WO2021238854A1 - Monoclonal antibody against sars-cov-2 spike protein, preparation method therefor, and application thereof - Google Patents

Abstract

Disclosed is a monoclonal antibody against SARS-CoV-2 spike protein, a preparation method therefor, and an application thereof. Also disclosed is a monoclonal antibody against SARS-CoV-2 spike protein, an amino acid sequence of a heavy chain variable region, and an amino acid sequence of a light chain variable region. The anti-SARS-CoV-2 spike protein monoclonal antibody can specifically bind to the S protein, so as to effectively block the S protein from binding to the ACE2 protein, specifically preventing the virus from infecting human cells. The anti-SARS-CoV-2 spike protein monoclonal antibody provides possible and convenient treatment and detection of the SARS-CoV-2 virus.

Description

Monoclonal antibody against SARS-CoV-2 spike protein, preparation method and application thereof Technical field

The invention belongs to the field of virus detection, diagnosis and treatment, and relates to an anti-SARS-CoV-2 spike protein monoclonal antibody. The invention also relates to a preparation method and application of the anti-SARS-CoV-2 spike protein monoclonal antibody.

Background technique

The new coronavirus is also called severe acute respiratory syndrome coronavirus-2 (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), which is composed of RNA nucleic acid and protein, etc. The disease caused by the virus is called the new coronavirus disease 2019 (coronavirus disease, 2019 COVID-19) [1-2]. Although the mortality rate of COVID-19 is significantly lower than that of SARS-CoV infection, the rate of human-to-human transmission of SARS-CoV-2 is higher. On March 11, 2020, the WHO announced that COVID-19 has caused a global pandemic [ 3].

Similar to SARS-CoV, SARS-CoV-2 also uses its highly glycosylated spike protein (S protein, S protein) to complete host cell receptor binding and virus infection in the form of a trimer [4 -6]. The S protein has two subunits, S1 and S2. The RBD (receptor-binding domain) region of the S1 subunit can recognize and bind to the angiotensin-converting enzyme 2 (hACE2) of the host cell, and the S2 subunit mediates the membrane fusion between the virus and the host cell[7 ]. And the binding of RBD to the hACE2 receptor may cause the S1 protein to fall off from the S2 protein, and promote S2-mediated virus-host membrane fusion and virus infection [8]. However, as of now, there is no approved preventive vaccine or treatment for COVID-19. Blocking monoclonal antibody (mAb) is one of the best drug candidates for neutralizing viral infections due to its extraordinary antigen specificity [9].

Timely and accurate diagnosis of SARS-CoV-2 infection is the key to providing patients with appropriate treatment, limiting the further spread of the virus and ultimately eliminating the virus from human society. At present, viral RNA detection based on PCR method is the most commonly used method to judge SARS-CoV-2 infection. However, RNA detection based on throat or nasopharyngeal swabs has a certain risk of false negatives [10]. At the same time, there are significant delays in early diagnosis and follow-up management, which poses a serious challenge for providing timely life support treatment and preventive quarantine. Therefore, in order to improve the timeliness and effectiveness of new coronavirus detection, new detection methods must be supplemented on the basis of PCR detection.

In summary, for SARS-CoV-2, the preparation of monoclonal neutralizing antibodies for S protein and the development of detection methods other than PCR technology are all urgent research content.

Summary of the invention

In one aspect, the present invention provides an anti-SARS-CoV-2 spike protein monoclonal antibody or functional fragment thereof, the antibody or functional fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein

(a) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3,

The HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 14, 20, 26, 32, 38, or 44, or the amino acid sequence shown includes at most three (for example, one, two or three) amino acid mutations. The HCDR2 includes an amino acid sequence selected from SEQ ID NO: 15, 21, 27, 33, 39, or 45 or a variant of the amino acid sequence including at most three amino acid mutations; the HCDR3 includes an amino acid sequence selected from SEQ ID NO: the amino acid sequence shown in 16, 22, 28, 34, 40, or 46 or a variant in which the shown amino acid sequence contains at most three amino acid mutations; and

(b) The light chain variable region includes LCDR1, LCDR2 and LCDR3,

The LCDR1 sequence includes an amino acid sequence selected from SEQ ID NO: 17, 23, 29, 35, 41, or 47 or a variant of the amino acid sequence including at most three amino acid mutations; the LCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 17, 23, 29, 35, 41, or 47. ID NO: The amino acid sequence shown in 18, 24, 30, 36, 42 or 48 or the amino acid sequence shown contains at most three amino acid mutations; the LCDR3 sequence includes SEQ ID NO: 19, 25, 31 The amino acid sequence shown in, 37, 43 or 49 or the shown amino acid sequence contains at most three amino acid mutations.

In some embodiments, the HCDR1 sequence includes an amino acid sequence selected from SEQ ID NO: 14, 20, 26, 32, 38, or 44, and the HCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 15, 21, 27 , 33, 39, or 45, the HCDR3 sequence includes an amino acid sequence selected from SEQ ID NO: 16, 22, 28, 34, 40, or 46; and the LCDR1 sequence includes an amino acid sequence selected from SEQ ID NO: 17, 23, 29, 35, 41 or 47, the LCDR2 sequence comprises an amino acid sequence selected from SEQ ID NO: 18, 24, 30, 36, 42 or 48, the LCDR3 The sequence includes an amino acid sequence selected from SEQ ID NO: 19, 25, 31, 37, 43, or 49.

