CN107298712B

CN107298712B - Fully humanized neutralizing antibody against middle east respiratory syndrome coronavirus

Info

Publication number: CN107298712B
Application number: CN201610232621.1A
Authority: CN
Inventors: 谭文杰; 周剑芳; 邓瑶; 秦堃; 卢帅; 余双庆; 刘雪; 王文玲; 舒跃龙
Original assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Current assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Priority date: 2016-04-14
Filing date: 2016-04-14
Publication date: 2020-04-07
Anticipated expiration: 2036-04-14
Also published as: CN107298712A

Abstract

本发明公开了全人源化抗中东呼吸综合征冠状病毒中和抗体。本发明公开的中和抗体，由重链和轻链组成，轻链由VL和CL组成，重链由VH和CH组成，VH和VL均由决定簇互补区和框架区组成，决定簇互补区由CDR1、CDR2和CDR3组成；VH的CDR1如序列1第26‑33位所示，CDR2如序列1第51‑58位所示，CDR3如序列1第97‑114位所示；VL的CDR1如序列3第26‑34位或序列6第26‑34位所示，CDR2如序列3第52‑54位或序列6第52‑54位所示，CDR3如序列3第91‑101位、序列4第91‑102位、序列5第91‑101位或序列6第91‑100位所示。The invention discloses a fully humanized neutralizing antibody against Middle East respiratory syndrome coronavirus. The neutralizing antibody disclosed in the present invention is composed of a heavy chain and a light chain, the light chain is composed of VL and CL, the heavy chain is composed of VH and CH, both VH and VL are composed of a determinant complementary region and a framework region, and the determinant complementary region It consists of CDR1, CDR2 and CDR3; the CDR1 of VH is shown in the 26-33rd position of the sequence 1, the CDR2 is shown in the 51-58th position of the sequence 1, and the CDR3 is shown in the 97-114th position of the sequence 1; the CDR1 of the VL is shown as Sequence 3 at position 26-34 or sequence 6 at position 26-34, CDR2 as sequence 3 at 52-54 or sequence 6 at 52-54, CDR3 as sequence 3 at 91-101, sequence 4 at position 52-54 shown at position 91-102, position 91-101 of sequence 5, or position 91-100 of sequence 6.

Description

Fully humanized neutralizing antibody against middle east respiratory syndrome coronavirus

Technical Field

The invention relates to a fully humanized neutralizing antibody against middle east respiratory syndrome coronavirus in the field of biomedicine.

Background

Middle east respiratory syndrome coronavirus (MERS-Cov) is another highly pathogenic coronavirus that occurs after severe acute respiratory syndrome coronavirus (SARS-Cov). Since the first report in month 9 of 2012, 1379 confirmed cases of MERS-Cov infection in the laboratory, of which 531 died, were reported globally to the world health organization by 29 days 6 of 2015. To date, MERS-Cov infection epidemic has spread to the middle east, Europe, North Africa, Asia, 26 countries and regions in the United states, especially the epidemic outbreak in Korea in 2015, resulting in 164 infections and 23 deaths from one patient infected after middle east travel, further suggesting that the virus has acquired limited human-to-human transmission. However, no effective vaccine or antiviral drug is available at present, and MERS-Cov infection is one of the serious challenges facing public health.

MERS-Cov virus binds to specific receptors on target cells via spikes (spike, S) on the surface of the virus, thereby invading the host cell. The S protein is also one of the important antigens inducing the body to produce neutralizing antibodies. Passive immunotherapy is an effective, classical strategy for the treatment and prevention of viral infections, and methods for making fully human antibodies for human use include: mouse monoclonal antibody humanization, phage display, yeast display and EBV transformed B cell screening to obtain the human antibody gene combined with the antigen. Neutralizing antibodies 1E9, 1F8, 3C12, 3B11, m336, which have been found and reported, were obtained by screening phage display libraries from non-immunized humans. Therefore, there is a need to develop further therapeutic antibodies, such as humanized or fully human antibodies, with good affinity and low side effects to meet the needs of clinical therapy.

Disclosure of Invention

The technical problem to be solved by the present invention is how to diagnose and/or treat and/or prevent middle east respiratory syndrome coronavirus or infection with the virus.

To solve the above technical problems, the present invention provides, first, an antibody consisting of a heavy chain consisting of a variable region VL and a constant region CL, and a light chain consisting of a variable region VH and a constant region CH, both of the VH and VL consisting of a determinant complementary region consisting of CDR1, CDR2 and CDR3 and a framework region; the amino acid sequence of CDR3 of VH is shown as 97 th-114 th position of sequence 1; the amino acid sequence of CDR1 of VH is shown in the 26 th to 33 th positions of sequence 1; the amino acid sequence of CDR2 of VH is shown in 51-58 of sequence 1;

the amino acid sequence of the CDR1 of VL is H1) or H2) below:

H1) positions 26-34 of the sequence 3;

H2) positions 26-34 of SEQ ID NO. 6;

the amino acid sequence of the CDR2 of the VL is I1) or I2) below:

I1) 52 th to 54 th of the sequence 3;

I2) 52 th to 54 th of the sequence 6;

the amino acid sequence of the CDR3 of the VL is any one of the following J1) -J4):

J1) 91 st-101 th of sequence 3;

J2) 91 st to 102 th of the sequence 4;

J3) 91-101 of SEQ ID NO. 5;

J4) 91-100 of the sequence 6.

