CN107056938B - Humanized high-affinity antibody 10K for resisting H7N9 avian influenza virus and application thereof - Google Patents

Abstract

The invention obtains the human source anti-H7N 9 avian influenza virus high affinity antibody 10K by screening based on the single cell sorting technology, and the amino acid sequences of the light chain and the heavy chain variable regions are respectively shown as SEQ ID No.2 and SEQ ID No. 5. The antibody specifically binds to H7N9 avian influenza virus 7 type hemagglutinin protein with high affinity, and can mediate killing (ADCC) of effector cells mainly comprising NK cells to H7N9 influenza virus infected cells. The antibody 10K can be used for developing a therapeutic drug for highly pathogenic avian influenza infection and can also be used for developing an antigen detection reagent for H7N9 influenza virus.

Description

Humanized high-affinity antibody 10K for resisting H7N9 avian influenza virus and application thereof

Technical Field

The invention relates to the technical fields of genetic engineering, single cell sorting technology and antibody library display, in particular to a humanized anti-H7N 9 avian influenza virus high-affinity antibody 10K and application thereof.

Background

Since 2013 in 2 months, human infection cases caused by avian influenza A virus H7N9 are discovered in the southeast China, and gradually spread to other provinces and cities in China from Shanghai and Anhui. By 2 months in 2017, 1079 influenza infections with H7N9 are reported in the whole country, the number of deaths is 417, and the mortality rate is as high as 38.6%. At present, no specific medicine for treating the high-pathotype avian influenza infection exists, and the curative effect of the existing nonspecific medicine neuraminidase inhibitor oseltamivir is only limited to the early stage of infection. Since the prophase symptoms of highly pathogenic H7N9 infection are not clearly distinguishable from the common flu, most of the infected individuals miss the optimal treatment time and progress to Acute Respiratory Distress Syndrome (ARDS) and multiple organ failure. The antigen of the highly pathogenic avian influenza virus has urgent need for rapid and sensitive diagnosis.

Monoclonal antibodies are highly homogeneous immunoglobulins produced by a single B cell clone directed against only a particular epitope. Due to small side effects, the humanized monoclonal antibody is not required to be humanized when being applied to human disease treatment, so that the loss of affinity in the process is avoided. The human monoclonal antibody plays a great role in the prevention and control process of infectious diseases, can neutralize viruses and mediate killing of effector cells to virus infected cells, and the protection effect of the human monoclonal antibody is fully proved in animal infection models of HIV, influenza virus, MERS virus, dengue fever virus, hantavirus, measles virus, RSV virus, rabies virus and the like. In addition, the monoclonal antibody with high affinity and high specificity is also a key component of a rapid detection reagent of pathogen.

Since 2008, single B-cell sorting and antibody gene direct amplification technology has become one of the major approaches to human antibody screening (Tiller et al, Journal of Immunological Methods 329, (2008), 112-124). In 2010, Wu et al (Science 329, (2010)856-861) used a flow cytometric sorting technique to sort single memory B cells with antigen specificity in HIV-infected peripheral blood, further used a reverse transcription PCR technique to directly amplify antibody genes VH and V kappa/V lambda of single cells, and then inserted the above gene fragments into a full-length IgG recombinant vector to perform eukaryotic cell transfection and expression purification, thereby successfully obtaining a famous HIV broad-spectrum neutralizing monoclonal antibody VRC 01. Because the technology can obtain antibody genes and recombinant antibody proteins from single cells in the shortest time and can ensure the original pairing of heavy chains and light chains of the antibodies (the antibody functions are optimal), the single cell sorting and antibody gene direct amplification technology quickly becomes a heavy tool in the field of antibody development. So far, the technology is successfully applied to screening broad-spectrum neutralizing monoclonal antibodies of HIV, influenza, MERS, Ebola, dengue fever and other viruses, and a plurality of high-efficiency antibodies obtained by the technology enter clinical drug research in sequence. The genetic engineering antibody from a single B cell brings new hope and broad prospect for the fields of rapid antigen detection and antibody pharmacy.

The number of the H7N 9-specific antibodies reported at home and abroad since the outbreak of the H7N9 influenza virus. Although the existing antibody HAs better capability of neutralizing H7N9 influenza virus, the research on the high and low affinity and subtype specificity of HA7 needs to be further carried out.

Disclosure of Invention

The invention aims to provide a human anti-H7N 9 avian influenza virus high-affinity antibody 10K and application thereof.

The concept of the invention is as follows: fully human antibodies are currently obtained mainly by antibody library display screening techniques and single B cell sorting techniques. Compared with single cell sorting method, the monoclonal antibody obtained by screening antibody library has lower probability of original light and heavy chain pairing, and the original light and heavy chain pairing of the antibody can maximize the function of the antibody. The light and heavy chains of the antibody gene obtained by the single cell sorting method are from original pairing theoretically, so the probability of obtaining the high-efficiency functional antibody by the single cell sorting method is far higher than that of an antibody library display screening method.

