阅读说明：本技术 一种中和新冠病毒新表位的全人单克隆抗体17-2及其应用 (Fully human monoclonal antibody 17-2 for neutralizing neoepitope of new coronavirus and application thereof ) 是由 庾蕾 温莺芬 郭文靖 于 2020-12-04 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种中和新冠病毒新表位的全人单克隆抗体17-2及其应用。本发明通过B细胞培养及单克隆抗体技术,成功筛选出针对S2的中和抗体17-2。本发明提供的抗体17-2,可与S蛋白及S2结合,其EC50分别为0.034μg/ml和0.038μg/mL,可有效中和SARS-CoV-2病毒。与其它表位的S1-RBD和S1-NTD抗体联合应用于SARS-CoV-2感染的预防和治疗。(The invention belongs to the technical field of biology, and particularly relates to a fully human monoclonal antibody 17-2 for neutralizing a neoepitope of a new coronavirus and application thereof. The invention successfully screens out the neutralizing antibody 17-2 aiming at S2 by B cell culture and monoclonal antibody technology. The antibody 17-2 provided by the invention can be combined with S protein and S2, the EC50 of the antibody is 0.034 mu g/mL and 0.038 mu g/mL respectively, and the antibody can effectively neutralize SARS-CoV-2 virus. Combined with S1-RBD and S1-NTD antibodies of other epitopes for the prevention and treatment of SARS-CoV-2 infection.)

1. A monoclonal antibody 17-2, wherein the heavy chain amino acid sequence of the monoclonal antibody 17-2 is shown as SEQ ID NO. 1; the light chain amino acid sequence of the monoclonal antibody 17-2 is shown as SEQ ID NO. 2.

2. The monoclonal antibody 17-2 of claim 1, wherein the nucleotide sequence encoding the heavy chain amino acid sequence is set forth in SEQ ID No. 3; the nucleotide sequence for coding the light chain amino acid sequence is shown as SEQ ID NO. 4.

3. A method for preparing the monoclonal antibody 17-2 of claim 1, comprising the steps of:

s1, recognition and sorting of memory B cells: separating and separating memory B cells from the mononuclear cells of the peripheral blood of the infected person, and separating the memory B cells by staining with a fluorescence labeled antibody;

s2, memory B cell culture and culture supernatant antibody screening: adding cell growth factors into a 96-well cell culture plate, then adding the memory B cells sorted in the step S1, culturing for 7-10 days, and screening the B cells in the culture supernatant of the B cells aiming at SARS-CoV-2S protein antibody by using capture ELISA;

s3, cloning of antibody: extracting RNA of the B cell finally screened in the step S2, performing reverse transcription to form cDNA, amplifying heavy chain and light chain variable region genes of the antibody by using nested PCR, performing enzyme digestion cloning to an antibody expression vector containing a human IgG1 constant region, and analyzing heavy chain and light chain variable region gene sequences by IMGT/V-Quest;

s4, specificity analysis of antibody: the constructed expression vectors with determined sequences are co-transfected into 293T cells, culture supernatants containing the antibodies are harvested after 5-6 days, purified by a proteinA affinity column, and the binding activity of the antibodies is measured by ELISA for capturing different virus membrane protein antigens.

4. The method of claim 3, wherein the fluorescent label in step S1 includes IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB, and CD45-KO, and the fluorescent label of memory B cells is IgD-IgM-CD27+ CD38 low.

5. The method of claim 3, wherein the cell growth factors of step S2 include CpG, IL21, IL2, and radiation-irradiated healthy human PBMCs.

6. The method of claim 3, wherein step S3 is performed by nested PCR using Phusion ultra fidelity DNA polymerase.

7. Use of the monoclonal antibody 17-1 of claim 1 in combination with other epitopic S1-RBD and S1-NTD antibodies in the manufacture of a medicament for the treatment of SARS-CoV-2 infection.

8. The use of claim 7, wherein the medicament further comprises normal saline for injection and pharmaceutically common adjuvants.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a fully human monoclonal antibody 17-2 for neutralizing a neoepitope of a new coronavirus and application thereof.

Background

Monoclonal antibodies (mAbs) are undoubtedly the most promising class of biological agents for diagnosis and therapy, and have been used in the treatment of tumors, autoimmune diseases, and infectious diseases. The development of rapid separation, high-throughput sequencing technology and structural biology of human monoclonal antibodies makes the antibodies an important means for rapidly dealing with new infectious diseases.