In the above antibody, the framework regions of both VH and VL may be derived from human.

In the antibody, the amino acid sequence of the VH is shown as 1 st to 125 th sites of a

sequence

1 or 1 st to 125 th sites of a sequence 2 in a sequence table, and the amino acid sequence of the VL is shown as 1 st to 111 th sites of a

sequence

3, 1 st to 112 th sites of a sequence 4, 1 st to 111 th sites of a sequence 5 or 1 st to 110 th sites of a sequence 6 in the sequence table.

In the above antibody, the CL and the CH may be both derived from human.

In the antibody, the amino acid sequence of the heavy chain is shown as a sequence 1 or a sequence 2 in a sequence table, and the amino acid sequence of the light chain is shown as a sequence 3, a sequence 4, a sequence 5 or a sequence 6.

In order to solve the above technical problems, the present invention also provides a derivative antibody of the antibody, wherein the derivative antibody is any one of the following a) to d):

a) a single chain antibody obtained by linking VH and VL of the antibody;

b) a fusion antibody comprising a) said single chain antibody;

c) a Fab comprising the VH and VL of the antibody;

d) an intact antibody comprising the VH and VL of said antibody.

In order to solve the technical problems, the invention also provides a biomaterial related to the antibody, wherein the biomaterial is any one of B1) to B20):

B1) a nucleic acid molecule encoding the antibody;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a recombinant cell line comprising the nucleic acid molecule of B1);

B10) a recombinant cell line comprising the expression cassette of B2);

B11) a nucleic acid molecule encoding the derivatized antibody;

B12) an expression cassette comprising the nucleic acid molecule of B11);

B13) a recombinant vector comprising the nucleic acid molecule of B11);

B14) a recombinant vector comprising the expression cassette of B12);

B15) a recombinant microorganism comprising the nucleic acid molecule of B11);

B16) a recombinant microorganism comprising the expression cassette of B12);

B17) a recombinant microorganism containing the recombinant vector of B13);

B18) a recombinant microorganism containing the recombinant vector of B14);

B19) a recombinant cell line comprising the nucleic acid molecule of B11);

B20) a recombinant cell line comprising the expression cassette of B12).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biological material, the expression cassette containing a nucleic acid molecule encoding the antibody according to B2) means a DNA capable of expressing the antibody in a host cell, and the DNA may include not only a promoter for promoting transcription of the antibody gene but also a terminator for terminating transcription of the antibody gene. Further, the expression cassette may also include an enhancer sequence. B12) The expression cassette containing the nucleic acid molecule encoding the derivative antibody is a DNA capable of expressing the derivative antibody in a host cell, and the DNA may include not only a promoter for initiating transcription of the derivative antibody gene but also a terminator for terminating transcription of the derivative antibody gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the antibody gene expression cassette can be constructed by using the existing expression vector.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above biological material, the recombinant vector can be p-11F11-H1, p-24F2-H1, p-11F11-L1, p-24F2-L1, p-24F2-L2 or p-24F 2-L3.

p-11F11-H1 is a recombinant vector obtained by replacing the DNA fragment between AgeI and SalI recognition sequences of the Ig gamma 1 expression vector with the DNA molecule shown in sequence 7. p-11F11-H1 can express heavy chain 11F11-H1 shown in sequence 1.

p-24F2-H1 is a recombinant vector obtained by replacing a DNA fragment between AgeI and SalI recognition sequences of an Ig gamma 1 expression vector with a DNA molecule shown in a sequence 8. The p-24F2-H1 can express the heavy chain 24F2-H1 shown in the sequence 2.

p-11F11-L1 is a recombinant vector obtained by replacing a DNA fragment between AgeI and XhoI recognition sequences of an Ig lambda expression vector with a DNA molecule shown in a sequence 9. The p-11F11-L1 can express the light chain 11F11-L1 shown in the sequence 3.

p-24F2-L1 is a recombinant vector obtained by replacing a DNA fragment between AgeI and XhoI recognition sequences of an Ig lambda expression vector with a DNA molecule shown in a sequence 10. The p-24F2-L1 can express light chain 24F2-L1 shown in sequence 4.

p-24F2-L2 is a recombinant vector obtained by replacing a DNA fragment between AgeI and XhoI recognition sequences of an Ig lambda expression vector with a DNA molecule shown in a sequence 11. p-24F2-L2 can express light chain 24F2-L2 shown in sequence 5.

p-24F2-L3 is a recombinant vector obtained by replacing a DNA fragment between AgeI and XhoI recognition sequences of an Ig lambda expression vector with a DNA molecule shown in a sequence 12. The p-24F2-L3 can express light chain 24F2-L3 shown in sequence 6.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. The recombinant cell can be

In the above biological material, the transgenic cell line is a non-propagating material. B9) The cell line containing B1) the nucleic acid molecule or B10) the recombinant cell line containing B2) the expression cassette may be any one of the following eight recombinant cell lines: introducing p-11F11-H1 and p-11F11-L1 into 293T cells to obtain recombinant cell lines, introducing p-11F11-H1 and p-24F2-L1 into 293T cells to obtain recombinant cell lines, introducing p-11F11-H1 and p-24F2-L2 into 293T cells to obtain recombinant cell lines, introducing p-11F11-H1 and p-24F2-L3 into 293T cells to obtain recombinant cell lines, introducing p-24F 3-H3 and p-11F 3-L3 into 293T cells to obtain recombinant cell lines, introducing p-24F 3-H3 and p-24F 3-L3 into 293T cells to obtain recombinant cell lines, and introducing p-24F 3-H3 and p-24F 3-L3 into 293T cells to obtain recombinant cell lines, the recombinant cell line obtained by introducing p-24F2-H1 and p-24F2-L3 into 293T cells.