In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the following sequences:

(a) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 14, 15, and 16 or the amino acid sequence shown respectively includes at most three amino acid mutation variants; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 14, 15, and 16 ID NO: The amino acid sequences shown in 17, 18 and 19 or the amino acid sequences shown respectively contain at most three amino acid mutations;

(b) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 20, 21 and 22 or the amino acid sequence shown respectively includes at most three amino acid mutation variants; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 20, 21 and 22 ID NO: The amino acid sequences shown in 23, 24, and 25 or the amino acid sequences shown respectively contain at most three amino acid mutations;

(c) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28 or the amino acid sequences shown respectively include up to three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 26, 27 and 28. ID NO: The amino acid sequences shown in 29, 30, and 31 or the amino acid sequences shown respectively contain at most three amino acid mutations;

(d) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 32, 33 and 34 or the amino acid sequence shown respectively comprises a variant of at most three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 32, 33 and 34 ID NO: The amino acid sequences shown in 35, 36 and 37 or the amino acid sequences shown respectively contain at most three amino acid mutations;

(e) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 38, 39 and 40 or the amino acid sequences shown respectively include up to three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: The amino acid sequence shown in 41, 42 and 43 or the amino acid sequence shown contains at most three amino acid mutations; or

(f) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 44, 45 and 46 or the amino acid sequence shown respectively comprises a variant of at most three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 44, 45 and 46. ID NO: The amino acid sequences shown in 47, 48, and 49 or the shown amino acid sequences contain at most three amino acid mutations.

In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the following sequences:

(a) The HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 14, 15, and 16, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 17, 18, and 19, respectively;

(b) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 23, 24 and 25;

(c) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 29, 30 and 31;

(d) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 32, 33 and 34 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 35, 36 and 37;

(e) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 38, 39 and 40 and the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 41, 42 and 43; or

(f) The HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 44, 45, and 46, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 47, 48, and 49, respectively.

In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the following sequences:

(a) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 14, 15, and 16, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 17, 18, and 19, respectively;

(b) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 20, 21, and 22, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 23, 24, and 25, respectively;

(c) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 26, 27, and 28, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 29, 30, and 31, respectively;

(d) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 32, 33, and 34, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 35, 36, and 37, respectively;

(e) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 38, 39 and 40, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 41, 42 and 43, respectively; or

(f) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 44, 45, and 46, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 47, 48, and 49, respectively.

In some embodiments, the heavy chain variable region sequence comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12; and the light chain The variable region sequence includes an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or 13. In some embodiments, the heavy chain variable region sequence comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, or 84% as shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acids Sequence; the light chain variable region sequence includes at least 80%, 81%, 82%, 83%, 84%, 85% of the amino acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or 13 , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence.

In some embodiments, the heavy chain variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12; the light chain variable region sequence comprises SEQ ID NO: 3, The amino acid sequence shown in 5, 7, 9, 11 or 13.

In some embodiments, the variable region of the heavy chain and the variable region of the light chain are selected from the following sequences:

(a) The heavy chain variable region contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 2 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, the light chain variable region comprises SEQ ID NO: The sequence shown in 3 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequence;

(b) The heavy chain variable region contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 4 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, the light chain variable region comprises SEQ ID NO: The sequence shown in 5 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequence;

(c) The heavy chain variable region contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 6 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, the light chain variable region comprises SEQ ID NO: The sequence shown in 7 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequence;

(d) The heavy chain variable region contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 8 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, the light chain variable region comprises SEQ ID NO: The sequence shown in 9 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequence;

(e) The heavy chain variable region contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 10 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, the light chain variable region comprises SEQ ID NO: The sequence shown in 11 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequence; or

(f) The heavy chain variable region contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 12 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, the light chain variable region comprises SEQ ID NO: The sequence shown in 13 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequence.

In some embodiments, the variable region of the heavy chain and the variable region of the light chain are selected from the following sequences:

(a) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 3;

(b) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 5;

(c) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 7;

(d) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 9;

(e) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 11; or

(f) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 13.

In a specific embodiment, the variable region of the heavy chain and the variable region of the light chain are selected from the following sequences:

(a) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3;

(b) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5;

(c) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7;

(d) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9;

(e) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 11; or

(f) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 13.

In another aspect, the present invention provides an isolated polynucleotide encoding the aforementioned anti-SARS-CoV-2 spike protein monoclonal antibody or functional fragment thereof.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the aforementioned anti-SARS-CoV-2 spike protein monoclonal antibody or functional fragment thereof, and encoding the anti-SARS-CoV -2 The nucleotide sequence of the light chain variable region of the spike protein monoclonal antibody or its functional fragment.

In another aspect, the present invention provides an expression vector containing the polynucleotide.

In another aspect, the present invention provides a host cell or a cell-free expression system comprising the expression vector.

In another aspect, the present invention provides a pharmaceutical composition comprising the anti-SARS-CoV-2 spike protein monoclonal antibody or functional fragment thereof and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides the application of the anti-SARS-CoV-2 spike protein monoclonal antibody or its functional fragments in the preparation of drugs for treating coronavirus. Also provided is the use of the anti-SARS-CoV-2 spike protein monoclonal antibody or functional fragments thereof for treating coronavirus infection diseases.

In another aspect, the present invention provides a kit for detecting coronavirus, which contains the monoclonal antibody or functional fragments thereof. The kit may involve monoclonal antibodies that bind to different epitopes, such as clones 5B7D7 and 5E10G8. The two monoclonal antibodies can be used as raw materials for ELISA kits for double antibody sandwich detection of antigens.

In some embodiments, the coronavirus is selected from SARS-CoV, MERS-CoV or SARS-CoV-2, preferably SARS-CoV-2. In other embodiments, the coronavirus is SARS-CoV-2.

In another aspect, the present invention provides a method for preparing an anti-SARS-CoV-2 spike protein monoclonal antibody or a functional fragment thereof, which comprises

1) Immunize an animal with the SARS-CoV-2 spike protein, and produce an immune response against the SARS-CoV-2 spike protein in the animal;

2) Take the hybridoma cells obtained by fusion of the spleen cells of the animal and myeloma cells, and screen them to obtain a positive clone that specifically recognizes the SARS-CoV-2 spike protein;

3) Subcloning the positive parent clone to obtain a stable hybridoma cell line;

4) Perform gene sequencing on the hybridoma cell line to obtain the variable region coding sequences of the heavy chain and light chain of the anti-SARS-CoV-2 spike protein antibody; and

5) Use the variable region coding sequence for recombinant antibody production to obtain a functional anti-SARS-CoV-2 spike protein monoclonal antibody.