SARS-CoV-2 is characterized by that it utilizes the spike protein of virus surface, i.e. S protein to mediate fusion of virus and cell membrane so as to make it invade host cell. The S protein includes two subunits, S1 and S2, and S1 is divided into N-terminal (NTD) and Receptor Binding (RBD) regions. When a virus is infected, the binding of S1-RBD and a receptor on a cell membrane firstly triggers S2 conformational change, so that the virus is fused with the cell membrane and enters a target cell. S1-RBD is considered to be the major protein inducing the production of neutralizing antibodies in the host. Therefore, in the face of a new coronavirus pneumonia epidemic situation, most researchers adopt a strategy of expressing RBD recombinant proteins to sort RBD specific B cells to obtain neutralizing antibodies. Such RBD antibodies have been reported in succession, and some have a strong ability to neutralize viruses (1.science.2020 Aug 21; 369(6506):956-963.2. Nature.2020Aug; 584(7821): 450-. However, the adoption of the strategy can lose some antibodies which are not directed to S1-RBD, and even lose antibodies which are produced by the body and have important antiviral effects. Such as antibodies against S2, may act by blocking fusion of the virus with the host cell membrane. Researchers have therefore changed the strategy to screen antibodies using protein S. Neutralizing antibodies against S1-NTD have been obtained by research teams at home and abroad from infection convalescent (1. Nature.2020Aug; 584(7821):450-456.2. science.2020Aug 7; 369(6504): 650-. However, no report on the acquisition of S2-neutralizing antibodies has been found so far.

The monoclonal antibody aiming at the single epitope is adopted as antiviral treatment, so that the phenomenon of virus escape, namely drug resistance, is easy to occur. Thus, 2-3 antibodies directed against different epitopes are often used in combination as antiviral therapeutic strategies. For example, ZMAP cocktail therapy with a cocktail of 3 mAbs (N Engl J Med.2016Oct 13; 375(15): 1448-.

Disclosure of Invention

Aiming at the defects generally existing in the prior art, the invention provides a fully human monoclonal antibody 17-2 for neutralizing a neoepitope of a new coronavirus and application thereof. The antibody 17-2 provided by the invention can be combined with S protein and S2, the EC50 of the antibody is 0.034 mu g/mL and 0.038 mu g/mL respectively, and the antibody can effectively neutralize SARS-CoV-2 virus. Combined with S1-RBD and S1-NTD antibodies of other epitopes for the prevention and treatment of SARS-CoV-2 infection.

In order to achieve the purpose, the invention adopts the technical scheme that:

a monoclonal antibody 17-2, the heavy chain amino acid sequence of said monoclonal antibody 17-2 is shown in SEQ ID NO. 1; the light chain amino acid sequence of the monoclonal antibody 17-2 is shown as SEQ ID NO. 2.

QLQLQESGPGLVKASETLSLTCTVSSGSVSSDSYYWSWIRQPPGKGLEWI GYIYYSGSTNYNPSLKSRVTMSVDTSKNQFSLKLDSVTAADTAAYYCARGPL GLYYYYMDVWGKGTTVTVSS(SEQ ID NO.1)；

DIQMTQSPSSLSASVGDRVTITCRASQSINNYLNWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFALTISGLQPEDFATYYCQESNSSPFTFGPGTKVD IK(SEQ ID NO.2)；

Preferably, the nucleotide sequence encoding the heavy chain amino acid sequence is shown as SEQ ID NO. 3; the nucleotide sequence for coding the light chain amino acid sequence is shown as SEQ ID NO. 4.

CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGGCTTCGGA GACCCTGTCCCTCACCTGCACTGTGTCTAGTGGCTCCGTCAGCAGTGATAG CTACTACTGGAGTTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGA TTGGATATATCTACTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGA GTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAG CTGGACTCTGTGACCGCTGCGGACACGGCCGCCTATTACTGTGCGAGAGGC CCACTAGGTCTGTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACG GTCACCGTCTCCTCA(SEQ ID NO.3)；

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG ACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAACAACTATTTAA ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTG CATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTG GGACAGATTTCGCTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAA CTTACTACTGTCAAGAGAGTAACAGTAGCCCATTCACTTTCGGCCCTGGGA CCAAAGTGGATATCAAA(SEQ ID NO.4)；

The invention also provides a method for preparing the monoclonal antibody 17-2, which comprises the following steps:

s1, recognition and sorting of memory B cells: separating and separating memory B cells from the mononuclear cells of the peripheral blood of the infected person, and separating the memory B cells by staining with a fluorescence labeled antibody;

s2, memory B cell culture and culture supernatant antibody screening: adding cell growth factors into a 96-well cell culture plate, then adding the memory B cells sorted in the step S1, culturing for 7-10 days, and screening the B cells in the culture supernatant of the B cells aiming at SARS-CoV-2S protein antibody by using capture ELISA;

s3, cloning of antibody: extracting RNA of the B cell finally screened in the step S2, performing reverse transcription to form cDNA, amplifying heavy chain and light chain variable region genes of the antibody by using nested PCR, performing enzyme digestion cloning to an antibody expression vector containing a human IgG1 constant region, and analyzing heavy chain and light chain variable region gene sequences by IMGT/V-Quest;

s4, specificity analysis of antibody: the constructed expression vector with determined sequence is co-transfected to 293T cells, culture supernatant containing the antibody is harvested after 5-6 days, and the antibody is purified by a proteinA affinity column and is captured by ELISA for detecting the binding activity of different virus membrane protein antigens.