To achieve the object of the present invention, the present inventors derived anti-H7N 9 avian influenza virus high affinity antibody 10K or an active fragment thereof, the amino acid sequences of the light and heavy chain hypervariable regions CDR1, CDR2 and CDR3 of the high affinity antibody 10K or an active fragment thereof are shown in the following table:

the amino acid sequence of the light chain variable region of the high affinity antibody 10K, i) is shown in SEQ ID No.2, or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids to the sequence; and

ii) the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.5, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence.

The full-length amino acid sequences of the light chain and the heavy chain of the high-affinity antibody 10K are respectively shown as SEQ ID No.3 and SEQ ID No. 6.

The invention also provides a gene encoding the high affinity antibody 10K. Wherein, the nucleotide sequences of the coding light chain variable region and the heavy chain variable region are respectively shown as SEQ ID No.1 and SEQ ID No. 4.

The invention also provides an expression cassette, an expression vector or a cloning vector comprising a nucleic acid comprising a gene sequence encoding said high affinity antibody 10K.

The invention also provides a host cell containing the encoding gene of the high-affinity antibody 10K, or the expression cassette and the vector.

The invention also provides a single-chain antibody ScFv or Fab antibody or a whole antibody immunoglobulin IgG obtained by modifying the high-affinity antibody 10K or the active fragment thereof.

The active fragment of the H7N9 influenza virus high-affinity antibody 10K is a Fab fragment of a human H7N9 affinity antibody 10K capable of binding with type 7 hemagglutinin protein.

The human anti-H7N 9 avian influenza virus high-affinity antibody 10K can be prepared by the following method: obtaining the variable region fragment of the hemagglutinin protein specific antibody of the H7N9 virus by utilizing a single memory B cell sorting and antibody gene direct amplification method, then constructing a eukaryotic transient expression vector of a complete IgG antibody by a genetic engineering method, and expressing and purifying the IgG protein.

The antibody is determined by specific gene sequences of hypervariable regions (CDRs) existing in variable regions of antibody light chain and heavy chain genes, and can obtain an effectively expressed functional antibody which is specifically combined with H7N9 influenza virus hemagglutinin protein in eukaryotic cells. It binds with high efficiency to the hemagglutinin protein type 7 (FIG. 3, FIG. 4) and has the function of mediating the killing (ADCC) of effector cells against H7N9 virus infected cells (FIG. 5).

The light chain and heavy chain genes of the H7N9 influenza virus high-affinity antibody 10K are derived from peripheral blood B cells of a patient infected by H7N9 avian influenza virus. The variable region sequence characteristics of the antibody are formed by the combination of three CDR region sequences corresponding to the light chain and heavy chain variable regions and the framework region sequences between the CDR regions, the light chain variable region gene of the antibody 10K belongs to the family IGKV3-20, and the heavy chain belongs to the family IGHV 5-51. The function of the antibody protein is determined by specific nucleotide sequences and complementary sequences thereof in complementarity determining regions CDR1, CDR2 and CDR3 of the light chain and heavy chain variable regions of the antibody gene, and 6 corresponding amino acid sequences of the CDR regions form a specific antigen binding region of the antibody, thereby determining the antigen binding characteristics of the antibody and the anti-H7N 9 avian influenza virus functional characteristics.

In addition, in consideration of the degeneracy of codons, for example, a gene sequence encoding a 10K variable region can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding an antibody having the same function. One skilled in the art can artificially synthesize and modify genes according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.

Further, the light chain variable region and the heavy chain variable region of the influenza H7N9 high affinity antibody 10K of the present invention may be recombined to form a Fab antibody of smaller molecular weight or a single chain antibody (ScFv) of smaller molecular weight. Fab antibodies and single chain antibodies also have the property of recognizing the surface antigen of H7N9 influenza virus. The antibody with small molecular weight has strong penetrating power and is easy to enter local tissues or cells to play a role.

The Fab antibody-encoding gene and the SCFV antibody-encoding gene can be cloned into an expression vector, and the Fab antibody and the single-chain antibody (ScFv) can be obtained by transforming a host and inducing expression.

The functional identification of the obtained IgG antibody 10K is carried out by methods such as SDS-PAGE, ELISA, in vitro virus neutralization experiments, antibody-dependent cell-mediated cytotoxicity killing (ADCC) experiments and the like, and the result shows that the molecular weight of the expressed and purified human IgG antibody 10K is in accordance with the expectation (figure 2), the expressed and purified human IgG antibody can be efficiently and specifically combined with the 7-type hemagglutinin protein (figure 3 and figure 4), and the antibody-dependent cell-mediated killing (ADCC) activity of effector cells on H7N9 virus infected cells (figure 5) is achieved.

