CN105367658B - Human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG and application thereof - Google Patents
Human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG and application thereof Download PDFInfo
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- CN105367658B CN105367658B CN201510870684.5A CN201510870684A CN105367658B CN 105367658 B CN105367658 B CN 105367658B CN 201510870684 A CN201510870684 A CN 201510870684A CN 105367658 B CN105367658 B CN 105367658B
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Abstract
Human anti-sialic acid binds antibody IgG of immunoglobulin-like lectin-9 and its application, the amino acid sequence of heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 9. The human antibody can be specifically combined with sialic acid binding immunoglobulin-like lectin-9; in vitro cell experiment results prove that the humanized anti-sialic acid binding immunoglobulin-like lectin-9 antibody can effectively induce apoptosis of sialic acid binding immunoglobulin-like lectin-9 positive cells and inhibit secretion of cytokines, so that the monoclonal antibody can be applied to clinical treatment and prevention of relevant diseases such as severe asthma, chronic obstructive pulmonary disease, autoimmune diseases, tumors and the like.
Description
Technical Field
The invention belongs to the technical field of monoclonal antibody medicines, and relates to a full-molecular human monoclonal antibody for resisting sialic acid combined with immunoglobulin-like lectin-9 (the surface biological acid-binding immunoglobulin-like lectin-9, Siglec-9), a coded DNA molecule thereof, and application of the full-molecular human monoclonal antibody in preparation of medicines for treating diseases such as severe asthma, chronic obstructive pulmonary disease, autoimmune diseases and tumors.
Background
Sialic acid-binding immunoglobulin-like lectins (siglecs) are important members of the immunoglobulin superfamily, and can mediate cell-cell or pathogen interactions by recognizing sialic acid-containing sugar chain structures. Recent studies show that the siglec family plays an important role in cell activation, proliferation and apoptosis, plays an important regulatory role in innate immunity and adaptive immunity, and can also participate in the regulation of immune tolerance.
Siglecs are members of the immunoglobulin superfamily, and the common structural feature of the molecules is the ability to bind sialic acid. Sialic acid binds to immunoglobulin-like lectins, is expressed on the surface of immune cells, and mediates inhibitory signals. Similar to other families of inhibitory immunoreceptors, such as killer immunoglobulin-like receptors and leukocyte immunoprotein-like receptors, siglecs are transmembrane molecules that contain immunoreceptor tyrosine inhibitory motifs, ITIMs, in the cytoplasmic tail, and immunoglobulin superfamily regions in the extracellular space. In contrast to other immunoglobulin superfamily, siglecs are characterized by their highly specific ligand sialylated carbohydrates, unlike most other immunoreceptor binding proteins. Humans have found 14 siglecs: sialoadhesin (siglec1CD169), CD22(siglec2), CD33(siglec3) and myelin-associated glycoprotein (MAG siglec4) and more recently CD 33-associated siglec (siglec 5-11). Most of the siglecs family is expressed in hematopoietic cells, except that siglec4 is expressed in neural cells.
Sialic acid binding immunoglobulin-like lectin 9(siglec9) is a type I transmembrane protein, an important member of the immunoglobulin superfamily, consisting mainly of an extracellular N-terminal V-region mediating sialic acid (SIA) recognition and two C-region immunoglobulin-like domains, a transmembrane region and an intracytoplasmic region containing two immunotyrosine inhibitory motifs (ITIMs). The membrane proximal motif ITIM is tyrosine phosphorylated, recruiting inhibitory phosphatases, such as Src homology region 2 including tyrosine phosphatase 1(SHP-1), SHP-2, and SH2 inositol phosphatase (SHIP), thereby mediating downstream tyrosine kinase phosphorylation and transmitting inhibitory or lethal signals intracellularly.
