CN113512117B - Antibody capable of binding CD206 and application thereof - Google Patents

Antibody capable of binding CD206 and application thereof Download PDF

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CN113512117B
CN113512117B CN202110782069.4A CN202110782069A CN113512117B CN 113512117 B CN113512117 B CN 113512117B CN 202110782069 A CN202110782069 A CN 202110782069A CN 113512117 B CN113512117 B CN 113512117B
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CN113512117A (en
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曾锐
廖文慧
郭毅
廖睿纯
邓璇
曾祥彬
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Tongji Hospital Affiliated To Tongji Medical College Of Huazhong University Of Science & Technology
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Wuhan Maikaojin Biotechnology Co ltd
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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Abstract

The invention relates to an antibody capable of binding to CD206, comprising a light chain variable region and a heavy chain variable region, wherein the sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NO:14-16, and the sequence of CDR1-3 of the light chain variable region is shown in SEQ ID NO: 17-19; also relates to the pharmaceutical use of the antibody and an antibody conjugated drug containing the antibody. The antibody can be specifically combined with the M2 type macrophage surface molecular marker CD206, and can be combined with serum albumin and cytotoxin PE, and the formed antibody coupling medicament can not only specifically identify the M2 type macrophage, but also maintain the killing effect on cells. Therefore, the compound can be used as an effective antitumor targeting drug.

