Antibody for neutralizing human infected H7N9 influenza A virus and application thereof
阅读说明：本技术 一种中和人感染h7n9甲型流感病毒的抗体及其用途 (Antibody for neutralizing human infected H7N9 influenza A virus and application thereof ) 是由 谢良志 孙春昀 张�杰 宋德勇 饶木顶 朱萍霞 李成红 于 2014-03-07 设计创作，主要内容包括：本发明涉及结合并中和人感染H7N9甲型流感病毒血凝素蛋白的人源化抗体及与其抗原结合的抗体片段,包括抗体重链和轻链可变区的核苷酸和氨基酸序列。本发明还涉及完整抗体在CHO细胞等体系中高效表达的方法。此外本发明还涉及所述抗体、相关血凝素蛋白抗原结合片段和表位在人感染H7N9甲型流感病毒感染的诊断、治疗和预防中的用途。(The present invention relates to humanized antibodies that bind to and neutralize human hemagglutinin protein of influenza a virus infected with H7N9 and antibody fragments that bind to its antigen, including the nucleotide and amino acid sequences of the variable regions of the heavy and light chains of the antibody. The invention also relates to a method for efficiently expressing the complete antibody in a CHO cell system and the like. The invention also relates to the use of said antibodies, associated hemagglutinin protein antigen-binding fragments and epitopes in the diagnosis, treatment and prevention of influenza a virus infection in humans with H7N 9.)
1. An antibody or antigen-binding fragment thereof that neutralizes H7-related influenza a virus, comprising a light chain CDR1 selected from the group consisting of seq id nos: SEQ ID NO:1 to 6; a light chain CDR2 selected from the group consisting of: SEQ ID NO: 7-10; a light chain CDR3 selected from the group consisting of: SEQ ID NO: 11-14;
but does not comprise the light chain CDR1 having the sequence SEQ ID NO. 2, the light chain CDR2 having the sequence SEQ ID NO. 8, and the light chain CDR3 having the sequence SEQ ID NO. 13.
2. An antibody or antigen-binding fragment thereof that neutralizes H7-related influenza a virus, comprising a heavy chain CDR1 selected from the group consisting of seq id nos: SEQ ID NO: 15; a heavy chain CDR2 selected from the following sequences: SEQ ID NO: 16-17; a heavy chain CDR3 selected from the following sequences: SEQ ID NO: 19.
3. the antibody and antigen binding fragment thereof neutralizing H7-related influenza a virus of claim 1 or 2 comprising a light chain comprising the amino acid sequence: CDRL1 is SEQ ID NO: 1. CDRL2 is SEQ ID NO: 7. CDRL3 is SEQ ID NO: 11; or CDRL1 is SEQ ID NO: 2. CDRL2 is SEQ ID NO: 8. CDRL3 is SEQ ID NO: 12; or CDRL1 is SEQ ID NO: 3. CDRL2 is SEQ ID NO: 7. CDRL3 is SEQ ID NO: 11; or CDRL1 is SEQ ID NO: 2. CDRL2 is SEQ ID NO: 8. CDRL3 is SEQ ID NO: 12; or CDRL1 is SEQ ID NO: 4. CDRL2 is SEQ ID NO: 9. CDRL3 is SEQ ID NO: 14; or CDRL1 is SEQ ID NO: 5. CDRL2 is SEQ ID NO: 10. CDRL3 is SEQ ID NO: 11; or CDRL1 is SEQ ID NO: 6. CDRL2 is SEQ ID NO: 8. CDRL3 is SEQ ID NO: 13.
4. an antibody and antigen binding fragment thereof according to any one of claims 1 to 3 which neutralises H7-related influenza A virus, comprising a heavy chain comprising the amino acid sequence: CDRH1 is SEQ ID NO: 15. CDRH2 is SEQ ID NO: 16. CDRH3 is SEQ ID NO: 19; or CDRH1 is SEQ ID NO: 15. CDRH2 is SEQ ID NO: 17. CDRH3 is SEQ ID NO: 19.