In some embodiments, the monoclonal antibody is a murine, chimeric, humanized, or human antibody. In some preferred embodiments, the monoclonal antibody is of murine origin. In other preferred embodiments, the monoclonal antibody is humanized.

In some embodiments, the monoclonal antibody may be IgG1 or IgG2. In some specific embodiments, the antibody comprises an IgG1 heavy chain constant region, and the IgG1 heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:50. In other specific embodiments, the antibody comprises an IgG2b heavy chain constant region sequence, and the IgG2b heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 51. In some embodiments, the monoclonal antibody comprises a Kappa light chain constant region. In some specific embodiments, the Kappa light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 52.

The monoclonal antibodies involved in the present invention can specifically bind to the epitope of the SARS-CoV-2 spike protein, and these monoclonal antibodies may have the same binding epitope or different binding epitopes. For example, clones 5E10G8, 4A1D10 and 10G6H5 have different binding epitopes, and clones 5B7D7, 6D11F2 and 11D5D3 have the same binding epitopes.

The anti-SARS-CoV-2 spike protein monoclonal antibody provided by the present invention can block the combination of SARS-CoV-2S protein and ACE2. _{In some embodiments, the IC 50} value of the monoclonal antibody blocking the binding of SARS-CoV-2S protein to ACE2 is about 0.001-10 μg/ml. _{In other embodiments, the IC 50} value of the monoclonal antibody blocking the binding of SARS-CoV-2S protein to ACE2 is about 0.01-1 μg/ml, preferably 0.10-0.5 μg/ml. _{In some specific embodiments, the IC 50} value of the monoclonal antibody blocking the binding of SARS-CoV-2S protein to ACE2 is about 0.05 μg/ml, 0.07 μg/ml, 0.09 μg/ml, 0.11 μg/ml, 0.13 μg/ml, 0.15μg/ml, 0.17μg/ml, 0.19μg/ml, 0.20μg/ml, 0.21μg/ml, 0.23μg/ml, 0.25μg/ml, 0.27μg/ml, 0.29μg/ml, 0.31 μg/ml, 0.33μg/ml, 0.35μg/ml, 0.37μg/ml, 0.39μg/ml or 0.41μg/ml.

The anti-SARS-CoV-2 spike protein monoclonal antibody provided by the present invention can prevent the SARS-CoV-2 pseudovirus from entering human ACE2 overexpression cells.

Beneficial effect

Compared with PCR detection, serological detection has the advantages of short detection cycle, high throughput and less workload. The anti-SARS-CoV-2 spike protein monoclonal antibody developed by the present invention can specifically bind to the S protein, and can effectively block the combination of the S protein and the ACE2 protein, and specifically prevent the virus from infecting human cells. In the pseudovirus neutralization experiment, it can reach the level of ACE2 protein tissue S protein invading human ACE2 overexpressing cells. In addition, the provided monoclonal antibodies can recognize 4 kinds of epitopes of the antigen, and the diversity of antibodies provides convenience for the development of detection kits.

Description of the drawings

Figure 1 is a graph showing the results of rat serum titer after immunization;

Figure 2 shows the SDS-PAGE identification result of the purified monoclonal antibody against SARS-CoV-2 spike protein;

Figure 3 is a graph showing the detection results of the affinity between the purified monoclonal antibody and the S recombinant protein;

Figure 4 is a diagram of the purified monoclonal antibody blocking the binding of S protein and ACE2 protein;

Figure 5 is a diagram of the purified monoclonal antibody preventing SARS-CoV-2 pseudovirus from entering human ACE2 overexpressing cells.

Figure 6 is a graph showing the pairing curve of 5B7D7 and 5E10G8 antibodies.

Detailed ways

The present invention relates to a functional virus SARS-CoV-2S protein antibody. The embodiments of the present invention will be described in detail below in conjunction with examples. Unless otherwise specified, the technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.

The term "new coronavirus" (SARS-CoV-2), also known as 2019-nCoV, belongs to the β-coronavirus, has an envelope, and the particles are round or elliptical, often pleomorphic, with a diameter of 60-140nm . Its genetic characteristics are significantly different from SARSr-CoV and MERSr-CoV. Studies have shown that it has more than 85% homology with bat SARS-like coronavirus (bat-SL-CoVZC45). When isolated and cultured in vitro, 2019-nCov can be found in human respiratory epithelial cells in about 96 hours, while isolation and culture in Vero E6 and Huh-7 cell lines takes about 6 days.

The term "antibody"" means an immunoglobulin molecule composed of four polypeptide chains (two heavy chains (H) and two light chains (L) are connected to each other by disulfide bonds (ie, "complete antibody molecules") ), and its multimers (such as IgM) or antigen-binding fragments thereof. Each heavy chain is composed of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (by the structural domains CH1, CH2, and CH3). Composition). Each light chain is composed of a light chain variable region ("LCVR or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR), with more conserved regions intervening called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the hydroxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the present invention, the FR of the antibody (or antigen-binding fragment thereof) may be the same as the human germline sequence or may be naturally or artificially modified.

The term "monoclonal antibody" refers to a uniform antibody that only targets a specific epitope. In contrast to a typical ordinary polyclonal antibody preparation that includes different antibodies directed against different epitopes (epitopes), each monoclonal antibody is directed against a single epitope on the antigen. The modifier "monoclonal" refers to the uniform characteristics of an antibody, and is not interpreted as an antibody that needs to be produced by any specific method. The monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods, or obtained by screening methods described elsewhere in the present invention.

