laboratory setups. Such fully automated systems can provide test results in 18 minutes for a single test (excluding time for sample collection, transport, and preparation), with a throughput of up to 300 tests / hour from a single analyzer, depending on the analyzer. Laboratory-based automated antigen assays eliminate manual handling and enable cost and error reduction through rapid turnaround time and high test throughput. [Overview of the project]

[0008] Summary of the Invention In a first aspect, the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that binds to the nucleocapsid protein of the SARS-CoV-2 virus (isolated), wherein the monoclonal antibody or antigen-binding fragment thereof a) 1.0E+05M determined by surface plasmon resonance -1 s -1 The association rate constant (k) exceeds the following value. a ) has, and / or b) 5.0E-04s determined by surface plasmon resonance -1 Dissociation rate constant (k) less than d ) has, and / or c) Having a half-life of 15 minutes or more t / 2diss determined by surface plasmon resonance, and / or d) It has a stoichiometric ratio of 1:1 or 1:2.

[0009] In a second aspect, the present invention relates to an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof a) Includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 1, 2, 3, 4, 5, and 6 respectively, b) Whether the antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, according to SEQ ID NOs: 1, 2, 3, 4, 5, and 6, bind to the same epitopes, or c) These antibodies compete for binding to the SARS-CoV-2 virus nucleocapsid protein with antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0010] In a third aspect, the present invention relates to an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof a) Each includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 17, 18, 19, 20, 21, and 22, respectively. b) Whether the antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, according to SEQ ID NOs. 17, 18, 19, 20, 21, and 22, bind to the same epitopes, or c) These antibodies compete for binding to the SARS-CoV-2 virus nucleocapsid protein with antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, as defined by SEQ ID NOs. 17, 18, 19, 20, 21, and 22.

[0011] In a fourth aspect, the present invention relates to an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof a) Each includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 33, 34, 35, 36, 37, and 38, b) Whether the antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, according to SEQ ID NOs. 33, 34, 35, 36, 37, and 38, bind to the same epitopes, or c) These antibodies, each containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs. 33, 34, 35, 36, 37, and 38 respectively, compete for binding to the SARS-CoV-2 virus nucleocapsid protein.

[0012] In a fifth aspect, the present invention relates to a kit comprising at least one antibody selected from the group of antibodies described above in relation to the first, second, third, or fourth aspect of the present invention.

[0013] In a sixth aspect, the present invention relates to a nucleic acid encoding an antibody selected from the group of antibodies described above in relation to the first, second, third, or fourth aspect of the present invention.

[0014] In a seventh aspect, the present invention relates to a host cell containing the nucleic acid described above in the sixth aspect of the present invention, and / or a host cell that produces the antibody described above in the first aspect of the present invention and the antibody described above in the second aspect.

[0015] In the eighth aspect, the present invention relates to a composition comprising at least one antibody selected from the group of antibodies described above in relation to the first, second, third, or fourth aspect of the present invention.

[0016] In a ninth aspect, the present invention relates to the use of an antibody according to the first, second, third, or fourth aspect of the present invention, or a kit according to the fifth aspect of the present invention, or a composition according to the eighth aspect of the present invention, for in vitro immunoassays. [Brief explanation of the drawing]

[0017] List of drawings [Figure 1A]Kinetic screening using exemplary kinetic signatures of antibody / N interactions. (A) Deselection after screening. [Figure 1B] Kinetic screening using exemplary kinetic signatures of antibody / N interactions. Further recommendations after screening. [Figure 2] Bonding constants for clones 5B6, 1G9, and 1.1.32. [Figure 3A] Antibody interactions with 1.2 nM, 3 nM, 11 nM, 33 nM, and 100 nM nucleocapsid proteins (NCPs) (black) overlaid with a Langmuir 1:1 binding model (gray). Despite the complex antibody-N binding behavior, kinetic quantification was facilitated with sufficiently high accuracy by using a binary Langmuir model with RMAX global. The complex binding behavior is likely induced by the base charge of the N protein. The highly stable N / M-1.1.32 antibody / antigen complex of kd 2.0E-05s-1 (see Figure 2) allowed for long-term dissociation phase monitoring. [Figure 3B] Antibody interactions with 1.2 nM, 3 nM, 11 nM, 33 nM, and 100 nM nucleocapsid proteins (NCPs) (black) overlaid with a Langmuir 1:1 binding model (gray). Despite the complex antibody-N binding behavior, kinetic quantification was facilitated with sufficiently high accuracy by using a binary Langmuir model with RMAX global. The complex binding behavior is likely induced by the base charge of the N protein. The highly stable N / M-1.1.32 antibody / antigen complex of kd 2.0E-05s-1 (see Figure 2) allowed for long-term dissociation phase monitoring. [Figure 3C]Antibody interactions with 1.2 nM, 3 nM, 11 nM, 33 nM, and 100 nM nucleocapsid proteins (NCPs) (black) overlaid with a Langmuir 1:1 binding model (gray). Despite the complex antibody-N binding behavior, kinetic quantification was facilitated with sufficiently high accuracy by using a binary Langmuir model with RMAX global. The complex binding behavior is likely induced by the base charge of the N protein. The highly stable N / M-1.1.32 antibody / antigen complex of kd 2.0E-05s-1 (see Figure 2) allowed for long-term dissociation phase monitoring. [Figure 4A] Exemplary sensorgram overlays for epitope binning experiments concerning complex formation between N and antibody pairs. Gray arrows indicate the start and stop of injection. 1) Primary antibody, 2) Blocking mixture, 3) N protein, 4) Primary antibody, 5) Secondary antibody, 6) Regeneration. A) Three sensorgram overlays showing 1G9 as the primary antibody and 5B6 as the secondary antibody form immune complexes with NCP. Two negative controls using 1G9 as the primary and secondary antibodies, and a second 1G9 as the buffer instead of the primary and secondary antibodies. Clearly, the positive reaction is undetectable in the run of both negative controls in time section 5. B) Two sensorgram overlays surprisingly demonstrate that 1G9 and 5B6 form a so-called bidirectional sandwich, showing two clearly separated and freely accessible epitope regions 2 and 4 (see Table 2). B) Using 5B6 as the primary antibody and 1G9 as the secondary antibody. As a control, the buffer was used instead of the secondary antibody that did not show a response in time section 5. [Figure 4B]Exemplary sensorgram overlays for epitope binning experiments concerning complex formation between N and antibody pairs. Gray arrows indicate the start and stop of injection. 1) Primary antibody, 2) Blocking mixture, 3) N protein, 4) Primary antibody, 5) Secondary antibody, 6) Regeneration. A) Three sensorgram overlays showing 1G9 as the primary antibody and 5B6 as the secondary antibody form immune complexes with NCP. Two negative controls using 1G9 as the primary and secondary antibodies, and a second 1G9 as the buffer instead of the primary and secondary antibodies. Clearly, the positive reaction is undetectable in the run of both negative controls in time section 5. B) Two sensorgram overlays surprisingly demonstrate that 1G9 and 5B6 form a so-called bidirectional sandwich, showing two clearly separated and freely accessible epitope regions 2 and 4 (see Table 2). B) Using 5B6 as the primary antibody and 1G9 as the secondary antibody. As a control, the buffer was used instead of the secondary antibody that did not show a response in time section 5. [Figure 5] The 14 antibodies, each with different kinetic properties, cover four distinct N-epitope regions. The numbers in the "Epitope Region" column indicate the epitope bin for each monoclonal antibody. [Figure 6] Epitope binning. Antibody 5B6 is shown as a representative antibody in an epitope binning matrix consisting of 14 test antibodies. Here, 196 antibody pairing combinations were analyzed. [Figure 7] Relative sensitivity (relSens) and relative specificity (relSpec) are defined as the percentage of positive agreement between two compared methods (here, SARS-CoV-2 PCR vs. Elecsys antigen test using our anti-nucleocapsid antibody). A comparison of two antibodies (A) 1.1.32+5B6) and three antibodies (B) 1.1.32+5B6+1G9) demonstrates higher sensitivity when using three antibodies. Relative specificity (relSpec) remains 100% in both scenarios.

[0018] Sequence List Sequence ID 1 Antibody 1.1.32:CDR-H1:TYVMH Sequence ID 2 Antibody 1.1.32:CDR-H2:YSDPYNGDSKDNENFKG Sequence ID 3 Antibody 1.1.32:CDR-H3:GFGNYLFYFDY Sequence ID No. 4 Antibody 1.1.32:CDR-L1:SASQDIRDYLN Sequence ID 5 Antibody 1.1.32:CDR-L2:YTSNLHS SEQ ID NO: 6 Antibody 1.1.32:CDR-L3:QQYSKLPYT SEQ ID NO: 7 Antibody 1.1.32:FR-H1:EVQLQQSGPELVKPGASVKMSCKASGYTFT Sequence ID 8 Antibody 1.1.32:FR-H2:WVKQKPGQGLEWIG Sequence ID 9 Antibody 1.1.32:FR-H3:KATLTSDKSSSTVYMELSSLTSEDSAVYYCAR Sequence ID No. 10 Antibody 1.1.32:FR-H4:WGQGTTLTVSS Sequence ID 11 Antibody 1.1.32:FR-L1:DIQMTQTTSSLSASLGDRVTISC Sequence ID 12 Antibody 1.1.32:FR-L2:WYQQKPDGTVKLLIY Sequence ID No. 13 Antibody 1.1.32:FR-L3:GVPSRFSGSGSGTDYSLTISNLEPEDIATYFC Sequence ID No. 14 Antibody 1.1.32:FR-L4:FGGGTKLEIK Sequence ID 15 Antibody 1.1.32: Heavy chain variable domain: EVQLQQSGPELVKPGASVKMSCKASGYTFTTYVMHWVKQKPGQGLEWIGYSDPYNGDSKDNENFKGKATLTSDKSSSTVYMELSSLTSEDSAVYYCARGFGNYLFYFDYWGQGTTLTVSS Sequence ID 16 Antibody 1.1.32: Light chain variable domain: DIQMTQTTSSLSASLGDRVTISCSASQDIRDYLNWYQQKPDGTVKLLIYYTSNLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCQQYSKLPYTFGGGTKLEIK Sequence ID 17 Antibody 5B6:CDR-H1:SYYMS Sequence ID 18 Antibody 5B6:CDR-H2:VMTAGGSTFYASWAKG Sequence ID 19 Antibody 5B6:CDR-H3:SIDTNYGSSI Sequence ID No. 20 Antibody 5B6:CDR-L1:QASEDIYTYLS Sequence ID No. 21 Antibody 5B6:CDR-L2:AASNLAS Sequence ID No. 22 Antibody 5B6:CDR-L3:QGDYYGSNYGLGT Sequence ID 23 Antibody 5B6:FR-H1:SQSVEESGGRLVTPGTPLTLTCTASGFSLS Sequence ID No. 24 Antibody 5B6:FR-H2:WVRQAPGKGLEWIG Sequence ID No. 25 Antibody 5B6:FR-H3:RFTISKTSTTVDLKITSPTTEDTATYFCAR Sequence ID No. 26 Antibody 5B6:FR-H4:WGPGTLVTVSL Sequence ID No. 27 Antibody 5B6:FR-L1:DVVMTQTPASMSEPVGGTVTIKC Sequence ID 28 Antibody 5B6:FR-L2:WYQQQSGQPPKVLIY Sequence ID No. 29 Antibody 5B6:FR-L3:GVSSRFKGSRSGTEYTLTISDLECADAATYYC SEQ ID NO: 30 Antibody 5B6:FR-L4:FGGGTEVVVK SEQ ID NO: 31 Antibody 5B6: Heavy chain variable domain: SQSVEESGGRLVTPGTPLTLTCTASGFSLSSYYMSWVRQAPGKGLEWIGVMTAGGSTFYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARSIDTNYGSSIWGPGTLVTVSL SEQ ID NO: 32 Antibody 5B6: Light chain variable domain: DVVMTQTPASMSEPVGGTVTIKCQASEDIYTYLSWYQQQSGQPPKVLIYAASNLASGVSSRFKGSRSGTEYTLTISDLECADAATYYCQGDYYGSNYGLGTFGGGTEVVVK SEQ ID NO: 33 Antibody 1G9:CDR-H1:TYAVN SEQ ID NO: 34 Antibody 1G9:CDR-H2:VIDGSGSTYYANWAKG SEQ ID NO: 35 Antibody 1G9:CDR-H3:GAGTDNFGNLNL SEQ ID NO: 36 Antibody 1G9:CDR-L1:QASESISSWLA SEQ ID NO: 37 Antibody 1G9:CDR-L2:RASTLAS Sequence ID No. 38 Antibody 1G9:CDR-L3:QQDYSTSNIDNT Sequence ID 39 Antibody 1G9:FR-H1:SQSVEESGGRLVTPGTPLTLTCTVSGFSLS Sequence ID No. 40 Antibody 1G9:FR-H2:WVRQAPGKGLEWIG Sequence ID No. 41 Antibody 1G9:FR-H3:RFTISKASTTVDLKITSPTTEDTATYFCAR Sequence ID No. 42 Antibody 1G9:FR-H4:WGPGTLVTVSS SEQ ID NO: 43 Antibody 1G9:FR-L1:DVVMTQTPASVEVAVGGTVTIKC Sequence ID 44 Antibody 1G9:FR-L2:WYQQKPGQPPKLLIY SEQ ID NO: 45 Antibody 1G9:FR-L3:GVPSRFKGSGSGTEYTLTISGVECADAATYYC SEQ ID NO: 46 Antibody 1G9:FR-L4:FGGGTEVVVK SEQ ID NO: 47 Antibody 1G9: Heavy chain variable domain: SQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYAVNWVRQAPGKGLEWIGVIDGSGSTYYANWAKGRFTISKASTTVDLKITSPTTEDTATYFCARGAGTDNFGNLNLWGPGTLVTVSS Sequence ID 48 Antibody 1G9: Light chain variable domain: DVVMTQTPASVEVAVGGTVTIKCQASESISSWLAWYQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEYTLTISGVECADAATYYCQQDYSTSNIDNTFGGGTEVVVK Sequence number 49 EcSlyD-EcSlyD-CoV-2-N(1-419):MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDE NLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHHHHDDGGGSGGGSGGG SGGGSGGGSGGGKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHHHHDDGGGSGGGSGGGSGGGSGGGSGGGMSDNGPQ NQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGN GGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFG MSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA [Modes for carrying out the invention]

[0019] Detailed description of the invention Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and that these may vary. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0020] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. In the event of any conflict between the definition or instruction of such incorporated reference and the definition or instruction cited herein, the text of this specification shall prevail.

