CN109021105B

CN109021105B - Human anti-sialic acid binding immunoglobulin-like lectin-7/9 antibody IgG and application thereof

Info

Publication number: CN109021105B
Application number: CN201810677486.0A
Authority: CN
Inventors: 王怡雯; 曹勇平; 朱进; 朱旭辉
Original assignee: Eastern Theater Disease Prevention And Control Center Of Pla
Current assignee: Eastern Theater Disease Prevention And Control Center Of Pla
Priority date: 2018-06-27
Filing date: 2018-06-27
Publication date: 2020-08-04
Anticipated expiration: 2038-06-27
Also published as: CN109021105A

Abstract

An antibody IgG of human anti-sialic acid combined immunoglobulin-like lectin-7/9 and application thereof, wherein the amino acid sequence of a 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. Experimental results prove that the humanized antibody can be specifically combined with Siglec-7 and Siglec-9 at the same time, and can effectively promote the killing effect of NK and other immune cells on tumor cells. Therefore, the monoclonal antibody of the invention can be applied to the relevant tumor immunotherapy of over-expression of Siglec-7 and Siglec9 binding ligands and the clinical treatment of relevant immune diseases and organ transplantation.

Description

Human anti-sialic acid binding immunoglobulin-like lectin-7/9 antibody IgG and application thereof

Technical Field

The invention belongs to the technical field of monoclonal antibody medicines, and relates to two human monoclonal antibody genes of sialic acid-binding immunoglobulin-like lectin-7/9 (sialic acid-binding immunoglobulin-like lectin-7/9, Siglec-7/9), protein molecules coded by the human monoclonal antibody genes and derivatives of the human monoclonal antibody genes, and application of the human monoclonal antibody in clinical treatment of tumors, organ transplantation, immune diseases and the like.

Background

Sialic acid-binding immunoglobulin-like lectins (siglecs) are important members of the immunoglobulin superfamily, which 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 be involved in the regulation of tumor 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 natural killer cell (NK cell) immunoglobulin-like receptors and leukocyte immunoprotein-like receptors. Siglecs are transmembrane molecules containing an Immunoreceptor Tyrosine Inhibition Motif (ITIMs) in the cytoplasmic tail, and an immunoglobulin superfamily region exists extracellularly. In contrast to other immunoglobulin superfamilies, Siglecs are characterized by their highly specific ligand sialylated carbohydrates, unlike most other immune receptor binding proteins. Humans have found 14 siglecs: sialoadhesin (Siglec-1, CD169), CD22(Siglec-2), CD33(Siglec-3) and myelin-associated glycoprotein (MAG, Siglec-4) and more recently CD 33-associated Siglec (Siglec-5-11). Most of the Siglec family is expressed in hematopoietic cells, except for Siglec-4 expressed in neural cells.

Siglec-7 is expressed predominantly on NK cells, T cells and monocytes. Siglec-7 is involved in a plurality of physiological processes such as differentiation, development and death of NK cells and is an important functional receptor for regulating innate immunity of the NK cells. The sialic acid polysaccharide ligand of human NK cell inhibitory receptor siglec-7 is widely expressed on tumor cells of different tissue sources, the expression of the siglec7 determines the NK cell sensitivity and cytotoxicity of human primitive NK cells against resistant tumor cells, and the anti-siglec-7/antibody can effectively improve the cytotoxicity of the NK cells. Siglec-7 on NK cells or ligands on malignant tumor cells are taken as targets, which probably proves that the tumor treatment strategy is good, and the strategy is used for expanding or complementing the existing immunotherapy method based on NK cells to enhance the anti-tumor immune activity of the NK cells. The anti-Siglec-7 antibody immunotherapy may have potential application value in the clinical treatment of organ transplantation, autoimmune diseases, tumors and other diseases.