The term "mutation" refers to a monoclonal antibody or a functional fragment thereof that contains one or more (several) amino acid residue changes at one or more (several) positions, that is, a polypeptide that is substituted, inserted, and/or deleted. Substitution refers to replacing the amino acid occupying a certain position with a different amino acid; deletion refers to removing the amino acid occupying a certain position; and insertion refers to adding 1-3 amino acids adjacent to the amino acid occupying a certain position and afterwards.

The term "isolated polynucleotide" refers to a polynucleotide that does not exist naturally in nature, including polynucleotides isolated from nature (including living organisms) through biological techniques, and also includes artificially synthesized polynucleotides. The isolated polynucleotide can be genomic DNA, cDNA, mRNA or other synthetic RNA, or a combination thereof. It should be pointed out that, based on the amino acid sequences of the heavy chain variable region and the light chain variable region provided herein, those skilled in the art can design a nucleus whose nucleotide sequences are not completely identical based on the codon degeneracy. Nucleotide sequences, but they all encode the same amino acid sequence. These modified nucleotide sequences are also included in the scope of the present invention.

When referring to polynucleotides, the term "vector" refers to any molecule (for example, nucleic acid, plasmid, virus, etc.) used to transfer nucleotide coding information into a host cell. The term "expression vector" or "expression cassette" refers to a vector suitable for expressing a target gene (nucleotide sequence to be expressed) in a host cell, and usually includes a target gene, a promoter, a terminator, a marker gene and other parts.

The term "host cell" refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a selected gene of interest. The term includes the offspring of the parent cell, regardless of whether the offspring is the same in morphology or genetic composition as the original parent cell, as long as the offspring has the selected target gene. Commonly used host cells include bacteria, yeast, and mammalian cells.

The term "antibody functional fragment" means the antigen-binding fragment and antibody analog of an antibody, which usually includes at least part of the antigen-binding region or variable region (for example, one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. For example, antibody fragments capable of binding to the Coronary Disease Spike (S) protein or part thereof, including but not limited to sdAb (single domain antibody), Fab (for example, the antibody is obtained by papain digestion), F(ab')2( For example, obtained by pepsin digestion), Fv or scFv (for example, obtained by molecular biology techniques).

The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersants, coatings, antibacterial and antifungal agents, isotonic and sustained release agents, and the like that are compatible with the administration of the drug. Suitable carriers are described in the standard reference documents in the latest edition of Remington’s Pharmaceutical Sciences, which are incorporated herein by reference in their entirety. Examples of suitable carriers or diluents include, but are not limited to, water, saline solution, ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and hydrophobic-aqueous media such as fixed oils can also be used. The use of media and agents for pharmaceutically active substances is well known in the art. Except for those conventional media or reagents that are incompatible with the active ingredients, its use in the ingredients can achieve the desired effect.

The term "amino acid substitution" refers to replacing an existing amino acid residue with a different amino acid residue in a predetermined (initial) amino acid sequence. Generally speaking, those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al., Molecular Biology of the Gene, The Benjamin/Cummings Pub.Co ., p. 224 (fourth edition, 1987)). Such exemplary substitutions are preferably carried out in accordance with the substitutions shown below:

Exemplary conservative amino acid substitutions

Original residue Conservative substitution Ala(A) Gly; Ser Arg(R) Lys; His Asn(N) Gln; His Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu

The "percent (%) amino acid sequence identity" of a peptide or polypeptide sequence is defined as comparing the sequences and introducing gaps when necessary to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Candidates The percentage of amino acid residues in the sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence. Sequence comparisons can be performed in a variety of ways within the skill of the art to determine percent amino acid sequence identity, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required to obtain the maximum comparison over the entire length of the sequence being compared.

When "administering" and "treatment" are used to refer to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means to combine exogenous drugs, therapeutic agents, diagnostic agents or compositions with animals, humans, and recipients. Contact with the treated person, cells, tissues, organs or biological fluids. "Administration" and "treatment" may refer to, for example, treatment methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting the agent with the cell and contacting the agent with a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" also mean the treatment of cells in vitro and ex vivo, for example, by reagents, diagnostic agents, binding compositions, or by other cells.

The term "subject" as used herein refers to an animal in need of alleviation, prevention and/or treatment of a disease or condition such as a viral infection, preferably a mammal, more preferably a human. The term includes human subjects who have a coronavirus such as SARS-CoV-2 infection or are at risk of having a coronavirus such as SARS-CoV-2 infection.

When referring to a pharmaceutical composition, the term "effective amount" as used herein refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals. "Pharmaceutically acceptable carrier" refers to a carrier used for administration, including various excipients, diluents and buffers, etc. These substances are suitable for administration to humans and/or animals without excessive adverse side effects, and at the same time It is suitable for maintaining the vitality of the drugs or active agents located therein.

Unless specifically stated otherwise, the use of the singular includes the plural. Unless specifically stated otherwise, the words "a" or "an" mean "at least one." The use of "or" means "and/or" unless stated otherwise. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more". In addition, the use of the term "including" and other forms such as "includes" and "included" is not limiting. In addition, unless specifically stated otherwise, terms such as "element" or "component" include elements or components that include one unit and elements and components that include more than one unit.

Unless otherwise specified, the methods and materials of the embodiments described below are all conventional products that can be purchased on the market. Those skilled in the art to which the present invention belongs will understand that the methods and materials described below are only exemplary and should not be regarded as limiting the scope of the present invention.

Example 1: Obtaining of SARS-CoV-2S protein antibody hybridoma cell lines and preparation of monoclonal antibodies

1) The animal immune antigen uses recombinant protein S-RBD-His (sequence shown in SEQ ID NO:1). Female Balb/c was subcutaneously immunized with 5μg S fusion protein. Subsequently, the immunization was repeated every other day to boost the mice for a total of 6 times. 5 mice titer after immunization 6 reached ¹⁰⁵ or more. The two mice (No. 1 and No. 2) exhibiting the highest antibody titers (Figure 1) were fused 4 days after the last immunization.