[0021] The elements of the present invention are described below. While these elements are listed in conjunction with specific embodiments, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood as supporting and encompassing any number of disclosures and / or embodiments combining the explicitly described embodiments with preferred elements. Furthermore, any permutations and combinations of all elements described in this application should be considered disclosed by the description of this application unless the context indicates otherwise.

[0022] definition It should be understood that the word "comprise," as well as variations such as "comprises" and "comprising," means the inclusion of a specified integer or process, or group of integers or processes, but not the exclusion of any other integer or process, or group of integers or processes.

[0023] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless otherwise explicitly indicated.

[0024] Concentration, quantity, and other numerical data may be expressed or presented in the form of “ranges” as specified herein. It should be understood that such range forms are used merely for convenience and conciseness, and therefore should be interpreted flexibly to include not only the numbers explicitly listed as boundaries of the range, but also all individual numbers or subranges contained within that range, as if each number and subrange were explicitly listed. For example, the numerical range “150 mg to 600 mg” should be interpreted to include not only the explicitly listed values ​​from 150 mg to 600 mg, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 150, 160, 170, 180, 190, ... 580, 590, 600 mg, and subranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges listing only one number. Furthermore, such interpretations should apply regardless of the scope or characteristics described.

[0025] When used in relation to a number, the term "approximately" means to encompass a range of numbers that have a lower limit 5% less than the given number and an upper limit 5% greater than the given number.

[0026] The “symptoms” of a disease are prominent indications of the disease in a tissue, organ, or organism having such a disease, and include, but are not limited to, pain, weakness, tenderness, tension, rigidity, and spasms of a tissue, organ, or individual. The “signs” or “signals” of a disease include, but are not limited to, changes or alterations, such as the presence or absence, increase or increase, decrease or decrease, of specific indicators such as biomarkers or molecular markers, or the onset, presence or exacerbation of symptoms. The symptoms of pain include, but are not limited to, unpleasant sensations that may be perceived as persistent or varying degrees of burning, throbbing, itching, or stinging pain.

[0027] The terms “disease” and “disorder” are used interchangeably herein and refer to abnormal medical conditions, particularly diseases or injuries that prevent a tissue, organ, or organism from efficiently performing its function. Typically, but not always, a disease is associated with specific symptoms or signs indicating the presence of such a disease. Thus, the presence of such symptoms or signs may indicate a tissue, organ, or organism that is diseased. Changes in these symptoms or signs may indicate the progression of such a disease. The progression of a disease is typically characterized by an increase or decrease in such symptoms or signs, which may indicate a “worsening” or “improvement” of the disease. A “worsening” of a disease is characterized by a decrease in the ability of a tissue, organ, or organism to efficiently perform its function, while a “improvement” of a disease is typically characterized by an increase in the ability of a tissue, organ, or organism to efficiently perform its function. A tissue, organ, or organism at “risk of developing” a disease is in a healthy state but indicates the possibility of the disease manifesting. Typically, risk of developing a disease is associated with early or mild signs or symptoms of such a disease. In such cases, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, infectious diseases, traumatic diseases, inflammatory diseases, skin conditions, endocrine disorders, intestinal diseases, neurological disorders, joint diseases, genetic disorders, autoimmune diseases, and various types of cancer.

[0028] The term "coronavirus" refers to a group of related viruses that cause disease in mammals and birds. In humans, coronaviruses cause respiratory infections that can range from mild to fatal. Mild illnesses include some cases of the common cold, while more deadly types can cause SARS, MERS, and COVID-19. Coronaviruses contain a positive-sense single-stranded RNA genome.

[0029] The viral envelope is formed by a lipid bilayer to which membrane (M), envelope (E), and spike (S) structural proteins are fixed. Within the envelope, multiple copies of the nucleocapsid (N) protein form a nucleocapsid, which is bound to the positive-sense single-stranded RNA genome in a continuous bead-on-string conformation. The genome contains Orfs 1a and 1b, which encode replicase / transcriptase polyproteins, followed by sequences encoding the spike (S)-envelope protein, envelope (E)-protein, membrane (M)-protein, and nucleocapsid (N)-protein. Interspersed among these reading frames are reading frames for different accessory proteins, which vary among different viral strains.

[0030] Several human coronaviruses are known, four of which cause fairly mild symptoms in patients: Human coronavirus NL63 (HCoV-NL63), α-CoV Human coronavirus 229E (HCoV-229E), α-CoV Human coronavirus HKU1 (HCoV-HKU1), β-CoV Human coronavirus OC43 (HCoV-OC43), β-CoV

[0031] HCoV-NL63, HCoV-229E, HCoV-HKU1, and HCoV-OC43 are often referred to as "common cold coronaviruses."

[0032] Three human coronaviruses can potentially cause severe symptoms: Middle East Respiratory Syndrome-related Coronavirus (MERS-CoV), β-CoV Severe acute respiratory syndrome coronavirus (SARS-CoV), β-CoV Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), β-CoV

[0033] SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). This strain is sometimes called the Wuhan virus because it was first discovered in Wuhan, China. SARS-CoV-2 is highly contagious to humans, and the World Health Organization (WHO) has designated the ongoing COVID-19 pandemic as a public health emergency of international concern. The earliest known case is thought to have been discovered on November 17, 2019. The SARS-CoV-2 sequence was first published on January 10, 2020 (Wuhan-Hu-1, GenBank accession number MN908947). Following the initial outbreak in Wuhan, the virus spread to all provinces of China, as well as to more than 150 other countries in Asia, Europe, North America, South America, Africa, and the Pacific. Symptoms include high fever, sore throat, dry cough, and wasting. In severe cases, pneumonia may develop.

[0034] The term “natural coronavirus” refers to coronaviruses occurring in nature, i.e., any coronavirus disclosed above. Natural coronaviruses are understood to contain all the protein and nucleic acid molecules present in naturally occurring viruses. Unlike natural coronaviruses, “viral fragments,” “virus-like particles,” or coronavirus-specific antigens contain only some, but not all, of the protein and nucleic acid molecules present in naturally occurring viruses. Therefore, such “viral fragments,” “virus-like particles,” or coronavirus-specific antigens are not infectious but can still elicit an immune response in patients. Thus, vaccination with coronavirus-specific viral fragments, coronavirus-specific virus-like particles, or coronavirus-specific antigens induces the production of antibodies against those viral fragments, virus-like particles, or antigens in patients.

[0035] The terms “measure,” “measure,” “detect,” or “determine,” or “decide,” include qualitative, semi-quantitative, or quantitative measurements. The term “detect presence” refers to a qualitative measurement that indicates presence or absence without a description of quantity (e.g., a yes or no description). The term “detect quantity” refers to a quantitative measurement (ng) in which an absolute number is detected. The term “detect concentration” refers to a quantitative measurement in which the quantity is determined in relation to a given volume (e.g., ng / ml).

[0036] As used herein, “patient” means any mammal, fish, reptile, or bird that can benefit from the determination or diagnosis described herein. In particular, “patient” is selected from the group consisting of laboratory animals (e.g., mice, rats, rabbits, or zebrafish), livestock (e.g., guinea pigs, rabbits, horses, donkeys, cattle, sheep, goats, pigs, chickens, camels, cattle, dogs, turtles, snakes, lizards, or goldfish), or primates including chimpanzees, bonobos, gorillas, and humans. It is particularly preferable that “patient” is human.

[0037] The terms “sample” or “target sample” are used interchangeably herein and refer to a part or fragment of a tissue, organ, or individual, which is usually smaller than such tissue, organ, or individual that is intended to represent the whole. For analysis, the sample provides information about the state of the tissue or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, fluid samples such as nasopharyngeal swabs, oropharyngeal swabs, blood, serum, plasma, synovial fluid, urine, saliva, and lymph, or solid samples such as tissue extracts, cartilage, bone, synovial membrane, and connective tissue. Analysis of a sample may be achieved visually or chemically. Visual analysis includes, but is not limited to, microscopic imaging or radiographic scanning of the tissue, organ, or individual that allows for morphological evaluation of the sample. Chemical analysis includes, but is not limited to, the detection of the presence or absence of certain indicators, or changes in their quantity or levels.

[0038] The term "host cell" refers to a cell that harbors a vector (e.g., plasmid or virus). Such host cells may be either prokaryotic cells (e.g., bacterial cells) or eukaryotic cells (e.g., fungal, plant, or animal cells). Host cells include both single-cell prokaryotes and eukaryotes (e.g., bacteria, yeast, and actinomycetes), as well as single cells of higher-order plants or animals when grown in cell culture.

[0039] The term "amino acid" generally refers to any monomer unit comprising a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group, and one or more side chains or groups of any of these groups, or analogs. Exemplary side chains include, for example, thiols, selenos, sulfonyls, alkyls, aryls, acyls, ketos, azides, hydroxyls, hydrazines, cyanos, halos, hydrazides, alkenyls, alkynyls, ethers, borates, boronates, phosphos, phosphonos, phosphines, heterocyclics, enones, imines, aldehydes, esters, thioacids, hydroxylamines, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids containing photoactivatable crosslinking agents, metal-linked amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids containing polyethylene glycol or polyether, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, aminothio acid-containing amino acids, and amino acids containing one or more toxic moieties. As used herein, the term “amino acid” includes the following 20 naturally occurring or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). Where an “X” residue is undefined, it should be defined as “any amino acid.” The structures of these 20 natural amino acids are, for example,This is shown in Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002). Further amino acids such as selenocysteine ​​and pyrrolysine may be genetically encoded (Stadtman (1996) “Selenocysteine,” Annu Rev Biochem. 65:83-100 and Ibba et al. (2002) “Genetic code: introducing pyrrolysine,” Curr Biol. 12(13):R464-R466). The term "amino acid" also includes unnatural amino acids, modified amino acids (e.g., those with modified side chains and / or backbones), and amino acid analogs. For example, Zhang et al. (2004) “Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells,” Proc.Natl.Acad.Sci.USA101(24):8882-8887, Anderson et al. (2004) “An expanded genetic code with a functional quadruplet codon,” Proc.Natl.Acad.Sci.USA101(20):7566-7571, Ikeda et al. (2003) “Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo,” Protein Eng.Des.Sel.16(9):699-706, Chin et al. (2003) “An Expanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James et al. (2001) “Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues,” Protein Eng.Des.Sel.14(12):983-991,Kohrer et al. (2001) “Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins,” Proc. Natl. Acad. Sci. USA98(25):14310-14315, Bacher et al. (2001) “Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol.183(18):5414-5425, Hamano-Takaku et al. (2000) “A Mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino Acid Azatyrosine More Efficiently than Tyrosine,” J. Biol. Chem. 275(51):40324-40328, and Budisa et al. See al. (2001) “Proteins with {beta}-(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids,” Protein Sci. 10(7):1281-1292. Amino acids can be incorporated into peptides, polypeptides, or proteins.

[0040] In the context of this invention, the term "peptide" refers to a short polymer of amino acids linked by peptide bonds. It has the same chemical (peptide) bonds as proteins, but is generally shorter in length. The shortest peptide is a dipeptide consisting of two amino acids linked by a single peptide bond. Tripeptides, tetrapeptides, pentapeptides, etc., can also exist. Typically, peptides have a length of up to 4, 6, 8, 10, 12, 15, 18, or 20 amino acids. Peptides have an amino terminus and a carboxyl terminus, unless they are cyclic peptides.

[0041] In relation to the present invention, the term "polypeptide" refers to a single chain of amino acids linked together by peptide bonds, typically containing at least about 21 amino acids, i.e., at least 21, 22, 23, 24, 25, etc. A polypeptide can be a single chain of a protein composed of two or more chains, or, if the protein is composed of a single chain, the protein itself.

[0042] In relation to various aspects of the present invention, the term "protein" refers to a molecule comprising one or more polypeptides that resume secondary and tertiary structures, and further to a protein composed of several polypeptides, i.e., several subunits, that form a quaternary structure. Proteins occasionally have non-peptide groups attached to them, which may be called prosthetic groups or cofactors.

[0043] In particular, the terms “peptide variant,” “polypeptide variant,” and “protein variant” are understood to refer to a peptide, polypeptide, or protein that differs from the peptide, polypeptide, or protein from which it is derived due to one or more changes in the amino acid sequence. The peptide, polypeptide, or protein from which a peptide, polypeptide, or protein variant is derived is also known as the parent peptide, polypeptide, or protein. Furthermore, the variants usable in this invention may also be derived from homologs, orthologues, or paralogs of the parent peptide, polypeptide, or protein, or from artificially constructed variants, insofar as the variant exhibits at least one biological activity of the parent peptide, polypeptide, or protein. Changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-terminal or C-terminal cleavage, or any combination thereof, which may occur at one or more sites. Peptide, polypeptide, or protein variants may exhibit up to 200 total changes in their amino acid sequence (i.e., exchanges, insertions, deletions, N-terminal cleavage, and / or C-terminal cleavage) (maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200). Amino acid exchanges may be conservative and / or non-conservative. Alternatively, or further, as used herein, “variant” may be characterized by some degree of sequence identity with the parent peptide, polypeptide, or protein from which it is derived. More precisely, in connection with the present invention, a peptide, polypeptide, or protein variant exhibits at least 80% sequence identity with its parent peptide, polypeptide, or protein. Sequence identity of a peptide, polypeptide, or protein variant extends over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids.

[0044] In this invention, the term "substitution" refers to replacing one amino acid with another. Therefore, the total number of amino acids remains the same. Deletion of an amino acid at a specific position or introduction of one (or more) amino acids at different positions are not explicitly included in the term "substitution."

[0045] The term "conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Such similarities include, for example, similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. In one embodiment, a conservative amino acid substitution is the substitution of one amino acid for another amino acid that falls into one of the following groups: (i) nonpolar (hydrophobic) amino acids, including alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, and methionine; (ii) polar neutral amino acids, including glycine, serine, threonine, cysteine, asparagine, and glutamine; (iii) positively charged (basic) amino acids, including arginine, lysine, and histidine; and (iv) negatively charged (acidic) amino acids, including aspartic acid and glutamic acid.

[0046] The term "specific binder" refers to a natural or non-natural molecule that specifically binds to a target. Examples of specific binders include, but are not limited to, proteins, peptides, and nucleic acids.