5. an antibody or antigen-binding fragment thereof that neutralizes H7-related influenza a virus, wherein the antibody comprises the amino acid sequence of SEQ ID NO:20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29; or comprises SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; or comprises SEQ ID NO:22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:31, a heavy chain variable region of the amino acid sequence of seq id no; or comprises SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ id no:32, a heavy chain variable region of the amino acid sequence of seq id no; or comprises SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 33; or comprises SEQ ID NO:25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34; or comprises SEQ ID NO:26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 35; or comprises SEQ id no:27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 36.
6. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is the rabbit antibody H7N9-R003(SEQ ID NO:20 and SEQ ID NO: 29), H7N9-R006(SEQ ID NO:21 and SEQ ID NO: 30), H7N9-R019(SEQ ID NO:22 and SEQ ID NO: 31), H7N9-R031(SEQ ID NO:23 and SEQ ID NO: 32), H7N9-RA401(SEQ ID NO:24 and SEQ ID NO: 33), H7N9-RA403(SEQ ID NO:25 and SEQ ID NO: 34), H7N9-RA595(SEQ ID NO:26 and SEQ ID NO: 35), H7N9-R021(SEQ ID NO:27 and SEQ ID NO: 36).
7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody is a monoclonal antibody, a purified antibody, an isolated antibody, a single chain antibody, Fab ', F (ab')2, Fv, scFv, or a scFv-based multi-chain antibody.
8. The antibody or antigen-binding fragment thereof of any one of claims 1-7 for use in the treatment of an influenza a virus infection.
9. An antibody or antigen-binding fragment thereof that neutralizes H7-related influenza a virus, which binds to the same epitope as the antibody of any one of the preceding claims.
10. The antibody or antigen-binding fragment thereof of claim 9, wherein the epitope is located on the head of the HA protein and is the region of the HA protein that binds to the sialyl sugar receptor, and specifically comprises 3 portions: i) the outermost beta-sheet (154-162aa) region of the HA1 head; ii) sialic acid receptor binds to the most important HA1 loop region (213-218 aa); iii) a 3-dimensional overhang region consisting of Gln53, Gln65, Arg81, Glu82 and Ser 84.
11. The antibody or antigen-binding fragment thereof of claim 9 or 10, wherein the epitope comprises the following amino acids: hemagglutinin trimer protein a chain: 53Q, 65Q, 81R, 82E, 84S, 127S, 130R, 181E, 184K, 213Q, 214V, 215N, 217L, 218S, hemagglutinin trimer protein B chain: 154Q, 156T, 158S, 160K, 162T and 198S.
12. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the H7-related influenza a virus is H7N 9.
13. A nucleic acid molecule comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of the preceding claims.
14. A vector comprising the nucleic acid molecule of claim 13.
15. A cell expressing the antibody or antigen-binding fragment thereof of any one of claims 1-12, or comprising the nucleic acid molecule of claim 13, or comprising the vector of claim 14.
16. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or comprising a nucleic acid molecule according to claim 13, or comprising a vector according to claim 14, a cell according to claim 15 in the preparation of (i) a medicament for the treatment of an H7-related influenza a virus infection; (ii) a vaccine detection agent; or (iii) H7-related influenza A virus infection.
17. The use of claim 16, wherein the H7-related influenza a virus is H7N 9.
Technical Field
The invention relates to a novel antibody series of hemagglutinin HA protein of A-type H7N9 influenza and humanized antibodies thereof, which can neutralize 100TCID under a smaller dose50The H7N9 virus infects MDCK cells to half, and protects mice under 10LD50 dose to achieve 90% death protection rate, so the antibody can be used for treating and preventing diseases caused by the H7N9 virus. The invention also relates to the binding epitope of the H7N9 hemagglutinin protein bound by the antibody and the amino acid bound by the antibody. The invention also relates to a method for producing the antibody in CHO cells by adopting a gene recombination technology.