Sialic acid binding immunoglobulin-like lectin 9(Siglec-9) consists of 463 amino acids, is a type I transmembrane protein, an important member of the immunoglobulin superfamily, consisting mainly of an extracellular N-terminal V-region that mediates recognition of sialic acid (SIA) and two C-region immunoglobulin-like domains, a transmembrane region and an intracytoplasmic region, containing two immunotyrosine inhibitory motifs (ITIMs) the membrane-proximal sequence ITIM tyrosine phosphorylation, recruitment of inhibitory phosphatases, such as Src homology region 2, including tyrosine phosphatase 1(SHP-1), SHP-2 and SH2 inositol phosphatase (srchomdoy 2domain binding kinase 5-phohatase, SHIP ip), mediating downstream tyrosine phosphorylation and transmitting inhibitory signals to the intracellular sigsiglec 637 and Siglec-9 have more than 80% homology in the extracellular, transmembrane and intracellular domains, but have a different pattern of Siglec-7 binding to the surface of cells and are expressed in the neutrophil, expressed as low-specific single stranded cells, CD-21, CD-C-7 and NK-9 are expressed in the cell subpopulation.

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-7/9 and application thereof.

The technical scheme is as follows: human anti-sialic acid binding immunoglobulin-like lectin-7/9 antibody IgG, V of said antibody_HThe complementarity determining regions of the strands have the following amino acid sequences of CDRs:

v shown in SEQ ID NO.3_H-CDR1；

V shown in SEQ ID NO.4_H-CDR2；

V shown in SEQ ID NO.5_H-CDR3；

And V of the antibody_LThe complementarity determining regions of the strands have the following amino acid sequences of CDRs:

v shown as SEQ ID NO.10_L-CDR1；

V shown in SEQ ID NO.11_L-CDR2；

V shown as SEQ ID NO.12_L-CDR3。

The human anti-sialic acid binds to the antibody IgG of the immunoglobulin-like lectin-7/9, and 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 the IgG antibody of immunoglobulin-like lectin-7/9, and 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-7/9 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-7/9 is shown in SEQ ID NO.6 as the nucleic acid sequence which codes for the constant region of the heavy chain, and in SEQ ID NO.13 as the nucleic acid sequence which codes for the constant region of the light chain.

The human anti-sialic acid antibody IgG combined with the immunoglobulin-like lectin-7/9 is applied to preparation of clinical treatment medicines for tumor immunotherapy, Siglec-7/9 related immune diseases and organ transplantation.

A medicine for tumor immunotherapy contains human IgG antibody and its derivatives as effective components, wherein the human IgG antibody binds to immunoglobulin-like lectin-7/9.

A medicine for preventing or treating autoimmune diseases contains human anti-sialic acid antibody IgG and its derivatives, which bind to immunoglobulin-like lectin-7/9.

A medicine for preventing or treating organ transplantation rejection contains human anti-sialic acid antibody IgG and its derivatives bound to immunoglobulin-like lectin-7/9 as effective component.

Has the advantages that: the invention provides a human monoclonal antibody with high specificity and high affinity to Siglec-7/9. The result of antibody function analysis shows that the human antibody can be specifically combined with Siglec-9 and Siglec-7; in vitro cell experiment results prove that the human anti-Siglec-7/9 antibody can effectively improve the killing effect of NK cells on tumor cells. Therefore, the monoclonal antibody of the invention can be applied to tumor immunotherapy and clinical treatment of Sigle-7/9 related immune diseases and organ transplantation.

Drawings

FIG. 1 is an SDS-PAGE detection map of purified Siglec-7/9 antibody; m is Marker; 1 control IgG; 2 Siglec-7/9 antibody IgG;

figure 2E L ISA detection of anti-Siglec-7/9 antibody;

FIG. 3 Western blot assay of anti-Siglec-7/9 antibodies.

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 Siglec-7 and 9 as target molecules, and prepares the human anti-Siglec-7/9 antibody IgG on the basis of 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. However, it should be understood that the illustrated embodiments are exemplary only, and should not be taken as limiting the scope of the 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 that all such changes and modifications as fall within the scope of the invention.

Example 1

Screening of Fab of human anti-Siglec-7/9 antibody

1) The solid phase screening E L ISA plates were coated with recombinant human Siglec-7 and Siglec-9 proteins from R & D, 2 μ g per well, washed, 3% BSA blocking solution added, washed, phage antibody library antibody added, and washed to remove unbound phage antibody.

2) 0.5% trypsin was added, specifically bound phage antibodies were eluted, and the helper phage VCSM13 was added for superinfection.

3) Repeating the screening steps, and carrying out five rounds of 'adsorption-elution-amplification' enrichment screening.