S-RBD-His amino acid sequence (SEQ ID NO:1):

2) Hybridoma fusion and screening

The mouse lymph nodes were extracted and homogenized to produce a single-cell suspension, and at the same time, a single-cell suspension of myeloma cells (SP2/0) was prepared. Electrofusion was used to fuse 5.95×10 ⁷ spleen cells with 2.975×10 ⁷ SP2/0 mouse myeloma cells. Resuspend the fused cells in 150ml of DMEM/10%FBS medium containing hybridoma selective agents thymidine, hypoxanthine and aminopterin, and transfer to 15×96 wells with a volume of 100μl with a pipette In the board. The plate was incubated in 5% CO ₂ at 37°C. After 7 days of incubation, start to test the presence of antibodies against S protein using the ELISA binding described below.

ELISA binding detection method:

Indirect ELISA was used to evaluate the binding ability of antibodies in the supernatant to S protein. The ELISA plate was coated with 100 μl/well of 1 μg/ml recombinant S protein in PBS at 4° C. overnight. The plate was washed with PBS-T (0.05% Tween) and blocked with 150 μl/well of PBST containing 1% BSA at 37°C for 1 hour. Then the blocking solution was discarded, 100 μl of hybridoma cell culture supernatant was added to each plate, and then incubated at 37°C for 1 hour. The plate was washed three times with PBST, and incubated with 100 μl/well of horseradish peroxidase-conjugated goat anti-mouse IgG (Fc-specific) secondary antibody (Jackson, 115-035-071) at 37°C for 0.5 hours. The plate was washed five times with PBST, then TMB color developing solution was added and incubated in the dark at room temperature for 13 minutes. The reaction was terminated by adding 50μl of 1M HCl stop solution (China National Pharmaceutical, 10011018). Use a microplate reader to read the plate at 450nm.

3) Hybridoma subcloning

The limiting dilution method was used for subcloning. Use a hemocytometer and serially dilute the cells in DMEM/10% FBS medium containing hybridoma cell selection agents thymidine, hypoxanthine and aminopterin to determine the number of cells until the cell density reaches 5-15 Cells/ml. For each hybridoma, transfer 200 μl of cell solution with a pipette to 96 wells with a density of 1-3 cells/well. After the culture was cultured in 5% CO2 at 37°C for 1 week, monoclonal cells were selected, and the supernatant was subjected to the above-mentioned ELISA combination to evaluate the presence of antibodies against the S protein, and the single cells with an OD value greater than 1.0 were selected. The cloned hole is amplified for subsequent experiments.

Example 2: Production of monoclonal antibodies based on hybridoma cells

After the above-mentioned cells were cultured and expanded, they were inoculated in shake flasks and cultured at 37°C for 7 days, and then the supernatant was collected for antibody purification. Before purification, the tubing and protein A column were depyrogenated with 0.2M NaOH. Re-equilibrate the column with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). Subsequently, the harvested cell culture supernatant was diluted 1:1 with 2× the above buffer and sterilized by filtration. Incubate the filtered supernatant and protein A column at room temperature for 2 hours. After washing the column with 1× the above buffer, elute the IgG with sterile 0.1M sodium citrate (pH 3.5), collect the eluate and use it for nine minutes One volume of sterile 1M Tris-HCl (pH 9) for neutralization. Under sterile conditions, the product buffer was exchanged for PBS (pH 7.4) to remove any elution buffer.

The purified antibody was analyzed by SDS-PAGE with a 10% precast gel (GenScript, M42012C) by BioRad electrophoresis system. The gel was stained with Estin 2.0 (GenScript, L00687R) and the molecular size and purity were estimated by comparing the stained band with the Protein Ladder (Takara, 3452). As shown in Figure 2, the purity of all antibodies was 99%.

Example 3: Sequencing of the variable region of monoclonal antibodies

After subtype identification of monoclonal antibodies using rapid ELISA mouse antibody subtype identification kit (Clonotyping System-HRP, Southern Biotech), use TRIzol (Life Technology, 15596-026) from 1×10 ⁶ to 5×10 ⁶ Total RNA was extracted from each hybridoma cell and reverse transcribed into cDNA using antibody subtype specific primers and universal primers (Prime ScriptTM 1st Strand cDNA Synthesis Kit, Takara). Subsequently, the mouse immunoglobulin heavy and light chain V-region fragments were amplified by RACE PCR (GenScript), and the resulting PCR fragments were subcloned into the pMD18-T vector system (Takara), and inserted using vector-specific primer pairs The fragments are sequenced. Finally, the unique V-region protein amino acid sequences of 6D11F2 (IgG1), 5E10G8 (IgG2b), 5B7D7 (IgG1), 4A1D10 (IgG2b), 10G6H5 (IgG1) and 11D5D3 (IgG1) were obtained.

6D11F2 heavy chain variable region amino acid sequence (SEQ ID NO: 2):

6D11F2 light chain variable region amino acid sequence (SEQ ID NO: 3):

5E10G8 heavy chain variable region amino acid sequence (SEQ ID NO: 4):

5E10G8 light chain variable region amino acid sequence (SEQ ID NO: 5):

5B7D7 heavy chain variable region amino acid sequence (SEQ ID NO: 6):

5B7D7 light chain variable region amino acid sequence (SEQ ID NO: 7):

4A1D10 heavy chain variable region amino acid sequence (SEQ ID NO: 8):

4A1D10 light chain variable region amino acid sequence (SEQ ID NO: 9):

10G6H5 heavy chain variable region amino acid sequence (SEQ ID NO: 10):

10G6H5 light chain variable region amino acid sequence (SEQ ID NO: 11):

11D5D3 heavy chain variable region amino acid sequence (SEQ ID NO: 12):