The nucleotide sequence of the antibody described in B1) or the derivative antibody described in B11) of the present invention can be easily mutated by a person of ordinary skill in the art using known methods such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more than 75% identity to the nucleotide sequence of the antibody of B1) or the antibody of B11) of the present invention, as long as they encode the antibody of B1) or the antibody of B11) of the derivative antibody and have the activity of the antibody or the derivative antibody, are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No.1 and/or SEQ ID No.2 and/or SEQ ID No. 3. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 75%, 80%, 85%, 90% or 95% or more.

In the biological material, the coding sequence of CDR1 of VH in the antibody is shown as 79-99 th position of sequence 7 in the sequence table;

the coding sequence of the CDR2 of the VH in the antibody is shown as position 154-174 of the sequence 7 in the sequence table;

the coding sequence of the CDR3 of the VH in the antibody is shown as position 292-342 of a sequence 7 in a sequence table;

the encoding sequence of the CDR1 of the VL in the antibody is h1) or h2) as follows:

h1) 79 th to 102 th of the sequence 9;

h4) 79 th to 102 th of the sequence 12;

the encoding sequence of the CDR2 of the VL in the antibody is i1) or i2) as follows:

i1) 157 th-162 bit of the sequence 9;

i4) 157 th-162 of the sequence 12;

the encoding sequence of the CDR3 of the VL in the antibody is any one of the following j1) -j 4):

j1) 274-303 of the sequence 9;

j2) 274-306 of the sequence 10;

j3) 274-303 of the sequence 11;

j4) 274-300 of the sequence 12.

In the biological material, the encoding sequence of VH in the antibody is shown as sequence 7 or sequence 8 in the sequence table;

the VL coding sequence in the antibody is shown as a sequence 9, a sequence 10, a sequence 11 or a sequence 12 in a sequence table.

In order to solve the above technical problems, the present invention also provides a product for diagnosing and/or treating and/or preventing a disease caused by middle east respiratory syndrome coronavirus, wherein the active ingredient of the product is the antibody, the derivative antibody or the biological material.

The above-mentioned product for diagnosing and/or treating and/or preventing a disease caused by middle east respiratory syndrome coronavirus may be a pharmaceutical, which may have the antibody, the derivative antibody or the biological material as an active ingredient alone, or may have a combination of the antibody, the derivative antibody or the biological material with other substances having an activity of diagnosing and/or treating and/or preventing a disease caused by middle east respiratory syndrome coronavirus as an active ingredient.

In order to solve the above technical problems, the present invention also provides a product for diagnosing and/or treating and/or preventing middle east respiratory syndrome coronavirus, wherein the active ingredient of the product is the antibody, the derivative antibody or the biological material.

The above-mentioned product for diagnosing and/or treating and/or preventing a middle east respiratory syndrome coronavirus may be a pharmaceutical, which may comprise the antibody, the derivative antibody or the biological material as an active ingredient alone, or may comprise the antibody, the derivative antibody or the biological material in combination with other substances having the activity of diagnosing and/or treating and/or preventing a middle east respiratory syndrome coronavirus as an active ingredient.

In order to solve the above technical problem, the present invention further provides any one of the following uses of the antibody, the derivative antibody, the biological material or the product:

x1 for use in the preparation of a reagent for diagnosing infection with, a medicament for treating, or a vaccine for preventing infection with, a middle east respiratory syndrome coronavirus;

x2, in the preparation of a reagent for diagnosing diseases caused by the middle east respiratory syndrome coronavirus, a medicine for treating the diseases caused by the middle east respiratory syndrome coronavirus or a vaccine for preventing the diseases caused by the middle east respiratory syndrome coronavirus;

x3, in the preparation of a reagent for diagnosing, a medicament for treating or a vaccine for preventing middle east respiratory syndrome coronavirus;

x4, for use in the diagnosis and/or treatment and/or prevention of middle east respiratory syndrome coronavirus infection;

x5, for use in the diagnosis and/or treatment and/or prevention of a disease caused by the middle east respiratory syndrome coronavirus;

x6, for use in the diagnosis and/or treatment and/or prevention of middle east respiratory syndrome coronavirus;

x7, and application in preparation of a medicine or preparation for neutralizing middle east respiratory syndrome coronavirus.

The fully humanized anti-MERS-Cov neutralizing antibodies 11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3 and 24F2-4 can recognize and bind MERS-CovS protein, and can prevent MERS-Cov from infecting susceptible cells. Wherein the half effective concentrations (EC50) of 11F1-1 and 24F2-2 binding to S protein are 1240ng/ml and 4.888ng/ml respectively; the MERS-Cov pseudovirus half inhibitory concentrations (IC50) for 100TCID50 were 20. mu.g/ml and 12.26ng/ml, respectively. The fully humanized anti-MERS-Cov neutralizing antibody has application value in diagnosis and/or treatment and/or prevention of middle east respiratory syndrome coronavirus.