Background
Influenza viruses are defined by the type of Hemagglutinin (HA) as two types a and B, type a can be subdivided into 16 subtypes, with group I existing with H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 subtypes and group II consisting of H3, H4, H7, H10, H14 and H15 subtypes. H1, H2 and H3 in types B and a cause a wider range of influenza diseases in humans, and H5, H7 and H9 subtypes cause sporadic severe infections and may cause new pandemics in humans. From 2 months to 6 months in 2013, people with outbreaks of high pathogenicity and high mortality in China infected with influenza A H7N9 virus, and 132 people in total, of which 43 died [ Rongbao G et al N Engl J Med.2013,368: 1888-. The virus is reassorted by H7 and N9 subtypes of virus that are shed in humans, and no corresponding immunoreactive memory is established in humans, so that severe respiratory distress syndrome can be triggered by the viral infection, leading to death of the patient [ Kageyama T et al, Euro SURvell.2013.18:20453 ]. The human seasonal influenza vaccine consists of A-H1N1, A-H3N2 and type-B3 subtypes, and the immune response established by a human body to the vaccine has no protective effect on H7N9 virus. Therefore, after the H7N9 patient is infected, exogenous antiviral drugs are urgently needed to inhibit the amplification of the virus in vivo. Although there is currently no transmission of H7N9 infection between humans, there have been several experiments that have shown that non-contact airborne transmission can occur between influenza-sensitive animals such as ferrets, pigs [ Herfst S, et al, science.2012; 336: 1534-41; zhu et al, science.2013.341(6142):183-186 ]. Autumn and winter are high-rise seasons of influenza, and effective drugs are needed to control and treat the emergence of new H7N9 influenza cases that may occur.
Currently, the H7N9 virus inhibiting drug is duffy (Oseltamivir) or an analogue thereof (Zanamivir), the small molecule drug is an enzyme activity inhibitor of the NA protein of the influenza virus, and the small molecule drug has the effect of avoiding or reducing the amplification of the virus so as to achieve the treatment effect of reducing the virus titer. The medicine has certain clinical effect, but the virus clearance rate is relatively slow, and duffy resistant strains have been reported, so that more ideal medicines are needed to quickly reduce the virus replication in vivo [ Yunwen Hu et al, Lancet.2013.381:2273-79 ].
It has been reported that influenza critically ill patients are rescued by serum from convalescent patients, and therefore neutralizing antibodies are also an antiviral drug under intense development for prevention and treatment. The hemagglutinin protein (HA), which is the trimeric protein HA0 consisting of HA1 and HA2 subunits and comprising more than 500 amino acids, is the primary target of broad-spectrum and subtype-specific antibodies. The HA1 head part is the binding part of the virus and the sialic acid receptor of the target cell, the virus can be adhered to the cell, the conformational change of the stem HA2 region can be triggered by the environment with low pH value, the virus can be fused with the cell membrane, when the virus is endocytosed in the cell, the genome of the virus is released to the cytoplasm, and the new virus can be replicated in the cell. Antibodies directed against HA can effectively block the process of viral infection of cells, thereby interrupting the viral replication chain. The regions of HA1 that bind sialic acid in the influenza receptor vary widely among subtypes, and the same subtype HAs evolved new variants in the HA1 region, thus, this site HAs no broad spectrum but is the most effective neutralization site [ Kida H et al, vaccine.1985,3,219-222 ]. The existing broad-spectrum antibody mainly aims at a relatively conserved HA2 region, and after the antibody is combined with an HA2 epitope, the allosteric of an HA protein can be prevented by steric hindrance to block the infection process of the virus. The antibody of M2 protein has also been reported to have protective effect in vivo, M2 is the ion channel protein on the surface of virus, and has only 24 amino acids on the surface of virus or cell, and when the virus infects cells, the cell surface antigen expresses a large amount of M2 protein. The anti-M2 antibody binds to virus-replicating cells and kills the cells by ADCC and CDC, thereby exerting a virus-inhibitory effect. Many different types of anti-hemagglutinin and anti-M2 antibodies are currently in development, a small number of antibodies have entered clinical phase I and II studies, and no antibody drug is currently on the market.
