WO2021233433A1 - Anti-sars-cov-2 spike protein monoclonal antibody - Google Patents

Abstract

The present application relates to the field of virus detection, diagnosis, and treatment, in particular, to an anti-SARS-CoV-2 spike (S) protein monoclonal antibody, a preparation method therefor, and an application thereof. The anti-SARS-CoV-2 S protein monoclonal antibody, and amino acid sequences of a heavy chain variable region and a light chain variable region thereof are provided. The provided anti-SARS-CoV-2 S protein monoclonal antibody can specifically bind to S protein, effectively block the binding of S protein to ACE2 protein, and specifically prevent the virus from infecting human cells. The provided anti-SARS-CoV-2 S protein monoclonal antibody provides possibility and convenience for the treatment and detection of SARS-CoV-2 virus.

Description

Anti-SARS-CoV-2 spike protein monoclonal antibody 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

Severe Acute Respiratory Syndrome Coronavirus 2 (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) is a positive-stranded single-stranded RNA virus with an envelope, belonging to the family of Coronavirus, type B coronavirus, belonging to Severe Acute Respiratory Syndrome Sign related coronavirus species. It can invade the human body through the upper respiratory tract of humans, and use ACE2 expressed on the surface of a variety of cells as receptors to achieve infection. The main infected organs include the lungs, heart, kidneys and other major organs. This resulted in an outbreak of COVID-19 (COVID-19) at the end of 2019. To date, more than 5 million COVID-19 cases and nearly 330,000 deaths have been reported to WHO.

Similar to SARS-CoV, SARS-CoV-2 also uses its highly glycosylated spike protein S (Spike protein, S protein) to complete host cell receptor binding and virus infection in the form of a trimer. The S protein has two subunits, S1 and S2. The receptor-binding domain (RBD) region of the S1 subunit can recognize and bind the host cell's angiotensin-converting enzyme 2 (hACE2). The S2 subunit mediates the membrane fusion between the virus and the host cell. Moreover, 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. Existing research shows that the main target of vaccine development against coronavirus is its S protein. However, as of now, no preventive vaccines or treatments against SARS-CoV-2 have been approved. Blocking monoclonal antibody (mAb) is one of the best drug candidates for neutralizing viral infections due to its extraordinary antigen specificity.

For SARS-CoV-2, the preparation of monoclonal neutralizing antibodies against S protein and the development of detection of antibodies against serum SARS-CoV-2 S protein are all urgent research content.

Summary of the invention

One aspect of the present invention provides a monoclonal antibody or functional fragment thereof against the spike protein of SARS-CoV-2, the antibody or functional fragment thereof 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 comprises an amino acid sequence selected from SEQ ID NO: 22, 28, 34, 40, 46, 52, 58, 64, 70, or 76 or the amino acid sequence shown includes at most three (e.g., one, two Or three) variants of amino acid mutations; the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 23, 29, 35, 41, 47, 53, 59, 65, 71 or 77 or the amino acid sequence shown comprises Variants with at most three (for example, one, two or three) amino acid mutations; the HCDR3 comprises a variant selected from SEQ ID NO: 24, 30, 36, 42, 48, 54, 60, 66, 72, or 78 The amino acid sequence shown or the amino acid sequence shown contains up to three (for example, one, two, or 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: 25, 31, 37, 43, 49, 55, 61, 67, 73 or 79 or the amino acid sequence shown includes at most three (e.g., one, two One or three) variants of amino acid mutations; the LCDR2 sequence comprises an amino acid sequence selected from SEQ ID NO: 26, 32, 38, 44, 50, 56, 62, 68, 74 or 80 or the amino acid sequence shown The sequence contains at most three (for example, one, two or three) variants of amino acid mutations; the LCDR3 sequence contains selected from SEQ ID NO: 27, 33, 39, 45, 51, 57, 63, 69, 75 Or the amino acid sequence shown in 81 or the amino acid sequence shown contains at most three (for example, one, two or three) amino acid mutation variants.