Drawings

FIG. 1 is 11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3 and 24F2-4 binding MERS-Cov S proteins.

FIG. 2 shows that 11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3 and 24F2-4 have a neutralizing effect on MERS-Cov pseudoviruses.

FIG. 3 is the median effective concentration of 11F11-1 and 24F2-2 binding S proteins (EC 50).

FIG. 4 shows MERS-Cov pseudovirus median inhibitory concentrations (IC50) of 11F11-1 and 24F2-2 against 100TCID 50.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of antibodies

This example provides 8 fully humanized neutralizing antibodies against middle east respiratory syndrome coronavirus (MERS-Cov) named 11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3, and 24F2-4, respectively, each of the 8 antibodies consisting of a heavy chain consisting of a variable region VL and a constant region CL, a heavy chain consisting of a variable region VH and a constant region CH, and a VH and a VL consisting of a determinant complementary region and a framework region, each of the determinant complementary region consisting of CDR1, CDR2, and CDR 3.

The heavy chains of 11F11-1, 11F11-2, 11F11-3 and 11F11-4 are the same and are named 11F 11-H1. The amino acid sequence of 11F11-H1 is shown as a sequence 1 in a sequence table, the sequence 1 consists of 434 amino acid residues, the 1 st to 125 th sites are the amino acid sequence of VH (named as 11F11-VH1) of 11F11-H1, the 26 th to 33 th sites are the amino acid sequence of CDR1 of 11F11-VH1, the 51 th to 58 th sites are the amino acid sequence of CDR2 of 11F11-VH1, and the 97 th to 114 th sites are the amino acid sequence of CDR3 of 11F11-VH 1. 11F11-VH1 can be encoded by the DNA molecule shown in sequence 7.

The light chains of 11F11-1, 11F11-2, 11F11-3 and 11F11-4 are different and are respectively 11F11-L1, 24F2-L1, 24F2-L2 and 24F 2-L3. The amino acid sequence of 11F11-L1 is shown as a sequence 3 in a sequence table, the sequence 3 consists of 192 amino acid residues, the 1 st to 111 th positions are the amino acid sequence of VHVL (named as 11F11-VL1) of 11F11-L1, the 26 th to 34 th positions are the amino acid sequence of CDR1 of 11F11-VL1, the 52 th to 54 th positions are the amino acid sequence of CDR2 of 11F11-VL1, and the 91 th to 101 th positions are the amino acid sequence of CDR3 of 11F11-VL 1. 11F11-VL1 can be coded by the DNA molecule shown in sequence 9. The amino acid sequence of 24F2-L1 is shown as a sequence 4 in a sequence table, the sequence 4 consists of 193 amino acid residues, the 1 st to 112 th positions are the amino acid sequence of VL (named as 24F2-VL1) of 24F2-L1, the 26 th to 34 th positions are the amino acid sequence of CDR1 of 24F2-VL1, the 52 th to 54 th positions are the amino acid sequence of CDR2 of 24F2-VL1, and the 91 th to 102 th positions are the amino acid sequence of CDR3 of 24F2-VL 1. 24F2-VL1 can be coded by the DNA molecule shown in the sequence 10. The amino acid sequence of 24F2-L2 is shown as a sequence 5 in a sequence table, the sequence 5 consists of 192 amino acid residues, the 1 st to 111 th positions are the amino acid sequence of VL (named as 24F2-VL2) of 24F2-L2, the 26 th to 34 th positions are the amino acid sequence of CDR1 of 24F2-VL2, the 52 th to 54 th positions are the amino acid sequence of CDR2 of 24F2-VL2, and the 91 th to 101 th positions are the amino acid sequence of CDR3 of 24F2-VL 2. 24F2-VL2 can be encoded by a DNA molecule as shown in sequence 11. The amino acid sequence of 24F2-L3 is shown as a sequence 6 in a sequence table, the sequence 6 consists of 191 amino acid residues, the 1 st to 110 th positions are the amino acid sequence of VL (named as 24F2-VL3) of 24F2-L3, the 26 th to 34 th positions are the amino acid sequence of CDR1 of 24F2-VL3, the 52 th to 54 th positions are the amino acid sequence of CDR2 of 24F2-VL3, and the 91 th to 100 th positions are the amino acid sequence of CDR3 of 24F2-VL 2. 24F2-VL3 can be encoded by the DNA molecule shown in sequence 12. The amino acids of CDR1 of 11F11-VL1, 24F2-VL1 and 24F2-VL2 are all the same, and the amino acids of CDR2 of 11F11-VL1, 24F2-VL1 and 24F2-VL2 are all the same.

The heavy chains of 24F2-1, 24F2-2, 24F2-3 and 24F2-4 are identical and are named 24F 2-H1. The amino acid sequence of 24F2-H1 is shown as a sequence 2 in a sequence table, the sequence 2 consists of 434 amino acid residues, the 1 st to 125 th positions are the amino acid sequence of VH of 24F2-H1 (named as 24F2-VH1), the 26 th to 33 th positions are the amino acid sequence of CDR1 of 24F2-VH1, the 51 th to 58 th positions are the amino acid sequence of CDR2 of 24F2-VH1, and the 97 th to 114 th positions are the amino acid sequence of CDR3 of 24F2-VH 1. 24F2-VH1 can be coded by DNA molecule shown in sequence 8. The

CDRs

1, 2 and 3 of 24F2-VH1 and 11F11-VH1 are all the same.