[0047] The term "antigen (Ag)" refers to a molecule or molecular structure bound by an antigen-specific antibody (Ab) or a B-cell antigen receptor (BCR). The presence of an antigen in the body usually triggers an immune response. In the body, each antibody is produced specifically so that it matches the antigen after contact with it by cells of the immune system, which enables accurate identification or matching of the antigen and the initiation of a personalized response. In most cases, an antibody can react to and bind to only one specific antigen. However, in some cases, antibodies can cross-react and bind to more than one antigen. Antigens are usually proteins, peptides (amino acid chains), and polysaccharides (monosaccharides / chains of single sugars) or combinations thereof.

[0048] The term "binding preference" or "binding preference" indicates that, under otherwise equivalent conditions, one of two alternative antigens or targets binds better than the other.

[0049] Typically, the term “antibody,” as used herein, refers to secretory immunoglobulins that lack a transmembrane region and can therefore be released into the bloodstream and body cavities. The type of heavy chain present defines the class of antibody (i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively), each playing a different role and directing the appropriate immune response to different types of antigens. Different heavy chains differ in size and composition and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the gastrointestinal tract, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, and inhibits colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389~417). IgD primarily functions as an antigen receptor on B cells not exposed to the antigen, and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437; Chen et al. (2009) Nat.Immunol.10:889-898). IgE is involved in allergic reactions by binding to allergens, which triggers histamine release from mast cells and basophils. IgE is also involved in protection against parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides the majority of antibody-based immunity against invading pathogens and is the only antibody isotype that can cross the placenta and provide passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four distinct IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum, with IgG1 being the most abundant (approximately 66%), followed by IgG2 (approximately 23%), IgG3 (approximately 7%), and IgG (approximately 4%). The biological profiles of different IgG classes are determined by the structure of their respective hinge regions. IgM is expressed on the surface of B cells in monomeric form and in secretory pentameric form, which has very high avidity.IgM is involved in eliminating pathogens in the early stages of B cell-mediated (humoral) immunity before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437). Antibodies are not only found as monomers, but are also well known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., bony fish IgM), or pentamers of five Ig units (e.g., mammalian IgM). Antibodies are typically constructed from four polypeptide chains, each containing two identical heavy chains and two identical light chains linked by disulfide bonds, resembling a "Y"-shaped macromolecule. Each chain contains several immunoglobulin domains, some of which are constant domains and others are variable domains. The immunoglobulin domains consist of a two-layer sandwich of 7 to 9 antiparallel chains arranged in two sheets. Typically, the heavy chain of an antibody contains four Ig domains, three of which are constant (CH domains: CHI, CH2, CH3) and one of which is a variable domain (VH). The light chain typically contains one constant Ig domain (CL) and one variable Ig domain (VL). For example, the human IgG heavy chain consists of four Ig domains linked from the N-terminus to the C-terminus in the order VwCH1-CH2-CH3 (also called VwCyl-Cy2-Cy3), while the human IgG light chain consists of two immunoglobulin domains linked from the N-terminus to the C-terminus in the order VL-CL, and is either kappa-type or lambda-type (VK-CK or VA-CA). For example, the constant chain of human IgG contains 447 amino acids. Throughout this specification and the claims, the numbering of amino acid positions of immunoglobulins is based on Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S., and Foeller, C., (1991) Sequences of proteins of immunological interest, 5. thThis is the "EU index" numbering as used in ed. USD Department of Health and Human Service, National Institutes of Health, Bethesda, MD. "EU index as used in Kabat" refers to the residue numbering of human IgG lEU antibodies. Therefore, in relation to IgG, the CH domains are as follows: "CHI" refers to amino acid positions 118-220 according to the EU index as used in Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index as used in Kabat; and "CH3" refers to amino acid positions 341-447 according to the EU index as used in Kabat.

[0050] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein synonymously to refer to an antibody in its substantially intact form, not to the antibody fragments described below. Specifically, these terms refer to an antibody having a heavy chain containing an Fc region.

[0051] Papain digestion of antibodies produces two identical antigen-binding fragments: one called the "Fab fragment" (also referred to as the "Fab moiety" or "Fab region"), each possessing a single antigen-binding site, and the remaining "Fc fragment" (also called the "Fc moiety" or "Fc region"), named to reflect its ability to readily crystallize. The crystal structure of the human IgG Fe region has been determined (Deisenhofer (1981) Biochemistry 20:2361~2370). In IgG, IgA, and IgD isotypes, the Fe region consists of two identical protein fragments derived from the CH2 and CH3 domains of the antibody's two heavy chains, while in IgM and IgE isotypes, the Fe region contains three heavy chain constant domains (CH2~4) in each polypeptide chain. Furthermore, smaller immunoglobulin molecules exist naturally or are artificially constructed. The term "Fab' fragment" refers to a Fab fragment that additionally contains the hinge region of an Ig molecule, while "F(ab')2 fragment" is understood to contain two Fab' fragments that are chemically linked or linked via disulfide bonds. "Single-domain antibodies (sdAb)" (Desmyter et al. (1996) Nat. Structure Biol. 3:803-811)) and "nanobodies" contain only a single VH domain, while "single-chain Fv (scFv)" fragments contain a heavy-chain variable domain linked to a light-chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). A divalent single-chain variable fragment (di-scFv) can be manipulated by linking two scFv (scFvA-scFvB). This can be done by generating a single peptide chain having two VH regions and two VL regions, obtaining a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is the creation of an scFv with a linker that is too short for the two variable regions to fold together, forcing the scFv to dimerize. Typically, a linker with a length of 5 residues is used to generate these dimers. This type is known as a "diabody".A shorter linker (one or two amino acids) between the VH and VL domains leads to the formation of a monospecific trimer, a so-called "tribody." Bispecific diabodies are formed by expression on chains having the sequences VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. A single-stranded diabody (scDb) contains VHA-VLB and VHB-VLA fragments (VHA-VLB-P-VHB-VLA) linked by a linker peptide (P) of 12 to 20 amino acids, preferably 14 amino acids. A "bispecific T cell engager (BiTE)" is a fusion protein consisting of two scFvs of different antibodies, one of which binds to T cells via the CD3 receptor and the other to tumor cells via tumor-specific molecules (Kufer et al. (2004) Trends Biotechnol. 22:238-244). The biaffinity retargeting molecule ("DART" molecule) is a diabody further stabilized by a C-terminal disulfide crosslink.

[0052] Therefore, the term “antibody fragment” refers to a portion of an intact antibody, preferably one containing its antigen-binding region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments; diabodies; sdAb, nanobodies, scFv, di-scFv, tandem scFv, triabodies, diabodies, scDb, BiTE, and DART.

[0053] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain. The variable domain of the heavy chain may be referred to as "VH," and the variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of the antibody and contain the antigen-binding site.

[0054] The term "variable" refers to the fact that the sequences of specific portions of the variable domain vary widely among antibodies and are used in the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions (HVRs) in both the light chain and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). The native heavy chain and light chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration, connected by three HVRs that link beta-sheet structures and, in some cases, form loops that form part of the beta-sheet structure. The HVRs within each chain are held in close proximity to one another by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domain is not directly involved in antibody binding to antigens, but it exhibits various effector functions, such as involvement in antibody-dependent cytotoxicity.

[0055] The "light chain" of an antibody (immunoglobulin) derived from any vertebrate species can be assigned to one of two distinctly different types called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0056] For the purposes of this specification, a "naked antibody" is an antibody that is not conjugated with any additional moieties, such as a cytotoxic moiety or label (e.g., radiolabeling).

[0057] As used herein, the terms “hypervariable region,” “HVR,” or “HV” refer to regions of antibody variable domains whose sequences are hypervariable and / or form structurally defined loops. Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the highest diversity among the six HVRs, and H3 in particular is thought to play a unique role in conferring superior specificity to antibodies. See, for example, Xu et al. Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). Several HVR descriptions are used and incorporated herein. HVRs that are Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Chothia, on the other hand, refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVR represents a compromise between Kabat's CDR and Chothia's structural loop and is used by Oxford Molecular's AbM antibody modeling software. “Contact” HVR is based on analysis of available complex crystal structures. The residues derived from each of these HVRs are shown below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0058] HVR may include "extended HVR" as follows: in VL, 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3); and in VH, 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). The variable domain residues are numbered according to Kabat et al. above for each of these extended HVR definitions.

[0059] A “framework” or “FR” residue is a variable domain residue other than an HVR residue as defined herein.

[0060] The light chain variable domain / sequence consists of a framework region (FR) and a complementarity-determining region (CDR) represented by formula I: FR-L1-CDR-L1-FR-L2-CDR-L2-FR-L3-CDR-L3-FR-L4

[0061] The heavy chain variable domain / sequence consists of FR and CDR represented by formula II: FR-H1-CDR-H1-FR-H2-CDR-H2-FR-H3-CDR-H3-FR-H4

[0062] The terms "Kabat-like variable domain residue numbering" or "Kabat-like amino acid position numbering" and variations thereof refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the antibodies edited by Kabat et al. supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c etc. according to Kabat). "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Thus, in relation to IgG, the CH domains are as follows: "CH1" refers to amino acid positions 118-220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index as in Kabat; "CH3" refers to amino acid positions 341-447 according to the EU index as in Kabat.

[0063] The term "binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (K D ). This chemical equilibrium is also the ratio of the "on rate" or "association rate constant" (k a ) to the "off rate" or "dissociation rate constant" (k d ). Two antibodies can have the same affinity, but one can have both a high on rate constant and an off rate constant, while the other can have both a low on rate constant and an off rate constant. The association rate constant k a[M-1 s-1] defines the antibody / antigen complex formation rate, while the dissociation rate constant [s-1] defines the antibody / antigen complex stability as decay per second. Recalculating according to the formula t / 2diss=ln(2) / (kd*60), the antibody / antigen complex half-life in minutes represents the descriptive parameter.

[0064] Affinity can be measured by common methods known in the art, including, but not limited to, surface plasmon resonance-based assays (e.g., the BIAcore assay as described in PCT application publication WO2005 / 012359); enzyme-linked immunosorbent assays (ELISA); and competitive assays (e.g., RIA). Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind rapidly to antigens and remain bound for longer periods. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of the present invention. Specific descriptive and exemplary embodiments for measuring binding affinity are described below.

[0065] k a Value and k dThe value can be measured using a surface plasmon resonance assay at 25°C with an immobilized antigen CM5 chip containing approximately 10 response units (RUs), for example, using a BIACORE®-2000 or BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, New Jersey), using methods well known in the art. Briefly, the carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of the coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, serially diluted Fab solutions (0.78 nM to 500 nM) are injected into PBS (PBST) containing 0.05% TWEEN20® surfactant at 25°C at a flow rate of approximately 25 μL / min. The association rate (k a ) and dissociation rate (k d The equilibrium dissociation constant (K) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir coupled model (BIAcore® Evaluation Software version 3.2). D ) is k d / k a It is calculated as a ratio. For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The on velocity obtained by the above surface plasmon resonance assay is 10 6 M -1 s -1If it exceeds this, the on rate can be determined using fluorescence quenching techniques, which measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band-passing) of 20 nM anti-antigen antibody (Fab type) (pH 7.2) in PBS at 25°C in the presence of increasing antigen concentrations, when measured with a spectrometer such as a spectrophotometer with stopped flow (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0066] As used herein, the term “monoclonal antibody” (mAb) refers to a monospecific antibody produced by identical immune cells, which are clones of a specific parent cell, and therefore all are reactive to the same epitope of a given target molecule. In contrast, “polyclonal antibodies” are produced from several different immune cells and therefore target different epitopes of a given target molecule. Thus, monoclonal antibodies have monovalent affinity, i.e., they bind to the same epitope, whereas polyclonal antibodies bind to several different epitopes of the same target. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are typically not contaminated with other immunoglobulins.

[0067] The modifier "monoclonal" indicates a characteristic of antibodies that are obtained from a substantially homogeneous antibody population, and should not be interpreted as meaning that the antibodies must be produced by a specific method. For example, monoclonal antibodies used in accordance with the present invention include, but are not limited to, hybridoma methods (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (e.g., see U.S. Patent No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al. al.,J.Mol.Biol.222:581-597(1992);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al.,J.Mol.Biol.340(5):1073-1093(2004);Fellouse,PNAS USA 101(34):12467-12472(2004); and Lee et al., J. Immunol. Methods See 284(1-2):119-132(2004), and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences (e.g., International Patent Application Publication Nos. 1998 / 24893; 1996 / 34096; 1996 / 33735; 1991 / 10741; Jakobovits et al., PNAS USA 90:2551(1993); Jakobovits et al., Nature 362:255-258(1993); Bruggemann et al., Year in Immunol.7:33 (1993); U.S. Patent No. 5,545,807; No. 5,545,806; No. 5,569,825; No. 5,625,126; No. 5,633,425; and No. 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and It can be fabricated using various techniques, including those described in Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0068] Antibodies may further include "effector groups" such as "tags" or "labels." The term "tag" refers to an effector group that provides an antibody with the ability to bind to or be bound to other molecules. Examples of tags include, but are not limited to, His tags attached to an antigen sequence to enable purification. Tags may also include partners in a bioaffin binding pair that allow the antigen to be bound by a second partner of the binding pair. The term "bioaffin binding pair" refers to two partner molecules (i.e., two partners in a pair) that have a strong affinity for binding to each other. Examples of partners in a bioaffin binding pair are a) biotin or biotin analog / avidin or streptavidin; b) hapten / anti-hapten antibody or antibody fragment (e.g., digoxin / anti-digoxin antibody); c) sugars / lectins; d) complementary oligonucleotide sequences (e.g., complementary LNA sequences); and generally e) ligand / receptor.

[0069] The term "label" refers to an effector group that enables the detection of an antigen. Labels include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical, or chemical labeling. Suitable labels exemplified include fluorescent dyes, luminescent or electrochemiluminescent complexes (e.g., ruthenium or iridium complexes), high electron-density reagents, and enzymatic labeling.