11D5D3 light chain variable region amino acid sequence (SEQ ID NO: 13):

IgG1 heavy chain constant region (SEQ ID NO: 50):

IgG2b heavy chain constant region (SEQ ID NO: 51):

Kappa light chain constant region (SEQ ID NO: 52):

Table 1 CDR region sequence of antibody

Example 4: ELISA competitive detection method

Competitive ELISA was used to evaluate the ability of the above cell culture supernatant to block the binding of S protein and ACE2 protein. The ELISA plate was coated with 100 μl/well of 1 μg/ml recombinant S protein in PBS at 4° C. overnight. The plate was washed with PBST (0.05% Tween) and blocked with 250 μl/well of PBST containing 1% BSA at 37°C for 2 hours. The blocking solution was then discarded, and 50 μl of the appropriate concentration of the supernatant to be tested was added to each test well. Then add 100ng/ml 50μl of recombinant ACE2-Fc protein (GenScript, Z03484) to each well, and incubate at 37°C for 1 hour. The plate was washed 3 times with PBST, and incubated with 100 μl/well of horseradish peroxidase-conjugated goat anti-mouse IgG (Fc-specific) secondary antibody (Jackson, 115-035-071) at 37°C for 0.5 hours. Finally, the plate was washed 4 times with PBST, and then TMB color developing solution (GenScript) was added and incubated in the dark at 25°C for 15 minutes. The reaction was terminated by adding 50μl of 1M HCl stop solution (China National Pharmaceutical, 10011018). Use a microplate reader to read the plate at 450nm. No antibody was added to the blank control group, which directly reflected the binding ability of ACE2 and S protein. If the antibody has a blocking effect on the binding of S protein and ACE2, then the detection value is lower than the control. Based on the control, the calculated blocking effect of each clone, for example, the blocking effect of 6D11F2 is (1-0.282/1.408)%=80%. The blocking effects of all antibodies obtained by the same calculation method are shown in Table 2.

Table 2 The blocking effect of each cloned cell supernatant on the binding of S protein and ACE2 protein

Example 5: Binding of monoclonal antibodies to viral S protein

Indirect ELISA is used to evaluate the binding ability of purified antibodies to S protein. The ELISA plate was coated with 100 μl/well of 0.5 μg/ml recombinant S protein in PBS at 4° C. overnight. The plate was washed with PBS-T (0.05% Tween) and blocked with 250 μl/well of PBST containing 1% BSA at 37°C for 2 hours. Then the blocking solution was discarded, and 100 μl of 1 μg/ml purified antibody was added to the first well, and diluted in a 3-fold gradient, a total of 11 test concentration gradients plus a blank well. Then incubate at 37°C for 1 hour. The plate was washed three times with PBST and incubated with 100 μl/well of horseradish peroxidase-conjugated goat anti-mouse IgG (Fc-specific) secondary antibody (Jackson, 115-035-071) at 37°C for 0.5 hours. The plate was washed four times with PBST, then TMB color developing solution (GenScript) was added and incubated at 25°C in the dark for 15 minutes. The reaction was terminated by adding 50μl of 1M HCl stop solution (China National Pharmaceutical, 10011018). Using a microplate reader to read the plate at 450nm, the EC ₅₀ detection curve of each clone is shown in Figure 3, and the EC ₅₀ value is shown in Table 3:

_{Table 3 EC 50} value of antibody binding to S protein

Example 6: Monoclonal antibody blocks the binding of S protein and ACE2 protein

The ELISA plate was coated with 100 μl/well of 0.5 μg/ml recombinant S protein in PBS at 4° C. overnight. The plate was washed with PBST (0.05% Tween) and blocked with 250 μl/well of PBST containing 1% BSA at 37°C for 2 hours. Then the blocking solution was discarded, and 50μl of 30μg/ml purified antibody to be tested was added to the first test well, and diluted in a 3-fold gradient, a total of 11 test concentration gradients plus a Blank well. Then add 50μl of recombinant human ACE2 protein (concentration of 0.15μg/ml) to each well, and incubate at 37°C for 1 hour. The plate was washed 3 times with PBST, and incubated with 100 μl/well of horseradish peroxidase-conjugated mouse anti-human IgG (Fc-specific) secondary antibody (Jackson, 115-035-071) at 37°C for 0.5 hour. Finally, the plate was washed 4 times with PBST, and then TMB color developing solution (GenScript) was added and incubated in the dark at 25°C for 15 minutes. The reaction was terminated by adding 50μl of 1M HCl stop solution (China National Pharmaceutical, 10011018). Use a microplate reader to read the plate at 450nm. As shown in Figure 3, there are 5 clones that have a good blocking effect on the binding of S protein and ACE2. The IC ₅₀ curve of each clone is shown in Figure 4, and the IC ₅₀ value is shown in Table 4.

_{Table 4 IC 50} value of antibody blocking the binding of S protein and ACE2 protein

Antibody IC ₅₀ (μg/ml) Antibody IC ₅₀ (μg/ml) 6D11F2 0.19 10G6H5 0.17 5B7D7 0.16 11D5D3 0.19 4A1D10 0.37 To To

Example 7: Functional detection of monoclonal antibodies

Purified monoclonal antibodies can prevent SARS-CoV-2 pseudovirus from entering human ACE2 overexpressing cells. Incubate the SARS-CoV-2 pseudovirus (GenScript) with two diluted antibody samples to be tested at 37°C for 1 hour. Make three replicate wells for each sample, and incubate the virus control and cell control wells at the same time. Collect the target cells to ^{a concentration of 4×10 5} cells/mL, and add 50 μL of the cell suspension to the 96-well cell culture plate. The final cell concentration in each well should be 20,000 cells/well. Add antibody and pseudovirus to the cells, _{incubate at 37°C under 5% CO 2} for 24 hours, add 70 μL of fresh medium the next day, and then _{continue to incubate at 37°C under 5% CO 2} for 24 hours. Carefully remove the medium from the cells to be tested. Rinse the cells with DPBS, taking care not to detach the attached cells. Remove as much PBS rinse as possible. Add 50 μL of lysis buffer to cover the cells and lyse at room temperature for 40 minutes. Transfer 40 μL of supernatant to a sterile opaque 96-well plate. Then the Bio-Glo ^™ (Promega, G7491) luciferase substrate working solution was added to the corresponding wells of the assay plate. EnVision (Perkin Elmer, EnVision 2105) was used to read the luminescence signal, and finally the data was analyzed as shown in Figure 5. The results showed that 6D11F2, 5B7D7, 10G6H5 and 11D5D3 are antibodies with neutralizing potential.