The composition of each antibody and the complementarity determining regions are shown in tables 1 and 2.

TABLE 1, 8 antibody heavy/light chain compositions

TABLE 2 antibody variable region sequences and characteristics

1. Preparation of recombinant vectors

The DNA fragment between the AgeI and SalI recognition sequences of the Ig gamma 1 expression vector (Efficient generation of monoclonal antibodies from simple human B cells RT-PCR and expression vector Thomas T et al.,. J Immunol methods.2008; 329(1-2): 112-124.) was replaced with the DNA molecule shown in sequence 7, and the other sequences were not changed, resulting in a recombinant vector designated p-11F 11-H1. p-11F11-H1 can express 11F11-H1 shown in sequence 1.

And replacing the DNA fragment between AgeI and SalI recognition sequences of the Ig gamma 1 expression vector with a DNA molecule shown in a sequence 8, and obtaining a recombinant vector without changing other sequences, wherein the recombinant vector is named as p-24F 2-H1. p-24F2-H1 can express 24F2-H1 shown in sequence 2.

The DNA fragment between the AgeI and XhoI recognition sequences of an Ig lambda expression vector (Efficient generation of monoclonal antibodies from simple human B cells RT-PCR and expression vector Thomas T et al.,. J Immunol methods.2008; 329(1-2): 112-124) was replaced with the DNA molecule shown in sequence 9, and the other sequences were not changed, to give a recombinant vector designated p-11F 11-L1. p-11F11-L1 can express 11F11-L1 shown in sequence 3.

And replacing a DNA fragment between AgeI and XhoI recognition sequences of the Ig lambda expression vector with a DNA molecule shown in a sequence 10, and obtaining a recombinant vector with the other sequences unchanged, wherein the recombinant vector is named as p-24F 2-L1. p-24F2-L1 can express 24F2-L1 shown in sequence 4.

And replacing a DNA fragment between AgeI and XhoI recognition sequences of the Ig lambda expression vector with a DNA molecule shown in a sequence 11, and obtaining a recombinant vector with the other sequences unchanged, wherein the recombinant vector is named as p-24F 2-L2. p-24F2-L2 can express 24F2-L2 shown in sequence 5.

And replacing a DNA fragment between AgeI and XhoI recognition sequences of the Ig lambda expression vector with a DNA molecule shown in a sequence 12, and obtaining a recombinant vector with the other sequences unchanged, wherein the recombinant vector is named as p-24F 2-L3. p-24F2-L3 can express 24F2-L3 shown in sequence 6.

2. Antibody suspension and purified antibody IgG preparation

2.1 preparation of cells

The 293T monolayer cells (ATCC, CRL-11268) with logarithmic growth period cultured in 10% FBS DMEM medium were taken out from the incubator, cell culture supernatant was poured out, washed once with Phosphate Buffered Saline (PBS), 1ml of pancreatin 0.25% was added, gently shaken to be spread evenly on a cell culture dish, pancreatin was poured out, and then placed in an incubator for about 1min to obtain pretreated 293T cells, pancreatin action was terminated with 10% FBS-containing DMEM medium, and counting was carried out. 6X 10 pretreated 293T cells were added to T75 cell culture flasks⁶Respectively, placing the culture flask at 37 deg.C and 5% CO₂Culturing overnight under the condition to obtain mixed cell suspension.

2.2 preparation of antibody fluids by transient transfection

Mu.g of p-11F11-H1 from step 1 and 9. mu.g of p-11F11-L1 from step 1 were added to 500. mu.l of antibiotic-free and serum-free DMEM medium, and the mixture was allowed to stand at room temperature for 5min, 72. mu.l of the transfection reagent Fugene HD (Roche,04709705001) was added, and the mixture was allowed to stand at room temperature for 20min to obtain a vector mixture. The vector mixture was then added to the mixed cell suspension of step 2.1 (when the vector mixture was mixed with the mixed cell suspension described above, this day was designated as transfection 0, and this day was designated as transfection 0), and the mixture was incubated at 37 ℃ with 5% CO₂Culturing under the condition, supplementing DMEM culture solution containing 10% FBS to 50ml in the 48-72 h of transfection, continuing culturing, and collecting cell supernatant on the 7 th day of transfection.

2.3 antibody purification

The cell supernatant from step 2.2 was centrifuged at 1500r/min for 4min to remove the precipitate, then filtered through a 0.45 μm filter, and the filtrate was collected and passed through a Protein A purification column (HiTrap Protein A HP, GE,170403-03) at a rate of 1-2 drops/sec to bind IgG in the filtrate to Protein A. Washing the purification column 2-3 times with PBS by centrifuging for 1min at 5000g, discarding the solution, adding 400 μ l of IgG eluent (the IgG eluent is a solution of glycine with concentration of 2.7 of 0.1M, pH obtained by adding glycine to water and adjusting pH with HCl) into the purification column, mixing gently, placing the column into a 1.5ml centrifuge tube into which 40 μ l of Tris-hydrochloric acid neutralization solution with pH 9.01M (the neutralization solution is a solution of Tris with concentration of 9.0 of 1M, pH obtained by adding Tris to water and adjusting pH with HCl) is added, centrifuging for 1min at 5000g, and obtaining the purified monoclonal antibody 11F11-1 solution (referred to as 11F11-1 solution for short), wherein the concentration of the antibody in the 11F11-1 solution is 1.2 mg/ml.