In some embodiments of the present invention, the monoclonal antibody or functional fragment thereof, wherein the HCDR1 sequence comprises SEQ ID NO: 22, 28, 34, 40, 46, 52, 58, 64, 70 Or the amino acid sequence shown in 76; the HCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 23, 29, 35, 41, 47, 53, 59, 65, 71, or 77; the HCDR3 sequence includes the selected From the amino acid sequence shown in SEQ ID NO: 24, 30, 36, 42, 48, 54, 60, 66, 72 or 78; and

The LCDR1 sequence includes an amino acid sequence selected from SEQ ID NO: 25, 31, 37, 43, 49, 55, 61, 67, 73, or 79; the LCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 26, 32 , 38, 44, 50, 56, 62, 68, 74 or 80; the LCDR3 sequence comprises an amino acid sequence selected from SEQ ID NO: 27, 33, 39, 45, 51, 57, 63, 69, 75 Or the amino acid sequence shown in 81.

In some embodiments of the present invention, in the monoclonal antibody or functional fragment thereof, 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 NO: 22, 23 and 24 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutations. And LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 25, 26, and 27 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutation variants ；

(b) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 28, 29 and 30 or the amino acid sequence shown includes at most three (for example, one, two or three) amino acid mutations. And LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 31, 32, and 33 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutation variants ；

(c) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 34, 35 and 36 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutations. And LCDR1, LCDR2, and LCDR3 respectively include the amino acid sequence shown in SEQ ID NO: 37, 38, and 39 or the shown amino acid sequence includes at most three (for example, one, two, or three) amino acid mutations ；

(d) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 40, 41 and 42 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutations. And LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 43, 44 and 45 or the shown amino acid sequence respectively include at most three (for example, one, two or three) amino acid mutation variants ；

(e) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 46, 47 and 48 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutations. And LCDR1, LCDR2, and LCDR3 respectively include the amino acid sequence shown in SEQ ID NO: 49, 50, and 51 or the shown amino acid sequence includes at most three (for example, one, two, or three) amino acid mutations ；

(f) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 52, 53 and 54 or the amino acid sequence shown includes at most three (for example, one, two or three) amino acid mutations. Body; and LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 55, 56 and 57 or the shown amino acid sequence respectively comprise at most three (for example, one, two or three) amino acid mutation variants ；

(g) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 58, 59 and 60 or the amino acid sequence shown includes at most three (for example, one, two or three) amino acid mutations. And LCDR1, LCDR2, and LCDR3 respectively include the amino acid sequence shown in SEQ ID NO: 61, 62, and 63 or the shown amino acid sequence includes at most three (for example, one, two, or three) amino acid mutations ；

(h) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 64, 65 and 66 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutations. And LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 67, 68, and 69 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutation variants ；

(i) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 70, 71 and 72 or the amino acid sequence shown includes at most three (for example, one, two or three) amino acid mutations. And LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 73, 74 and 75 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutation variants ;or

(j) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 76, 77 and 78 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutations. And LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 79, 80, and 81 or the amino acid sequence shown respectively includes at most three (for example, one, two or three) amino acid mutation variants .

In some preferred embodiments of the present invention, in the monoclonal antibody or functional fragment thereof, 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 sequences shown in SEQ ID NOs: 22, 23 and 24, and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27;

(b) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 31, 32 and 33;

(c) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 34, 35 and 36 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 37, 38 and 39;

(d) The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 40, 41 and 42, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 43, 44 and 45, respectively;

(e) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 and the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 49, 50 and 51;

(f) The HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 52, 53, and 54 respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 55, 56 and 57, respectively;

(g) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 58, 59 and 60 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 61, 62 and 63;

(h) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 64, 65 and 66 and the LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 67, 68 and 69 respectively;

(i) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 70, 71 and 72 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 73, 74 and 75; or

(j) The HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 76, 77, and 78, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 79, 80, and 81, respectively.

In some embodiments of the present invention, the monoclonal antibody or functional fragment thereof, wherein the heavy chain variable region sequence includes SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, The amino acid sequence shown in 16, 18 or 20 has at least 80% (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical amino acid sequences; and

The light chain variable region sequence comprises at least 80% (for example, 80%, 81%, and the amino acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21). 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%) identical amino acid sequence.

In some embodiments of the present invention, the monoclonal antibody or functional fragment thereof, wherein the heavy chain variable region sequence comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 , 18 or 20; and the light chain variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21.