Description

Antibody capable of binding CD206 and application thereof
Technical Field
The invention belongs to the field of antitumor drugs, and particularly relates to an antibody capable of combining human or mouse CD206 and application thereof.
Background
Some gene mutations are responsible for tumor production. Chemotherapy, while inhibiting the growth of tumor cells, often can also damage normal tissues and cells, resulting in impaired physical health or induction of tumor gene mutations, leading to tumor recurrence. Thus, targeted therapies are becoming a research hotspot.
An Antibody-Drug Conjugates (ADC) -specific Antibody is conjugated to a cytotoxic Drug, targeting the Drug to a specific antigen, thereby killing the Drug against cells expressing the corresponding antigen. ADCs are generally composed of "warhead" drugs (cytotoxic drugs), antibodies, and conjugated chain 3 moieties. Stable binding ADCs are capable of pulling drugs towards target cells, preventing their degradation on peripheral blood and normal cell surfaces. Therefore, under the same administration dosage, the stable ADC can keep the effective killing concentration to the target cells in vivo for a long time like a slow release agent without releasing redundant toxin, so that the ADC has more efficient killing effect and less adverse reaction in the target tissue.
In order to achieve an efficient and specific killing of tumor cells, on the one hand, an antigen associated with the tumor cells is determined, and on the other hand, specific antibodies against the antigen are prepared and screened, and the antibodies can be used for coupling cytotoxic drugs without affecting their specificity and affinity.
Tumor-associated macrophages include two subtypes, M1 and M2, wherein M2-type tumor-associated macrophages promote tumor growth in association with a poor prognosis for various tumors, whereas M1-type tumor-associated macrophages have anti-tumor function. Therefore, M2 type tumor-associated macrophages can be used as specific targets for intervention, T cell-based tumor immunotherapy is combined, and M1 type macrophages with anti-tumor activity are effectively reserved.
We have found during the course of the study that the surface-specific expression of the receptor CD206 by M2-type tumor-associated macrophages can be used as a target antigen. To achieve specific killing against M2 tumor-associated macrophages, we prepared antibodies using CD206 and constructed antibody-conjugated drugs.
Disclosure of Invention
To solve the above problems, the present invention provides an antibody capable of binding to CD206, comprising a light chain variable region and a heavy chain variable region, wherein the sequences of CDRs 1-3 of the heavy chain variable region are as set forth in SEQ ID NO:14-16, and the sequence of CDR1-3 of the light chain variable region is shown in SEQ ID NO: 17-19.
In a specific embodiment, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4 or a mutant thereof.
In a specific embodiment, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4 or a mutant thereof.
In a specific embodiment, the sequence of the antibody is shown in SEQ ID NO. 10.
The invention also provides application of the antibody in preparing an anti-tumor drug or a drug for specifically killing M2 type macrophages.
The invention also provides an antibody coupling drug, which comprises the antibody as an N-terminal domain.
In a specific embodiment, the antibody-conjugated drug further comprises a polypeptide having cytotoxicity as a C-terminal domain.
In a specific embodiment, the sequence of the polypeptide having cytotoxicity is as shown in SEQ ID NO. 12.
In a specific embodiment, the sequence of the antibody-conjugated drug is shown in SEQ ID NO. 13.
The antibody obtained by screening can be specifically combined with the M2 type macrophage surface molecular marker CD206, and can be combined with serum albumin and cytotoxin PE, and the formed antibody coupling medicament can not only specifically identify M2 type macrophages, but also maintain the killing effect on cells. Therefore, the compound can be used as an effective antitumor targeting drug.
Drawings
FIG. 1 is a graph showing fluorescence intensity statistics of supernatants of 8 hybridoma cell lines by flow assay;
FIG. 2 is a flow chart of the flow detection of different processed macrophage cell lines;
FIG. 3 is a statistical screenshot of flow cytometry detection after incubation of M1 and M2 macrophages with fluorescently labeled cytotoxins at 4deg.C;
FIG. 4 is a statistical screenshot of flow cytometry detection after incubation of M1 and M2 macrophages with fluorescently labeled cytotoxins at 37 ℃;
FIG. 5 is a photomicrograph of a six well plate cytotoxicity assay;
FIG. 6 is a statistical plot of OD450 in CCK-8 experiments.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention.
1. Screening and preparation of antibodies
The solid phase synthesis method synthesizes CD206 polypeptide according to the corresponding sequence, and HPLC and MASS quality inspection results show that the synthesized polypeptide has correct sequence and purity of more than 90 percent, and the total amount meets the requirement. CD206 is crosslinked with MBS-KLH linker to form CD206-KLH covalent complexes as immunogens.
Mice were immunized with the above immunogens in the following amounts: at the time of priming, 100 μg antigen plus an equal volume of Freund's complete adjuvant was injected into each mouse; at boost, each mouse was injected with 50 μg antigen plus an equal volume of Freund's incomplete adjuvant. After 5 times of booster immunization, the antiserum is prepared from mouse tail vein blood, ELISA detection shows that the antiserum has higher titer, and mouse spleen cells are taken for fusion screening.
Spleen cells of immunized mice were fused with resuscitated SP2/0 cells as follows: myeloma cells and spleen cells were mixed in a ratio of 1:3-1:10, and subjected to cell fusion procedures in standard procedures, followed by culture in HAT-DMEM complete medium, after which hybridoma cells were seen 3 days after fusion, 1/2HAT complete medium was changed on day 7, and 1/2HT medium was changed on day 8. Screening was started about 10 days after fusion. Cell supernatants were aspirated at 100 ul/well and subjected to indirect ELISA assays. Based on ELISA results, positive wells were judged. The second review was performed to further confirm the positive wells.