According to the method of the above steps 2.2 and 2.3, the solution of the purified monoclonal antibody 11F11-2 (abbreviated as 11F11-2 solution) is obtained by replacing p-11F11-L1 with p-24F2-L1, and the concentration of the antibody in the 11F11-2 solution is 1 mg/ml.

According to the method of the steps 2.2 and 2.3, the p-11F11-L1 is replaced by the p-24F2-L2, and other steps are not changed, so that a solution of the purified monoclonal antibody 11F11-3 (which is called 11F11-3 solution for short) is obtained, wherein the concentration of the antibody in the 11F11-3 solution is 1.008 mg/ml.

According to the method of the above steps 2.2 and 2.3, the p-11F11-L1 is replaced by p-24F2-L3, and the other steps are not changed, so that a solution of the purified monoclonal antibody 11F11-4 (referred to as 11F11-4 solution) is obtained, wherein the concentration of the antibody in the 11F11-4 solution is 1.3 mg/ml.

According to the method of the above steps 2.2 and 2.3, the p-11F11-H1 is replaced by p-24F2-H1, and the other steps are not changed, so that a solution of the purified monoclonal antibody 24F2-1 (referred to as 24F2-1 solution) is obtained, wherein the concentration of the antibody in the 24F2-1 solution is 1.67 mg/ml.

According to the method of the steps 2.2 and 2.3, the p-11F11-H1 is replaced by p-24F2-H1, the p-11F11-L1 is replaced by p-24F2-L1, and other steps are not changed, so that the solution of the purified monoclonal antibody 24F2-2 (referred to as 24F2-2 solution) is obtained, and the concentration of the antibody in the 24F2-2 solution is 5.2 mg/ml.

According to the method of the steps 2.2 and 2.3, the p-11F11-H1 is replaced by p-24F2-H1, the p-11F11-L1 is replaced by p-24F2-L2, and other steps are not changed, so that the solution of the purified monoclonal antibody 24F2-3 (the solution is simply called 24F 2-3) is obtained, and the concentration of the antibody in the 24F2-3 solution is 1.41 mg/ml.

According to the method of the steps 2.2 and 2.3, the p-11F11-H1 is replaced by p-24F2-H1, the p-11F11-L1 is replaced by p-24F2-L3, and other steps are not changed, so that the solution of the purified monoclonal antibody 24F2-4 (the solution is simply called 24F2-4) is obtained, and the concentration of the antibody in the 24F2-4 solution is 1.426 mg/ml.

2.4 preparation of control liquid

Adding 50 μ L/well of DMEM medium without antibiotics or serum into a U-shaped bottom 96-well plate, respectively adding 0.05 μ g of each plasmid carrying heavy chain genes and/or each plasmid carrying light chain genes (p-11F 11-H1, p-24F2-H1, p-11F11-L1, p-24F2-L1, p-24F2-L2 or p-24F2-L3 of step 1), simultaneously setting the plasmid carrying heavy chain genes only and the plasmid carrying light chain genes only and blank wells without DNA as controls, then adding 0.4 μ L/well of transfection reagent Fugene HD, standing at room temperature for 20min, 6 × 10T 293 cells⁴And/well, culturing a 96-well plate at 37 ℃ under the condition of 5% CO2 for 48H, and collecting cell supernatant to obtain a solution only containing 11F11-H1, 24F2-H1, 11F11-L1, 24F2-L1, 24F2-L2 or 24F 2-L3. Blank wells containing no DNA were also used as blanks.

Example 2, 8 antibodies of example 1 can bind to MERS-Cov S protein

1. The 8 antibodies of example 1 can bind to MERS-Cov S protein

Binding of 8 antibodies (i.e., 11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3 and 24F2-4) obtained in example 1 to MERS-Cov S protein was measured, using a solution containing an anti-influenza virus H7 subtype-specific antibody H7 (Beijing Hopkinson. Hokkah Biotech Co., Ltd.) having a neutralizing activity and only 11F11-H1, 24F2-H1, 11F11-L1, 24F2-L1, 24F2-L2 and 24F2-L3 of example 1 as a negative control, using the blank control of example 1 as a positive control, using MERS-Cov as a positive control after being diluted with recovery phase serum 1:300, and repeating the experiment three times, respectively. The method comprises the following specific steps:

coating MERS-Cov S protein (Beijing Yi Qiao Shen Biotechnology Co., Ltd.) on ELISA plate, 1 μ g/ml, 2 wells per sample, 100 μ l per well, and overnight at 4 ℃; PBST plate washing, 3 times; blocking with 1% BSA at 200. mu.l/well, 37 ℃ for 2 h; PBST washing plate, 3 times, each hole adding an antibody suspension (11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3 or 24F2-4 solution) or a control liquid 100 μ l, 37 ℃,1 h; PBST plate washing, 3 times; addition of HRP labelingDiluting goat anti-human IgG (Fc specific, 1mg/ml) at a ratio of 1:10000, washing the plate with PBST for 3 times at 37 deg.C, adding TMB color developing solution 100 μ l, reacting in dark, adding 2M H₂SO₄Aqueous solution, 50. mu.l per well; the OD450 nm was measured (OD450), and the results are shown in FIG. 1 and Table 3.