In some embodiments of the present invention, the monoclonal antibody or functional fragment thereof, wherein the heavy chain variable region and the light chain variable region are selected from the following sequences:

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

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

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

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

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

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

(g) The heavy chain variable region contains at least 80% (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%) of the sequence shown in SEQ ID NO: 14 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical amino acid sequence, the light chain variable region Contains at least 80% of the sequence shown in SEQ ID NO: 15 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical amino acid sequences;

(h) The heavy chain variable region contains at least 80% (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%) of the sequence shown in SEQ ID NO: 16 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical amino acid sequence, the light chain variable region Contains at least 80% of the sequence shown in SEQ ID NO: 17 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical amino acid sequences;

(i) The heavy chain variable region contains at least 80% (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%) of the sequence shown in SEQ ID NO: 18 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical amino acid sequence, the light chain variable region Contains at least 80% of the sequence shown in SEQ ID NO: 19 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical amino acid sequence; or

(j) The heavy chain variable region contains at least 80% (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%) of the sequence shown in SEQ ID NO: 20 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical amino acid sequence, the light chain variable region Contains at least 80% of the sequence shown in SEQ ID NO: 21 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical amino acid sequences.

In some preferred embodiments of the present invention, the monoclonal antibody or functional fragment thereof, wherein the heavy chain variable region and the light chain variable region 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;

(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;

(g) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 15;

(h) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 17;

(i) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19; or

(j) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 20, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 21.

In some embodiments of the present invention, the monoclonal antibody is a rabbit monoclonal antibody, or a humanized antibody or a fully human antibody based on the rabbit monoclonal antibody.

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

In one embodiment of the present invention, the polynucleotide of the present invention comprises a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody or functional fragment thereof, and a nucleotide sequence encoding the monoclonal antibody or functional fragment thereof The nucleotide sequence of the light chain variable region.

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

In a further aspect, the present invention provides a host cell or 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.

The present invention further 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.

In some embodiments of the present invention, the coronavirus is selected from SARS-CoV, MERS-Cov or SARS-Cov-2, preferably SARS-Cov-2.

In another aspect, the present invention provides a kit for detecting coronavirus, which contains the anti-SARS-CoV-2 spike protein monoclonal antibody or functional fragment thereof of the present invention.

In some embodiments of the present invention, the coronavirus is selected from SARS-CoV, MERS-Cov or SARS-Cov-2, preferably SARS-Cov-2.

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 of 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, the ACE2 protein can prevent the S protein from invading human ACE2 overexpressing cells. The monoclonal antibody of the present invention can also be used for the detection of SARS-CoV-2S-RBD cells. The monoclonal antibody provided by the present invention can recognize 4 kinds of epitopes of the antigen, and the diversity of the antibody provides convenience for the development of the detection kit.

Description of the drawings

Figure 1: The results of rabbit serum titer detection after immunization.

Figure 2: Rabbit monoclonal antibodies (BS-R2B2, BS-R2B17, 4G6, 12D3, 39G6) specifically bind to S recombinant protein.

Figure 3: Rabbit monoclonal antibody (BS-R2B2, BS-R2B17, 4G6, 12D3, 39G6) blocking the binding of S protein and ACE2 protein.

Figure 4: Rabbit monoclonal antibodies (BS-R2B2, BS-R2B17) prevent SARS-CoV-2 pseudovirus from entering human ACE2 overexpressing cells.

Figure 5: Rabbit monoclonal antibodies (BS-R2B17, 12D3) specifically bind to cell lines expressing S protein by flow cytometry.

Figure 6: Pairing curve diagram of monoclonal antibody BS-R2B30 and BS-R1B8.

the term

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" is intended to refer to an immunoglobulin molecule consisting 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 (for example, 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 (composed of domains CH1, CH2, and CH3). 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), There is a more conserved region inserted in between called the framework region (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 "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 cores 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 an antigen-binding fragment of an antibody and antibody analogs, which usually include 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, antibody 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 drug administration. 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 the replacement of existing amino acid residues with different amino acid residues 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 person being treated, cells, tissues, organs or biological fluids. "Administration" and "treatment" can refer to, for example, treatment methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting the reagent with the cell and contacting the reagent 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 for administration, including various excipients, diluents and buffers, etc. These substances are suitable for human and/or animal administration without excessive adverse side effects, and at the same time It is suitable for maintaining the vitality of the drug or active agent 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 as well as elements and components that include more than one unit.