The invention also provides application of the high-affinity antibody 10K or the active fragment thereof in preparing a medicament for preventing or treating diseases caused by the H7N9 avian influenza virus, particularly respiratory diseases.

The invention also provides application of the high-affinity antibody 10K or the active fragment thereof in preparation of an H7N9 avian influenza virus antigen detection reagent or detection kit.

The invention further provides a medicament, a detection reagent or a detection kit containing the high-affinity antibody 10K or the active fragment thereof.

The invention adopts a multicolor fluorescence labeled flow cytometry sorting method to obtain 7-type hemagglutinin protein antigen specificity memory B cells, utilizes a reverse transcription kit to obtain cDNA of a single B cell, utilizes an antibody specificity primer to amplify antibody gene variable region fragments, utilizes a genetic engineering technology to construct a eukaryotic IgG expression vector, and utilizes a cell engineering technology to obtain purified target IgG antibody 10K through transient transfection and an antibody purification technology; the antibody product which has high affinity and can specifically recognize the H7N9 influenza virus and mediate and kill the H7N9 virus infected cells is obtained by utilizing the obtained humanized high affinity H7N9 avian influenza virus genetic engineering antibody variable region gene in the form of Fab antibody, single chain antibody gene and full-length IgG antibody in prokaryotic cells, eukaryotic cells (including yeast cells) and any recombinant protein expression system and expressing and producing the antibody, or any other gene containing the antibody gene after modification on the basis of the variable region gene, so that the antibody product can be used for developing H7N9 influenza virus antigen rapid detection kits or preparing specific antibody drugs for clinically preventing and treating diseases caused by the H7N9 avian influenza virus, such as acute respiratory infectious diseases.

The antibody 10K provided by the invention can be efficiently combined with H7N9 influenza virus hemagglutinin protein HA₇Directed against HA₇EC of protein affinity₅₀0.00698 μ g/ml (0.0465nM) (fig. 3). Specifically, antibody 10K has an OD of 0.00698. mu.g/ml (0.0465nM) in an ELISA assay for affinity detection with hemagglutinin protein type 7₄₅₀The value may still reach half the maximum value. The antibody provided by the invention can be used for developing an H7N9 influenza virus antigen detection reagent.

The antibody 10K provided by the invention can specifically recognize H7N9 influenza virus hemagglutinin protein HA₇. The affinity of IgG10K for hemagglutinin proteins of H1N1, H5N1 and H7N9 influenza viruses was tested by ELISA assay, and it was found that IgG10K specifically recognizes hemagglutinin protein of H7N9, EC₅₀0.00698 ug/ml (0.0465nM) but had no binding capacity for the hemagglutinin proteins of H1N1 and H5N1 influenza viruses, indicating HA₇Subtype-specific antibodies (FIG. 4). The antibody provided by the invention can be used for developing an H7N9 influenza virus antigen detection reagent.

The antibody 10K provided by the invention can effectively mediate killing of effector cells (such as NK cells) to H7N9 infected cells. Specifically, the percentage of 10 μ g/ml 10K antibody mediated killing of MDCK cells infected with H7N9 reached 43.5% (fig. 5). The antibody provided by the invention can be used for treating patients infected by H7N9 influenza virus.

Drawings

FIG. 1 is a schematic diagram of the platform of the fully human monoclonal antibody technology based on single cell sorting and antibody gene amplification.

FIG. 2 shows the molecular weight and purity of the antibodies detected by SDS-PAGE electrophoresis of the IgG10K of the invention, both denatured and non-denatured.

FIG. 3 is an example of the present invention in which IgG10K is directed against HA₇Protein affinity assay results (ELISA). Wherein, HA₇The protein was coated on ELISA plates at a concentration of 1. mu.g/ml and IgG10K was diluted in a 4-fold gradient starting at 333.3 nm. When OD is reached₄₅₀The concentration of antibody 10K at half of the maximum value is determined as its concentration against HA₇EC of (1)₅₀The value is obtained. IgG9114L was also tested as a negative control by gradient dilution with HA₇IgG9114L is the broad-spectrum neutralizing antibody CR9114 (references: Dreyfus C, LaurenNS, Kwaks T, Zuijdest D, Khayat R, EkiertDC, et al. Highlyconcerved protectedpeptides on flubenza B viruses. science 2012Sep 14; 337(6100): 1343-8).