[0070] Sandwich immunoassays are widely used for the detection of analytes of interest. In such assays, the analyte is "sandwiched" between a first antibody and a second antibody. Typically, a sandwich assay requires that the capture and detection antibodies bind to different, non-overlapping epitopes on the analyte of interest. By appropriate means, such a sandwich complex is measured, thereby quantifying the analyte. In a typical sandwich assay, a first antibody, bound to or capable of binding to a solid phase, and a second antibody, detectably labeled, each bind to the analyte at different, non-overlapping epitopes. The binder specific to the first analyte (e.g., an antibody) is either covalently or passively bound to the solid surface. The solid surface is typically glass or a polymer, with the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be particles, tubes, beads, microplate discs, or any other surface suitable for performing the immunoassay. The binding process is well known in the art and generally consists of crosslinking covalent bonding or physical adsorption, and the polymer-antibody complex is washed in preparation for the test sample. Aliquots of the sample to be tested are then added to the solid-phase complex and incubated for a sufficient period (e.g., 2–40 minutes or overnight if more convenient) and under appropriate conditions (e.g., room temperature to 40°C, e.g., 25°C to 37°C (including both ends)) to allow binding between the first antibody or capture antibody and the corresponding antigen. Following the incubation period, the solid phase containing the first antibody or capture antibody and the antibody-bound antigen may be washed and incubated with a secondary antibody or labeled antibody that binds to another epitope on the antigen. The second antibody is bound to a reporter molecule used to demonstrate the binding of the second antibody to the complex of the first antibody and the antigen of interest.

[0071] A highly versatile alternative sandwich assay format involves the use of a solid phase coated with the first partner of the binding pair, for example, microparticles coated with paramagnetic streptavidin. Such microparticles are incubated with an analyte-specific binder conjugated to the second partner of the binding pair (e.g., a biotinylated antibody), a sample suspected of containing or containing an analyte with the second partner of the binding pair conjugated to the analyte-specific binder, and a detectably labeled second analyte-specific binder. As will be apparent to those skilled in the art, these components are incubated under appropriate conditions for a period sufficient to conjugate the analyte, the analyte-specific binder (conjugated to the second partner of the binding pair), and the labeled antibody via the first partner of the binding pair to the solid phase microparticles. Such assays may optionally include one or more washing steps.

[0072] The term “detectably labeled” encompasses labels that can be detected directly or indirectly. A directly detectable label either provides a detectable signal, or the label interacts with a second label to modify a detectable signal provided by a first or second label, for example, to give FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al “Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids,” J.Chem.Soc., Perkin-Trans.1(1997)1051-1058) provide detectable signals and are generally applicable to labeling. In one embodiment, “detectably labeled” refers to a label that provides or can induce the provision of a detectable signal, i.e., a fluorescent label, an luminescent label (e.g., a chemiluminescent or electrochemiluminescent label), an radioactive label, or a metal chelate label, respectively.

[0073] Numerous labels (also referred to as dyes) are available, which can generally be classified into the following categories, all of which are grouped together, and each of them represents an embodiment of the present disclosure.

[0074] (a) Fluorescent dyes Fluorescent dyes have been described, for example, by Briggs et al. "Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058).

[0075] Fluorescent labels or phosphors include rare earth chelates (europium chelate), fluorescein-type labels (including FITC, 5-carboxyfluoroceine, and 6-carboxyfluoroceine), rhodamine-type labels (including TAMRA), dansyl, lysamine, cyanine, phycoerythrin, Texas Red, and their analogues. Fluorescent labels can be attached to aldehyde groups contained within target molecules using the techniques disclosed herein. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0076] (b) Luminescent dye Luminescent dyes or labels can be further classified into chemiluminescent dyes and electrochemiluminescent dyes.

[0077] Different classes of chemiluminescent labels include systems based on luminol, acridinium compounds, coelenterazine and its analogs, dioxetane, peroxyoxalic acid, and peroxyoxalic acid derivatives. For immunodiagnostic procedures, acridinium-based labels are primarily used (a detailed overview is provided in Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0078] The main relevant labels used as electrochemiluminescence labels are ruthenium and iridium-based electrochemiluminescent complexes, respectively. Electrochemiluminescence (ECL) has proven to be very useful for analytical applications as a highly sensitive and selective method. ECL combines the analytical advantages of chemiluminescence analysis (absence of background light signal) with the ease of reaction control by applying electrode potential. Generally, ruthenium complexes, particularly [Ru(Bpy)3]2+ (emitting photons at approximately 620 nm) regenerated using TPA (tripropylamine) in the liquid phase or liquid-solid interface, are used as ECL labels.

[0079] Electrochemiluminescence (ECL) assays provide highly sensitive and accurate measurements of the presence and concentration of an analyte of interest. Such techniques utilize labels or other reactants that can be induced to emit light when electrochemically oxidized or reduced in a suitable chemical environment. This electrochemiluminescence is caused by a voltage applied to the working electrode in a specific manner at a specific time. The light produced by the label is measured to indicate the presence or amount of the analyte. For a more complete description of such ECL techniques, see U.S. Patents 5,221,605, 5,591,581, 5,597,910, PCT Publications WO90 / 05296, WO92 / 14139, WO90 / 05301, WO96 / 24690, US95 / 03190, US97 / 16942, and US96. See also / 06763, PCT application publication WO95 / 08644, PCT application publication WO96 / 06946, PCT application publication WO96 / 33411, PCT application publication WO87 / 06706, PCT application publication WO96 / 39534, PCT application publication WO96 / 41175, PCT application publication WO96 / 40978, PCT / US97 / 03653 and U.S. Patent Application 08 / 437,348 (U.S. Patent No. 5,679,519). Also see the 1994 review by Knight, et al. (Analyst, 1994, 119:879-890) on the analytical applications of ECL and the references cited in the review. In one embodiment, the method according to this specification is carried out using electrochemiluminescence labeling.

[0080] In recent years, iridium-based ECL labels have also been described (International Publication No. 2012107419).

[0081] (c) The radioactive label shall be a radioactive isotope (radionic nuclide), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 131Bi.

[0082] (d) Metal chelate complexes suitable as labels for imaging and therapeutic purposes are well known in the art (U.S. Patent Application Publication No. 2010 / 0111861; U.S. Patents No. 5,342,606; U.S. Patents No. 5,428,155; U.S. Patents No. 5,316,757; U.S. Patents No. 5,480,990; U.S. Patents No. 5,462,725; U.S. Patents No. 5,428,139; U.S. Patents No. 5,385,893; U.S. Patents No. 5,739,294; U.S. Patents No. 5,750,660; U.S. Patents No. 5,834,461; Hnatowich et al, J. Immunol. Methods 65(1983)147-157; Meares et al, Anal. Biochem. 142(1984)68-78; Mirzadeh et al, Bioconjugate Chem.1(1990)59-65;Meares et al,J.Cancer(1990),Suppl.10:21-26;Izard et al,Bioconjugate Chem.3(1992)346-350;Nikula et al,Nucl.Med.Biol.22(1995)387-90;Camera et al. al,Nucl.Med.Biol.20(1993)955-62;Kukis et al,J.Nucl.Med.39(1998)2105-2110;Verel et al.,J.Nucl.Med.44(2003)1663-1670;Camera et al. al,J.Nucl.Med.21(1994)640-646;Ruegg et al,Cancer Res.50(1990)4221-4226;Verel et al,J.Nucl.Med.44(2003)1663-1670;Lee et al,Cancer Res.61(2001)4474-4482;Mitchell,et al,J.Nucl.Med.44(2003)1105-1112;Kobayashi et al Bioconjugate Chem.10(1999)103-111;Miederer et al,J.Nucl.Med.45(2004)129-137;DeNardo et al,Clinical Cancer Research 4(1998)2483-90;Blend et al,Cancer Biotherapy&Radiopharmaceuticals18(2003)355-363;Nikula et al J.Nucl.Med.40(1999)166-76;Kobayashi et al al, J. Nucl. Med. 39 (1998) 829-36; Mardirossian et al, Nucl. Med. Biol. 20 (1993) 65-74; Roselli et al, Cancer Biotherapy & Radiopharmaceuticals, 14 (1999) 209-20). .

[0083] As used herein, “particle” means a small, localized object to which physical properties such as volume, mass, or average size can be attributed. Thus, particles may be symmetrical, spherical, essentially spherical, or spherical in shape, or they may be irregular, asymmetrical in shape or form. The size of particles can vary. The term “fine particles” refers to particles having diameters in the nanometer and micrometer range.

[0084] The fine particles as defined herein above may contain, or consist of, any suitable material known to those skilled in the art, for example, inorganic or organic materials, or essentially consist of them. Typically, they contain, consist of, or essentially consist of metals or alloys of metals, or organic materials, or carbohydrate elements. Examples of materials envisioned for fine particles include agarose, polystyrene, latex, polyvinyl alcohol, silica, and ferromagnetic metals, alloys, or hybrids. In one embodiment, the fine particles are magnetic or ferromagnetic metals, alloys, or hybrids. In further embodiments, the material may have specific properties, for example, hydrophobic or hydrophilic. Such fine particles are typically dispersed in aqueous solutions, retaining a small negative surface charge while keeping the particles separated and avoiding nonspecific cluster formation.

[0085] In one embodiment of the present invention, the microparticles are paramagnetic microparticles, and the separation of the particles in the measurement method according to the present disclosure is facilitated by magnetic force. A magnetic force is applied to extract paramagnetic or magnetic particles from a solution / suspension and to retain them as needed, and the liquid of the solution / suspension can be removed, and the particles can be washed, for example.

[0086] A “kit” is any product (e.g., a package or container) comprising at least one reagent, such as a drug for treating a disorder, or a probe for specifically detecting the biomarker gene or protein of the present invention. The kit is preferably encouraged, distributed, or sold as a unit for carrying out the method of the present invention. Typically, the kit may further include a carrier means partitioned to receive one or more container means, such as vials and tubes, under strict control. In particular, each container means contains one of the distinct elements used in the method of the first embodiment. The kit may further include one or more other containers containing further materials, including but not limited to buffers, diluents, filters, needles, syringes, and accompanying documentation with instructions for use. Labels may be presented on the container to indicate that the composition is intended for a specific use, and may also indicate instructions for either in vivo or in vitro use. Computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disk), or directly on a computer or data processing device. Furthermore, the kit may contain standard amounts of biomarkers, as described elsewhere in this specification, for calibration purposes.

[0087] "Package insert" is used to refer to the instructions typically included in the commercial packaging of a therapeutic drug or drug, which contain information about the indications, use, dosage, administration, contraindications of such therapeutic drug or drug, other therapeutic drugs to be used in combination with the packaged product, and / or warnings regarding their use.

[0088] Embodiment Currently available PCR-format diagnostic assays for detecting SARS-CoV-2 virus in patient samples require several hours for results to become available. Therefore, they are insufficient to meet the high demand for coronavirus testing in the ongoing pandemic. Rapid point-of-care antigen tests offer much faster results but often lack the sensitivity and / or specificity required for reliable diagnosis. To meet the high demand for reliable diagnostic results in the pandemic, we have developed a high-throughput antigen assay using highly specific antibodies.

[0089] In a first aspect, the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that binds to the nucleocapsid protein of the SARS-CoV-2 virus (isolated), wherein the monoclonal antibody or antigen-binding fragment thereof a) 1.0E+05M determined by surface plasmon resonance -1 s -1 The association rate constant (k) exceeds the following value. a ) has, and / or b) 5.0E-04s determined by surface plasmon resonance -1 Dissociation rate constant (k) less than d ) has, and / or c) Having a half-life of 15 minutes or more t / 2diss determined by surface plasmon resonance, and / or d) It has a stoichiometric ratio of 1:1 or 1:2.

[0090] In certain embodiments, the antibody is 1.5E+05M -1 s -1 Exceeding 2.0E+05M -1 s -1 The association rate constant (k) exceeds the following value. a ) has. In certain embodiments, the antibody is 3.0E+05M -1 s -1 Exceeding 4.0E+05M -1 s -1 The association rate constant (k) exceeds the following value.a ) has. In certain embodiments, the antibody is 5.0E+05M -1 s -1 The association rate constant (k) exceeds the following value. a ) has.

[0091] In certain embodiments, the antibody is 5.0E-04s -1 Less than, especially 3.0E-04s -1 Dissociation rate constant (k) less than d ) has. In a particular embodiment, the antibody is 2.0E-04s -1 Less than, especially 1.0E-04s -1 Dissociation rate constant (k) less than d ) has. In a particular embodiment, the antibody is 2.0E-05s -1 Dissociation rate constant (k) less than d ) has.

[0092] In certain embodiments, the antibody has an antibody / antigen complex half-life of 25 minutes or more t / 2diss, particularly 40 minutes or more t / 2diss. In certain embodiments, the antibody has an antibody / antigen complex half-life of 50 minutes or more t / 2diss, particularly 75 minutes or more t / 2diss. In certain embodiments, the antibody has an antibody / antigen complex half-life of 100 minutes or more t / 2diss. In certain embodiments, the antibody has an antibody / antigen complex half-life of 200 minutes or more t / 2diss.

[0093] In certain embodiments, the antibody is 3.4E+05M -1 s -1 The association rate constant (k a ) and 2.0E-05s -1 The dissociation rate constant (k d ) has. In a particular embodiment, the antibody has an antibody / antigen complex half-life of 579 minutes t / 2diss.

[0094] In certain embodiments, the antibody is 2.0E+05M -1 s -1 The association rate constant (k a ) and 2.4E-04s -1 The dissociation rate constant (k d) has. In certain embodiments, the antibody has an antibody / antigen complex half-life of 48 minutes t / 2diss.

[0095] In certain embodiments, the antibody is 1.8E+05M -1 s -1 The association rate constant (k a ) and 1.2E-04s -1 The dissociation rate constant (k d ) has. In certain embodiments, the antibody has an antibody / antigen complex half-life of 93 minutes t / 2diss.

[0096] In certain embodiments, the antibody has a sequence described in any of the following embodiments 2 to 4.

[0097] In several embodiments, the antibody or antigen-binding fragment of the present invention is an isolated antibody or antigen-binding fragment. Therefore, the antibody or antigen-binding fragment is a purified antibody or antigen-binding fragment. Purification of the antibody can be achieved by methods well known in the art, such as size exclusion chromatography (SEC). Therefore, the antibody or antigen-binding fragment is assumed to be isolated from the cells from which the antibody was produced. In some embodiments, the isolated antibody or antigen-binding fragment is purified to more than 70% by weight of the antibody, for example, when measured by the Lowry method, and in some embodiments to more than 80%, 90%, 95%, 96%, 97%, 98%, or 99% by weight of the antibody. In one preferred embodiment, the isolated antibody or antigen-binding fragment according to the present invention is purified to a purity of more than 90%, when determined by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection.