Despite decades of research, rapid identification of the most effective emergency drugs for emergencies, such as H7N9 virus outbreak, remains a very crucial and challenging task, and thus it is necessary to identify the most effective drugs through various channels (chemical, traditional and biological).
Disclosure of Invention
The invention mainly relates to an antibody and an antigen binding fragment thereof for binding human H7N9 influenza A virus hemagglutinin protein, wherein the antibody is obtained by screening H7N9(A/Anhui/1/2013) hemagglutinin protein from an antibody library constructed from spleen and bone marrow tissues of rabbits immunized by H7N7A/Netherlands/219/2003 and H7N9(A/Anhui/1/2013) hemagglutinin protein HA, and the binding epitope of the antibody is a region on the surface of HA1 bound with sialic acid. Accordingly, the invention encompasses antibodies and antigen binding fragments thereof that neutralize human infection with H7N9 and related H7 influenza a virus infection.
In a specific embodiment of the invention, the invention comprises an antibody and antigen-binding fragments thereof that neutralizes influenza virus infection in humans with
In one embodiment of the invention, the antibodies and antigen binding fragments thereof encompassed by the invention include light chain CDR1 having the amino acid sequence of SEQ ID NOS: 1-6, light chain CDR2 having the amino acid sequence of SEQ ID NOS: 7-10 and light chain CDR3 having the amino acid sequence of SEQ ID NOS: 11-14, wherein the antibodies neutralize influenza A virus. In another embodiment of the invention, the invention encompasses antibodies and antigen binding fragments thereof that include heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 15, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 16-18, and heavy chain CDR3 having SEQ ID NO. 19, wherein the antibodies neutralize human infection with
In one embodiment of the invention, the antibodies and antigen-binding fragments thereof encompassed by the invention have a light chain CDR1 homology in the range of 61% to 100%, a light chain CDR2 homology in the range of 62% to 100%, and a light chain CDR3 homology in the range of 80% to 100%. In another embodiment of the invention, the heavy chain CDR1 has a homology in the range of 100%, the heavy chain CDR2 has a homology in the range of 87% to 100%, and the heavy chain CDR3 has a homology in the range of 100%. Wherein the antibody neutralizes human infection with influenza A H7N9 virus.
In one embodiment of the invention, the invention comprises an antibody and antigen binding fragments thereof, wherein the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 29; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO. 21 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 30; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO. 22 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 31; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO. 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 32; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO. 24 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 33; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO. 25 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 34; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO. 26 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 35; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36; or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO 28 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 37; and wherein the antibody neutralizes human influenza A infection with H7N9 and is H7N9-R002 and variants thereof H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N 9-R021.
In one embodiment of the invention, the invention comprises an antibody and antigen binding fragments thereof, wherein the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID No. 38 and a heavy chain variable region comprising the nucleotide sequence of SEQ ID No. 39; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO. 40 and a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO. 41; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO. 42 and a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO. 43; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO. 44 and a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO. 45; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO. 46 and a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO. 47; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO. 48 and a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO. 49; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO. 50 and a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO. 51; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO 52 and a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO 53; or the antibody comprises a light chain variable region comprising the nucleotide sequence of SEQ ID NO:54 and a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO: 55; and wherein the antibody neutralizes human influenza A infection with H7N9 and is H7N9-R002 and variants thereof H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N 9-R021.
In another embodiment of the invention, the invention comprises an antibody or antigen binding fragment thereof that neutralizes human infection with influenza A H7N9 virus, wherein said antibody is derived from a library of rabbit antibodies to the hemagglutinin protein of H7N7(A/Netherlands/219/2003), wherein the antibodies are humanized antibodies, and wherein the antibodies are humanized by design from rabbit antibodies, and wherein the antibodies are produced by expression in eukaryotic cells.