Preferably, the fluorescence labeling in step S1 includes IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB and CD45-KO, and the fluorescence labeling of memory B cells is IgD-IgM-CD27+ CD38 low.

Preferably, the cell growth factors in step S2 include CpG, IL21, IL2, radiation-irradiated healthy human PBMCs.

Preferably, step S3 is performed by performing nested PCR amplification using Phusion ultra-fidelity DNA polymerase.

The invention also provides application of the monoclonal antibody 17-1 and S1-RBD and S1-NTD antibody mixture of other epitopes in preparation of a medicine for treating SARS-CoV-2 infection.

Preferably, the medicine also comprises normal saline for injection and auxiliary materials commonly used in pharmacy.

Compared with the prior art, the monoclonal antibody 17-2 provided by the invention has the following advantages:

(1) the strategy for obtaining the monoclonal antibody 17-2 provided by the invention is combined with a B cell culture and antibody cloning technology, and can separate a conformation dependent functional antibody which exists in vivo and is difficult to imitate in vitro;

(2) the strategy for obtaining the monoclonal antibody 17-2 provided by the invention does not need to use an antigen to label the memory B cells in the separation process, so that the strategy is not limited by a labeled antigen and can simultaneously screen antibodies combined with different proteins;

(3) according to the monoclonal antibody 17-2 provided by the invention, the heavy chain and the light chain of the antibody are paired with each other and are original to a naturally-generated antibody, the affinity is high, and the monoclonal antibody is more suitable for practical application.

Drawings

FIG. 1 is a diagram showing the construction results of heavy and light chain expression vectors for monoclonal antibody 17-2;

FIG. 2 is a graph showing the results of the binding reaction between the monoclonal antibody 17-2 and the virus S protein;

FIG. 3 is a graph showing the results of a pseudovirus neutralization test using monoclonal antibody 17-2;

FIG. 4 is a schematic of B cell culture and antibody cloning strategy.

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The flow cytometer is a Beckmann Coulter MoFlo Astrios EQ ultra-high-speed flow cytometry sorting system and can be purchased from Shanghai Zealand instruments and Equipment Co., Ltd; the cDNA Synthesis kit is SuperScript III First Strand Synthesis System, which is purchased from Invitrogen, USA and is numbered # 18080051; the fluorescent labeled antibodies IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB and CD45-KO are all available from the national medicine group chemical reagent company, Inc.; the cell growth factors CpG, IL21, IL2, and radiation-irradiated PBMC were all available from Guangzhou Haohjin Biotech, Inc.

Example 1 recognition and sorting of memory B cells

The specific process of recognition and sorting of memory B cells is as follows:

1.1 peripheral blood mononuclear cell separation: collecting peripheral vein EDTA anticoagulation of Zika infected patients in convalescent period, separating peripheral blood mononuclear cells by density gradient centrifugation method, and subpackaging 5 × 10⁶Putting the tube in liquid nitrogen for freezing;

1.2 fluorescent-labeled antibody staining: peripheral blood mononuclear cells were placed in a water bath at 37 ℃ and washed 3 times with PBS buffer, followed by staining with the addition of antibody, incubation for 15min in the dark at room temperature, and after washing with PBS buffer, 400 μ L of PBS buffer was added to suspend the cell up-flow instrument, which is a beckmann coulter MoFlo Astrios EQ ultra high-speed flow cytometry sorting system, staining with fluorescently labeled antibody: setting 9 analysis tubes, adding corresponding single fluorescent labeled antibody into the tubes 1 to 7 in the table, adding 7 mixed fluorescent labeled antibodies into the tube 9, and dyeing the sample tube as the tube 9 with a blank tube with cells only;

TABLE 1 combination of fluorescently labeled antibodies in each tube during memory B cell sorting

Pipe number	Dyeing pipe arrangement	Antibodies and fluorescence of labels	Volume (μ L)	Fluorescent channel
					1	Single dyeing	IgD-FITC	10	FL1
2	Single dyeing	CD19-ECD	5	FL3
					3	Single dyeing	7-AAD	10	FL4
4	Single dyeing	CD27-PC7	5	FL5
					5	Single dyeing	CD38-APC A750	2	FL8
6	Single dyeing	IgM-PB	5	FL9
					7	Single dyeing	CD45-KO	5	FL10
8	Blank tube	Without addition of antibody
					9	Sample tube	Mixture of 7 kinds of antibodies

1.3 sorting of memory B cells: the samples were analyzed on the machine, and live CD45 positive leukocytes were gated out based on 7-AAD and CD 45. B cells were circled according to CD 19. Definition of IgD in the B-cell population which is double-negative for IgM and IgD^-IgM^-CD27⁺CD38^lowThe population is memory B cells, which are sorted into 96-well cell culture plates containing cell culture fluid, 25-50 cells per well.