FIG. 4 shows the result of the affinity detection (ELISA method) of IgG10K against HA proteins of different subtypes and different fragments in the present invention. IgG10K was incubated at a concentration of 0.1. mu.g/ml. H7 is coated Influenza A H7N9(A/Shanghai/2/2013) hemagglutinin protein 1 μ g/ml (Acrobiosystems, cat # HA 9-V5227); h1 is coated Influenza AH1N1(A/Beijing/22808/2009) hemagglutinin protein 1 μ g/ml (Beijing Yinqiao Hibiscus Biotechnology Co., Ltd., cat # 40035-V08H-100), H5 is coated Influenza A H5N1(A/Common magpie/Hong Kong/2256/2006) hemagglutinin protein 1 μ g/ml (Beijing Yi Qianzhou Biotechnology Co., Ltd., cat # 11700-V08H-100).

FIG. 5 shows the ADCC activity of IgG10K against H7N9 infected cells in the present invention. After SP17 virus infects MDCK cells for 48h, 10 mug/ml antibody is added to adsorb the cells, the supernatant is removed, the healthy human peripheral blood lymphocytes are added, and the lactate dehydrogenase activity (OD) of the supernatant is detected after culturing for 4h₄₉₂). Each antibody was set with 6 test replicates, 4 negative control replicates (no antibody added) and 4 positive control replicates(lysis solution was added) and the average was used to calculate cell killing activity with ADCC% 100 × (OD)_{492 test well}-OD_{492 negative control wells})/(OD_{492 positive control}-OD_{492 negative control})。

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 human high affinity antibody 10K against H7N9 avian influenza virus and method for preparation thereof

1. Labelling of antigens

HA was purchased from Acrobiosystems (HA9-V5227) Inc₇The protein was diluted to 0.5-2mg/ml with PBS, and then biotin (EZ-Link) from Thermal company was used^TMSulfo-NHS-LC-Biotin, 21335) HA was prepared according to the kit protocol₇Protein labeling (the ratio of the number of molecules is protein: biotin is 1: 20-100), incubating for 0.5-2h at room temperature in dark place, centrifuging with 10KD centrifugal semi-permeable column (Merck Millipore, UFC501096) at 8000g for 4-6 times, supplementing with sterile PBS, removing the excess biotin molecules, and labeling HA₇The protein molecule will be used for screening HA₇Specific memory B cells.

2. Antigen-specific memory B cell sorting and reverse transcription

Peripheral blood mononuclear cells from H7N 9-infected patients at convalescent stage were isolated, washed once with PBS, and then resuspended to 10% in 1% BSA in PBS (PBSA)⁶～10⁸mL, biotinylated HA was added first₇Protein was brought to a concentration of 10 μ g/ml, mixed well and incubated at 4 ℃ for half an hour, then washed once with PBS, resuspended PBMCs with the same volume of PBSA, and then mixed according to a 1: 50 to volume ratio of 50, mouse anti-human CD19(APC-H7), mouse anti-human IgG (APC), mouse anti-human IgM (Percp-cy5.5) and mouse anti-human CD27(FITC) were purchased from biolegend, while mixing them as follows: 200 to 7AAD (Percp-cy5.5) and Jackson immun from Invitrogen corporationThe streptoavidin-PE (016. sup. sand. 110. sup. sand. 084) from olab was mixed and incubated at 4 ℃ for half an hour. PBS was washed twice and PBSA was resuspended for cell sorting. Sorting of CD19+, IgM-, IgG +, CD27+, 7aad-, HA-from patient PBMCs using BD FACS ariaIII₇PE + Positive cells, 1 cell per well, were treated with 5. mu.l of resuspension buffer (SuperScript)^TMIII cell direct cDNA Synthesis System, invitrogen 18080-.

3. Amplification of variable regions of antibody genes

The heavy chain variable region (VH) and light chain variable region (VK/VL) of the antibody gene were amplified using primers and amplification protocols described by Tiller, 2008, published in J Immunol Methods. VH, VK and VL are amplified in two rounds, firstly 3-5 mul cDNA is taken as a template, first round amplification is carried out by using antibody variable region gene leader sequence specific primers, specifically, primers 1-4 and primers 45 and 46 are used for amplifying heavy variable region (VH); primers 17, 18, 19 and primer 51 amplify the Kappa chain variable region (VK) and primers 31-37 and primer 57 amplify the Lambda chain variable region (VL). Then, 5 mul of products of the first round of amplification is used as a template to carry out second round of amplification by utilizing nested primers with enzyme cutting sites, and specifically, primers 5-16 and primers 48-50 are used for amplifying heavy chain variable regions (VH); primers 21-30 and 53-56 amplify the Kappa chain variable region (VK), and primers 38-43 and 58 amplify the Lambda chain variable region (VL). The PCR system used Super HiFi PCR Mix (KT212)2 XPCR Mix from Tiangen. The PCR procedure was: 3min at 95 ℃; 30sec at 95 ℃, 30sec at 58-60 ℃, 1min at 72 ℃ and 50 cycles; 10min at 72 ℃; 10min at 4 ℃. The amplified product was electrophoresed on 1.5% agarose gel (120V, 40min) to cut a variable region fragment of about 400 bp. In order to avoid false positives due to contamination, the cDNA in wells without sorted cells was used as a template for the first round of PCR and the reaction without added first round PCR product was used as a negative control for the second round of PCR. And (3) pairing the amplified heavy chain variable region and the light chain variable region, and performing the next enzyme digestion and vector construction only if the pairing of the heavy chain variable region and the light chain variable region is amplified simultaneously.