Detailed ways

Unless otherwise specified, the methods and materials of the embodiments described below are conventional products that can be purchased on the market. Those skilled in the art to which the present invention pertains 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: Preparation of anti-SARS-CoV-2 S protein rabbit monoclonal antibody based on single B cell platform

(1) Recombinant protein S-RBD-His (sequence shown in SEQ ID NO:1) is used as the animal immune antigen. Female New Zealand white rabbits were immunized with 200μg S-RBD-His. Subsequently, the immunization was repeated every 2 weeks to boost the New Zealand white rabbits for a total of 3 times. 3 New Zealand white rabbits titer after three immunizations are up to ¹⁰⁵ or more. One rabbit (7316) showing the highest antibody titer (Figure 1) was subjected to single B cell selection or fusion 4 days after the last immunization.

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

(2) Enrichment and screening of single B cells

The rabbit spleen was extracted and homogenized to produce a single cell suspension. The plasma B cells were enriched according to the following steps, and single B cells that could secrete SARS-CoV-2 S protein antibody were sorted using the Beacon platform. a. Resuspend the cells in 0.5ml PBS and add 50μl (5%) goat serum to block for 5min; b. Add 5μg/ml biotin-anti-mouse IgG polyclonal antibody and incubate at room temperature for 15min; c. Continue to add 100μl Selection Cocktail (Stemcell ); d. Mix and incubate at room temperature for 15 minutes; e. According to EasySep ^TM (Stem Cell. Technologies Inc.) Biotin Positive protocol (https://www.stemcell.com/easysep-biotin-positive-selection-kit-ii.html ) Obtain plasma B cells; f. Put the obtained plasma B cells on the Beacon machine and use OptoSelect 14,000 chips; g. Screen positive clones on the chip: Use Antigen Beads to screen positive clones; h. Export: Will be screened The positive clones were exported to a 96-well plate and quickly stored in a -80°C refrigerator.

(3) Single B cell RNA extraction and expression plasmid construction

Refer to

The cDNA Recovery for Cell Exports protocol of the platform (https://www.berkeleylights.com/systems/beacon/) extracts RNA and reverse transcribes it into cDNA. Using the cDNA as a template, the method of homologous recombination is used to amplify the light chain. The DNA fragments of variable region + constant region and heavy chain variable region + constant region are inserted into pcDNA expression vectors to form expression plasmids.

Example 2: Preparation of anti-SARS-CoV-2 S protein rabbit monoclonal antibody based on the hybridoma platform

(1) Hybridoma fusion and screening

The rabbit spleen of step (1) of Example 1 was 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. The spleen cells: SP2/0=2:1 were fused using an electrofusion device. The fused cells were resuspended in 150ml of DMEM/10%FBS medium containing hybridoma cell selection agents thymidine, hypoxanthine and aminopterin, and transferred to 15×96 wells with a volume of 100μl with a pipette In the board. The plates were incubated in 5% CO ₂ at 37°C. After 10 days of culture, the indirect ELISA described below was used to screen hybridomas that could secrete antibodies against the S protein, and the methods described below were used for RNA extraction and PCR amplification.

(2) Indirect ELISA 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 200 μl/well of PBST containing 1% BSA at 37° C. for 0.5 hour. Then the blocking solution was discarded, 100 μl of hybridoma cell culture supernatant was added to each plate, and then incubated at room temperature for 1 hour. The plate was washed three times with PBST, and incubated with 100 μl/well of horseradish peroxidase-conjugated goat anti-rabbit IgG (Fab-specific) (GenScript) at 37°C for 0.5 hours. The plate was washed five times with PBST, then TMB color developing solution (GenScript) was added and incubated in the dark at room temperature for 15 minutes. The reaction was terminated by adding 50μl of 1M HCl stop solution (Sigma). Use a microplate reader to read the plate at 450nm.

(3) RNA extraction and construction of expression plasmid

RNA was extracted by Trizol method and reverse transcribed into cDNA. Using cDNA as template, the method of homologous recombination was used to amplify DNA fragments containing light chain variable region + constant region and heavy chain variable region + constant region, and insert them separately In the pcDNA expression vector, an expression plasmid is formed.

