CN110066336B - anti-CD 47 monoclonal antibody, fragment and medical application thereof - Google Patents
anti-CD 47 monoclonal antibody, fragment and medical application thereof Download PDFInfo
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Abstract
A CD47 antibody or antigen-binding fragment thereof, comprising any 1 CDR region sequence or mutated sequence selected from the group consisting of an antibody heavy chain variable region HCDR sequence as set forth in SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, a nitrogen source; the sequence of the LCDR of the antibody light chain variable region is shown as SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81. the antibody of the invention is a CD47 monoclonal antibody with high affinity and high specificity, has blocking function, does not cause biological functions such as erythrocyte agglutination and the like, and has good medicinal development value.
Description
Technical Field
The invention relates to a high-affinity monoclonal antibody capable of specifically binding to CD47, in particular to a preparation method of the monoclonal antibody, a variable region sequence of the monoclonal antibody, and application of the antibody in a tumor treatment process.
Background
CD47 is also known as integrin-associated protein (IAP), CD47 is a 5-transmembrane glycoprotein with a molecular weight of 50KD and is a member of the immunoglobulin superfamily. From a steric structure perspective, the CD47 molecule is composed of the extracellular domain of IgV, 5 highly hydrophobic transmembrane segments, and a shorter intracellular domain. Since the discovery, CD47 is confirmed to be a plurality of tumor cell surface antigens, and is highly expressed on the surfaces of a plurality of human tumor cells, such as human ovarian cancer, acute myelogenous leukemia, chronic conformal cell leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphocytoma, bladder cancer, breast cancer and other malignant tumor cells, high expression of CD47 is found, and high expression of CD47 is negatively correlated with prognosis and survival rate of patients, and the published research shows that CD47 can be used as a potential target for treating cancers. CD47 interacts with signal-regulatory proteins, thrombospondin, integrins to mediate cell proliferation, apoptosis, and phagocytosis regulation. CD47 interacts with signal-regulatory protein alpha as a receptor and ligand, and a CD47-SIRP alpha complex is formed. The SIRP alpha belongs to an immunoglobulin superfamily, is mainly expressed on the surfaces of dendritic cells, macrophages and neutrophils, the tail part of the SIRP alpha is combined with immunoreceptor tyrosine inhibitor motif immune-based inhibition motifs (ITIMs), and CD47-SIRP alpha, so that the tail part of the SIRP alpha intracellular segment is phosphorylated, and further SHP1 and SHP2 are recruited to conduct an immunosuppressive signal, thereby regulating the phagocytosis of the macrophages, and the tumor cells are subjected to the mechanism to avoid the monitoring of an immune system by highly expressing CD47 to 'eat me' signals. The anti-CD 47 antibody, the polypeptide for blocking the interaction between CD47 and SIRPa, the fusion protein and other methods are used for blocking the interaction between CD47 and SIRPa, and the method has the significance of targeted therapy. The antibodies and fusion proteins for blocking the interaction between CD47 and SIRPa have been distributed by a plurality of pharmaceutical companies, and at present, the antibodies and fusion proteins are clinically Hu5F9-G4 of Forty Seven, CC-90002 of Celgene and TTI-621 of Trillium, TJC4 of a habitat organism, IBI188 of a messenger organism, SHR-1603 of a constant medicine and the like.
The invention takes recombinant human CD47 extracellular domain (rhECD47) protein as antigen, and prepares the anti-CD 47 specific monoclonal antibody with high affinity and high blocking effect by a hybridoma technology and a rapid screening system, and in vitro experiments verify that the obtained anti-CD 47 monoclonal antibody has good biological activity and anti-tumor activity.
Disclosure of Invention
The present invention provides a new nucleotide sequence and corresponding amino acid sequence of CD47 monoclonal antibody.
In one aspect, the invention provides an antibody or functional fragment thereof that specifically binds to CD 47. Wherein the antibody comprises a heavy chain and a light chain. (I) Wherein the antibody heavy chain comprises SEQ ID NO: 58. 59 and 60, or SEQ ID NO: 64. 65 and 66, or SEQ ID NO: 70. 71 and 72, or SEQ ID NO: 73. 74 and 75 or SEQ ID NO: 76. 77 and 78, HCDR1, HCDR2 and HCDR 3; (II) wherein the antibody light chain comprises SEQ ID NO: 61. 62 and 63, or SEQ ID NO: 67. 68 and 69, or SEQ ID NO: 79. 80 and 81, LCDR1, LCDR2 and LCDR 3.