TABLE 3 binding of 8 antibodies to MERS-Cov S protein

The results showed that neither the light chain control alone could bind to the S protein nor had neutralizing activity, whereas the heavy chain 11F11-VH1 control alone could bind to the S protein, with enhanced binding to the S protein when combined with the light chains (11F1-VL1, 24F2-VL1, 24F2-VL2, 24F2-VL 3); the single heavy chain 24F2-VH2 can bind to the S protein, and when combined with the light chain (11F1-VL1, 24F2-VL1, 24F2-VL2, 24F2-VL3), the binding strength with the S protein is enhanced. Whereas the control anti-influenza virus H7 mab did not bind to the S protein. The fully humanized anti-MERS-Cov neutralizing antibodies 11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3 and 24F2-4 of the invention can recognize and bind MERS-CovS protein.

2. 11F11-1 and 24F2-2 binding to half the effective concentration of S protein (EC50)

The experiment was repeated three times:

diluting the 11F11-1 solution of example 1 with PBS to obtain 11F11-1 solutions with 11F11-1 concentrations of 10000, 1000, 500, 100, 80, 50, 30, 10, 5, 3, 1 and 0ng/ml respectively; diluting the 24F2-2 solution of example 1 with PBS to obtain 24F2-2 solutions with 24F2-2 concentrations of 10000, 1000, 500, 100, 80, 50, 30, 10, 5, 3, 1 and 0ng/ml respectively; an anti-influenza virus H7 subtype specific antibody H7 (Beijing Yiqian Shenzhou Biotechnology Co., Ltd.) having a neutralizing activity was diluted with PBS to obtain H7 solutions having H7 concentrations of 10000, 1000, 500, 100, 80, 50, 30, 10, 5, 3, 1 and 0ng/ml, respectively, as a control.

According to the method of step 1, "adding one antibody suspension (11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3 or 24F2-4 solution) or 100. mu.l of a control liquid per well" was replaced with "adding one concentration of the above antibody solution (11F11-1 solution, 24F2-2 solution or H7 solution, one antibody per well) per well at 100. mu.l per well", and the other steps were not changed, detecting OD450 (FIG. 3 and Table 4), and calculating EC50 by Graphpad prism 5.0 software analysis.

TABLE 4, 11F11-1 and 24F2-2 OD450 values of S-binding proteins ((mean. + -. standard deviation)

The results showed that the half-effective concentrations of 11F1-1 and 24F2-2 binding S proteins (EC50) were 1240ng/ml and 4.888ng/ml, respectively.

Example 3 detection of neutralizing Activity of antibody

The neutralizing activity of 8 antibodies (11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3 and 24F2-4) obtained in example 1 against MERS-Cov pseudovirus was measured, and the experiment was repeated twice using an anti-influenza virus H7 subtype-specific antibody H7 (Beijing-Yi-Qiao-Shen Biotechnology Co., Ltd.) having the neutralizing activity as a control. The method comprises the following specific steps:

3.1MERS-Cov pseudoviruses (MERS-Cov PPs) preparation and infectious titer determination

The packaging plasmid pNL4-3, Luc.R-E (NIH AIDS research & reference reagent,3418) and plasmid pVRC-SY (expression purification and serological detection application analysis of different fragments of HcovNL63N protein) for expressing MERS-CoV S protein are mixed at a ratio of 4:1, transfection reagents (multifect, Qiagen, p2010-1) are added and fully mixed, the mixture is placed at room temperature for 15 minutes to form a DNA-transfection reagent compound, 293FT cells (ATCC, CBP60438) are transfected, the transfected cells are cultured in a 5% carbon dioxide incubator at 37 ℃ for 10 hours, the culture is continued for 10% and the supernatant is collected after 48 hours, the supernatant is centrifuged at 2500rpm for 5 minutes to obtain DMEM cell residues, the supernatant is liquid containing MERS-Cov PPs, and the liquid is frozen and stored at-70 ℃ after subpackaging.

Frozen MERS-CovPPs were removed with serum-free DMEM at 1:10 dilutions were followed by serial dilutions of 5 titers at 5 fold ratio. 96-well culture plates inoculated with Huh7.5 cells (ATCC, CBP60202) on the previous day were washed twice with serum-free DMEM, 100. mu.l of each plate was added to the cell surface, 8 wells per titer were made, and 8 wells were set as negative controls. After incubation at 37 ℃ for 18 hours, the solutions were changed, and after 48 hours 70. mu.l of Bright Glo luciferase assay reagent (Promega) was added to each well, and the readings were read by a GLOMAX96 microplate luminescence detector, which was positive by more than 2.5 times the reading of the negative well, and TCID50 was calculated according to the Reed-Much equation.