[0098] In some embodiments, the antibody or its antigen-binding fragment is a naked antibody or a naked antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment further includes a tag or label. In certain embodiments, the tag allows the antibody or its antigen-binding fragment to be directly or indirectly bound to a solid phase. In certain embodiments, the tag is a partner of a bioaffine binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxin, hapten, or complementary oligonucleotide sequences (particularly complementary LNA sequences). In certain embodiments, the tag is biotin.

[0099] In certain embodiments, the label enables the detection of an antibody or its antigen-binding fragment. In certain embodiments, the label is an electrochemiluminescent ruthenium or iridium complex. In certain embodiments, the electrochemiluminescent ruthenium complex is a negatively charged electrochemiluminescent ruthenium complex. In certain embodiments, the label is a negatively charged electrochemiluminescent ruthenium complex present in the antigen in stoichiometric amounts from 1:1 to 15:1. In certain embodiments, the stoichiometric ratios are 2:1, 2.5:1, 3:1, 5:1, 10:1, or 15:1.

[0100] In a second aspect, the present invention relates to an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof a) Includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 1, 2, 3, 4, 5, and 6 respectively, b) Whether the antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, according to SEQ ID NOs: 1, 2, 3, 4, 5, and 6, bind to the same epitopes, or c) These antibodies compete for binding to the SARS-CoV-2 virus nucleocapsid protein with antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0101] In certain embodiments, the antibody or its antigen-binding fragment comprises a CDR containing the sequences specifically listed above (i.e., without amino acid mutations).

[0102] In certain embodiments, the antibody or its antigen-binding fragment comprises one or more CDRs having sequence mutations in the sequence described above. In certain embodiments, the sequence mutation comprises one or two, particularly one, amino acid changes. In certain embodiments, one or two amino acid changes are, independently of each other, amino acid deletions, amino acid additions, or amino acid substitutions. In certain embodiments, the amino acid substitution is a conservative amino acid substitution.

[0103] In certain embodiments, the antibody or antigen-binding fragment of the second embodiment is further, a) Each includes FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to sequence numbers 7, 8, 9, 10, 11, 12, 13, and 14, respectively, b) Whether they bind to the same epitopes as antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 7, 8, 9, 10, 11, 12, 13, and 14, respectively, or c) These antibodies compete for binding to the SARS-CoV-2 virus nucleocapsid protein with antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4, respectively, as specified by SEQ ID NOs. 7, 8, 9, 10, 11, 12, 13, and 14.

[0104] In certain embodiments, the antibody or its antigen-binding fragment comprises an FR containing the sequences specifically listed above (i.e., without amino acid mutations).

[0105] In certain embodiments, the antibody or its antigen-binding fragment contains one or more FRs having sequence mutations in the sequence described above. In certain embodiments, the sequence mutations include up to five, in particular one, two, three, four, or five, amino acid changes. In certain embodiments, the up to five, in particular one, two, three, four, or five, amino acid changes are, independently of each other, amino acid deletions, amino acid additions, or amino acid substitutions. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0106] In certain embodiments, the antibody or antigen-binding fragment of the second embodiment is a) comprising a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 15 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 16, b) The antibody contains a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 15 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 16, and binds to the same epitope. or c) The antibody containing a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 15 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 16 competes with the antibody for binding to the SARS-CoV-2 virus nucleocapsid protein.

[0107] In certain embodiments, the antibody or its antigen-binding fragment includes the sequences specifically listed above, i.e., a heavy chain variable domain and a light chain variable domain without amino acid mutations.

[0108] In certain embodiments, the antibody or its antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain having sequence variations of the sequences listed above. In certain embodiments, the variant sequence is at least 85% identical to the sequences specifically listed above. In a further embodiment, the identity is at least 90%. In a further embodiment, the identity is at least 95%, and in particular at least 98%.

[0109] In certain embodiments, the antibody or its antigen-binding fragment binds to the nucleocapsid protein of the SARS-CoV-2 virus, and the antibody or its antigen-binding fragment a) 1.0E+05M determined by surface plasmon resonance -1 s -1 The association rate constant (k) exceeds the following value. a ) has, and / or b) 5.0E-04s determined by surface plasmon resonance -1 Dissociation rate constant (k) less than d ) has, and / or c) Having a half-life of 15 minutes or more t / 2diss determined by surface plasmon resonance, and / or d) It has a stoichiometric ratio of 1:1 or 1:2.

[0110] In certain embodiments, the antibody is 1.5E+05M -1 s -1 Exceeding 2.0E+05M -1 s -1 The association rate constant (k) exceeds the following value. a ) has. In certain embodiments, the antibody is 3.0E+05M -1 s -1 Exceeding 4.0E+05M -1 s -1 The association rate constant (k) exceeds the following value. a ) has. In certain embodiments, the antibody is 5.0E+05M -1 s -1 The association rate constant (k) exceeds the following value. a ) has.

[0111] In certain embodiments, the antibody is 5.0E-04s -1 Less than, especially 3.0E-04s -1 Dissociation rate constant (k) less than d ) has. In a particular embodiment, the antibody is 2.0E-04s -1 Less than, especially 1.0E-04s -1 Dissociation rate constant (k) less than d) and in certain embodiments, the antibody has a dissociation rate constant (k -1 less than 2.0E-05 s d ).

[0112] In certain embodiments, the antibody has an antibody / antigen complex half-life time of t / 2diss of 25 minutes or more, particularly 40 minutes or more. In certain embodiments, the antibody has an antibody / antigen complex half-life time of t / 2diss of 50 minutes or more, particularly 75 minutes or more. In certain embodiments, the antibody has an antibody / antigen complex half-life time of t / 2diss of 100 minutes or more. In certain embodiments, the antibody has an antibody / antigen complex half-life time of t / 2diss of 200 minutes or more.

[0113] In certain embodiments, the antibody has an association rate constant (k -1 s -1 ) of 3.4E+05 M a and a dissociation rate constant (k -1 ) of 2.0E-05 s d . In certain embodiments, the antibody has an antibody / antigen complex half-life time of t / 2diss of 579 minutes.

[0114] In several embodiments, the antibody or antigen-binding fragment of the present invention is an isolated antibody or antigen-binding fragment. Thus, the antibody or antigen-binding fragment is a purified antibody or antigen-binding fragment. Purification of the antibody can be achieved by methods well known in the art such as size exclusion chromatography (SEC). Thus, it is assumed that the antibody or antigen-binding fragment is isolated from the cells in which the antibody was produced. In some embodiments, the isolated antibody or antigen-binding fragment is purified to more than 70% by weight of the antibody, in some embodiments 80%, 90%, 95%, 96%, 97%, 98% or 99% by weight, as measured by, for example, the Lowry method. In a preferred embodiment, the isolated antibody or antigen-binding fragment according to the present invention is purified to a purity of more than 90% as determined by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection.

[0115] In some embodiments, the antibody or its antigen-binding fragment is a naked antibody or a naked antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment further includes a tag or label. In certain embodiments, the tag allows the antibody or its antigen-binding fragment to be directly or indirectly bound to a solid phase. In certain embodiments, the tag is a partner of a bioaffine binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxin, hapten, or complementary oligonucleotide sequences (particularly complementary LNA sequences). In certain embodiments, the tag is biotin.

[0116] In certain embodiments, the label enables the detection of an antibody or its antigen-binding fragment. In certain embodiments, the label is an electrochemiluminescent ruthenium or iridium complex. In certain embodiments, the electrochemiluminescent ruthenium complex is a negatively charged electrochemiluminescent ruthenium complex. In certain embodiments, the label is a negatively charged electrochemiluminescent ruthenium complex present in the antigen in stoichiometric amounts from 1:1 to 15:1. In certain embodiments, the stoichiometric ratios are 2:1, 2.5:1, 3:1, 5:1, 10:1, or 15:1.

[0117] In a third aspect, the present invention relates to an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof a) Each includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 17, 18, 19, 20, 21, and 22, respectively. b) Whether the antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, according to SEQ ID NOs. 17, 18, 19, 20, 21, and 22, bind to the same epitopes, or c) These antibodies compete for binding to the SARS-CoV-2 virus nucleocapsid protein with antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, as defined by SEQ ID NOs. 17, 18, 19, 20, 21, and 22.

[0118] In certain embodiments, the antibody or its antigen-binding fragment comprises a CDR containing the sequences specifically listed above (i.e., without amino acid mutations).

[0119] In certain embodiments, the antibody or its antigen-binding fragment comprises one or more CDRs having sequence mutations in the sequence described above. In certain embodiments, the sequence mutation comprises one or two, particularly one, amino acid changes. In certain embodiments, one or two amino acid changes are, independently of each other, amino acid deletions, amino acid additions, or amino acid substitutions. In certain embodiments, the amino acid substitution is a conservative amino acid substitution.

[0120] In certain embodiments, the antibody or antigen-binding fragment of the third embodiment is further, a) Each includes FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to sequence numbers 23, 24, 25, 26, 27, 28, 29, and 30, b) Whether they bind to the same epitopes as antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 23, 24, 25, 26, 27, 28, 29, and 30, respectively, or c) These antibodies compete for binding to the SARS-CoV-2 virus nucleocapsid protein with antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4, respectively, as defined by SEQ ID NOs. 23, 24, 25, 26, 27, 28, 29, and 30.

[0121] In certain embodiments, the antibody or its antigen-binding fragment comprises an FR containing the sequences specifically listed above (i.e., without amino acid mutations).

[0122] In certain embodiments, the antibody or its antigen-binding fragment comprises one or more FRs having sequence mutations in the above sequence.

[0123] In certain embodiments, the sequence mutation includes up to five amino acid changes, particularly one, two, three, four, or five amino acid changes. In certain embodiments, the up to five amino acid changes, particularly one, two, three, four, or five amino acid changes, are independently amino acid deletions, amino acid additions, or amino acid substitutions. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0124] In certain embodiments, the antibody or antigen-binding fragment of the third embodiment is a) comprising a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 31 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 32, b) The antibody contains a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 31 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 32, and binds to the same epitope. or c) The antibody containing a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 31 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 32 competes with the antibody for binding to the SARS-CoV-2 virus nucleocapsid protein.

[0125] In certain embodiments, the antibody or its antigen-binding fragment includes the sequences specifically listed above, i.e., a heavy chain variable domain and a light chain variable domain without amino acid mutations.

[0126] In certain embodiments, the antibody or its antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain having sequence variations of the sequences listed above. In certain embodiments, the variant sequence is at least 85% identical to the sequences specifically listed above. In a further embodiment, the identity is at least 90%. In a further embodiment, the identity is at least 95%, and in particular at least 98%.

[0127] In certain embodiments, the antibody or its antigen-binding fragment binds to the nucleocapsid protein of the SARS-CoV-2 virus, and the antibody or its antigen-binding fragment a) 1.0E+05M determined by surface plasmon resonance-1 s -1 having an association rate constant (k a ) greater than and / or b) having a dissociation rate constant (k -1 ) less than 5.0E-04 s determined by surface plasmon resonance d and / or and / or c) having a half-life time of t / 2diss of at least 15 minutes determined by surface plasmon resonance and / or d) having a stoichiometric ratio of 1:1 or 1:2

[0128] <> In certain embodiments, the antibody has an association rate constant (k -1 s -1 ) greater than 1.5E+05 M -1 s -1 , particularly greater than 2.0E+05 M a s -1 . In certain embodiments, the antibody has an association rate constant (k -1 ) greater than 3.0E+05 M -1 s -1 , particularly greater than 4.0E+05 M a s -1 . In certain embodiments, the antibody has an association rate constant (k -1 ) greater than 5.0E+05 M a s

[0129] In certain embodiments, the antibody has a dissociation rate constant (k -1 -1 ) less than 5.0E-04 s, particularly less than 3.0E-04 s -1 s d In certain embodiments, the antibody has a dissociation rate constant (k ) less than 2.0E-04 s, particularly less than 1.0E-04 s -1 s -1 In certain embodiments, the antibody has a dissociation rate constant (k d ) less than 2.0E-05 s -1 s d

[0130] In certain embodiments, the antibody has an antibody / antigen complex half-life of 25 minutes or more t / 2diss, particularly 40 minutes or more t / 2diss. In certain embodiments, the antibody has an antibody / antigen complex half-life of 50 minutes or more t / 2diss, particularly 75 minutes or more t / 2diss. In certain embodiments, the antibody has an antibody / antigen complex half-life of 100 minutes or more t / 2diss. In certain embodiments, the antibody has an antibody / antigen complex half-life of 200 minutes or more t / 2diss.

[0131] In certain embodiments, the antibody is 1.8E+05M -1 s -1 The association rate constant (k a ) and 1.2E-04s -1 The dissociation rate constant (k d ) has. In certain embodiments, the antibody has an antibody / antigen complex half-life of 93 minutes t / 2diss.

[0132] In several embodiments, the antibody or antigen-binding fragment of the present invention is an isolated antibody or antigen-binding fragment. Therefore, the antibody or antigen-binding fragment is a purified antibody or antigen-binding fragment. Purification of the antibody can be achieved by methods well known in the art, such as size exclusion chromatography (SEC). Therefore, the antibody or antigen-binding fragment is assumed to be isolated from the cells from which the antibody was produced. In some embodiments, the isolated antibody or antigen-binding fragment is purified to more than 70% by weight of the antibody, for example, when measured by the Lowry method, and in some embodiments to more than 80%, 90%, 95%, 96%, 97%, 98%, or 99% by weight of the antibody. In one preferred embodiment, the isolated antibody or antigen-binding fragment according to the present invention is purified to a purity of more than 90%, when determined by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection.

[0133] In some embodiments, the antibody or its antigen-binding fragment is a naked antibody or a naked antigen-binding fragment. In several embodiments, the antibody or its antigen-binding fragment further includes a tag or label. In certain embodiments, the tag allows the antibody or its antigen-binding fragment to be directly or indirectly bound to a solid phase. In certain embodiments, the tag is a partner of a bioaffine binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxin, hapten, or complementary oligonucleotide sequences (particularly complementary LNA sequences). In certain embodiments, the tag is biotin.

[0134] In certain embodiments, the label enables the detection of an antibody or its antigen-binding fragment. In certain embodiments, the label is an electrochemiluminescent ruthenium or iridium complex. In certain embodiments, the electrochemiluminescent ruthenium complex is a negatively charged electrochemiluminescent ruthenium complex. In certain embodiments, the label is a negatively charged electrochemiluminescent ruthenium complex present in the antigen in stoichiometric amounts from 1:1 to 15:1. In certain embodiments, the stoichiometric ratios are 2:1, 2.5:1, 3:1, 5:1, 10:1, or 15:1.