Siglec-9 is highly expressed in neutrophils and monocytes, NK cells, CD4+And CD8+Certain subpopulations of T cells are under-expressed. Siglec-9 can be expressed on the cell membrane surface of the immature granulocyte in the middle and later stages of the differentiation of the bone marrow hematopoietic stem cells, and the Siglec-9 is involved in a plurality of physiological processes such as differentiation, development and death of neutrophils and is an important functional receptor for regulating the innate immunity of the neutrophils. The lung tissue massive eosinophil infiltration with neutrophilia is an important pathophysiological characteristic of severe asthma, and the anti-Siglec-9 antibody substitution treatment combines PCD and PCD, and has potential treatment value in the treatment of severe asthma, chronic obstructive pulmonary disease, autoimmune diseases, tumors and other diseases.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides human anti-sialic acid antibody IgG combined with immunoglobulin-like lectin-9 and application thereof.
The technical scheme is as follows: human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG, V of said antibodyLThe complementarity determining regions of the strands have the following amino acid sequences of CDRs:
v shown as SEQ ID NO.10L-CDR1；
V shown in SEQ ID NO.11L-CDR2；
V shown as SEQ ID NO.12L-CDR3；
And V of the antibodyHThe complementarity determining regions of the strands have the following amino acid sequences of CDRs:
v shown in SEQ ID NO.3H-CDR1；
V shown in SEQ ID NO.4H-CDR2；
V shown in SEQ ID NO.5H-CDR3。
The human anti-sialic acid binds to IgG antibody of immunoglobulin-like lectin-9, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 9. And conservative variant obtained by conservative mutation of the sequence through one or more amino acid addition, deletion, substitution and modification.
The human anti-sialic acid binds to IgG antibody of immunoglobulin-like lectin-9, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 7; the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 14. And conservative variant obtained by conservative mutation of the sequence through one or more amino acid addition, deletion, substitution and modification.
The nucleotide sequence of the antibody IgG for coding the human anti-sialic acid combined immunoglobulin-like lectin-9 is shown in SEQ ID NO.1, and the nucleotide sequence of the coding light chain variable region is shown in SEQ ID NO. 8.
The nucleotide sequence of the IgG antibody which codes for the human anti-sialic acid to combine with the immunoglobulin-like agglutinin-9 is shown in SEQ ID NO.6, and the nucleotide sequence of the light chain constant region is shown in SEQ ID NO. 13.
The application of the human anti-sialic acid antibody IgG combined with the immunoglobulin-like lectin-9 in preparing the medicines for preventing or treating severe asthma, chronic obstructive pulmonary disease, autoimmune diseases and/or tumors is disclosed.
A medicine for preventing or treating severe asthma contains human anti-sialic acid antibody IgG conjugated with immunoglobulin-like lectin 9 as effective component.
A medicine for preventing or treating chronic obstructive pulmonary disease contains human anti-sialic acid antibody IgG capable of binding to immunoglobulin-like lectin 9 as effective component.
A medicine for preventing or treating autoimmune diseases contains human anti-sialic acid antibody IgG capable of binding to immunoglobulin-like lectin 9 as effective component.
A medicine for preventing or treating tumor contains human IgG antibody with sialic acid binding immunoglobulin-like lectin-9 as effective component.
Has the advantages that: the invention provides a human monoclonal antibody which has high protection, high specificity and good affinity and is used for resisting sialic acid and combining with immunoglobulin-like lectin-9. The acting energy of the full-molecular humanized antibody can be identified, and the result shows that the humanized antibody can be specifically combined with sialic acid combined immunoglobulin-like lectin-9; in vitro cell experiment results prove that the humanized anti-sialic acid binding immunoglobulin-like lectin-9 antibody can effectively induce apoptosis of sialic acid binding immunoglobulin-like lectin-9 positive cells and inhibit secretion of cytokines, so that the monoclonal antibody can be applied to clinical treatment and prevention of relevant diseases such as severe asthma, chronic obstructive pulmonary disease, autoimmune diseases, tumors and the like.
Drawings
FIG. 1 SDS-PAGE detection of antibody IgG after purification;
FIG. 2 ELISA detection of anti-Siglec-9 antibodies;
FIG. 3 Western blot detection of anti-Siglec-9 antibodies;
FIG. 4 detection of affinity of anti-Siglec-9 antibody binding to antigen;
FIG. 5 anti-Siglec-9 antibody-induced neutrophil apoptosis assay;
FIG. 6 FACS detection of anti-Siglec-9 antibody induced neutrophil ROS.