Two rounds of subcloning were performed on the rescreened positive well cells. A stably positive hybridoma cell line was detected as a cell from which monoclonal antibodies were finally prepared, and expanded. The subtypes of each supernatant were separately assayed using the monoclonal antibody subtype identification kit of Southern Biotech in the united states. And (3) through two rounds of subcloning and rechecking, determining the positive cell strain and the subtype thereof, and further identifying the binding force between the antibody contained in the supernatant of the positive cell strain and the CD206 and the endocytic capacity after the binding with the CD 206. 8 positive cell lines and the subtypes thereof were determined. The hybridoma supernatant was used to incubate with IL-4 induced differentiated M2-type tumor-associated macrophages, and the fluorescence intensity was measured by overcurrent, which showed that the 8# cell line (6D5, igG 1) had higher fluorescence intensity and could be used to target M2-type tumor-associated macrophages (FIG. 1). Flow-through assays were performed using undifferentiated macrophage lineage (RAW), IL-4 incubated RAW, and IL-4 and 6D5 supernatant incubated RAW, respectively, and the resulting flow patterns are shown in FIG. 2, which shows that antibodies in hybridoma supernatants have a strong affinity for M2-type tumor-associated macrophages.
The hybridoma cell line 6D5 is cultivated, RNA is extracted, and cDNA is prepared by reverse transcription. The designed and synthesized mouse IgG specific primer group is used for sequencing.
The result shows that the DNA coding sequence of the heavy chain of the antibody corresponding to the cell strain 6D5 is shown as SEQ ID NO. 1, the corresponding amino acid sequence is shown as SEQ ID NO. 2, the DNA coding sequence of the heavy chain variable region is shown as SEQ ID NO. 3, the corresponding amino acid sequence is shown as SEQ ID NO. 4 (wherein, the 26 th to 33 th amino acids are CDR1, the sequence is shown as SEQ ID NO. 14, the 51 st to 58 th amino acids are CDR2, the sequence is shown as SEQ ID NO. 15, and the 97 th to 104 th amino acids are CDR3, and the sequence is shown as SEQ ID NO. 16). The DNA coding sequence of the light chain of the antibody is shown as SEQ ID NO. 5, the corresponding amino acid sequence is shown as SEQ ID NO. 6, the DNA coding sequence of the light chain variable region is shown as SEQ ID NO. 7, the corresponding amino acid sequence is shown as SEQ ID NO. 8 (wherein, 27 th to 32 th amino acids are CDR1, the sequence is shown as SEQ ID NO. 17, 50 th to 62 th amino acids are CDR2, the sequence is shown as SEQ ID NO. 18, 89 th to 97 th amino acids are CDR3, and the sequence is shown as SEQ ID NO. 19).
2. Construction of Single chain antibodies
Based on the above sequences, we mutated both serine at position 44 of the heavy chain variable region and glycine at position 100 of the light chain variable region to cysteine, by which mutation the stability of the finally formed single chain antibody was increased. The mutated heavy chain variable region and the light chain variable region are then joined by a flexible polypeptide chain (SEQ ID NO: 9) to give a single chain antibody having the sequence shown in SEQ ID NO: 10. According to ELISA experiments, the single-chain antibody has good affinity and specificity to CD 206.
3. Coupling of targeted drugs
Albumin binding domains (ABD, sequence shown in SEQ ID NO: 11) were used to extend serum half-life. Exotoxin PE38X8 of pseudomonas aeruginosa PE38 inhibits the function of the protein by catalyzing irreversible ribosylation of elongation factor 2, thereby exerting a cytotoxic effect in the cytoplasm, mediating target cell death. Deletion of the PE38X8 native cell binding domain and removal of the T cell epitope results in a truncated form of PE38X8, hereinafter PE (SEQ ID NO: 12), with reduced immunogenicity for multiple administration to the tumor site.
And (3) connecting the single-chain antibody with PE in the direction from the N end to the C end by using the flexible connecting peptide to obtain the alpha CD206-PE.
And (3) sequentially connecting the single-chain antibody, the ABD and the truncated PE38X8 from the N end to the C end by using a flexible connecting peptide to obtain the antibody coupling medicament (the amino acid sequence is shown as SEQ ID NO: 13) targeting the M2 type tumor-associated macrophage, wherein the alpha CD206-ABD-PE is prepared.
The above proteins (PE, αCD206-PE and αCD 206-ABD-PE) were used to construct expression vectors, and E.coli was used for expression.
4. Endocytosis and binding of cells
The constructed proteins PE and alpha CD206-PE are treated with Zip Alexa Fluor TM 488Rapid Antibody Labeling Kit (Thermo Fisher Scientific) markers; inoculating the macrophage line RAW into two 12-pore plates, wherein one 12-pore plate is added with LPS to induce the M1 type macrophages to differentiate, and the other 12-pore plate is added with IL-4 to induce the M2 type macrophages to differentiate; after differentiation was completed, labeled PE and αcd206-PE were added to six wells of a 12-well plate, respectively, and PBS containing 0.1% fetal bovine serum was added, and incubated at 4 ℃ for 30min; the reaction was then stopped by adding PBS, centrifuged at 1200rpm for 6min, and washed twice. The MFI of the incubation mixture was then determined with a flow cytometer. The results are shown in FIG. 3, in which αCD206-PE cell surface binding occurs but internalization is inhibited. After incubation, the average fluorescence intensity (MFI) of M1 macrophages was less than M2 macrophages. In contrast, M1 and M2 macrophages incubated with labeled "PE" had lower MFI.
The same experiment was repeated, except that the temperature of incubation of the cytotoxin was 37 ℃. As a result, as shown in FIG. 4, the surface binding and internalization of the αCD206-PE with the M1 type and M2 type macrophages can be performed simultaneously. After incubation, the average fluorescence intensity (MFI) of M1 macrophages was less than M2 macrophages. In contrast, M1 and M2 macrophages incubated with labeled "PE" had lower MFI.
The above results show that αcd206-PE is more prone to M2 type macrophage binding than PE and internalization is unaffected at 37 ℃.
5. Cytotoxicity test
5.1 six well plate experiments