3.2MERS-Cov PPs neutralization assay

3.2.1

Mu.l of the 11F11-1 solution of example 1 was mixed with 50. mu.l of MERS-Cov PPs containing 100TCID50, incubated at 37 ℃ for 1 hour, added to the surface of Huh7.5 cells (ATCC, CBP60202), incubated at 37 ℃ for 18 hours, and then the solution was changed, and 70. mu.l of Bright Glo luciferase assay reagent was added to each well after 48 hours, and luciferase activity was assayed by Luminescence assay using GLOMAX96 microwell plates (RLU). H7 and the solutions of example 1 containing only 11F11-H1, 24F2-H1, 11F11-L1, 24F2-L1, 24F2-L2 and 24F2-L3 were used as controls.

According to the method, the 11F11-1 solution is replaced by 11F11-2 solution, 11F11-3 solution, 11F11-4 solution, 24F2-1 solution, 24F2-2 solution, 24F2-3 solution and 24F2-4 solution respectively, and the neutralization effect of each antibody on MERS-Cov PPs is detected without changing other steps.

The results are shown in FIG. 2 and Table 5.

TABLE 5 neutralization of MERS-Cov PPs by 8 antibodies

The results showed that 8 antibodies (11F11-1, 11F11-2, 11F11-3, 11F11-4, 24F2-1, 24F2-2, 24F2-3 and 24F2-4) obtained in example 1 all had neutralizing activity against MERS-Cov pseudovirus.

3.2.2

Diluting the 11F11-1 solution of example 1 with PBS to obtain 11F11-1 solutions with 11F11-1 concentrations of 10000, 2500, 625, 156.25, 39.063, 9.766, 2.441 and 0ng/ml respectively; diluting the 24F2-2 solution of example 1 with PBS to obtain 24F2-2 solutions with 24F2-2 concentrations of 10000, 2500, 625, 156.25, 39.063, 9.766, 2.441 and 0ng/ml respectively; h7 was diluted with PBS to give H7 solutions of H7 concentration 10000, 2500, 625, 156.25, 39.063, 9.766, 2.441 and 0ng/ml, respectively, as negative controls.

According to the method in the step 3.2.1, the 11F11-1 solution is replaced by the 11F11-1 solution with different concentration and the 24F2-2 solution with different concentration respectively, and the neutralization efficiency of the 11F11-1 and the 24F2-2 to MERS-Cov PPs is detected without changing other steps. Different concentrations of H7 solution were used as controls. PBS was used as blank control.

The neutralizing activity was calculated using the following formula: neutralization efficiency (neutralization Rate) NR ═ RLU1-RLU2)/RLU 1X 100%

Wherein, the average reading value of luciferase of the PPs blank control hole is RLU1, and the reading value of luciferase obtained by incubating the antibody to be detected and the PPs is RLU 2. IC50 was calculated by analysis with Graphpad prism 5.0 software.

Neutralization experiments performed similarly to the S binding experiments, i.e., neither the light chain control alone inhibited entry of pseudovirus into susceptible cells huh7.5, the heavy chain 24F2-VH2 alone partially neutralized pseudovirus infection, and the neutralization activity was significantly increased when combined with light chains (11F1-VL1, 24F2-VL1, 24F2-VL2, 24F2-VL 3). 11F1-1 and 24F2-2 showed 20. mu.g/ml and 12.26ng/ml of MERS-Cov pseudovirus half inhibitory concentration (IC50) to 100TCID50 respectively (FIG. 4), and H7 showed no neutralization effect on MERS-Cov pseudovirus.

Claims

1. A fully humanized anti-middle east respiratory syndrome coronavirus neutralizing antibody consisting of a heavy chain consisting of a variable region VL and a constant region CL and a light chain consisting of a variable region VH and a constant region CH, characterized in that: the amino acid sequence of the VH is shown as 1-125 th site of a sequence 1 or 1-125 th site of a sequence 2 in a sequence table, and the amino acid sequence of the VL is shown as 1-111 th site of a sequence 3, 1-112 th site of a sequence 4, 1-111 th site of a sequence 5 or 1-110 th site of a sequence 6 in the sequence table.

2. The biomaterial related to the antibody of claim 1, being any one of B1) to B8):

B1) a nucleic acid molecule encoding the antibody of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism comprising the recombinant vector of B4).

3. The biomaterial of claim 2, wherein:

the encoding sequence of the VH of the antibody of claim 1 is shown as the sequence 7 or the sequence 8 in the sequence table;

the antibody of claim 1, wherein the coding sequence of VL is represented by sequence 9, sequence 10, sequence 11 or sequence 12 in the sequence table.

4. Product for the diagnosis and/or treatment and/or prevention of a disease caused by the middle east respiratory syndrome coronavirus characterized in that: the active ingredient of the product is the antibody of claim 1 or the biological material of claim 2 or 3.

5. Product for the diagnosis and/or treatment and/or prevention of middle east respiratory syndrome coronavirus characterized in that it comprises: the active ingredient of the product is the antibody of claim 1 or the biological material of claim 2 or 3.

6. Use of any of the antibody of claim 1, the biological material of claim 2 or 3, or the product of claim 4 or 5 for:

x1, in the preparation of a reagent for diagnosing middle east respiratory syndrome coronavirus infection or a medicament for treating middle east respiratory syndrome coronavirus infection;

x2, in the preparation of a reagent for diagnosing or treating a middle east respiratory syndrome coronavirus;

x3, and application in preparation of a medicine or preparation for neutralizing middle east respiratory syndrome coronavirus.