Detailed Description
The heavy chain variable region and the light chain variable region of the antibody are both human antibodies, and are called full-molecule human antibodies because they are linked to the constant regions of human antibodies.
The invention takes sialic acid combined immunoglobulin-like lectin-9 as a target molecule, and prepares human anti-sialic acid combined immunoglobulin-like lectin-9 antibody IgG on the basis of a phage antibody library technology. The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It should be understood that the illustrated embodiments are exemplary only, and are not intended to limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit of the invention, and it is intended to cover all such changes and modifications as fall within the scope of the invention.
Example 1
Screening of Fab of human-derived anti-sialic acid-binding immunoglobulin-like lectin-9 antibody
1) Coating the solid phase screening ELISA plate with purified recombinant human sialic acid binding immunoglobulin-like lectin-9 protein, 2 μ g per well, washing, adding blocking solution, washing, adding phage antibody library antibody, and washing to remove unbound phage antibody.
2) Trypsin was added to elute specifically bound phage antibodies and the helper phage VCSM13 was added for superinfection.
3) Repeating the screening steps, and carrying out four rounds of 'adsorption-elution-amplification' enrichment screening.
4) The phage obtained by the last round of screening and proliferation is diluted and then paved on an ampicillin culture plate added with 100 mu g/mL for culture overnight, 80 single colonies are picked up in a cell culture plate and cultured overnight by shaking.
5) After overnight, 5. mu.L of the suspension was transferred from each well of the first plate to the second plate and cultured with shaking.
6) Adding helper phage VCSM13 for superinfection, and shaking for culture; centrifuging, resuspending the precipitate in the culture medium, and culturing overnight with shaking.
7) Centrifuging, taking supernatant, performing ELISA detection, measuring the absorbance of each well at 450nm and 650nm, and calculating the absorbance of each well according to A450 nm-A650 nm. When the P/N value (Positive/Negative) is more than 6, the strain is a Positive monoclonal phage strain.
8) And (3) analyzing the nucleic acid sequence of the positive clone, wherein the nucleic acid sequence of the coding heavy chain variable region is shown as SEQ ID NO.1 in the sequence table, and the nucleic acid sequence of the coding light chain variable region is shown as SEQ ID NO.8 in the sequence table, so that the Fab with the correct gene sequence is obtained.
Preparation of human anti-sialic acid-binding immunoglobulin-like lectin-9 antibody IgG
1) Designing primers of Infusion PCR according to the variable region sequences of the obtained antibodies
The heavy chain PCR amplification primer and the light chain PCR amplification primer of the antibody are designed according to the Infusion PCR principle, the primers need to comprise 15bp bases on an expression vector and at least 15bp bases inserted into a target fragment, and the bases inserted into the target fragment are designed according to the principle of common primer design.
Heavy and light chain PCR amplification primers:
heavy chain amplification primers:
HF:5’-GGTGTCCACTCGCTACAGGTGCAGCTGGTGCAGTCT-3’
HR:5’-GCCCTTGGTGGATGCTGAGGAGACGGTGAC-3’
light chain amplification primers:
L5F:5’-ACAGACGCTCGCTGCTCCTATGAGCTGACTCAGCCACCT-3’
L5R：5’-GTTGGCCTTGGGCTGTAGGACGGTCAGCCT-3’
2) amplification of human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG heavy chain, light chain
And (3) amplifying heavy chain and light chain genes of the full-molecular human antibody by respectively using the prepared human Fab as a template and the related upstream and downstream primers of the heavy chain and the light chain.
①PCR
Reaction system:
reaction conditions are as follows:
and (3) performing ② 2% agarose gel electrophoresis, observing a target band under ultraviolet, cutting the gel and recovering.
③ gel recovery kit purifies the target DNA fragment and elutes with deionized water.