The method comprises the following steps: macrophage cell line RAW was inoculated into two six-well plates, cultured at 37℃for 8h for adherence, and then 2.5. Mu.L of IL-4 (20 ng/. Mu.L) was added to the first row of wells of the six-well plates, respectively, for stimulation for 48 hours, to differentiate into M2 type macrophages. PE was added to one of the two six well plates and the other was added to the alpha CD206-PE. FIG. 5 shows the third and fourth days after addition of cytotoxin, showing that the cell number is relatively stable in wells without IL-4, and that the more cells are killed over time in IL-4-induced wells. Since RAW cells have not been induced to M2 type cells for only 8 hours prior to the addition of cytotoxin, the aCD 206-PE has no killing effect on these cells. However, over time, more and more cells are induced to M2 type cells and are replaced by αCD206-PE. In the six-well plate with PE, this phenomenon does not exist. Therefore, the coupling of the alpha CD206 and PE has no influence on the killing power of PE and the specificity of antibody, and the coupling medicine formed by the alpha CD206 and the PE can specifically kill M2 type macrophages without obvious killing effect on undifferentiated RAW cells.
5.2CCK8 reagent enzyme-labeled instrument detection experiment
The method comprises the following steps: RAW cells were seeded into 96-well plates (10,000 cells per well), stimulated with IL-4 for 48h, incubated with cytotoxin for 48h, and then incubated with 10 μl CCK8 reagent per well for 48h. Absorbance at 450nm was measured using a microplate reader. As shown in FIG. 6, the toxicity of PE was not affected by the coupling of the single-chain antibody and ABD.
The experiment shows that the single-chain antibody of the invention does not affect ABD and PE, and the constructed ADC can specifically kill M2 type macrophages in vitro.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> WUHANMAIKAISHEN Biotechnology Co.Ltd
<120> an antibody capable of binding to CD206 and use thereof
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catggaaaga gccttgagtg gattggaggt attaatccta acaatggtgg tactagctac 180
aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240
atggacctcc gcagcctgac atctgaggat tctgcagtct attactgtgc ctccactacg 300
gctggggagt actggggcca aggcaccact ctcacagtct cctcggctaa aacgacaccc 360
ccatctgtct atccactggc ccctggatct gctgcccaaa ctaactccat ggtgaccctg 420
ggatgcctgg tcaagggcta tttccctgag ccagtgacag tgacctggaa ctctggatcc 480
ctgtccagcg gtgtgcacac cttcccagct gtcctgcagt ctgacctcta cactctgagc 540
agctcagtga ctgtcccctc cagcacctgg cccagcgaga ccgtcacctg caacgttgcc 600
cacccggcca gcagcaccaa ggtggacaag aaaattgtgc ccagggattg tggttgtaag 660
ccttgcatat gtacagtccc agaagtatca tctgtcttca tcttcccccc aaagcccaag 720
gatgtgctca ccattactct gactcctaag gtcacgtgtg ttgtggtaga catcagcaag 780
gatgatcccg aggtccagtt cagctggttt gtagatgatg tggaggtgca cacagctcag 840
acgcaacccc gggaggagca gttcaacagc actttccgct cagtcagtga acttcccatc 900
atgcaccagg actggctcaa tggcaaggag ttcaaatgca gggtcaacag tgcagctttc 960
cctgccccca tcgagaaaac catctccaaa accaaaggca gaccgaaggc tccgcaggtg 1020
tacaccattc cacctcccaa ggagcagatg gccaaggata aagtcagtct gacctgcatg 1080
ataacagact tcttccctga agacattact gtggagtggc agtggaatgg gcagccagcg 1140
gagaactaca agaacactca gcccatcatg gacacagatg gctcttactt cgtctacagc 1200
aagctcaatg tgcagaagag caactgggag gcaggaaata ctttcacctg ctctgtgtta 1260
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Val Arg Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
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Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr
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Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly
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Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys
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Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
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Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
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Ser Thr Thr Ala Gly Glu Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
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Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly
115 120 125
Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys
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Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu
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Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
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Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu
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Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp
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Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
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Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp
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Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn
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Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp
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Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro
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Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala
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Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp
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Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile
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Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn
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Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys
385 390 395 400
Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys
405 410 415
Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu
420 425 430
Ser His Ser Pro Gly Lys
435
<210> 3
<211> 345
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gaggttcggc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60
tcctgcaaga cttctggata cacattcact gaatacacca tgcactgggt gaagcagagc 120
catggaaaga gccttgagtg gattggaggt attaatccta acaatggtgg tactagctac 180
aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240
atggacctcc gcagcctgac atctgaggat tctgcagtct attactgtgc ctccactacg 300
gctggggagt actggggcca aggcaccact ctcacagtct cctcg 345
<210> 4
<211> 114