4. Construction of transient eukaryotic expression vectors

Transient expression vectors were constructed using the antibody gene transient expression vector system described in J Immunol Methods, published by Tiller in 2008. The system contains 3 vectors which are respectively used for expressing IgG1 heavy chain, Kappa chain and Lambda chain and are respectively named as IgH (Access Number DQ407610), IgK (Access Number rDQ407610) and IgL (Access Number FJ 517647). According to the system, the antibody capable of obtaining full-length IgG1 by eukaryotic cell transfection can be obtained by carrying out double enzyme digestion, connection and transformation on the vector and the variable region fragment with the enzyme digestion site. The heavy chain vector and the fragment are subjected to double enzyme digestion by AgeI and SalI, the kappa chain vector and the fragment are subjected to double enzyme digestion by AgeI and BsiwI, and the Lambda chain vector and the fragment are subjected to double enzyme digestion by AgeI and XhoI. After transforming escherichia coli, selecting monoclonals, sequencing vector insertion sequences by using a 5' absense primer (primer 44), selecting 3-5 bacterial monoclonals for sequencing in each transformation, and translating the insertion sequences and the vector into complete antibody fragments with consistent three sequencing results, wherein the insertion sequences are different from the original vector restriction enzyme sequence and are successfully constructed as the vector.

Primers (5 '-3') used for antibody gene amplification:

1L-VH 1ACAGGTGCCCACTCCCAGGTGCAG

2L-VH 3AAGGTGTCCAGTGTGARGTGCAG

3L-VH 4/6CCCAGATGGGTCCTGTCCCAGGTGCAG

4L-VH 5CAAGGAGTCTGTTCCGAGGTGCAG

5AgeI VH1CTGCAACCGGTGTACATTCCCAGGTGCAGCTGGTGCAG

6AgeI VH1/5CTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGCAG

7AgeI VH3CTGCAACCGGTGTACATTCTGAGGTGCAGCTGGTGGAG

8AgeI VH3–23CTGCAACCGGTGTACATTCTGAGGTGCAGCTGTTGGAG

9AgeI VH4CTGCAACCGGTGTACATTCCCAGGTGCAGCTGCAGGAG

10AgeI VH 4–34CTGCAACCGGTGTACATTCCCAGGTGCAGCTACAGCAGTG

11AgeI VH 1–18CTGCAACCGGTGTACATTCCCAGGTTCAGCTGGTGCAG

12AgeI VH 1–24CTGCAACCGGTGTACATTCCCAGGTCCAGCTGGTACAG

13AgeI VH3–33CTGCAACCGGTGTACATTCTCAGGTGCAGCTGGTGGAG

14AgeI VH 3–9CTGCAACCGGTGTACATTCTGAAGTGCAGCTGGTGGAG

15AgeI VH4–39CTGCAACCGGTGTACATTCCCAGCTGCAGCTGCAGGAG

16AgeI VH 6–1CTGCAACCGGTGTACATTCCCAGGTACAGCTGCAGCAG

17L Vκ1/2ATGAGGSTCCCYGCTCAGCTGCTGG

18L Vκ3CTCTTCCTCCTGCTACTCTGGCTCCCAG

19L Vκ4ATTTCTCTGTTGCTCTGGATCTCTG

20Pan VκATGACCCAGWCTCCABYCWCCCTG

21AgeI Vκ1–5CTGCAACCGGTGTACATTCTGACATCCAGATGACCCAGTC

22AgeI Vκ1–9TTGTGCTGCAACCGGTGTACATTCAGACATCCAGTTGACCCAGTCT

23AgeI Vκ1D–43CTGCAACCGGTGTACATTGTGCCATCCGGATGACCCAGTC

24AgeI Vκ2–24CTGCAACCGGTGTACATGGGGATATTGTGATGACCCAGAC

25AgeI Vκ2–28CTGCAACCGGTGTACATGGGGATATTGTGATGACTCAGTC

26AgeI Vκ2–30CTGCAACCGGTGTACATGGGGATGTTGTGATGACTCAGTC

27Age Vκ3–11TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACACAGTC

28Age Vκ3–15CTGCAACCGGTGTACATTCAGAAATAGTGATGACGCAGTC

29Age Vκ3–20TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACGCAGTCT

30Age Vκ4–1CTGCAACCGGTGTACATTCGGACATCGTGATGACCCAGTC

31L Vλ1GGTCCTGGGCCCAGTCTGTGCTG

32L Vλ2GGTCCTGGGCCCAGTCTGCCCTG

33L Vλ3GCTCTGTGACCTCCTATGAGCTG

34L Vλ4/5GGTCTCTCTCSCAGCYTGTGCTG

35L Vλ6GTTCTTGGGCCAATTTTATGCTG

36L Vλ7GGTCCAATTCYCAGGCTGTGGTG

37L Vλ8GAGTGGATTCTCAGACTGTGGTG

38AgeI Vλ1CTGCTACCGGTTCCTGGGCCCAGTCTGTGCTGACKCAG

39AgeI Vλ2CTGCTACCGGTTCCTGGGCCCAGTCTGCCCTGACTCAG

40AgeI Vλ3CTGCTACCGGTTCTGTGACCTCCTATGAGCTGACWCAG

41AgeI Vλ4/5CTGCTACCGGTTCTCTCTCSCAGCYTGTGCTGACTCA

42AgeI Vλ6CTGCTACCGGTTCTTGGGCCAATTTTATGCTGACTCAG

43AgeI Vλ7/8CTGCTACCGGTTCCAATTCYCAGRCTGTGGTGACYCAG

44Ab sense GCTTCGTTAGAACGCGGCTAC

45CγCH1GGAAGGTGTGCACGCCGCTGGTC

46CμCH1GGGAATTCTCACAGGAGACGA

47IgG(internal)GTTCGGGGAAGTAGTCCTTGAC

48Sall JH 1/2/4/5TGCGAAGTCGACGCTGAGGAGACGGTGACCAG

49Sall JH 3TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG

50Sall JH 6TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG

51Cκ543GTTTCTCGTAGTCTGCTTTGCTCA

52Cκ494GTGCTGTCCTTGCTGTCCTGCT

53BsiWI Jκ1/4GCCACCGTACGTTTGATYTCCACCTTGGTC

54BsiWI Jκ2GCCACCGTACGTTTGATCTCCAGCTTGGTC

55BsiWI Jκ3GCCACCGTACGTTTGATATCCACTTTGGTC

56BsiWI Jκ5GCCACCGTACGTTTAATCTCCAGTCGTGTC

57CλCACCAGTGTGGCCTTGTTGGCTTG

58XhoI CλCTCCTCACTCGAGGGYGGGAACAGAGTG

5. preparation and purification of monoclonal antibodies

2ml of E.coli containing a vector which succeeded in the construction was inoculated into 200ml of 2YT medium (Trypton16g/L, Yeastextract 10g/L, NaCl 5g/L, Ampicillin 100. mu.g/ml) and cultured at 37 ℃ and 220rpm for 16 hours. Centrifuging at 6000g for 15min to collect thallus, and extracting plasmid according to the kit (Thermal Co.) (

HiPure plasmid maxiprep Kit, K210006) and filtering and sterilizing for later use. By using

FreeStyle^TM293Expression Medium (Thermal,12338018) culture based on CO at 37 ℃₂The shaking incubator cultures 293F (Thermal, R79007) cells, which are grown to a density of 1.0 × 10 according to the S-shaped growth curve⁶At/ml, transfection was performed. Each 30ml of 293F cells was transfected with 37.5ug of antibody plasmid, where the heavy and light chain plasmid mass ratio was 2: 3, after transfection cells in 8% CO₂Shaking culture at 125rpm in a shaking incubator at 37 deg.C for 96-120h, and centrifuging to collect supernatant. Monoclonal IgG antibodies were purified according to the protocol using Protein G Agarose Beads (13103-PNAE-RN) from Beijing Yinqiao Shenzhou. The antibody was eluted with 0.2M glycine solution pH2.2, immediately neutralized with 1/20 volumes of 1M Tris-HCl (pH9.2), and then centrifuged 4-6 times at 8000g using a 10KD semipermeable spin column (Merck Millipore, UFC501096), supplemented with sterile PBS, and the excess glycine molecules were removed. Nanodrop spectrophotometer detection of OD₂₈₀The antibody concentration was determined by filtration through a 0.22 μm filter, sterilized, dispensed and stored at-20 ℃ or-80 ℃.

The platform diagram of the fully human monoclonal antibody technology based on single cell sorting and antibody gene amplification is shown in figure 1.