4) The phage obtained from the last round of selection and proliferation were diluted and plated on ampicillin plates added with 100. mu.g/m L for overnight culture, and 60 single colonies were picked up on the cell culture plates and cultured overnight with shaking.

5) After overnight, 5. mu. L of the bacterial 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 E L ISA detection, measuring absorbance values of 450nm and 650nm of each well, and calculating the absorbance value of each well according to A450 nm-A650 nm, wherein when the P/N value (Positive/Negative) is more than 4, 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-Siglec-7/9 antibody IgG

1) Designing primers of Infusion PCR according to the variable region sequences of the obtained antibodies

Heavy chain PCR amplification primers HF and HR and light chain PCR amplification primers L F and L R of the antibody are designed according to the principle of Infusion PCR, 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.

HF:5’-CAGGTGTCCACTCGCAGATCCCGCTGAAGG-3’

HR:5’-GGCCCTTGGTGGATGCTGAGGAGACGGTGACC-3’

LF:5’-TTACAGACGCTCGCTGCGACATCGTGATGACC-3’

LR:5’-GTGCAGCCACCGTACGTTTGATCTCCACCTTGG-3’

2) Amplification of human anti-Siglec-7/9 IgG heavy chain and light chain

And (3) amplifying heavy chain and light chain genes of the 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-C L Ig-h kappa (available from Invivogen) contain the base coding sequences for the heavy and light chain constant regions of human origin of IgG1, the specific nucleotide sequence coding for the heavy chain constant region is shown in SEQ ID No.7, and the specific nucleotide sequence coding for the light chain constant region is shown in SEQ ID No. 14.

① pFUSE-CHIg-hG1 pFUSE-C L Ig-h kappa template carrier 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.

4) Infusion PCR recombinant expression plasmid

Reaction system:

reaction conditions are as follows: incubate at 50 ℃ for 15 min.

Transforming 5 mu L reaction liquid into competent bacteria, spreading on a plate with corresponding resistance, picking out clone the next day, sequencing, preserving strain for clone with correct sequencing result, enlarging culture, and extracting plasmid.

5) Expression of human anti-Siglec-9 antibody IgG

① mu.m of the Opti-MEM medium was added 250. mu.g of L pFUSE-CHIg-hG1-H (i.e., 50. mu.g) to 1m L, 250. mu.g of L pFUSE-C L Ig-H kappa-L (i.e., 50. mu.g) to 1m L, 200. mu.m of L293 Fectin to 2.8m L, and the three mixtures were allowed to stand at room temperature for 5 min.

② mixing the two plasmid mixed solutions evenly, adding 500 mu 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 the cells with 293F Expression Medium, counting and calculating the ratio of cell activity with trypan blue, sucking 100 × 10⁶The cells were grown in flasks and were brought to 94m L using 293F Expression Medium.

③ 20min, 6m L of DNA, 293Fectin complex was added to 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 human anti-Siglec-7/9 antibody IgG

The collected cell supernatant was filtered through a 0.22 μm filter while the balance and the eluate were filtered through a membrane filter, and purified by an AKATA purifier according to the standard procedure for Protein A purification, loaded at 1m L/min, and eluted at 1.5m L/min.

Functional activity identification of human anti-Siglec-7/9 antibody IgG

1) Enzyme linked immunosorbent assay

Recombinant human sialic acid-binding immunoglobulin-like lectin-9 and recombinant human sialic acid-binding immunoglobulin-like lectin-7 were diluted with coating solution (0.1M carbonate buffer, pH 9.6) to 2. mu.g/M L-coated E L ISA 96 well plates, 100. mu. L was added to each well overnight at 4 ℃, PBST (PBS containing 0.5% Tween20) was blocked with 5% skimmed milk-wash buffer and incubated at 37 ℃ for 2h, after 5 PBST washes, 100. mu. L PA21 antibody (2. mu.g/M L starting concentration, 14 concentration gradient dilutions) was added to each well at 37 ℃ for 2h, goat anti-human secondary antibody 100. mu. L/well diluted at 1:2000 was added to the wells and incubated at 37 ℃ for 1h, peroxidase substrate solution 100. mu. L/well was added to each well and after 10 min at room temperature the reaction was stopped with 2M sulfuric acid, colorimetric detection was performed on the machine using 450 nm/nm 690 as a result, human anti-Sig 7/9-binding protein binding to both the lec binding protein and the binding ability of IgG 48325.