[0135] In a fourth aspect, the present invention relates to an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof a) Each includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 33, 34, 35, 36, 37, and 38, b) Whether the antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, according to SEQ ID NOs. 33, 34, 35, 36, 37, and 38, bind to the same epitopes, or c) These antibodies, each containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs. 33, 34, 35, 36, 37, and 38 respectively, compete for binding to the SARS-CoV-2 virus nucleocapsid protein.

[0136] In certain embodiments, the antibody or its antigen-binding fragment comprises a CDR containing the sequences specifically listed above (i.e., without amino acid mutations).

[0137] In certain embodiments, the antibody or its antigen-binding fragment comprises one or more CDRs having sequence mutations in the sequence described above. In certain embodiments, the sequence mutation comprises one or two, particularly one, amino acid changes. In certain embodiments, one or two amino acid changes are, independently of each other, amino acid deletions, amino acid additions, or amino acid substitutions. In certain embodiments, the amino acid substitution is a conservative amino acid substitution.

[0138] In certain embodiments, the antibody or antigen-binding fragment of the fourth embodiment is further: a) Each includes FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to sequence numbers 39, 40, 41, 42, 43, 44, 45, and 46, b) Whether they bind to the same epitopes as antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, and 46, respectively, or c) These antibodies, each comprising FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, and 46 respectively, compete for binding to the SARS-CoV-2 virus nucleocapsid protein.

[0139] In certain embodiments, the antibody or its antigen-binding fragment comprises an FR containing the sequences specifically listed above (i.e., without amino acid mutations).

[0140] In certain embodiments, the antibody or its antigen-binding fragment contains one or more FRs having sequence mutations in the sequence described above. In certain embodiments, the sequence mutations include up to five, in particular one, two, three, four, or five, amino acid changes. In certain embodiments, the up to five, in particular one, two, three, four, or five, amino acid changes are, independently of each other, amino acid deletions, amino acid additions, or amino acid substitutions. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0141] In certain embodiments, the antibody or antigen-binding fragment of the fourth embodiment is a) comprising a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 47 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 48, b) The antibody contains a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 47 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 48, and binds to the same epitope. or c) The antibody containing a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 47 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 48 competes with the antibody for binding to the SARS-CoV-2 virus nucleocapsid protein.

[0142] In certain embodiments, the antibody or its antigen-binding fragment includes the sequences specifically listed above, i.e., a heavy chain variable domain and a light chain variable domain without amino acid mutations.

[0143] In certain embodiments, the antibody or its antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain having sequence variations of the sequences listed above. In certain embodiments, the variant sequence is at least 85% identical to the sequences specifically listed above. In a further embodiment, the identity is at least 90%. In a further embodiment, the identity is at least 95%, and in particular at least 98%.

[0144] In certain embodiments, the antibody or its antigen-binding fragment binds to the nucleocapsid protein of the SARS-CoV-2 virus, and the antibody or its antigen-binding fragment a) 1.0E+05M determined by surface plasmon resonance -1 s -1 The association rate constant (k) exceeds the following value. a ) has, and / or b) 5.0E-04s determined by surface plasmon resonance -1 Dissociation rate constant (k) less than d ) has, and / or c) Having a half-life of 15 minutes or more t / 2diss determined by surface plasmon resonance, and / or d) It has a stoichiometric ratio of 1:1 or 1:2.

[0145] In certain embodiments, the antibody is 1.5E+05M -1 s -1 Exceeding 2.0E+05M -1 s -1 The association rate constant (k) exceeds the following value. a ) has. In certain embodiments, the antibody is 3.0E+05M -1 s -1 Exceeding 4.0E+05M -1 s -1 The association rate constant (k) exceeds the following value. a ) has. In certain embodiments, the antibody is 5.0E+05M -1 s -1 The association rate constant (k) exceeds the following value. a ) has.

[0146] In certain embodiments, the antibody is 5.0E-04s -1 Less than, especially 3.0E-04s -1 Dissociation rate constant (k) less than d ) has. In a particular embodiment, the antibody is 2.0E-04s -1 Less than, especially 1.0E-04s -1 Dissociation rate constant (k) less than d) has. In a particular embodiment, the antibody is 2.0E-05s -1 Dissociation rate constant (k) less than d ) has.

[0147] In certain embodiments, the antibody has an antibody / antigen complex half-life of 25 minutes or more t / 2diss, particularly 40 minutes or more t / 2diss. In certain embodiments, the antibody has an antibody / antigen complex half-life of 50 minutes or more t / 2diss, particularly 75 minutes or more t / 2diss. In certain embodiments, the antibody has an antibody / antigen complex half-life of 100 minutes or more t / 2diss. In certain embodiments, the antibody has an antibody / antigen complex half-life of 200 minutes or more t / 2diss.

[0148] In certain embodiments, the antibody is 2.0E+05M -1 s -1 The association rate constant (k a ) and 2.4E-04s -1 The dissociation rate constant (k d ) has. In certain embodiments, the antibody has an antibody / antigen complex half-life of 48 minutes t / 2diss.

[0149] In several embodiments, the antibody or antigen-binding fragment of the present invention is an isolated antibody or antigen-binding fragment. Therefore, the antibody or antigen-binding fragment is a purified antibody or antigen-binding fragment. Purification of the antibody can be achieved by methods well known in the art, such as size exclusion chromatography (SEC). Therefore, the antibody or antigen-binding fragment is assumed to be isolated from the cells from which the antibody was produced. In some embodiments, the isolated antibody or antigen-binding fragment is purified to more than 70% by weight of the antibody, for example, when measured by the Lowry method, and in some embodiments to more than 80%, 90%, 95%, 96%, 97%, 98%, or 99% by weight of the antibody. In one preferred embodiment, the isolated antibody or antigen-binding fragment according to the present invention is purified to a purity of more than 90%, when determined by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection.

[0150] In some embodiments, the antibody or its antigen-binding fragment is a naked antibody or a naked antigen-binding fragment. In several embodiments, the antibody or its antigen-binding fragment further includes a tag or label. In certain embodiments, the tag allows the antibody or its antigen-binding fragment to be directly or indirectly bound to a solid phase. In certain embodiments, the tag is a partner of a bioaffine binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxin, hapten, or complementary oligonucleotide sequences (particularly complementary LNA sequences). In certain embodiments, the tag is biotin.

[0151] In certain embodiments, the label enables the detection of an antibody or its antigen-binding fragment. In certain embodiments, the label is an electrochemiluminescent ruthenium or iridium complex. In certain embodiments, the electrochemiluminescent ruthenium complex is a negatively charged electrochemiluminescent ruthenium complex. In certain embodiments, the label is a negatively charged electrochemiluminescent ruthenium complex present in the antigen in stoichiometric amounts from 1:1 to 15:1. In certain embodiments, the stoichiometric ratios are 2:1, 2.5:1, 3:1, 5:1, 10:1, or 15:1.

[0152] In a fifth embodiment, the present invention relates to a kit comprising at least one antibody selected from the group of antibodies described above for the first, second, third, or fourth embodiment of the present invention. Accordingly, in an embodiment, the kit may comprise the antibody described above for the first embodiment of the present invention. In a further embodiment, the kit may comprise the antibody described above for the second embodiment of the present invention. In a further embodiment, the kit may comprise the antibody described above for the third embodiment of the present invention. In a further embodiment, the kit may comprise the antibody described above for the fourth embodiment of the present invention.

[0153] In certain embodiments, the kit further comprises a second antibody selected from the group of antibodies described above for the first, second, third, or fourth aspects of the present invention.

[0154] Therefore, in embodiments, the kit may include the antibody described above for the first embodiment of the present invention and the antibody described above for the second embodiment. In further embodiments, the kit may include the antibody described above for the first embodiment of the present invention and the antibody described above for the third embodiment. In further embodiments, the kit may include the antibody described above for the first embodiment of the present invention and the antibody described above for the fourth embodiment. In further embodiments, the kit may include the antibody described above for the second embodiment of the present invention and the antibody described above for the third embodiment. In further embodiments, the kit may include the antibody described above for the second embodiment of the present invention and the antibody described above for the fourth embodiment. In further embodiments, the kit may include the antibody described above for the third embodiment of the present invention and the antibody described above for the fourth embodiment.

[0155] In certain embodiments, the kit includes the antibody described above in relation to the second aspect of the present invention and the antibody described above in relation to the third aspect of the present invention.

[0156] In certain embodiments, the kit further includes a third antibody selected from the group of antibodies described above for the first, second, third, or fourth embodiment of the present invention. Thus, in embodiments, the kit may include the antibody described above for the first embodiment of the present invention, the antibody described above for the second embodiment, and the antibody described above for the third embodiment. In further embodiments, the kit may include the antibody described above for the first embodiment of the present invention, the antibody described above for the second embodiment, and the antibody described above for the fourth embodiment.

[0157] In further embodiments, the kit may include the antibody described above in relation to the second embodiment of the present invention, the antibody described above in relation to the third embodiment, and the antibody described above in relation to the fourth embodiment. In further embodiments, the kit may include the antibody described above in relation to the first embodiment of the present invention, the antibody described above in relation to the third embodiment, and the antibody described above in relation to the fourth embodiment. In further embodiments, the kit may include the antibody described above in relation to the first embodiment of the present invention, the antibody described above in relation to the second embodiment, and the antibody described above in relation to the fourth embodiment.

[0158] In certain embodiments, the kit includes the antibody described above in relation to the second aspect of the present invention, the antibody described above in relation to the third aspect, and the antibody described above in relation to the fourth aspect. In the sixth aspect, the present invention relates to a nucleic acid encoding an antibody selected from the group of antibodies described above in relation to the first, second, third, or fourth aspect of the present invention.

[0159] In a seventh aspect, the present invention relates to a host cell containing the nucleic acid described above in the sixth aspect of the present invention, and / or a host cell that produces the antibody described above in the first aspect of the present invention and the antibody described above in the second aspect.

[0160] In a preferred embodiment, the host cell is a hybridoma cell. Furthermore, the host cell can be any type of cell line that can be manipulated to produce antibodies according to the present invention. For example, the host cell can be an animal cell, particularly a mammalian cell. In one embodiment, HEK293 (human fetal kidney cell) or CHO (Chinese hamster ovary) cells, such as HEK293-F cells used in the Examples section, are used as the host cell. In another embodiment, the host cell is a non-human animal or mammalian cell.

[0161] The host cell preferably contains at least one polynucleotide encoding the antibody of the present invention or a fragment thereof. In certain embodiments, the host cell contains a nucleic acid according to the sixth aspect of the present invention. In particular, the host cell contains at least one polynucleotide encoding the light chain of the antibody of the present invention and at least one polynucleotide encoding the heavy chain of the antibody of the present invention. The polynucleotide(s) are operably linked to a suitable promoter.

[0162] In an eighth aspect, the present invention relates to a composition comprising at least one antibody selected from the group of antibodies described above for the first, second, third, or fourth aspect of the present invention. Accordingly, in an embodiment, the composition may comprise the antibody described above for the first aspect of the present invention. In a further embodiment, the composition may comprise the antibody described above for the second aspect of the present invention. In a further embodiment, the composition may comprise the antibody described above for the third aspect of the present invention. In a further embodiment, the composition may comprise the antibody described above for the fourth aspect of the present invention.

[0163] In certain embodiments, the composition further comprises a second antibody selected from the group of antibodies described above for the first, second, third, or fourth aspect of the present invention.

[0164] Therefore, in embodiments, the composition may include the antibody described above for the first aspect of the present invention and the antibody described above for the second aspect. In further embodiments, the composition may include the antibody described above for the first aspect of the present invention and the antibody described above for the third aspect. In further embodiments, the composition may include the antibody described above for the first aspect of the present invention and the antibody described above for the fourth aspect. In further embodiments, the composition may include the antibody described above for the second aspect of the present invention and the antibody described above for the third aspect. In further embodiments, the composition may include the antibody described above for the second aspect of the present invention and the antibody described above for the fourth aspect. In further embodiments, the composition may include the antibody described above for the third aspect of the present invention and the antibody described above for the fourth aspect.

[0165] In certain embodiments, the composition includes the antibody described above in relation to the second aspect of the present invention and the antibody described above in relation to the third aspect of the present invention.

[0166] In certain embodiments, the composition further comprises a third antibody selected from the group of antibodies described above for the first, second, third, or fourth embodiment of the present invention. Thus, in embodiments, the composition may include the antibody described above for the first embodiment of the present invention, the antibody described above for the second embodiment, and the antibody described above for the third embodiment. In further embodiments, the composition may include the antibody described above for the first embodiment of the present invention, the antibody described above for the second embodiment, and the antibody described above for the fourth embodiment.

[0167] In further embodiments, the composition may include the antibody described above in relation to the second aspect of the present invention, the antibody described above in relation to the third aspect, and the antibody described above in relation to the fourth aspect. In further embodiments, the composition may include the antibody described above in relation to the first aspect of the present invention, the antibody described above in relation to the third aspect, and the antibody described above in relation to the fourth aspect. In further embodiments, the composition may include the antibody described above in relation to the first aspect of the present invention, the antibody described above in relation to the second aspect, and the antibody described above in relation to the fourth aspect.

[0168] In certain embodiments, the composition includes the antibody described above in relation to the second aspect of the present invention, the antibody described above in relation to the third aspect, and the antibody described above in relation to the fourth aspect.

[0169] In certain embodiments, the composition is a diagnostic composition. Therefore, in certain embodiments, it is for diagnostic purposes.

[0170] In a ninth aspect, the present invention relates to the use of antibodies or antigen-binding fragments of the first, second, third, or fourth aspect of the present invention, or a kit of the fifth aspect of the present invention or a composition of the eighth aspect of the present invention, for in vitro immunoassays. In certain aspects, the immunoassay is a heterologous immunoassay.

[0171] In a tenth aspect, the present invention relates to an in vitro method for detecting the presence of SARS-CoV-2 virus in a sample obtained from a patient, wherein the method is: a) Incubate the sample with at least one antibody or antibody-conjugated fragment that binds to the SARS-CoV-2 nucleocapsid, thereby generating a complex between the at least one antibody or antibody-conjugated fragment and the SARS-CoV-2 nucleocapsid. b) Optionally, the formed composite can be immobilized on a solid phase, particularly on fine particles. c) detecting the presence of the SARS-CoV-2 virus in the sample by detecting the complex formed in step a), and the above procedure is performed.