Example 3: Sequencing of variable region of rabbit monoclonal antibody and recombinant production of antibody

1) Transform the expression plasmids constructed in Example 1 and Example 2 into competent cells, shake the bacteria for 3-4 hours, directly pipette 100 μL of bacterial solution into the plate, cover the plate, and take it to a bench shaker for 3 minutes . Take the coated plate back to the ultra-clean workbench, and then place the plate in a 37°C water-proof constant temperature incubator overnight for 12-16 hours. Pick a single colony of shake bacteria, and sequence. Finally, the unique V-region nucleotide/protein sequences of clones BS-R2B2, BS-R2B7, BS-R1B8, BS-R1B12, BS-R2B15, BS-R2B17, BS-R2B30, 4G6, 12D3 and 39G6 were obtained. The amino acid sequence of the variable region of the antibody is as follows:

BS-R2B2 heavy chain variable region amino acid sequence (SEQ ID NO: 2):

BS-R2B2 light chain variable region amino acid sequence (SEQ ID NO: 3):

BS-R2B7 heavy chain variable region amino acid sequence (SEQ ID NO: 4):

BS-R2B7 light chain variable region amino acid sequence (SEQ ID NO: 5):

BS-R1B8 heavy chain variable region amino acid sequence (SEQ ID NO: 6):

BS-R1B8 light chain variable region amino acid sequence (SEQ ID NO: 7):

BS-R2B12 heavy chain variable region amino acid sequence (SEQ ID NO: 8):

BS-R2B12 light chain variable region amino acid sequence (SEQ ID NO: 9):

BS-R2B15 heavy chain variable region amino acid sequence (SEQ ID NO: 10):

BS-R2B15 light chain variable region amino acid sequence (SEQ ID NO: 11):

BS-R2B17 heavy chain variable region amino acid sequence (SEQ ID NO: 12):

BS-R2B17 light chain variable region amino acid sequence (SEQ ID NO: 13):

BS-R2B30 heavy chain variable region amino acid sequence (SEQ ID NO: 14):

BS-R2B30 light chain variable region amino acid sequence (SEQ ID NO: 15):

4G6 heavy chain variable region amino acid sequence (SEQ ID NO: 16):

Amino acid sequence of 4G6 light chain variable region (SEQ ID NO: 17):

12D3 heavy chain variable region amino acid sequence (SEQ ID NO: 18):

12D3 light chain variable region amino acid sequence (SEQ ID NO: 19):

39G6 heavy chain variable region amino acid sequence (SEQ ID NO: 20):

39G6 light chain variable region amino acid sequence (SEQ ID NO: 21):

The CDR sequences of the antibody heavy chain and light chain are shown in Table 1.

Table 1: CDR region sequence of antibody

2) The above plasmids were co-transfected into HEK293-6E (ATCC) cells and cultured in a shake flask at 37°C for 6 days, and then the supernatant was collected for antibody purification. The column was re-equilibrated 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 for 2 hours at room temperature. After washing the column with 1× the above buffer, use sterile 0.1M sodium citrate (pH 3.5) to elute IgG, collect the eluate and use One-half volume of sterile 1M Tris-HCl (pH9) for neutralization. Under sterile conditions, the product buffer was exchanged for PBS (pH 7.4) to remove any elution buffer and concentrate the sample. After concentration, the antibody was quantified by OD280nm using an extinction coefficient Ec (0.1%) of 1.43.

3) The purified antibody was analyzed by SDS-PAGE with a 10% precast gel (GenScript) on the BioRad electrophoresis system. The gel was stained with Estin2.0 (GenScript) and the molecular size and purity were estimated by comparing the stained band with Protein Ladder (GenScript).