Example 2 monoclonal antibody 10K against HA₇EC of protein₅₀Measurement of

Dilution of HA with ELISA coating₇The protein was brought to 1. mu.g/ml, and then 50. mu.l per well was coated on ELISA plates (Corning, 3690) overnight at 4 ℃. PBST washing plate, 5% skim milk PBS blocking more than 2 h. Monoclonal antibodies were added to the blocked ELISA plates after 4-fold gradient dilution with 5% skim milk in PBS starting at 50. mu.g/ml. A total of 10 gradients were set, two replicates, wells without antibody were negative controls, and gradient dilutions of IgG9114L were added (i.e., CR9114, Dreyfus C, Laursen NS, Kwaks T, Zuijdiest D, Khayat R, Ekiert DC, et alnserved protective epitopes on underfluenza B viruses.science 2012Sep 14; 337(6100) 1343-8) is an antibody control. Goat anti-human IgGFc-HRP (1:10000, Jackson immunolab,109-₄₅₀The value is obtained. The OD was plotted by averaging the duplicate wells for each gradient₄₅₀Concentration curves, curve fitting according to the sigmoidal dose response model of GraphPad Prism, and calculating the OD₄₅₀The concentration of antibody corresponding to the half of the maximum value (i.e., 1.6), which is the antibody directed against HA₇EC of (1)₅₀The value is obtained.

Example 3 determination of the neutralizing Activity of monoclonal antibody 10K against H7N9(A/Shenzhen/SP17/2014(H7N 9)))

Inoculating MDCK cells on a 96-well cell culture plate, culturing until the cell density is about 70% -90%, and washing with PBS for two times for later use; 3-fold dilution of the monoclonal antibodies to be detected (100. mu.g/ml start) in a 96-well microtiter plate, with 4-well replicates per antibody to be detected, 8 gradients; TCID based on virus₅₀The virus was diluted to a titer of 200TCID₅₀100 mul; mixing 60 mul of diluted virus liquid with 60 mul of diluted monoclonal antibody sample, and incubating in an incubator at 37 ℃ for 2h to ensure that the antigen and the antibody fully act; then 100. mu.l of the virus plasma mixture was placed in washed 96-well MDCK cells, infected in a 37 ℃ incubator for 1 hour, 150. mu.l of MEM medium supplemented with TPCK pancreatin was used to replace the virus solution, and the mixture was placed in a 37 ℃ CO system₂The incubator is cultured for 72h, CPE is observed, and the result of CPE is confirmed by an erythrocyte agglutination test. Distance ratios were calculated according to the Reed-Muench method, using concentrations capable of inhibiting infection in half-well MDCK cells as a measure of the IC of neutralizing activity of the monoclonal antibody₅₀The value is obtained.

Example 4 detection of antibody-dependent cell-mediated cytotoxicity killing (ADCC) Activity of monoclonal antibody 10K

Inoculating MDCK cells on a 96-well cell culture plate, culturing until the cell density is about 70% -90%, and washing twice with PBS; with 1000TCID₅₀Viral load per well infected MDCK cells per well in 5% CO₂Then, the supernatant was removed and 170. mu.l of a virus growth medium (MEM culture) was added theretoThe gene [ Thermal, 11095-080) contained 1% penicillin-streptomycin diabody (Thermal,15140163) and 0.1-0.5. mu.g/ml TPCK pancreatin (Sigma, T1426)]Is placed in CO at 37 DEG C₂Culturing for 48h in a cell culture box with the concentration of 5 percent; the virus-infected cells were washed twice with PBS, and MEM medium (Thermal, 11095-₂Culturing in 5% cell culture box for 30min, removing supernatant, washing with PBS once, removing supernatant, adding 200 μ l of 5 × 10-containing solution per well⁵MEM medium from healthy human PBMC, incubated at 37 ℃ in CO₂The cells were cultured for 4 hours in a 5% cell culture chamber. The Lactate Dehydrogenase (LDH) activity in the culture supernatant was measured using a non-radioactive cytotoxicity assay kit (G1780) from Promega according to the protocol. Enzyme-linked immunosorbent assay (OD) reading₄₉₂Wells with no or no antibody associated with influenza added were negative controls and wells with kit lysate (no PBMC added) were positive controls, ADCC% ═ 100 × (OD)_{492 test well}-OD_{492 negative control wells})/(OD_{492 positive control}-OD_{492 negative control})。

The antibody 10K provided by the invention can be efficiently combined with H7N9 influenza virus hemagglutinin protein HA₇Directed against HA₇EC of protein affinity₅₀0.00698 μ g/ml (0.0465nM) (fig. 3). Specifically, antibody 10K has an OD of 0.00698. mu.g/ml (0.0465nM) in an ELISA assay for affinity detection with hemagglutinin protein type 7₄₅₀The value may still reach half the maximum value. The antibody provided by the invention can be used for developing an H7N9 influenza virus antigen detection reagent.