The invention provides antibodies comprising an antibody or functional fragment capable of specifically binding to CD47, (I) the antibody heavy chain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 39. 43, 47, 51 and 55; (II) the antibody light chain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41. 45, 49, 53 and 57.
In another aspect, the invention provides nucleotide sequences capable of encoding antibodies and functional fragments that specifically bind CD47, wherein (I) the nucleotide sequences encoding the heavy chain of the antibody comprise a nucleotide sequence having the amino acid sequence of SEQ ID NO: 38. 42, 46, 50, 54; (II) the nucleotide sequence encoding the light chain of the antibody has the sequence shown in SEQ ID NO: 40. 44, 48, 52.
Preferably, the antibody of the invention is selected from clones 2D1,2D14,2D27,2D47,2D 51.
In a preferred embodiment of the invention, the antigen-specific binding fragment may be a Fab, scFv, (Fab') 2.
The invention takes recombinant human CD47 extracellular region protein (purchased from Beijing Yiqiao Shenzhou) as immunogen, and is mixed with a mouse rapid immune adjuvant in equal volume to immunize a Balb/c mouse, the serum titer is detected after three times of immunization, the cell fusion is carried out on mouse spleen cells and sp2/0-Ag14 myeloma cells which meet the fusion requirement, the hybridoma cell strains which can stably secrete anti-CD 47 and have the function of blocking biological function antibody are obtained by HAT condition culture and screening, stably express CD47 protein HEK293 cell ELISA screening and blocking function rapid screening system screening, after subcloning and expanding culture, the heavy chain and light chain variable region sequences of the antibody are respectively regulated and sequenced by a molecular cloning method. The ascites is produced by injecting hybridoma cells into the abdominal cavity of the mouse, and then the monoclonal antibody is prepared by the purification of protein A/G affinity chromatography. A series of in vitro experiments prove that the antibody is a high-affinity and high-specificity CD47 monoclonal antibody, has a blocking function, does not cause biological functions such as erythrocyte agglutination and the like, and has good medicinal development value.
The beneficial effects of the invention include:
the invention successfully prepares the CD47 monoclonal antibody with good specificity, high affinity and strong blocking effect, and in vitro experiments show that the antibody can effectively block the interaction between CD47 and SIRPa. In vitro hemagglutination experiments showed that the antibody did not cause hemagglutination. In vitro phagocytosis experiments show that the antibody can effectively promote the phagocytosis of macrophages. In vitro experiments show that the anti-CD 47 antibody has good medicinal development value.
Drawings
FIG. 1 is a graph showing the results of serum titer determination of immunized mice.
FIG. 2 shows SDS-PAGE identification of purified antibody, wherein the denatured and reduced antibody has an obvious heavy chain band at 50kDa, an obvious light chain band at 25kDa and the denatured and non-reduced antibody has a single band at 150kDa, so that the antibody has a molecular weight corresponding to the size of the antibody and a purity of 95% or higher.
FIG. 3 is a schematic diagram showing the result of subtype identification of monoclonal antibodies.
FIG. 4 is a schematic diagram of flow cytometry detection of binding of antibody 2D1 to CD47 antigen native to the surface of K562 cells.
FIG. 5 is a schematic diagram of flow cytometry detection of binding of antibody 2D14 to the native CD47 antigen on the surface of K562 cells.
FIG. 6 is a schematic diagram of flow cytometry detection of binding of antibody 2D27 to CD47 antigen native to the surface of K562 cells.
FIG. 7 is a schematic diagram of the detection of the binding of antibody 2D51 to the CD47 antigen native to the surface of K562 cells by flow cytometry.
FIG. 8 is a schematic diagram of flow cytometry detection of binding of antibody 2D47 to CD47 antigen native to the surface of K562 cells.
FIG. 9 monoclonal antibody 2D1 blocks binding of CD47 to SIRPa, and an IC50 curve fit plot.
FIG. 10 monoclonal antibody 2D14 blocks binding of CD47 to SIRPa, and an IC50 curve fit plot.