Example 4: 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 purified antibody of 1 μg/ml was added to the first well, and diluted in a 3-fold gradient, a total of 11 test concentration gradients and 1 zero well. Then incubate for 1 hour at room temperature. The plate was washed three times with PBST, and incubated with 100 μl/well of horseradish peroxidase-conjugated goat anti-rabbit IgG (Fc specific) (GenScript) at 37°C for 0.5 hours. The plate was washed 4 times with PBST, then TMB color developing solution (GenScript) was added and incubated in the dark at room temperature for 15 minutes. The reaction was terminated by adding 50 μl of 1M HCl stop solution. Use a microplate reader to read the plate at 450nm. _{The EC 50 of} each clone is as follows:

BS-R2B2: EC ₅₀ =0.96ng/ml; BS-R2B7:EC ₅₀ =2.046ng/ml;

BS-R2B12: EC ₅₀ = 1.56ng/ml; BS-R2B15: EC ₅₀ = 2.021ng/ml;

BS-R2B17: EC ₅₀ =1.68ng/ml; BS-R2B30: EC ₅₀ =0.96ng/ml;,

BS-R1B8: EC ₅₀ =2.67ng/ml; 12D3:EC ₅₀ =0.90ng/ml;

4G6: EC ₅₀ = 1.28 ng/ml; 39G6: EC ₅₀ = 1.17 ng/ml.

The results of an exemplary rabbit monoclonal antibody specifically binding to S recombinant protein are shown in FIG. 2.

Example 5: 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 human ACE2 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 50 μl of 3μg/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 and 1 zero well. Then add 50 μl of biotin-labeled S protein (concentration of 0.15 μg/ml) to each well, and incubate for 1 hour at 37°C. The plate was washed 3 times with PBST, and incubated with 100 μl/well of streptavidin HRP (SA-HRP, GenScript) at 37°C for 10 minutes. Finally, the plate was washed 5 times with PBST, and 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 50 μl of 1M HCl stop solution. 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 of} each clone is as follows:

BS-R2B2: IC ₅₀ = 123.1ng/ml; BS-R2B17: IC ₅₀ = 171.0ng/ml;

12D3: IC ₅₀ =119.6ng/ml; 4G6: IC ₅₀ =182.3ng/ml;

39G6: IC ₅₀ =208.6ng/ml.

Example 6: Functional detection of monoclonal antibodies

Incubate the SARS-CoV-2 pseudovirus (Genscript) with 2 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 add the Bio-Glo ^TM Luciferase Substrate Working Solution to the corresponding wells of the assay plate. Use EnVision to read the luminescence signal, and finally analyze the data. The results of the BS-R2B2 and BS-R2B17 pseudovirus neutralization experiments are shown in Figure 4. The antibody can effectively prevent the SARS-CoV-2 pseudovirus from entering the human ACE2 overexpressing cells.

Example 7: Flow Cytometry Detection of Monoclonal Antibodies

The SARS-CoV-2 S-RBD plasmid was transfected into 293F cells by the PEI method. After 60 hours, the cells were collected and washed with PBS. The cells were fixed with 4% PFA for 15 minutes at 4°C, and the membrane was permeabilized with 0.5 mg/ml saponin for 15 minutes at room temperature. Then wash the cells, add anti-SARS-CoV-2 S-RBS antibody (10μg/ml), and incubate at 4°C for 40 minutes. Incubate with Alexa Flour 488 conjugated goat anti-rabbit IgG (3μg/ml) secondary antibody at 4°C for 30 minutes. The cells were analyzed by flow cytometry, and the data was analyzed by FlowJob analysis software. Among them, 7 clones can be used for flow cytometry. As shown in Figure 5, clonal antibodies such as BS-R2B17 and 12D3 can be used to detect SARS-CoV-2 S-RBD 293F cells overexpressing SARS-CoV-2. 97.8% of overexpressing SARS-CoV-2 S-RBD 293F cells can be specifically bound by BS-R2B17, indicating that the prepared rabbit monoclonal antibodies against SARS-CoV-2 S-RBD can be used to express SARS-CoV-2 S- Detection of RBD cells.