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Val Arg Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
1 5 10 15
Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr
20 25 30
Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly
35 40 45
Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys
50 55 60
Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
65 70 75 80
Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
85 90 95
Ser Thr Thr Ala Gly Glu Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
100 105 110
Ser Ser
<210> 5
<211> 645
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
gatgttgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60
atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acagaaacca 120
ggacaatctc ctaaactact gatttactcg gcatcctacc ggtacactgg agtccctgat 180
cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgtgcaggct 240
gaagacctgg cagtttatta ctgtcagcaa cattatagta ctccgtacac gttcggaggg 300
gggaccaagc tggaaatgaa acgcgcagat gctgcaccaa ctgtatccat cttcccacca 360
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 600
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag 645
<210> 6
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Asp Val Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 7
<211> 321
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gatgttgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60
atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acagaaacca 120
ggacaatctc ctaaactact gatttactcg gcatcctacc ggtacactgg agtccctgat 180
cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgtgcaggct 240
gaagacctgg cagtttatta ctgtcagcaa cattatagta ctccgtacac gttcggaggg 300
gggaccaagc tggaaatgaa a 321
<210> 8
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Asp Val Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys
100 105
<210> 9
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 10
<211> 237
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Glu Val Arg Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Met His Trp Val Lys Gln Ser His Gly Lys Cys Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Thr Thr Ala Gly Glu Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Val Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser
130 135 140
Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser
145 150 155 160
Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
165 170 175
Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe
180 185 190
Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val
195 200 205
Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr
210 215 220
Pro Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu Met Lys
225 230 235
<210> 11
<211> 46
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly
1 5 10 15
Val Ser Asp Phe Tyr Lys Arg Leu Ile Asn Lys Ala Lys Thr Val Glu
20 25 30
Gly Val Glu Ala Leu Lys Leu His Ile Leu Ala Ala Leu Pro
35 40 45
<210> 12
<211> 218
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser
1 5 10 15
Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His
20 25 30
Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr
35 40 45
Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg
50 55 60
Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp
65 70 75 80
Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg
85 90 95
Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser
100 105 110
Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu
115 120 125
Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg
130 135 140
Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr
145 150 155 160
Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala
165 170 175
Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser
180 185 190
Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser
195 200 205
Gln Pro Gly Lys Pro Pro Lys Asp Glu Leu
210 215
<210> 13
<211> 531
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Glu Val Arg Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Met His Trp Val Lys Gln Ser His Gly Lys Cys Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Thr Thr Ala Gly Glu Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Val Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser
130 135 140
Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser
145 150 155 160
Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
165 170 175
Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe
180 185 190
Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val
195 200 205
Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr
210 215 220
Pro Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu Met Lys Gly Gly Gly
225 230 235 240
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Ala Glu Ala
245 250 255
Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Phe
260 265 270
Tyr Lys Arg Leu Ile Asn Lys Ala Lys Thr Val Glu Gly Val Glu Ala
275 280 285
Leu Lys Leu His Ile Leu Ala Ala Leu Pro Gly Gly Gly Gly Ser Gly
290 295 300
Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Thr Gly Ala Glu Phe Leu
305 310 315 320
Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp
325 330 335
Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly
340 345 350
Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser
355 360 365
Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile
370 375 380
Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr
385 390 395 400
Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala
405 410 415
Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg
420 425 430
Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg
435 440 445
Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro
450 455 460
Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala
465 470 475 480
Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn
485 490 495
Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala
500 505 510
Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys
515 520 525
Asp Glu Leu
530
<210> 14
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Gly Tyr Thr Phe Thr Glu Tyr Thr
1 5
<210> 15
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Ile Asn Pro Asn Asn Gly Gly Thr
1 5
<210> 16
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Ala Ser Thr Thr Ala Gly Glu Tyr
1 5
<210> 17
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Gln Asp Val Ser Thr Ala
1 5
<210> 18
<211> 3
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Ser Ala Ser
1
<210> 19
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Gln Gln His Tyr Ser Thr Pro Tyr Thr
1 5