2)Western blot

The neutrophil lysate supernatant was subjected to 10% SDS-PAGE and electroporated onto nitrocellulose membrane, which was incubated with 2. mu.g/m L antibody for 1h at room temperature, and HRP-goat anti-human IgG (Beijing Cedar) and EC L luminescence kit (Pierce, Inc., USA) were diluted 1:2000 and exposed to a gel imaging system (Bio-Rad, Inc.).

The results are shown in FIG. 3: the human antibody IgG specifically binds to both Siglec-7 and 9 proteins.

3) Detection of NK cell killing induced by antibody

Taking 20m L venous blood of healthy volunteers, anticoagulating heparin, adding 1m L RosetteSep antibody compound (1m L whole blood/50 m L RosetteSep antibody compound), incubating for 20min at room temperature, adding PBS with equal volume and containing 2% FBS, mixing, placing on ficoll lymphocyte separation liquid with equal volume, centrifuging for 20min at 2000rpm at room temperature, sucking cells between PBS and a plasma layer, and repeatedly washing twice with PBS for later use.

Using K562 as target cell in logarithmic growth phase, adjusting the concentration of target cell to 5 × 10⁴M L, inoculating in 96-well plate, 100 μ L/well, adding effector cells according to effective target ratio of 5:1, 10:1 and 20:1, 100 μ L/well, setting up antibody dry group, adding anti-Siglec-7/9 antibody of 10 μ g/m L, adding in 5% CO₂Culturing for 24h in the incubator, adding 15 μ L CCK-8 reagent into each well, incubating for 2h, detecting OD value at 450nm wavelength with microplate reader, establishing simple effector cells andthe killing rate was calculated by taking the average of 3 replicate wells, using a single target cell well. Compared with the experimental group without the antibody, the killing capacity of the antibody to NK cells is respectively increased by 14.13%, 18.93% and 37.54%

The cell experiment results show that the humanized anti-Siglec-7/9 antibody IgG has good immunological activity, can remarkably enhance the killing effect of NK cells on tumor cells, and shows that the antibody can be used for clinical treatment of tumor immunotherapy, Siglec-7/9 related immune diseases and organ transplantation.

Sequence listing

<110> military medical research institute of south Beijing military district of China's people liberation army

<120> human anti-sialic acid binding immunoglobulin-like lectin-7/9 antibody IgG and application thereof

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<211>366

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

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tcctgcaagg cctctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaaatat 180

gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240

atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagatacttt 300

gatcgatacg gcagttggag tctttgggac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210>2

<211>122

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Asp Arg Tyr Gly Ser Trp Ser Leu Trp Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>3

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Gly Tyr Thr Phe Thr Gly Tyr Tyr

1 5

<210>4

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Ile Asn Pro Asn Ser Gly Gly Thr

1 5

<210>5

<211>15

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<213> Artificial Sequence (Artificial Sequence)

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Ala Arg Tyr Phe Asp Arg Tyr Gly Ser Trp Ser Leu Trp Asp Tyr

1 5 10 15

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<211>990

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<213> Artificial Sequence (Artificial Sequence)

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gcatccacca agggcccatc tgtcttcccc ctggccccat cctccaagag cacctctggc 60