Example 8: Monoclonal antibody epitope identification

Competitive ELISA was used to evaluate the epitope of purified antibodies. 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 a pair (50 μl of one labeled biotin test antibody and 50 μl (10 μg/ml) of purified antibody) were added to each well for competition experiment. Then incubate at 37°C for 1 hour. The plate was washed 3 times with PBST, and incubated with 100 μl/well of streptavidin HRP (SA-HRP, GenScript) at 37°C for 15 minutes. 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. Stop the reaction by adding 50μl of 1M HCl stop solution. Use a microplate reader to read the plate at 450nm. Judgment criteria: For example, the OD of the self-competitive well of the supernatant BS-R2B30 is 0.545. Compared with the value of 0 well, the difference of the OD value is close to 1.0, which has obvious competitive effect. When the difference between the OD value of the supernatant BS-R2B12 and the supernatant BS-R2B30-HRP mixture is about 0.9, there is a clear competitive effect. Therefore, it can be determined that the supernatant BS-R2B30 and the supernatant BS-R2B12 recognize the same epitope. The difference between the OD value of the supernatant BS-R2B17 and the supernatant BS-R2B30-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 BS-R2B17 and the supernatant BS-R2B30 recognize different epitopes of the antigen. BS-R2B30, BS-R2B12 is an epitope (epitope 1), BS-R2B17 is an epitope (epitope 2) BS-R2B2, 12D3, 4G6, 39G6 is an epitope (epitope 3), BS- R1B8 is an epitope (epitope 4).

Example 9: Paired detection of monoclonal antibodies

Sandwich ELISA is used to evaluate the paired detection of purified antibodies. The ELISA plate was coated with 100 μl/well of 1 μg/ml purified antibody 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 a series of diluted SARS-CoV-2 S-RBD of different concentrations were added to each well, and then incubated at 37°C for 1 hour, and the plate was washed with PBS-T (0.05% Tween). Subsequently, a biotin-labeled antibody was added, and then incubated at 37°C for 0.5 hours, washed with PBST 3 times, and incubated with 100 μl/well of streptavidin HRP (SA-HRP, GenScript) at 37°C for 15 minutes. 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. Stop the reaction by adding 50μl of 1M HCl stop solution. Use a microplate reader to read the plate at 450nm. The pairing results are shown in Table 2 and Figure 6. The pairing of BS-R2B2 and BS-R2B30 can be used for kit development, used for virus antigen detection, and can be used for clinical virus detection of SARS-CoV-2 patients with a sensitivity of 0- 20pg/ml.

Table 2: Pairing results of BS-R2B2 and BS-R2B30

Claims (17)

1. A monoclonal antibody or functional fragment thereof against the spike protein of SARS-CoV-2, said antibody or functional fragment thereof 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 comprises an amino acid sequence selected from SEQ ID NO: 22, 28, 34, 40, 46, 52, 58, 64, 70, or 76 or a variant of the amino acid sequence that contains at most three amino acid mutations; The HCDR2 includes an amino acid sequence selected from SEQ ID NO: 23, 29, 35, 41, 47, 53, 59, 65, 71, or 77 or a variant of the amino acid sequence including at most three amino acid mutations; HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 24, 30, 36, 42, 48, 54, 60, 66, 72, or 78 or a variant of the amino acid sequence shown with 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: 25, 31, 37, 43, 49, 55, 61, 67, 73, or 79 or a variant of the amino acid sequence that includes at most three amino acid mutations; The LCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 26, 32, 38, 44, 50, 56, 62, 68, 74, or 80 or a variant of the amino acid sequence that includes at most three amino acid mutations; The LCDR3 sequence includes an amino acid sequence selected from SEQ ID NO: 27, 33, 39, 45, 51, 57, 63, 69, 75, or 81 or a variant of the amino acid sequence including at most three amino acid mutations.
2. 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: 22, 28, 34, 40, 46, 52, 58, 64, 70, or 76; the HCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 23, 29 , 35, 41, 47, 53, 59, 65, 71 or 77; the HCDR3 sequence comprises an amino acid sequence selected from SEQ ID NO: 24, 30, 36, 42, 48, 54, 60, 66, 72 Or the amino acid sequence shown in 78; and