Claims (8)

1. An antibody that binds CD206 comprising a light chain variable region and a heavy chain variable region, wherein the sequences of CDRs 1-3 of the heavy chain variable region are set forth in SEQ ID NOs: 14-16, the sequences of CDRs 1-3 of the light chain variable region are set forth in SEQ ID NOs: 17-19.
2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4 and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 8.
3. The antibody of claim 1, wherein the sequence is set forth in SEQ ID NO. 10.
4. Use of the antibody of any one of claims 1-3 in the manufacture of a medicament for specifically killing M2-type macrophages.
5. An antibody-conjugated drug comprising the antibody of any one of claims 1-3 as an N-terminal domain.
6. The antibody conjugated drug of claim 5, further comprising a polypeptide having cytotoxicity as a C-terminal domain.
7. The antibody conjugated drug according to claim 6, wherein the sequence of the polypeptide having cytotoxicity is shown in SEQ ID NO. 12.
8. The antibody conjugated drug of any of claims 5-7, wherein the sequence is shown in SEQ ID No. 13.
CN202110782069.4A 2021-07-12 2021-07-12 Antibody capable of binding CD206 and application thereof Active CN113512117B (en)

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WO2018227023A1 (en) * 2017-06-07 2018-12-13 Silverback Therapeutics, Inc. Antibody construct conjugates
CN110872348A (en) * 2018-09-03 2020-03-10 长春金赛药业有限责任公司 Humanized anti-CD 47 monoclonal antibody and application thereof
CN111484553A (en) * 2020-05-18 2020-08-04 上海普铭生物科技有限公司 Screening method of pH-dependent antibody of targeting complement protein C5

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EP3360898A1 (en) * 2017-02-14 2018-08-15 Boehringer Ingelheim International GmbH Bispecific anti-tnf-related apoptosis-inducing ligand receptor 2 and anti-cadherin 17 binding molecules for the treatment of cancer

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WO2018227023A1 (en) * 2017-06-07 2018-12-13 Silverback Therapeutics, Inc. Antibody construct conjugates
CN110872348A (en) * 2018-09-03 2020-03-10 长春金赛药业有限责任公司 Humanized anti-CD 47 monoclonal antibody and application thereof
CN111484553A (en) * 2020-05-18 2020-08-04 上海普铭生物科技有限公司 Screening method of pH-dependent antibody of targeting complement protein C5

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