Example 8: Monoclonal antibody epitope identification

Competitive ELISA was used to evaluate the epitope of cell culture supernatant. The ELISA plate was coated with 100 μl/well of 1 μg/ml recombinant S protein in PBS at 37° C. for 2 hours. The plate was washed with PBS-T (0.05% Tween) and blocked with 250 μl/well of PBST containing 1% BSA at 37°C for 1 hour. Then the blocking solution was discarded, and 50 μl of cell supernatant pre-mixed with horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson, 115-035-071) secondary antibody was added to each well. 50μl of clear stock solution. Then incubate at 37°C for 1 hour. The plate was washed 4 times with PBST, and then TMB color developing solution (GenScript) was added and incubated in the dark at 25°C for 15 minutes. Stop the reaction by adding 50μl of 1M HCl stop solution (GenScript). Use a microplate reader to read the plate at 450nm. Clone 5E10G8 is an epitope (epitope 1), clones 6D11F2, 5B7D7 and 11D5D3 are an epitope (epitope 2), clone 4A1D10 is an epitope (epitope 3), clone 10G6H5 is an epitope (epitope 4) ). After the first round of epitope classification, we get the preliminary classification results. On this basis, we select one antibody for each epitope plus the remaining unclassified antibodies for the second round of classification. The judgment logic of epitope classification Yes: A, B, and C represent different clones. If the OD of the self-competitive well of the supernatant A is 0.545, the difference in OD value is close to 1.0 compared with the value of the blank well, which has a significant competitive effect. When the difference between the OD value of the supernatant B and the supernatant A-HRP mixture is about 0.9, there is an obvious competitive effect. Therefore, it can be determined that supernatant A and supernatant B recognize the same antigenic determinant. The difference between the OD value of the supernatant C and the supernatant A-HRP mixture is not significantly different from the value of 0 wells, and there is no competitive effect. Therefore, it can be determined that the supernatant C and the supernatant A recognize different epitopes of the antigen. Finally, the classification results of all tested antibodies are obtained, as shown in Table 5.

Table 5 Classification of antibody binding epitopes

Epitope 1 Epitope 2 Epitope 3 Epitope 4 5E10G8 5B7D7 4A1D10 10G6H5 N/A 6D11F2 N/A N/A N/A 11D5D3 N/A N/A

Example 9: Paired detection of monoclonal antibodies

The ELISA plate was coated with 100 μl/well of 2.5 μg/ml unlabeled purified antibody (such as 5B7D7) in PBS at 4° C. overnight. The plate was washed with PBST (0.05% Tween) and blocked with 250 μl/well of PBST containing 1% BSA at 37°C for 2 hours. Then the blocking solution was discarded, and 100 μl of recombinant S protein at a 2-fold dilution concentration of 20 ng/ml and 0 ng/ml were added to the first well, and incubated at 37° C. for 1 hour. Wash the plate 4 times with PBST, add 1 μg/ml of a biotin-labeled antibody (such as Biotin-5E10G8) to the plate wells, add 100 μl to each well, and incubate at 37°C for 1 hour. The plate was washed 4 times with PBST, and incubated with 100 μl/well of streptavidin HRP (SA-HRP, GenScript) at 37°C for 15 minutes. Finally, the plate was washed 4 times with PBST, and then TMB color developing solution (GenScript) was added and incubated at 25°C in the dark for 15 minutes. Stop the reaction by adding 50μl of 1M HCl stop solution (GenScript). Use a microplate reader to read the plate at 450 nm, and the specific OD values are shown in Table 6. We use one of the antibodies (such as 5B7D7) to coat the plate according to 2.5ug/ml, then add different concentrations of detection agent to react, and finally add a biotin-labeled detection antibody (such as 5E10G8-biotin), and use SA-HRP for The reaction develops color. The value of the well with the antigen of 0 is selected to be lower than 0.1, and the value of the detection antibody has a good linear relationship with the decrease of the antigen gradient (R value is greater than 0.99). We judge that these two antibodies are a relatively good pair of antibodies and can be used as double antibodies Development of ELISA kits for sandwich detection of antigens. The recommended paired antibodies are 5B7D7 and 5E10G8, and their sensitivity can reach 0-40pg/ml, as shown in Figure 6.