   The LCDR1 sequence includes an amino acid sequence selected from SEQ ID NO: 25, 31, 37, 43, 49, 55, 61, 67, 73, or 79; the LCDR2 sequence includes an amino acid sequence selected from SEQ ID NO: 26, 32 , 38, 44, 50, 56, 62, 68, 74 or 80; the LCDR3 sequence comprises an amino acid sequence selected from SEQ ID NO: 27, 33, 39, 45, 51, 57, 63, 69, 75 Or the amino acid sequence shown in 81.
3. 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: 22, 23 and 24 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: 22, 23 and 24. ID NO: The amino acid sequences shown in 25, 26 and 27 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: 28, 29 and 30 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: 28, 29 and 30 ID NO: The amino acid sequences shown in 31, 32, and 33 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 sequence shown in SEQ ID NOs: 34, 35 and 36 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: 34, 35 and 36. ID NO: The amino acid sequences shown in 37, 38 and 39 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: 40, 41 and 42 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: 40, 41 and 42 ID NO: The amino acid sequences shown in 43, 44 and 45 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 sequence shown in SEQ ID NOs: 46, 47 and 48 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: 46, 47 and 48. ID NO: The amino acid sequences shown in 49, 50 and 51 or the amino acid sequences shown respectively contain at most three amino acid mutations;

   (f) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 52, 53 and 54 or the amino acid sequence shown comprises a variant of at most three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 52, 53 and 54 ID NO: The amino acid sequences shown in 55, 56 and 57 or the shown amino acid sequences each contain at most three amino acid mutations;

   (g) The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 58, 59 and 60 or the amino acid sequences shown respectively include up to three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 58, 59 and 60. ID NO: The amino acid sequences shown in 61, 62 and 63 or the amino acid sequences shown respectively contain at most three amino acid mutations;

   (h) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 64, 65 and 66 or the amino acid sequences shown respectively comprise at most three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: 64, 65 and 66. ID NO: The amino acid sequence shown in 67, 68 and 69 or the amino acid sequence shown contains at most three amino acid mutations;

   (i) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 70, 71 and 72 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: 70, 71 and 72 ID NO: The amino acid sequence shown in 73, 74 and 75 or the amino acid sequence shown contains at most three amino acid mutations; or

   (j) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 76, 77 and 78 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: 76, 77 and 78 ID NO: 79, 80, and 81 amino acid sequence shown or the amino acid sequence shown contains at most three amino acid mutation variants.
4. 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 respectively comprise the amino acid sequences shown in SEQ ID NOs: 22, 23 and 24, and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27;

   (b) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 31, 32 and 33;

   (c) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 34, 35 and 36 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 37, 38 and 39;

   (d) The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 40, 41 and 42, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 43, 44 and 45, respectively;

   (e) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 and the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 49, 50 and 51;

   (f) The HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 52, 53, and 54 respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 55, 56 and 57, respectively;

   (g) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 58, 59 and 60 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 61, 62 and 63;

   (h) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 64, 65 and 66 and the LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 67, 68 and 69 respectively;

   (i) The HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 70, 71 and 72 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 73, 74 and 75; or

   (j) The HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 76, 77, and 78, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 79, 80, and 81, respectively.
5. The monoclonal antibody or functional fragment thereof according to any one of claims 1 to 4, wherein the heavy chain variable region sequence comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 , 16, 18 or 20 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, 13, 15, 17, 19 or 21.
6. The monoclonal antibody or functional fragment thereof according to claim 5, wherein:

   The heavy chain variable region sequence includes the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20; and the light chain variable region sequence includes SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 amino acid sequence.
7. The monoclonal antibody or functional fragment thereof according to claim 5, 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;

   (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;

   (g) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 14, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 15. % Identical amino acid sequence;

   (h) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 16, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 17. % Identical amino acid sequence;

   (i) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 18, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 19. % Identical amino acid sequence; or

   (j) The heavy chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 20, and the light chain variable region includes an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 21. % Identical amino acid sequence.
8. 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;

   (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;

   (g) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 15;

   (h) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 17;

   (i) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19; or

   (j) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 20, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 21.
9. An isolated polynucleotide encoding the anti-SARS-CoV-2 spike protein monoclonal antibody or functional fragment thereof according to any one of claims 1-8.
10. 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.
11. An expression vector comprising the polynucleotide according to claim 9 or 10.
12. A host cell or a cell-free expression system comprising the expression vector according to claim 11.
13. 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.
14. 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.
15. The use according to claim 14, wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-Cov or SARS-Cov-2, preferably SARS-Cov-2.
16. A kit for detecting coronavirus, said kit comprising the monoclonal antibody or functional fragments thereof according to any one of claims 1-8.
17. 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.

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