In another embodiment, the invention comprises a nucleic acid molecule comprising a polynucleotide of an antibody of the invention and antigen binding fragments thereof. In another aspect, the invention includes a vector comprising a nucleic acid molecule of the invention or a cell expressing an antibody or antigen-binding fragment thereof of the invention. In yet another aspect, the invention includes hemagglutinin proteins and antibody binding epitopes comprising antibodies or antigen binding fragments thereof that bind to the invention, and immunogenic polypeptides from which the antibodies can be obtained that are isofunctional.
The invention further includes a pharmaceutical composition comprising an antibody of the invention and antigen binding fragments thereof, a nucleotide molecule of the invention, a vector comprising a nucleic acid molecule of the invention, a cell expressing an antibody or antibody fragment of the invention, or an immunogenic polypeptide of the invention, and a pharmaceutically acceptable diluent or carrier.
The antibody and antigen binding fragment thereof of the present invention, the nucleotide sequence of the present invention, the vector comprising the nucleotide sequence of the present invention, the cell expressing the vector of the present invention, the epitope comprising the antibody or antibody fragment binding thereto of the present invention and the immunogenic polypeptide thereof, or the pharmaceutical composition of the present invention (i) in the preparation of a medicament for the treatment and prevention of human infection with influenza a virus of
In another aspect, the invention includes a method of reducing infection in a human by influenza a virus infection H7N9, or reducing the risk of infection in a human by influenza a virus infection H7N9, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody, or antigen-binding antibody fragment thereof, of the invention.
In another aspect, the invention includes an epitope that specifically binds to an antibody or antigenic fragment thereof of the invention for use in: (i) preparing a medicament for treating human infection with influenza a
Drawings
FIG. 1: hemagglutination inhibitory activity of rabbit-derived anti-H7N9 antibody against H7N9(A/Anhui/1/2013) influenza virus hemagglutinin HA protein (2. mu.g/mL). 1a is a photograph of agglutination of the hemagglutination plate, and 1b is an analysis of agglutination data of the antibody.
FIG. 2: hemagglutination inhibitory activity of humanized H7N9 antibody H7N9-H002 and original rabbit antibody against 4H 7N9(A/Anhui/1/2013, A/Shanghai/1/2013, A/Hangzhou/1/2013, A/Pigeon/Shanghai/S1069/2013) influenza virus hemagglutinin HA proteins.
FIG. 3: micro-neutralizing (MN) activity of humanized H7N9 antibody H7N9-H002 and original rabbit antibody against H7N9(A/Anhui/1/2013) virus.
FIG. 4: humanized H7N9 antibody H7N9-H002 variable region sequence; 4 a: the light chain variable region nucleotide and amino acid sequences of humanized antibodies, 4 b: the heavy chain variable region nucleotide and amino acid sequences of the humanized antibodies.
FIG. 5: the animal protective effect of humanized H7N9 antibody H7N9-H002 in the flu-preventing group; 5 a: survival of mice, 5 b: body weight loss ratio of mice. The antibody dose of the preventive group was 1mg/kg, 3mg/kg, 10mg/kg and 20mg/kg, respectively, and the virus was H7N9 (A/Anhui/1/2013).
FIG. 6: the animal protection effect of humanized H7N9 antibody H7N9-H002 in influenza treatment group; 6 a: survival of mice, 6 b: body weight loss ratio of mice. The antibody dose in the treatment group was 20mg/kg, the administration time was 3 hours, 1 day and 3 days after challenge, and the virus was H7N9 (A/Anhui/1/2013).
FIG. 7: animal protection effect of humanized H7N9 antibody H7N9-H002 in influenza treatment group, 7 a: survival of mice, 7 b: body weight loss ratio of mice. The antibody dose in the treatment group was 40mg/kg, the administration time was 1 day and 3 days after challenge, and the virus was H7N9 (A/Anhui/1/2013).
FIG. 8: structural schematic diagram of HA protein of H7N9(A/Anhui/1/2013) influenza virus hemagglutinin: the epitope to which humanized H7N9 antibody H7N9-H002 binds the HA protein is indicated by blue; the epitope to which sialic acid of the HA protein receptor binds is shown in red; the portion of the antibody binding epitope that intersects the sialic acid binding epitope is indicated in yellow.