[0172] In one embodiment, the method described above does not involve the collection of a sample from the subject. Rather, a sample obtained from the subject (for example, under the supervision of the attending physician) is provided. For example, the sample can be provided by delivering the sample to a laboratory that performs detection of the presence of the SARS-CoV-2 virus in the sample.

[0173] In certain embodiments, at least one antibody or antibody-conjugated fragment is an antibody or antibody-conjugated fragment of the first, second, third, and / or fourth embodiment of the present invention.

[0174] In one embodiment, the sample is incubated with the antibody described above for the first aspect of the present invention in step a). In a further embodiment, the sample is incubated with the antibody described above for the second aspect of the present invention. In a further embodiment, the sample is incubated with the antibody described above for the third aspect of the present invention. In a further embodiment, the sample is incubated with the antibody described above for the fourth aspect of the present invention.

[0175] In certain embodiments, the sample is further incubated in step a) with a second antibody selected from the group of antibodies described above for the first, second, third, or fourth aspects of the present invention.

[0176] In certain embodiments, the sample is incubated with two antibodies that bind to the nucleocapsid of SARS-CoV-2 in step a). As will be apparent to those skilled in the art, the sample can be exposed to the first and second antibodies for a sufficient amount of time and under conditions to form a first anti-SARS-CoV-2 N-antibody / SARS-CoV-2 N-antigen / second anti-SARS-CoV-2 N-antibody complex in any desired order (i.e., the first antibody first, then the second antibody, or the second antibody first, then the first antibody, or simultaneously). As anyone skilled in the art will readily understand, establishing appropriate or sufficient time and conditions for the formation of a complex between a specific anti-SARS-CoV-2N antibody and the SARS-CoV-2N antigen / analyte (= anti-SAR-CoV-2N complex), or for the formation of a secondary complex or sandwich complex (= first anti-SAR-CoV-2N antibody / SARS-CoV-2N antigen / second anti-SAR-CoV-2N antibody complex) containing a first antibody (anti-SAR-CoV-2N antibody), the SARS-CoV-2N antigen (analyte), and a second anti-SAR-CoV-2N antibody, is merely routine experimentation.

[0177] The detection of anti-SARS-CoV-2 N-antibody / SARS-CoV-2 N-antigen complexes can be carried out by any suitable means. The detection of a first anti-SARS-CoV-2 N-antibody / SARS-CoV-2 N-antigen / second anti-SARS-CoV-2 N-antibody complex can be carried out by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0178] Thus, in an embodiment, the sample is incubated in step a) with the antibody described above for the first aspect of the present invention and the antibody described above for the second aspect. In a further embodiment, the sample is incubated with the antibody described above for the first aspect of the present invention and the antibody described above for the third aspect. In a further embodiment, the sample is incubated with the antibody described above for the first aspect of the present invention and the antibody described above for the fourth aspect. In a further embodiment, the sample is incubated with the antibody described above for the second aspect of the present invention and the antibody described above for the third aspect. In a further embodiment, the sample is incubated with the antibody described above for the second aspect of the present invention and the antibody described above for the fourth aspect. In a further embodiment, the sample is incubated with the antibody described above for the third aspect of the present invention and the antibody described above for the fourth aspect.

[0179] In a particular embodiment, the sample is incubated in step a) with the antibody described above for the second aspect of the present invention and the antibody described above for the third aspect.

[0180] In a particular embodiment, the sample is further incubated in step a) with a third antibody selected from the group of antibodies described above for the first, second, third or fourth aspect of the present invention. Thus, in an embodiment, the sample is incubated with the antibody described above for the first aspect of the present invention, the antibody described above for the second aspect, and the antibody described above for the third aspect. In a further embodiment, the sample is incubated with the antibody described above for the first aspect of the present invention, the antibody described above for the second aspect, and the antibody described above for the fourth aspect.

[0181] In a further embodiment, the sample is incubated in step a) with the antibodies described above for the second aspect of the invention, the antibodies described above for the third aspect, and the antibodies described above for the fourth aspect. In a further embodiment, the sample is incubated with the antibodies described above for the first aspect of the invention, the antibodies described above for the third aspect, and the antibodies described above for the fourth aspect. In a further embodiment, the sample is incubated with the antibodies described above for the first aspect of the invention, the antibodies described above for the second aspect, and the antibodies described above for the fourth aspect.

[0182] In certain embodiments, the sample is incubated in step a) with the antibodies described above for the second aspect of the invention, the antibodies described above for the third aspect, and the antibodies described above for the fourth aspect.

[0183] In embodiments, the first antibody can be immobilized on a solid phase and the second antibody is labeled with a detectable label. In embodiments, the detectable label is a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye. In embodiments, the antibody capable of being immobilized on the solid phase is tagged, in particular, together with a partner of a bioaffinity binding pair, particularly biotin or a complementary LNA sequence.

[0184] In embodiments, the first antibody is labeled with a detectable label and the second antibody can be immobilized on a solid phase. In embodiments, the detectable label is a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye. In embodiments, the antibody capable of being immobilized on the solid phase is tagged, in particular, together with a partner of a bioaffinity binding pair, particularly biotin or a complementary LNA sequence.

[0185] In the embodiment, the first antibody can be immobilized on a solid phase, the second antibody is labeled with a detectable label, and the third antibody is labeled with a detectable label. In the embodiment, the detectable label is a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye. In the embodiment, the antibody that can be immobilized on a solid phase is tagged in particular with a bioaffin-binding partner, particularly biotin or a complementary LNA sequence.

[0186] In the embodiment, the first antibody is labeled with a detectable label, the second antibody can be immobilized on a solid phase, and the third antibody is labeled with a detectable label. In the embodiment, the detectable label is a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye. In the embodiment, the antibody that can be immobilized on a solid phase is tagged in particular with a bioaffin-binding partner, particularly biotin or a complementary LNA sequence.

[0187] In some embodiments, the method is an enzyme-linked immunosorbent assay (ELISA), an electrochemiluminescence immunoassay (ECLIA), or a radioimmunoassay (RIA). In some specific embodiments, the method is the ELICA method.

[0188] In certain embodiments, the patient sample is a fluid sample, particularly a body fluid sample. In certain embodiments, the sample is selected from the group consisting of nasopharyngeal swabs, oropharyngeal swabs, sputum, saliva, whole blood, serum, or plasma. In certain embodiments, the sample is selected from the group consisting of nasopharyngeal swabs, oropharyngeal swabs, sputum, or saliva. In certain embodiments, the sample is a nasopharyngeal swab or an oropharyngeal swab. In embodiments, the sample is an in vitro sample, i.e., it is analyzed in vitro and not returned to the body. In certain embodiments, the method for detecting the presence of the SARS-CoV-2 virus has a sensitivity of less than 10 pg / ml. In certain embodiments, the method has a sensitivity of less than 5 pg / ml, particularly less than 3 pg / ml. In certain embodiments, the method has a sensitivity of less than 500 fM, less than 100 fM, less than 50 fM, or less than 35 fM.

[0189] In certain embodiments, the patient is an experimental animal, livestock, or primate. In certain embodiments, the patient is a human patient.

[0190] In the embodiment, if the nucleocapsid of COVID-19 is detected in the patient's sample, the patient is selected for treatment of SARS-CoV-2 (i.e., SARS-CoV-2 infection).

[0191] In further embodiments, the present invention relates to the following items.

[0192] 1. A monoclonal antibody (isolated) that binds to the nucleocapsid protein of the SARS-CoV-2 virus, or an antigen-binding fragment thereof, a) 1.0E+05M determined by surface plasmon resonance -1 s -1 The association rate constant (k) exceeds the following value. a ) has, and / or b) 5.0E-04s determined by surface plasmon resonance -1 Dissociation rate constant (k) less than d ) has, and / or c) t for 15 minutes or more determined by surface plasmon resonance / 2diss Having a half-life time, and / or d) Having a stoichiometric ratio of 1:1 or 1:2, (Isolated) monoclonal antibody or its antigen-binding fragment.

[0193] 2.a) Includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 1, 2, 3, 4, 5, and 6 respectively, b) Whether the antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, according to SEQ ID NOs: 1, 2, 3, 4, 5, and 6, bind to the same epitopes, or c) Antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs. 1, 2, 3, 4, 5, and 6 respectively, compete for binding of the SARS-CoV-2 virus to the aforementioned nucleocapsid protein. The isolated monoclonal antibody or its antigen-binding fragment described in item 1.

[0194] 3.a) Each includes FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to sequence numbers 7, 8, 9, 10, 11, 12, 13, and 14, respectively. b) Whether they bind to the same epitopes as antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 7, 8, 9, 10, 11, 12, 13, and 14, respectively, or c) Antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 7, 8, 9, 10, 11, 12, 13, and 14 respectively, compete for binding of the SARS-CoV-2 virus to the aforementioned nucleocapsid protein. The isolated monoclonal antibody or antigen-binding fragment described in item 2.

[0195] 4.a) A heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 15 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 16, b) The antibody contains a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 15 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 16, and binds to the same epitope. or c) An antibody comprising a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 15 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 16 competes with an antibody for binding to the nucleocapsid protein of the SARS-CoV-2 virus. An isolated monoclonal antibody or antigen-binding fragment as described in any of items 1 to 3.

[0196] 5. a) Comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 according to SEQ ID NOs: 17, 18, 19, 20, 21 and 22 respectively, b) Binding to the same epitope as an antibody comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 according to SEQ ID NOs: 17, 18, 19, 20, 21 and 22 respectively, Or c) Competing with an antibody comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 according to SEQ ID NOs: 17, 18, 19, 20, 21 and 22 respectively for binding to the said nucleocapsid protein of the SARS-CoV-2 virus, The isolated monoclonal antibody or antigen-binding fragment according to item 1.

[0197] 6. a) Comprising FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3 and FR-L4 according to SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29 and 30 respectively, b) Binding to the same epitope as an antibody comprising FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3 and FR-L4 according to SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29 and 30 respectively, Or c) Competing with an antibody comprising FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3 and FR-L4 according to SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29 and 30 respectively for binding to the said nucleocapsid protein of the SARS-CoV - 2 virus, The isolated monoclonal antibody or antigen-binding fragment according to item 5.

[0198] 7. a) Comprising a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 31 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 32, b) The antibody contains a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 31 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 32, and binds to the same epitope. or c) An antibody comprising a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 31 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 32 competes with an antibody for binding to the nucleocapsid protein of the SARS-CoV-2 virus. An isolated monoclonal antibody or antigen-binding fragment as described in item 1, 5, or 6.

[0199] 8.a) Each includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 33, 34, 35, 36, 37, and 38, b) Whether the antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, according to SEQ ID NOs. 33, 34, 35, 36, 37, and 38, bind to the same epitopes, or c) Antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs. 33, 34, 35, 36, 37, and 38 respectively, compete for binding of the SARS-CoV-2 virus to the nucleocapsid protein. The isolated monoclonal antibody or antigen-binding fragment described in item 1.

[0200] 9.a) Each includes FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to sequence numbers 39, 40, 41, 42, 43, 44, 45, and 46, respectively. b) Whether they bind to the same epitopes as antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, and 46, respectively, or c) Antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, and 46 respectively, compete for binding of the SARS-CoV-2 virus to the nucleocapsid protein. The isolated monoclonal antibody or antigen-binding fragment described in item 8.

[0201] 10.a) A heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 47 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 48, b) The antibody contains a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 47 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 48, and binds to the same epitope. or c) An antibody comprising a heavy chain variable domain having the amino acid sequence according to SEQ ID NO: 47 and a light chain variable domain having the amino acid sequence according to SEQ ID NO: 48 competes with an antibody for binding to the nucleocapsid protein of the SARS-CoV-2 virus. An isolated monoclonal antibody or antigen-binding fragment as described in item 1, 8, or 9.

[0202] 11. A kit comprising at least one antibody described in sections 2-4, optionally a second antibody described in sections 5-7, and optionally a third antibody described in sections 8-10.

[0203] 12. A nucleic acid encoding an antibody as described in any of items 1 to 10.

[0204] 13. A host cell that contains the nucleic acid described in item 12 and / or produces an antibody described in any of items 1 to 10.

[0205] 14. A composition comprising an antibody as described in any of items 1 to 10.

[0206] 15. Use of an antibody described in any one of sections 1 to 10, a kit described in section 11, or a composition described in section 14 for in vitro immunoassays.

[0207] 16. An in vitro method for detecting the presence of SARS-CoV-2 virus in a sample obtained from a patient, a) Incubating the sample with at least one antibody or antibody-conjugated fragment thereof that binds to the nucleocapsid of SARS-CoV-2, or at least one antibody or antibody-conjugated fragment thereof as described in any of items 1 to 10, thereby generating a complex between the antibody and the nucleocapsid of SARS-CoV-2, b) Optionally, the formed composite is immobilized on a solid phase, particularly on fine particles. c) A method comprising detecting the presence of the SARS-CoV-2 virus in the sample.

[0208] 17. The method according to any one of items 16 to 18, wherein the patient's sample is selected from the group consisting of nasopharyngeal swabs, oropharyngeal swabs, sputum, saliva, etc.

[0209] 18. A method for detecting the presence of the SARS-CoV-2 virus, having a sensitivity of less than 10 pg / ml, according to any of items 16-19.

[0210] The following examples and figures are provided to aid in understanding the present invention, and the true scope of the invention is set forth in the appended claims. It is understood that modifications to the procedures described may be made without departing from the spirit of the invention. [Examples]

[0211] Example 1: Antibody generation To generate highly specific antibodies against the SARS-CoV-2 N protein, the inventors immunized New Zealand white rabbits and NMRI mice with the full-length N protein and then screened for nucleocapsid protein-binding antibodies. Immunogen: SARS-CoV-2 nucleocapsid, full length, untagged, expressed in Escherichia coli (E. coli). Screening reagent: Full-length biotinylated SARS-CoV-2 nucleocapsid.

[0212] The generation of nucleocapsid antigens used for immunization and screening is described in detail here: European Patent No. 20171154.6; European Patent No. 20178739.7; European Patent No. 20173315.1.