Table 6

references

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[Corrected according to Rule 26 21.06.2021]
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Claims (18)

An anti-SARS-CoV-2 spike protein monoclonal antibody or functional fragment thereof, said antibody or functional fragment comprising a heavy chain variable region and a light chain variable region, wherein (a) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, The HCDR1 includes an amino acid sequence selected from SEQ ID NO: 14, 20, 26, 32, 38, or 44 or a variant of the amino acid sequence including at most three amino acid mutations; the HCDR2 includes an amino acid sequence selected from SEQ ID NO: : The amino acid sequence shown in 15, 21, 27, 33, 39, or 45 or a variant of the shown amino acid sequence containing at most three amino acid mutations; the HCDR3 includes SEQ ID NO: 16, 22, 28, 34, The amino acid sequence shown by 40 or 46 or the amino acid sequence shown contains at most three amino acid mutations; and (b) The light chain variable region includes LCDR1, LCDR2 and LCDR3, The LCDR1 sequence includes an amino acid sequence selected from SEQ ID NO: 17, 23, 29, 35, 41, or 47 or a variant of the amino acid sequence including at most three amino acid mutations; the LCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 17, 23, 29, 35, 41, or 47. ID NO: The amino acid sequence shown in 18, 24, 30, 36, 42 or 48 or the amino acid sequence shown contains at most three amino acid mutations; the LCDR3 sequence includes SEQ ID NO: 19, 25, 31 The amino acid sequence shown in, 37, 43 or 49 or the shown amino acid sequence contains at most three amino acid mutations. The monoclonal antibody or functional fragment thereof according to claim 1, wherein: The HCDR1 sequence includes an amino acid sequence selected from SEQ ID NO: 14, 20, 26, 32, 38, or 44; the HCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 15, 21, 27, 33, 39, or 45 The amino acid sequence shown; the HCDR3 sequence includes an amino acid sequence selected from SEQ ID NO: 16, 22, 28, 34, 40, or 46; and The LCDR1 sequence includes an amino acid sequence selected from SEQ ID NO: 17, 23, 29, 35, 41, or 47; the LCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 18, 24, 30, 36, 42 or 48 The amino acid sequence shown; the LCDR3 sequence includes an amino acid sequence selected from SEQ ID NO: 19, 25, 31, 37, 43, or 49. The monoclonal antibody or functional fragment thereof according to claim 1 or 2, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the following sequences: (a) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 14, 15, and 16 or the amino acid sequence shown respectively includes at most three amino acid mutation variants; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 14, 15, and 16 ID NO: The amino acid sequences shown in 17, 18 and 19 or the amino acid sequences shown respectively contain at most three amino acid mutations; (b) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 20, 21 and 22 or the amino acid sequence shown respectively includes at most three amino acid mutation variants; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 20, 21 and 22 ID NO: The amino acid sequences shown in 23, 24, and 25 or the amino acid sequences shown respectively contain at most three amino acid mutations; (c) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28 or the amino acid sequences shown respectively include up to three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 26, 27 and 28. ID NO: The amino acid sequences shown in 29, 30, and 31 or the amino acid sequences shown respectively contain at most three amino acid mutations; (d) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 32, 33 and 34 or the amino acid sequence shown respectively comprises a variant of at most three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 32, 33 and 34 ID NO: The amino acid sequences shown in 35, 36 and 37 or the amino acid sequences shown respectively contain at most three amino acid mutations; (e) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 38, 39 and 40 or the amino acid sequences shown respectively include up to three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: The amino acid sequence shown in 41, 42 and 43 or the amino acid sequence shown contains at most three amino acid mutations; or (f) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 44, 45 and 46 or the amino acid sequence shown respectively comprises a variant of at most three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 44, 45 and 46. ID NO: The amino acid sequences shown in 47, 48, and 49 or the shown amino acid sequences contain at most three amino acid mutations. The monoclonal antibody or functional fragment thereof according to claim 3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the following sequences: (a) The HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 14, 15, and 16, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 17, 18, and 19, respectively; (b) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 23, 24 and 25; (c) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 29, 30 and 31; (d) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 32, 33 and 34 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 35, 36 and 37; (e) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 38, 39 and 40 and the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 41, 42 and 43; or (f) The HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 44, 45, and 46, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 47, 48, and 49, respectively. The monoclonal antibody or functional fragment thereof according to any one of claims 1 to 4, wherein the heavy chain variable region sequence comprises the sequence shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12. The amino acid sequence has an amino acid sequence with at least 80% identity; and The light chain variable region sequence comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or 13. The monoclonal antibody or functional fragment thereof according to claim 5, wherein: The heavy chain variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12; and the light chain variable region sequence comprises SEQ ID NO: 3, 5, 7, The amino acid sequence shown in 9, 11 or 13. The monoclonal antibody or functional fragment thereof according to any one of claims 1 to 6, wherein the variable region of the heavy chain and the variable region of the light chain are selected from the following sequences: (a) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 2, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 3. % Identical amino acid sequence; (b) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 4, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 5; % Identical amino acid sequence; (c) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 6, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 7 % Identical amino acid sequence; (d) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 8, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 9 % Identical amino acid sequence; (e) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 10, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 11. % Identical amino acid sequence; or (f) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 12, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 13 % Identical amino acid sequence. The monoclonal antibody or functional fragment thereof according to claim 7, wherein the variable region of the heavy chain and the variable region of the light chain are selected from the following sequences: (a) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 3; (b) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 5; (c) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 7; (d) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 9; (e) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 11; or (f) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 13. An isolated polynucleotide encoding the anti-SARS-CoV-2 spike protein monoclonal antibody or functional fragment thereof according to any one of claims 1 to 8. The polynucleotide of claim 9, wherein the polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody or a functional fragment thereof, and encoding the monoclonal antibody Or the nucleotide sequence of the light chain variable region of its functional fragment. An expression vector comprising the polynucleotide according to claim 9 or 10. A host cell or a cell-free expression system comprising the expression vector according to claim 11. A pharmaceutical composition comprising the monoclonal antibody or functional fragment thereof according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. Use of the monoclonal antibody or functional fragments thereof according to any one of claims 1 to 8 in the preparation of drugs for the treatment of coronaviruses. According to the application of claim 14, the coronavirus is selected from SARS-CoV, MERS-CoV or SARS-CoV-2, preferably SARS-CoV-2. A kit for detecting coronavirus, said kit comprising the monoclonal antibody or functional fragments thereof according to any one of claims 1-8. The kit according to claim 16, wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV or SARS-CoV-2, preferably SARS-CoV-2. The monoclonal antibody or functional fragment thereof according to any one of claims 1 to 8, wherein the antibody is murine, chimeric, humanized or human.

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