FIG. 9: data for ELISA binding of humanized H7N9 antibody H7N9-H002 to HA protein of different strains of
FIG. 10: data for ELISA binding of humanized H7N9 antibody H7N9-H002 to H7-class viral HA.
Detailed Description
Detailed Description
The present invention is based on the discovery and isolation of an animal-derived antibody that specifically neutralizes the enhanced immune maturation of the H7N9 human infectious virus from individual animals immunized with the H7N7 (hemagglutinin HA protein is highly homologous to the H7N9 virus) or H7N9 virus hemagglutinin protein. The neutralizing activity of these antibodies against H7N9 virus is expected and the hemagglutinin protein epitope recognized by the antibodies should be part of the H7N9 virus subunit vaccine.
In one aspect, the invention provides antibodies and antigen-binding fragments thereof that neutralize human influenza a virus infection with H7N9, which in one embodiment can bind to a variety of partially variant H7N9 influenza viruses.
In another aspect of the invention, the invention provides neutralizing antibodies against HA of influenza a virus infected with H7N9 from different humans, and antigen binding fragments thereof. In one embodiment, the antibodies and antigen binding fragments thereof of the invention specifically bind to a sialic acid receptor binding epitope of influenza a virus,
Humanized monoclonal antibodies, CHO, 293 transfected cell lines expressing the antibodies of the invention and nucleotide sequences encoding the antibodies of the invention are included within the scope of the invention.
As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to any fragment of an antibody of the invention that retains the antigen-binding activity of the antibody, as well as fragments that are 60% similar. Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fab ', F (ab')2, Fv, or scFv. As used herein, the term "antibody" includes antibodies and antigen-binding fragments thereof.
As used herein, "neutralizing antibody" refers to an antibody that can neutralize, i.e., prevent, inhibit, reduce, hinder, or interfere with the ability of a virus to initiate and/or maintain a host against infection. The terms "neutralizing antibody" or "… neutralizing antibody" are used interchangeably herein. These antibodies may be used alone or as a prophylactic or therapeutic agent in combination with a NA inhibitor or a vaccine or the like in a suitable formulation, as described herein, and include any modification or improvement based on the antibodies.
The antibodies and antigen binding fragments of the invention have high affinity and high neutralizing potency. The concentration of the antibody of the present invention required to neutralize 50% of influenza a virus may be, for example, 200ng/ml or less. In one example, a concentration of about 25ng/ml of an antibody of the present invention is required to neutralize 50% of influenza A virus of 100TCID50A/Anhui/1/2013 virus, with antibody affinities of both nM (10) grades-9M)。
Antibodies of the invention
The invention provides an antibody which HAs specific binding to a receptor binding region in the HA1 globular head of HA of H7N9 and H7 viruses, and the high-affinity binding of the antibody to the H7N9 virus can prevent the binding of the virus to the receptor, thereby preventing the virus from invading cells and inhibiting the replication and spread of the virus.
Exemplary antibodies of the invention include rabbit antibodies H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N9-R021, and H7N 9-R002.
The CDRs of the antibody heavy chain are referred to as H-CDR1, H-CDR2, and H-CDR3, respectively, and similarly, the CDRs of the antibody light chain are referred to as L-CDR1, L-CDR2, and L-CDR3, respectively. The positions of the CDR amino acids are defined according to the IMGT numbering system as CDR1-IMGT positions 27-38, CDR2-IMGT positions 56-65 and CDR3-IMGT positions 105-117.
Tables 1 and 2 provide the amino acid sequences of the 6 CDRs of the heavy and light chains, respectively, of an exemplary antibody of the invention.
Table 1: amino acid sequence of light chain CDR1-3 of H7N9 rabbit neutralizing antibody
a: all CDR sequence homologies were normalized to H7N9-R002
Table 2: amino acid sequence of heavy chain CDR1-3 of H7N9 rabbit neutralizing antibody
a: all CDR sequence homologies were normalized to H7N9-R002
In one embodiment, the antibody or antibody fragment of the invention comprises at least one CDR having a sequence with at least 95% sequence homology to any one of SEQ ID NOs 1-19, wherein said antibody neutralizes human infected H7N9 influenza a virus.