3) Double restriction enzyme IgG expression plasmid
The IgG expression plasmids pFUSE-CHIg-hG1, pFUSE-CLIg-hk (available from Invivogen) contain the base-encoding sequences of the heavy and light chain (Kappa) constant regions of human origin of IgG1, the specific nucleotide sequence encoding the heavy chain constant region being shown in SEQ ID NO.7 and the specific nucleotide sequence encoding the light chain constant region being shown in SEQ ID NO. 14.
① pFUSE-CHIg-hG1 pFUSE-CLIg-hk template vector double enzyme digestion
Reaction system:
reaction conditions are as follows: the cleavage was carried out overnight at 37 ℃.
② 1% agarose gel electrophoresis, ultraviolet cutting gel recovery.
③ gel recovery kit purifies the target DNA fragment and elutes with deionized water.
4) Infusion PCR recombinant expression plasmid
Reaction system:
reaction conditions are as follows: incubate at 50 ℃ for 15 min.
5 mul of reaction solution was taken to transform competent bacteria, spread on the corresponding resistant plates, and the next day clones were picked for sequencing. And cloning and preserving strains with correct sequencing results, carrying out amplification culture, and extracting plasmids.
5) Expression of human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG
① mu.L of pFUSE-CHIg-hG1-H (i.e., 50. mu.g) was put in 1mL of Opti-MEM medium, 250. mu.L of pFUSE-CLIg-hk-L (i.e., 50. mu.g) was put in 1mL of Opti-MEM medium, 200. mu.L of 293Fectin was put in 2.8mL of Opti-MEM medium, and the three mixtures were allowed to stand at room temperature for 5 min.
② mixing the two plasmid mixed solutions evenly, adding 500 μ L Opti-MEM culture Medium, mixing evenly, adding the mixed solution of the transfection reagent 293Fectin directly, mixing evenly, standing for 20min, treating 293F cells, centrifuging 293F cells, resuspending with 293F Expression Medium, counting, calculating cell activity ratio with trypan blue, sucking 100 × 106The cells were placed in a flask and were brought to a volume of 94mL using 293F Expression Medium.
③ 20min was completed and 6mL of the DNA, 293Fectin complex was added to the prepared 293F cells.
④ the cells were cultured in a shaker incubator with 8% CO2Cell supernatants were collected after 6 days at 120rmp, 37 ℃.
6) Purification of IgG antibody of human anti-sialic acid binding immunoglobulin-like lectin-9
The collected cell supernatant was filtered through a 0.22 μm filter while the balance and the eluate were filtered through a membrane. The Protein A was purified using an AKATA purifier according to standard procedures for Protein A purification, loading at 1mL/min, and eluting at 1.5 mL/min. Results IgG antibodies were successfully expressed and purified. The results of SDS-PAGE are shown in FIG. 1.
Functional Activity identification of human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG
1) Enzyme linked immunosorbent assay
Diluting recombinant human sialic acid binding immunoglobulin-like lectin-9 with coating solution (0.1M carbonate buffer, pH9.6) to 2. mu.g/mL coated ELISA 96-well plate, adding 100. mu.L per well, and standing overnight at 4 deg.C; PBST (PBS containing 0.5% Tween20) 5% skimmed milk-wash buffer blocked and incubated at 37 ℃ for 2 h; after 5 PBST washes, 100. mu.L of PA21 antibody (2. mu.g/mL starting concentration, 14 concentration gradient dilutions) was added to each well for 2h at 37 ℃; mixing the raw materials in a ratio of 1: adding 100 mu L/hole of 2000 diluted goat anti-human secondary antibody into the hole, and incubating for 1h at 37 ℃; the peroxidase substrate color developing solution is 100 mu L/hole, the reaction is stopped by 2M sulfuric acid after 10 minutes at room temperature, and the double-wavelength 450nm/690nm is adopted for machine detection and color comparison. As a result, as shown in FIG. 2, human anti-sialic acid-binding IgG immunoglobulin-like lectin 9 antibody bound to Siglec9 protein.
2)Western blot
The neutrophil lysate supernatant was subjected to 10% SDS-PAGE and transferred electrically to a nitrocellulose membrane, which was incubated with 2. mu.g/mL of antibody at room temperature for 1h, and HRP-goat anti-human IgG (Pico, Beijing) and ECL luminescence kit (Pierce, USA) were diluted 1:2000 and exposed to a gel imaging system (Bio-Rad).