The antibody 10K provided by the invention can specifically recognize H7N9 influenza virus hemagglutinin protein HA₇. The affinity of IgG10K for hemagglutinin proteins of H1N1, H5N1 and H7N9 influenza viruses was tested by ELISA assay, and it was found that IgG10K specifically recognizes hemagglutinin protein of H7N9, EC₅₀0.00698 ug/ml (0.0465nM) but had no binding capacity for the hemagglutinin proteins of H1N1 and H5N1 influenza viruses, indicating HA₇Subtype-specific antibodies (FIG. 4). The antibody provided by the invention can be used for development of H7N9 influenza virus antigen detection reagentAnd (4) sending.

The antibody 10K provided by the invention can effectively mediate killing of effector cells (such as NK cells) to H7N9 infected cells. Specifically, the percentage of 10 μ g/ml 10K antibody mediated killing of MDCK cells infected with H7N9 reached 43.5% (fig. 5). The antibody provided by the invention can be used for treating patients infected by H7N9 influenza virus.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Shenzhen Pulan technology Limited

<120> human anti-H7N 9 avian influenza virus high-affinity antibody 10K and application thereof

<130>KHP171113095.8

<160>6

<170>PatentIn version 3.3

<210>1

<211>328

<212>DNA

<213> antibody 10K light chain variable region DNA sequence

<400>1

gaaattgtgt tgacacagtc tccaggcacc ctgtcattgt ctccagggga cagagccacc 60

ctctcctgca gggccagtga gactgttagc agcggtttca tagcctggta ccaacagaaa 120

cctggccagt ctcccaggct cctcatctat ggtgcatcca gcagggccgc tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccataaa cgggctggag 240

cctgaagatt ttgcagtcta ttactgtcac cagtatggtt cctcacctcc gttcactttt 300

ggccagggga ccaacctgga catcaaac 328

<210>2

<211>110

<212>PRT

<213> antibody 10K light chain variable region

<400>2

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Asp Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Thr Val Ser Ser Gly

20 25 30

Phe Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Gly Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ser Ser Pro

8590 95

Pro Phe Thr Phe Gly Gln Gly Thr Asn Leu Asp Ile Lys Arg

100 105 110

<210>3

<211>216

<212>PRT

<213> full length of antibody 10K light chain

<400>3

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Asp Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Thr Val Ser Ser Gly

20 25 30

Phe Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Gly Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ser Ser Pro

85 90 95

Pro Phe Thr Phe Gly Gln Gly Thr Asn Leu Asp Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>4

<211>366

<212>DNA

<213> antibody 10K heavy chain variable region DNA sequence

<400>4

gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60

tcctgtaagg gttctggata cagctttacc aactactgga tcggctgggt gcgccagatg 120

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 180

agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcaa caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc gagggggtgg 300

cataacgcat actactactt tggtatggac gtctggggcc aagggaccac ggtcaccgtc 360

tcttca 366

<210>5

<211>122

<212>PRT

<213> antibody 10K heavy chain variable region

<400>5

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Asn Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Trp His Asn Ala Tyr Tyr Tyr Phe Gly Met Asp Val Trp

100 105 110

Gly Gln Gly ThrThr Val Thr Val Ser Ser

115 120

<210>6

<211>452

<212>PRT

<213> full length of heavy chain of antibody 10K

<400>6

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Asn Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Trp His Asn Ala Tyr Tyr Tyr Phe Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

Claims (10)

1. The human anti-H7N 9 avian influenza virus high affinity antibody 10K or an active fragment thereof, wherein the amino acid sequences of the light and heavy chain hypervariable regions CDR1, CDR2 and CDR3 of the antibody 10K or an active fragment thereof are shown in the following table:

2. the high affinity antibody 10K according to claim 1, wherein the amino acid sequence of the light chain variable region is represented by SEQ ID No.2 and the amino acid sequence of the heavy chain variable region is represented by SEQ ID No. 5.

3. A gene encoding the high affinity antibody 10K of claim 2.

4. The gene of claim 3, wherein the nucleotide sequences encoding the light chain variable region and the heavy chain variable region are shown in SEQ ID No.1 and SEQ ID No.4, respectively.

5. An expression cassette, an expression vector or a cloning vector comprising a nucleic acid comprising the gene sequence of claim 3 or 4.

6. A host cell comprising the gene of claim 3 or 4, or the expression cassette or vector of claim 5.

7. The high affinity antibody 10K or an active fragment thereof of claim 1 or 2, which is a single chain antibody ScFv or Fab antibody or a whole immunoglobulin IgG.

8. Use of the high affinity antibody 10K or an active fragment thereof according to claim 1 or 2 for the preparation of a medicament for the prevention or treatment of a disease caused by H7N9 avian influenza virus.

9. Use of the high affinity antibody 10K or an active fragment thereof according to claim 1 or 2 for preparing an antigen detection reagent or a detection kit for avian influenza virus H7N 9.

10. A medicament, a detection reagent or a detection kit comprising the high affinity antibody 10K or an active fragment thereof according to claim 1 or 2.

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