Exemplary antibodies of the invention include rabbit antibodies H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N9-R021, H7N9-R002 and humanized antibodies H7N 9-H002. in one embodiment, the amino acid sequences of the light chain (VL) and heavy chain variable regions (VH) of the antibodies of the invention and antibody fragments are set forth in SEQ ID NO:28 and SEQ ID NO:37, and the amino acid sequences of the light chain variable regions (VL) and heavy chain variable regions (VH) of the humanized antibodies are set forth in SEQ ID NO:56 and SEQ ID NO: 57.
In one embodiment, the antibodies and antibody fragments thereof of the invention comprise a light chain variable region having an amino acid sequence that is about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO 28. In another embodiment, the antibodies and antibody fragments thereof of the invention comprise a heavy chain variable region having an amino acid sequence that is about 92%, 95%, 97%, 98%, 99% or 100% identical to any of the sequences set forth in SEQ ID NO 37.
In one embodiment, the antibodies and antibody fragments thereof of the invention comprise a light chain variable region having an amino acid sequence that is about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 56. In another embodiment, the antibodies and antibody fragments thereof of the invention comprise a heavy chain variable region having an amino acid sequence that is about 92%, 95%, 97%, 98%, 99% or 100% identical to any of the sequences set forth in SEQ ID NO. 57.
In one embodiment, the antibodies and antibody fragments thereof of the invention comprise a light chain variable region having an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to any of
Table 3: sequences of the light and heavy chain variable regions of the H7N9 rabbit neutralizing antibody
a: all sequence homologies of the variable regions were normalized to H7N9-R002
The invention also includes an antibody or antibody fragment thereof that competes with, i.e., binds to the same epitope as, an antibody of the invention or an antibody fragment thereof. Including but not limited to H7N9-R003, H7N9-R006, H7N9-R019, H7N9-R031, H7N9-RA401, H7N9-RA403, H7N9-RA595, H7N9-R021, H7N9-R002 and humanized antibodies H7N 9-H002.
The antibodies of the invention may be of any isotype (e.g., IgA, IgG, IgM, i.e., alpha, gamma or mu heavy chain), but are predominantly IgG. In the IgG isotype, the antibody may be of the IgG1, IgG2, IgG3 or IgG4 subclasses. The antibodies of the invention may have a kappa or lambda light chain.
Preparation of antibodies
The antibodies of the invention may be prepared by any method known in the art. For example, the sequence of the antibody is inserted into a corresponding eukaryotic expression vector, and transfected into cells, such as a technology for obtaining the antibody by adopting 293 cell transient expression and a technology for obtaining the antibody by adopting CHO cell stable expression. High density cell culture techniques are commonly used to obtain high yields of antibodies.
Antibody purification techniques, including techniques for making pharmaceutical grade antibodies, are also known in the art. Antibodies can be purified by various chromatographic methods such as centrifugation, filtration, affinity, charge, molecular weight, hydrophobicity, etc.
The manner of preparation of the antibody fragments of the invention is also known in the art and includes enzymatic digestion with pepsin or papain, or the antibody fragments may be obtained by cloning or expressing the sequences of portions of the heavy or light chains. Antibody fragments may include scFv, Fab ', F (ab')2And Fv fragments. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies.
Any suitable host cell/vector system may be used for the expression of the DNA sequences encoding the antibody molecules of the invention and fragments thereof. Suitable host systems include E.coli, yeast cells, insect cells and mammalian host cells. Mammalian host cells include, but are not limited to, CHO, HEK293, per.c6, NSO,
The antibody of the present invention can be prepared by the following steps: i) expressing the nucleic acid sequences of the invention in a suitable host cell, and ii) isolating the expressed antibody product, iii) purifying the antibody.