ggcacagctg ccctgggctg cctggtgaag gactacttcc ctgagcctgt gacagtgtcc 120

tggaactctg gcgccctgac cagcggcgtg cacaccttcc ctgctgtgct ccagtcctct 180

ggcctgtact ccctgagcag cgtggtgaca gtgccatcca gcagcctggg cacccagacc 240

tacatctgca atgtgaacca caagcccagc aacaccaagg tggacaagcg ggtggagccc 300

aagtcctgtg acaagaccca cacctgcccc ccatgccccg cccctgagct gctgggcggc 360

ccatctgtct tcctgttccc ccccaagccc aaggacaccc tgatgatctc ccggaccccc 420

gaggtgacct gtgtggtggt ggatgtgagc catgaggacc ccgaggtgaa gttcaactgg 480

tatgtggatg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 540

agcacctacc gggtggtgag cgtgctgaca gtgctgcatc aggactggct gaatggcaag 600

gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccattgagaa gaccatctcc 660

aaggccaagg gccagccccg ggagccccag gtctacaccc tgcccccctc ccgggaggag 720

atgaccaaga accaggtgag cctgacctgc ctggtgaagg gcttctaccc cagcgacatt 780

gctgtggagt gggagagcaa cggccagcct gagaacaact acaagaccac cccccctgtg 840

ctggactctg atggctcctt cttcctgtac agcaagctga cagtggacaa gagccggtgg 900

cagcagggca atgtcttctc ctgctctgtg atgcatgagg ccctgcacaa ccactacacc 960

cagaagagcc tgtccctgtc ccccggcaag 990

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 5560

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>8

<211>345

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<213> Artificial Sequence (Artificial Sequence)

<400>8

gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

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tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180

gaatcggggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcaggacca gatctatagt 300

tctctcccgg tgactttcgg cggagggacc aaggtggaga tcaaa 345

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<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

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

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asp

85 90 95

Gln Ile Tyr Ser Ser Leu Pro Val Thr Phe Gly Gly Gly Thr Lys Val

100 105110

Glu Ile Lys

115

<210>10

<211>12

<212>PRT

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Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr

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<210>11

<211>3

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

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Trp Ala Ser

1

<210>12

<211>11

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Gln Asp Gln Ile Tyr Ser Ser Leu Pro Val Thr

1 5 10

<210>13

<211>336

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<213> Artificial Sequence (Artificial Sequence)

<400>13

cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagcttgcc cgtcacaaag 300

agcttcaaca ggggaagagt gttagttcta gataat 336

<210>14

<211>112

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>14

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg GlyArg Val Leu Val Leu Asp Asn

100 105 110

<210>15

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

caggtgtcca ctcgcagatc ccgctgaagg 30

<210>16

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

ggcccttggt ggatgctgag gagacggtga cc 32

<210>17

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

ttacagacgc tcgctgcgac atcgtgatga cc 32

<210>18

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

gtgcagccac cgtacgtttg atctccacct tgg 33

Claims

1. Human anti-sialic acid antibody IgG binding to immunoglobulin-like lectin-7/9, characterized in that:

v of the antibody_HThe chain has the following amino acid sequences of CDRs:

v shown in SEQ ID NO.3_H-CDR1；

V shown in SEQ ID NO.4_H-CDR2；

V shown in SEQ ID NO.5_H-CDR3；

V of the antibody_LThe chain has the following amino acid sequences of CDRs:

v shown as SEQ ID NO.10_L-CDR1；

V shown in SEQ ID NO.11_L-CDR2；

V shown as SEQ ID NO.12_L-CDR3。

2. Human anti-sialic acid antibody IgG binding to immunoglobulin-like lectin-7/9, characterized in that: the amino acid sequence of the 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.

3. The human anti-sialic acid antibody IgG according to claim 1 or 2, which binds to immunoglobulin-like lectin-7/9, characterized in that: 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 as SEQ ID NO. 14.

4. Nucleic acid encoding the human anti-sialic acid binding immunoglobulin-like lectin-7/9 IgG of claim 2, wherein the nucleic acid sequence encoding the heavy chain variable region is SEQ ID No.1 and the nucleic acid sequence encoding the light chain variable region is SEQ ID No. 8.

5. Nucleic acid encoding the human anti-sialic acid binding immunoglobulin-like lectin-7/9 IgG of claim 3, wherein the nucleic acid encoding the heavy chain constant region has the sequence shown in SEQ ID No.6 and the nucleic acid encoding the light chain constant region has the sequence shown in SEQ ID No. 13.

6. Use of the human anti-sialic acid IgG antibody binding to immunoglobulin-like lectin 7/9 of claim 1 in the manufacture of a medicament for the clinical treatment of cancer immunotherapy, autoimmune diseases or organ transplantation.

7. A clinical therapeutic agent for tumor immunotherapy, autoimmune diseases, or organ transplantation, characterized in that the active ingredient is the human anti-sialic acid antibody IgG binding to immunoglobulin-like lectin-7/9 of claim 1.