[0213] Immunotherapy revealed that various individual rabbit and mouse IgG clones specifically reacted with the SARS-CoV-2-derived N protein, but not with other coronaviruses (common cold coronaviruses and MERS). The specificity of these antibodies against the N protein was demonstrated by ELISA assays and SPR Biacore analysis of B cell supernatant and mouse hybridoma supernatant, respectively (not shown).

[0214] Example 2: Antibody SPR screening All SPR experiments were performed using the SlyD-SlyD-tagged nucleocapsid full-length protein (SEQ ID NO: 49: aa 1-419 nucleocapsid protein + 2xSlyD tag; molecular weight: 85 kDa) as described in detail in European Patent Publication Nos. 20171154.6, 20178739.7, and 20173315.1.

[0215] Kinetic screening of the generated antibodies was performed at 37°C using GE Healthcare BIAcore® 8K+, 8K, and B4000 instruments. Biacore CM-5 series S sensors were attached to the instruments and pre-conditioned according to the manufacturer's instructions.

[0216] The system buffer was PBS-NT (11 mM PO4 pH 8.0, 500 mM NaCl, 2.7 mM KCl, 0.05% Tween 20). 1 mg / mL CMD (carboxymethyl dextran, Fluka) was added to the system buffer and used as the sample buffer for preparing the dilution series.

[0217] Rabbit or mouse species-specific antibody capture systems were immobilized on the sensor surface. These included 30 μg / ml NaAc pH 4.5 polyclonal goat anti-rabbit IgG Fc capture antibody GARbFcγ (111-005-046, Jackson Immuno Research) or 30 μg / ml NaAc pH 5.0 polyclonal goat anti-mouse Fc-y capture antibody PAK<M-IgG(Fcy)> Z(115-005-071) was amine-coupled using EDC / NHS chemistry according to the manufacturer's instructions. Finally, ligand densities of 10,000 RU to 15,000 RU were obtained. The free activated carboxyl groups were saturated with 1 M ethanolamine at pH 8.5.

[0218] Rabbit or mouse antibody (IgG 150kDa) solution was diluted with sample buffer and injected at a rate of 5 μl / min or 10 μl / min for 2 minutes. Antibody capture level (CL) in resonance units (RU) was monitored.

[0219] A 150 nM analyte, SlyD-SlyD-N protein, was injected into pre-captured anti-NCP antibody at 30 or 40 μL / min at 37°C. The analyte association phase was monitored for 3–5 minutes. The antibody / N protein complex dissociation phase was monitored for 5, 10, or 14 minutes. After each measurement cycle, the capture system was regenerated by injecting 10 mM glycine buffer pH 2.0 and pH 2.25 at 20 μL / min for 60 seconds.

[0220] The kinetic signature was monitored by BIAcore® 8K Control-SW V3.0.11.15423 and evaluated by BIAcore® Insight Evaluation SW V3.0.11.15423 (B4000 Control SW V1.1 and Evaluation SW V1.1, respectively).

[0221] Kinetic data were interpreted by reporting point evaluation. The stability of the antibody / antigen complex was compared using two reporting points: Analyte Binding Late (BL), a response signal recorded immediately before the end of N protein analyte injection, and Stability Late (SL), a signal recorded immediately before the end of the dissociation phase.

[0222] According to the Langmuir model, the dissociation rate constant k d (s -1 ) calculate the formula t / 2diss =ln(2) / (k d The antibody / antigen complex half-life was calculated in minutes according to *60).

[0223] The molar ratio and bond stoichiometry were calculated using the following formulas. MR=B(antigen)*MW(antibody) / (MW(antigen)*CL(antibody)).

[0224] Example 3: Kinetic characterization of SARS-CoV-2 N antibody Monoclonal rabbit and mouse nucleocapsid antibodies selected by kinetic screening were further characterized.

[0225] Measurements were performed using BIAcore® 8K and 8K+ instruments. N protein concentration series from 1.2 nM to 300 nM were injected at a rate of 30 to 60 μL / min. The association phase was monitored for 3 to 5 minutes, and the dissociation phase was monitored at 37°C for 5 to 60 minutes.

[0226] For the kinetic characterization of clones 5B6, 1G9, and 1.1.32, the system sample buffer was as described above, but supplemented with 2 mg / mL (bovine serum albumin) BSA. Kinetic rate constant and dissociation equilibrium constant K D These values ​​were calculated using a Langmuir 1:1 fitted model from BIAcore® Insight Evaluation SW V3.0.11.15423, or using a Langmuir 1:1 fitted model from Scrubber-SW V2.0c.

[0227] The results of SPR kinetic screening and characterization of representative N antibodies are shown in Figures 1, 2, and 3, respectively.

[0228] All antibodies that meet the inventors' stringent selection criteria are >1.0E+05M -1 s -1 Fast meeting speed in the range (k a ) and 5.0E-04s -1 Dissociation rate less than (k d ) is shown. All antibodies exhibit affinity in the nanomolar and sub-nanomolar ranges, respectively. Figure 1 shows examples of antibodies that met the selection criteria defined above (Figure 1B) and antibodies that were deselected without further investigation because they showed a kinetic signature unsuitable for the inventors' purposes (Figure 1A). Antibody 1.1.32 is characterized by a high affinity of 0.06 nM ± 5.1% for N. 1G9 has an affinity of 1.2 nM ± 0.3%, but for 5B6, K D The concentration is 0.7 nM ± 1.4% (Figure 2). The interactions of antibodies 5B6, 1G9, and 1.1.32 with nucleocapsid proteins (NCPs) at different concentrations of 1.2 nM, 3 nM, 11 nM, 33 nM, and 100 nM were determined in pairs, and the Langmuir 1:1 binding models (see Figures 3A, B, and C, respectively) were superimposed.

[0229] Conclusion: As a result of nucleocapsid immunization, we produced rabbit and mouse monoclonal IgG that is specific to the SARS-CoV-2 nucleocapsid but does not react with N proteins derived from common cold coronaviruses or MERS. This is supported by the results of Biacore SPR and immunoassay analysis (see examples below).

[0230] A total of 13,248 rabbit antibodies and 21,504 mouse antibodies were pre-screened using nucleocapsid-targeted ELISA. 3,427 rabbit and mouse antibodies were tested in SPR experiments. 157 clones with kinetic properties meeting the Elecsys-platform criteria were identified. 60 rabbit and mouse clones identified by kinetic screening... <n>The antibody's binding to the N protein was further characterized kinetically.

[0231] Example 4: Sandwich composite formation experiment Antibody / antigen sandwich formation experiments were performed at 25°C using a GE Healthcare BIAcore™ 8K+ instrument. The Biacore 2 D-PEG sensor surface was mounted on the instrument and pre-conditioned according to the manufacturer's instructions. Rabbit or mouse antibody capture systems were used as described. The activation time for the EDC / NHS mixture was 30 seconds. The capture system was immobilized at a maximum of 400 RU. The sensor was saturated as described. The system buffer was PBS-NT (11 mM PO4 pH 8.0, 500 mM NaCl, 2.7 mM KCl, 0.05% (w / v) Tween 20). System buffer supplemented with 1 mg / mL CMD (carboxymethyl dextran, Fluka) was used as the sample buffer. Rabbit or mouse N mAbs were tested at 25°C for sandwich complex formation with full-length N (aa 1~419).

[0232] The primary antibody supernatant was diluted and captured on each Fc2 channel for 2 minutes at 10 μL / min. The capture system was blocked with 1 μM KN-IgG or a mouse-specific antibody blocking cocktail at 30 μL / min for 3 minutes. Subsequently, a double injection of 75 nM nucleocapsid protein (SlyD-SlyD tagged N protein full length) was performed for 3 minutes as the initial injection, and the primary antibody supernatant was repeatedly injected at 30 μL / min for 2 minutes, diluted 1:20 to 1:50. The secondary antibody solution was diluted and injected for 3 minutes, followed by a dissociation time of 5 minutes at 30 μL / min.

[0233] The assay was performed as described above.

[0234] The stability of the immune complex was evaluated using the SW extension "Epitope Binning" of BIAcore® Insight Evaluation SW V3.0.11.15423. Sandwich complex formation experiments were interpreted by reporting point evaluation. Immune complex stability was characterized using two reporting points: capture level (CL) (signal recorded immediately after completion of primary antibody capture) and early analyte stability (signal recorded immediately after completion of secondary antibody injection). Epitope accessibility was quantified as the molar ratio (MR) by forming the quotient between the resonance unit of the secondary antibody binding response signal and the primary antibody capture level.

[0235] By combining information from four different experiments, we were able to identify four distinct N-epitope regions. Fourteen antibodies with different kinetic properties cover four different nucleocapsid epitope regions (see Figures 4 and 5). The numbers in the "Epitope Region" column indicate the epitope bin of each monoclonal antibody.

[0236] Example 5: Application to electrochemiluminescence immunoassay (ECLIA) An ELICA assay using nucleocapsid antibodies was established to detect SARS-CoV-2 nucleocapsid antigen in patient samples. Recombinant nucleocapsids, inactivated virus lysates, and patient samples were used in Elecsys (c) The performance of anti-N antibodies on the platform was tested. Kinetic profiles and epitope binning SPR data (see above) served as a basis for selecting candidate antibodies for assay development. To address the best sandwich-forming antibody pairs on the Elecsys platform with inactivated virus lysates (see Figure 6), 50 different combinations were used in this Elecsys assay setup. <n> Antibodies were tested. After identifying the most promising antibody pairings, patient samples were evaluated for SARS-CoV-2 PCR testing. Results obtained from the Elecsys assay of patient samples were compared with those from the PCR assay. Two antibodies, 1.1.32 and 5B6, were identified as the best antibody pair with a relative sensitivity (relSens) of 20% and a relative specificity (relSpec) of 100%. A third antibody, 1G9, could achieve signal amplification to increase the relative sensitivity of the assay to 26%. The calculations of relSens and relSpec compared to the SARS-CoV-2 PCR test are shown in Figure 7.< / n> < / n>

Claims

1. A monoclonal antibody or its antigen-binding fragment that binds to the nucleocapsid protein of the SARS-CoV-2 virus, a) 1.0E+05M determined by surface plasmon resonance －１ s －１ The association rate constant (k) exceeds the following value. ａ ) has and / or b) 5.0E-04s determined by surface plasmon resonance －１ Dissociation rate constant (k) less than ｄ ) has and / or c) t of 15 minutes or more determined by surface plasmon resonance ／２ｄｉｓｓ Having a half-life time, and / or d) Having a stoichiometric ratio of 1:1 or 1:2, Here, (i) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 1, 2, 3, 4, 5, and 6 respectively, (ii) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 17, 18, 19, 20, 21, and 22, respectively, (iii) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 33, 34, 35, 36, 37, and 38 respectively, The monoclonal antibody or its antigen-binding fragment, comprising the above.

2. a) Each includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to Sequence IDs 1, 2, 3, 4, 5, and 6, b) Whether the antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs: 1, 2, 3, 4, 5, and 6 respectively bind to the same epitope, or c) Antibodies comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs: 1, 2, 3, 4, 5, and 6 respectively, compete for the binding of SARS-CoV-2 virus to the nucleocapsid protein. The monoclonal antibody or antigen-binding fragment according to claim 1.

3. a) Each includes FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to sequence numbers 7, 8, 9, 10, 11, 12, 13, and 14, b) Whether the antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4, respectively, as defined in SEQ ID NOs. 7, 8, 9, 10, 11, 12, 13, and 14, bind to the same epitopes, or c) Antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 7, 8, 9, 10, 11, 12, 13, and 14 respectively, compete for the binding of SARS-CoV-2 virus to the nucleocapsid protein. The monoclonal antibody or antigen-binding fragment according to claim 2.

4. a) Each includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 17, 18, 19, 20, 21, and 22, b) Whether they bind to the same epitope as antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs. 17, 18, 19, 20, 21, and 22, respectively, or c) Antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs. 17, 18, 19, 20, 21, and 22, respectively, compete for the binding of SARS-CoV-2 virus to the nucleocapsid protein. The monoclonal antibody or antigen-binding fragment according to claim 1.

5. a) Each includes FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to sequence numbers 23, 24, 25, 26, 27, 28, 29, and 30, b) Whether the antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4, respectively, as defined in SEQ ID NOs. 23, 24, 25, 26, 27, 28, 29, and 30, bind to the same epitopes, or c) Antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 23, 24, 25, 26, 27, 28, 29, and 30 respectively, compete for the binding of SARS-CoV-2 virus to the nucleocapsid protein. The monoclonal antibody or antigen-binding fragment according to claim 4.

6. a) Each includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to sequence numbers 33, 34, 35, 36, 37, and 38, b) The antibodies containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, bind to the same epitope, or c) Antibodies comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 according to SEQ ID NOs. 33, 34, 35, 36, 37, and 38 respectively, compete for the binding of SARS-CoV-2 virus to the nucleocapsid protein. The monoclonal antibody or antigen-binding fragment according to claim 1.

7. a) Each includes FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to sequence numbers 39, 40, 41, 42, 43, 44, 45, and 46, b) Whether the antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4, respectively, according to SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, and 46, bind to the same epitopes, or c) Antibodies containing FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4 according to SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, and 46 respectively, compete for the binding of SARS-CoV-2 virus to the nucleocapsid protein. The monoclonal antibody or antigen-binding fragment according to claim 6.

8. A kit comprising at least one antibody according to claim 2.

9. A nucleic acid encoding the antibody according to claim 1.

10. A host cell comprising the nucleic acid described in claim 9.

11. A composition comprising the antibody described in claim 1.

12. An in vitro method for detecting the presence of SARS-CoV-2 virus in a sample obtained from a patient, a) Contacting the sample with at least one antibody or antibody-binding fragment described in claim 1, thereby forming a complex between the antibody and the nucleocapsid of SARS-CoV-2, b) Depending on the circumstances, the formed composite may be immobilized on a solid phase. c) To detect the presence of the SARS-CoV-2 virus in the sample, Methods that include...

13. The method according to claim 12, wherein the sample from the patient is a nasopharyngeal swab or an oropharyngeal swab.

14. The method according to claim 12, wherein the method for detecting the presence of the SARS-CoV-2 virus has a sensitivity of less than 10 pg / ml.

15. The method according to claim 12, wherein the solid phase includes fine particles.

16. The method according to claim 12, wherein the method is an enzyme-linked immunoassay (ELISA), an electrochemiluminescence immunoassay (ECLIA), or a radioimmunoassay (RIA).

17. The method according to claim 12, wherein the patient is a human patient.

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