Antibody epitopes
The antibodies of the invention can be used to assay the epitopes to which they bind. The present inventors have found that neutralizing H7N9 influenza a virus infection antibodies bind to an epitope on the HA protein. In one embodiment, the antibody HAs been analyzed for binding epitopes to sialic acid binding regions in the globular head of HA. The epitope bound by the antibody of the invention may be a linear contiguous or conformationally discontinuous sequence of amino acids.
The epitopes recognized by the antibodies of the invention can have a variety of uses. Epitopes and their mimotopes in purified or synthetic form may be used to enhance the immune response (i.e., as vaccines or for antibody preparation for other uses), or to screen sera for antibodies immunoreactive with the epitope or an immunological epitope thereof.
The epitopes of the invention may also be used to screen for ligands that bind to the epitope. Such ligands include, but are not limited to, antibodies (of different species), antibody fragments, peptides, and other similar viral proteins that can block an epitope and thus prevent infection.
The epitopes of the invention may also be used in the development and application of diagnostic tools. Such applications include direct testing and post-labeling use.
Pharmaceutical composition
The invention provides a pharmaceutical composition comprising an antibody and/or antibody fragment of the invention and/or a nucleic acid encoding such an antibody and/or an epitope recognized by an antibody of the invention. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The carrier itself should not induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, and inactive viral particles.
The pharmaceutical compositions of the present invention generally have a pH of 5.5-8.5, in some embodiments, the pH may be 7.4, and the pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen-free and isotonic with respect to human body fluids.
The pharmaceutical composition may comprise an effective amount of one or more antibodies of the invention and/or comprise an epitope polypeptide that binds to an antibody of the invention, i.e., an amount sufficient to treat, ameliorate or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect.
Medical treatment and use
The antibodies and antibody fragments or derivatives and variants thereof of the invention are useful for the treatment, prevention and diagnosis of influenza a virus infection in humans with H7N9 infection.
The present invention provides i) an antibody, antibody fragment or variant thereof and derivatives thereof; ii) the mode of production of the antibody of the invention; iii) an epitope capable of binding to an antibody of the invention; or iv) a ligand, preferably an antibody capable of binding an epitope that binds to an antibody of the invention, useful in therapy.
In one embodiment, an antibody, antibody fragment, epitope, or composition of the invention is administered to a subject in need of treatment and prevention. Such subjects include, but are not limited to, patients infected with influenza a virus H7N9, subjects at risk of infection with influenza a virus H7N9 or susceptible to infection with influenza a virus H7N9, e.g., immunocompromised subjects. Due to the broad spectrum protective effect of the antibodies provided in the present invention against viruses within subtypes, the antibodies of the present invention may have similar quality and prophylactic effects against influenza antibodies of other H7 series.
The antibodies or antibody fragments of the invention can be used for passive immunization or active immunization.
The antibodies and fragments thereof described in the present invention may also be used in kits for diagnosing H7N9 for influenza infection. Furthermore, epitopes capable of binding to the antibodies of the invention may be used in kits to monitor the efficiency of vaccination programs by detecting the presence of protective anti-H7N9 influenza a virus antibodies. The antibodies, antibody fragments or variants and derivatives thereof of the present invention may also be used in kits to monitor the production of vaccines with desired immunogenicity.
The invention also provides a method of preparing a medicament comprising the step of admixing a monoclonal antibody and one or more pharmaceutically acceptable carriers. The steps involving obtaining (e.g., by expression and purification) monoclonal antibodies, mixing them with the pharmaceutical carrier can be performed at different times, with different personnel at different locations.
The antibody of the invention is screened from a rabbit antibody immunized by HA protein, and a medicinal antibody is obtained by humanization modification. Constructing a eukaryotic expression vector, transfecting a CHO cell line, carrying out high-density culture expression on the cell, and producing the antibody by separation and purification. Optimization of the antibody, including codon optimization and antibody affinity maturation optimization. The present invention covers all nucleic acids, vectors, sequences, antibodies, etc. used and prepared in these steps.