The results are shown in FIG. 3: the human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG specifically binds to Siglec-9 protein.
3) Affinity assays
According to the isoelectric point of the antigen and the protocol of Biacore-X100 control soft, the coupling condition is optimized, and sodium acetate is selected as the coupling dilution buffer solution in slope optimization. Antigen samples were diluted to 25. mu.g/mL with this buffer and coupled to CM5 chips. The preset coupling level was 1500 RU. Antigen samples were then diluted with Running buffer at pH7.4 to a range of concentrations of 0uM, 5nM, 10nM 20nM, 40nM, 80 nM. The sample injection time is set to 180s, the dissociation time is set to 10min, and the regeneration buffer is 50mM pH2.2 Gly-HCl. The on-board test was performed according to the protocol of BiacoreX100 control soft. The affinity assay results are shown in FIG. 6, with KD of 7.380X 10-10M。
4) Detection of antibody-induced neutrophil apoptosis
Neutrophils were isolated using Ficoll isolate, pre-treated with GM-CSF for 30 min at 25ng/mL, and Siglec-9mAb at 10. mu.g/mL (B10101) and negative control added, respectively. After 8 hours of action, the cells were stained with Annexin-V and PI and examined by flow cytometry. The result shows that the antibody can induce the apoptosis of the neutrophil granulocytes, and the apoptosis rate reaches 21.15%.
5) ROS detection in cells:
5mL of venous blood is adopted, and the neutrophilic granulocyte is separated from a Ficoll separating medium and divided into three groups: negative control group, GM-CSF group and GM-CSF + siglec-9mAb20 μ g/mL group, each acted for 2 hours. The culture medium was added with 100ng/mL LPS and allowed to act for 1 hour. According to the instructions, add Sigma company ROS probe, 37 degrees were incubated for 30 minutes, ice PBS washed cells three times, flow cytometry detection. The results show that antibody treatment significantly increased ROS levels in neutrophils.
The animal experiment results show that the anti-human sialic acid binding immunoglobulin-like lectin-9 IgG antibody has good immunological activity and can induce apoptosis increase of neutrophils, and the antibody can be used for treating diseases such as severe asthma, chronic obstructive pulmonary disease, autoimmune diseases and tumors.
Sequence listing
<110> Zhu, in
<120> human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG and application thereof
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Claims (1)
1. The application of human anti-sialic acid combined immunoglobulin-like lectin-9 antibody IgG in preparing a medicament for inhibiting neutrophils is disclosed, wherein the amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID No. 2; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9; the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO. 7; the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 14.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510870684.5A CN105367658B (en) | 2015-12-02 | 2015-12-02 | Human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG and application thereof |
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| CN1371916A (en) * | 2001-02-28 | 2002-10-02 | 第二军医大学免疫学研究所 | Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use |
| CN101035807A (en) * | 2004-06-09 | 2007-09-12 | 泰勒公司 | Diagnosis and treatment of SIGLEC-6 associated diseases |
| CN102803293A (en) * | 2009-10-06 | 2012-11-28 | 阿莱斯亚生物疗法股份有限公司 | SIGLEC 15 antibodies in treating bone loss-related disease |
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| CN1371916A (en) * | 2001-02-28 | 2002-10-02 | 第二军医大学免疫学研究所 | Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use |
| CN101035807A (en) * | 2004-06-09 | 2007-09-12 | 泰勒公司 | Diagnosis and treatment of SIGLEC-6 associated diseases |
| CN102803293A (en) * | 2009-10-06 | 2012-11-28 | 阿莱斯亚生物疗法股份有限公司 | SIGLEC 15 antibodies in treating bone loss-related disease |
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| Siglec-9, a Novel Sialic Acid Binding Member of the Immunoglobulin Superfamily Expressed Broadly on Human Blood Leukocytes;Jiquan Q. Zhang等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20000508;第275卷(第29期);摘要，第22122页左栏最后1段至右栏第1段 * |
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