Example 2 memory B cell culture and screening of culture supernatant antibodies

CpG, IL21, IL2, radiation-irradiated healthy human PBMC, and EBV-containing B95.8 cell culture supernatant were added to 96-well cell culture plates containing memory B cells and cultured for 10 days. The B cell culture supernatants were screened for the presence of antibodies against SARS-CoV-2S protein by capture ELISA.

EXAMPLE 3 cloning of the antibody

3.1cDNA Synthesis: for B cells in which antibodies against SARS-CoV-2S protein are present in the supernatant, RNA is extracted and then reverse transcribed into cDNA. cDNA Synthesis was performed using a kit (SuperScript III First Strand Synthesis System, Invitrogen, # 18080051).

3.2 nested PCR amplification of the heavy and light chain variable region genes of the antibody: PCR primer sequences and specific PCR amplification procedures are described in the literature references (J Immunol methods.2008Jan 1; 329(1-2):112-24.) and Phusion ultra-Fidelity DNA polymerase (Phusion High-Fidelity PCR Master Mix with GC Buffer, NEB, # M0532s) was used for the reactions.

3.3 heavy and light chain variable region PCR products of the antibody were cloned by restriction enzymes (VH, AgeI/SalI, VK, AgeI/Xhol, VL, AgeI/BsiWI) into an antibody expression vector containing the constant region of human IgG1, see FIG. 1. The measured gene sequences of the heavy chain and light chain variable regions are analyzed by IMGT/V-Quest.

Example 4 analysis of the specificity of the antibodies

4.1 production of antibodies: co-transfecting 293T cells with the constructed heavy chain and light chain vectors of the antibody with determined sequences, and harvesting culture supernatant containing the antibody after 6 days;

4.2 purification of the antibody: purifying the culture supernatant containing the antibody by a proteinA affinity column, and determining the protein content;

4.3 binding reaction of antibody with S protein and neutralization assay:

the 17-2 antibody binds to S and S2 proteins with EC50 at 0.034. mu.g/mL and 0.038. mu.g/mL, respectively. Does not bind to the S1 protein, see fig. 2. The pseudovirus neutralization experiment system shows that the antibody can effectively neutralize SARS-CoV-2, and the IC50 is 0.5874 mu g/mL, which is shown in figure 3.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> eighth national hospital in Guangzhou City

<120> fully human monoclonal antibody 17-2 neutralizing neoepitope of new coronavirus and application thereof

<130> 2020.12.4

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 122

<212> PRT

<213> 17-2 heavy chain amino acid sequence (17-2 heavy chain amino acid sequence)

<400> 1

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Ala Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Ser Gly Ser Val Ser Ser Asp

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asp Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr

85 90 95

Cys Ala Arg Gly Pro Leu Gly Leu Tyr Tyr Tyr Tyr Met Asp Val Trp

100 105 110

Gly Lys Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 2

<211> 107

<212> PRT

<213> 17-2 light chain amino acid sequence (17-2 light chain amino acid sequence)

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Glu Ser Asn Ser Ser Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 3

<211> 366

<212> DNA

<213> Nucleotide sequence encoding the 17-2 heavy chain (Nucleotide sequence encoding 17-2 heavy chain)

<400> 3

cagctgcagc tgcaggagtc gggcccagga ctggtgaagg cttcggagac cctgtccctc 60

acctgcactg tgtctagtgg ctccgtcagc agtgatagct actactggag ttggatccgg 120

cagcccccag ggaagggact ggagtggatt ggatatatct actacagtgg gagcaccaac 180

tacaacccct ccctcaagag tcgagtcacc atgtcagtag acacgtccaa gaaccagttc 240

tccctgaagc tggactctgt gaccgctgcg gacacggccg cctattactg tgcgagaggc 300

ccactaggtc tgtactacta ctacatggac gtctggggca aagggaccac ggtcaccgtc 360

tcctca 366

<210> 4

<211> 321

<212> DNA

<213> Nucleotide sequence encoding 17-2 light chain (Nucleotide sequence encoding 17-2 light chain)

<400> 4

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattaac aactatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcgctctca ccatcagcgg tctgcaacct 240

gaagattttg caacttacta ctgtcaagag agtaacagta gcccattcac tttcggccct 300

gggaccaaag tggatatcaa a 321