FIG. 11 monoclonal antibody 2D47 blocks binding of CD47 to SIRPa, and an IC50 curve fit plot.
FIG. 12 monoclonal antibody 2D51 blocks binding of CD47 to SIRPa, and an IC50 curve fit plot.
FIG. 13 monoclonal antibody 2D27 blocks binding of CD47 to SIRPa, and an IC50 curve fit plot.
FIG. 14 monoclonal antibody 2D1 binds to recombinant CD47 protein, EC50 curve fit plot.
FIG. 15 monoclonal antibody 2D14 binds to recombinant CD47 protein, EC50 curve fit plot.
FIG. 16 monoclonal antibody 2D27 binds to recombinant CD47 protein, EC50 curve-fit plot.
Fig. 17 monoclonal antibody 2D47 binds to recombinant CD47 protein, EC50 curve fit plot.
FIG. 18 monoclonal antibody 2D51 binds to recombinant CD47 protein, EC50 curve fit plot.
FIG. 19 is a graph showing the dependence of the concentration of monoclonal antibody 2D1 on CD47/HEK293 cells.
FIG. 20 is a graph showing the dependence of the concentration of monoclonal antibody 2D14 on CD47/HEK293 cells.
FIG. 21 is a graph showing the dependence of the concentration of monoclonal antibody 2D27 on CD47/HEK293 cells.
FIG. 22 is a graph showing the dependence of the concentration of monoclonal antibody 2D47 on CD47/HEK293 cells.
FIG. 23 is a graph showing the dependence of the concentration of monoclonal antibody 2D51 on CD47/HEK293 cells.
FIG. 24 shows PCR amplification of heavy and light chain genes of monoclonal antibodies 2D1,2D14 and 2D 27.
FIG. 25 shows PCR amplification of heavy and light chain genes for monoclonal antibody 2D47 and 2D 51.
FIG. 26 is a schematic diagram showing the hemagglutination results of the CD47 monoclonal antibody.
FIG. 27 is a graph showing the results of the CD47 chimeric antibody on macrophage phagocytosis of K562 cells.
Detailed Description
The present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the present invention. The monoclonal antibody and the simple modification of the preparation method thereof provided by the invention belong to the protection scope of the invention.
The experimental procedures used in the examples listed below are all conventional procedures unless otherwise specified.
Example 1 mouse immunization
The recombinant human CD47-FC (12283-H02H, Beijing Yiqian Shenzhou) protein is dissolved in PBS buffer solution with the concentration of 0.5 mu g/mu l, 25 mu g (50 mu l) of antigen and an equal volume of QuickAntibody-Mouse5W (KX 0210042, Beijing Boolong immune technology Co., Ltd.) immune adjuvant are fully mixed, and the hind leg calf muscle is injected into BAL b/c (Shanghai Sphere-BiKai laboratory animals Co., Ltd., license number:) with the age of 5-8 weeks, and each injection is 100 mu l. The total time of 3-5 immunizations is 14 days, the tail vein of the mouse is sampled 10 days after 3 immunizations, and the serum titer of the mouse is determined by an indirect ELISA method by using an antibody diluent to dilute the serum from 1:1000 times. And selecting a mouse with high specificity and high titer of serum antibodies as a fusion candidate mouse. After three times of immunization, the serum titer of the mice reaches more than 1:100,000, and the mice are considered as fusible mice, and are subjected to impact immunization, and the measurement result of the serum titer of the immunized mice is shown in figure 1.
EXAMPLE 2 preparation of hybridoma cell lines
1. Cell fusion
The cell fusion is carried out by the polyethylene glycol method. The specific operation is as follows:
recovering sp2/0-Ag14 myeloma cells, and after sp2/0-Ag14 is recovered and passaged for 2 times, the cell survival rate is more than or equal to 95 percent, the cell state is considered to be good, and the fusion is suitable. The cells are expanded and cultured before cell fusion, the cells are replaced by fresh culture medium after the cells reach logarithmic growth period before cell fusion, and the cell fusion is started after four hours. After the third immunization of the mice, the serum titer reaches 1:100000, which is regarded as reaching the fusion standard, and the mice are subjected to impact immunization. Selecting a mouse with good antigen specificity and highest titer from immune serum, performing impact immunization according to 3 days before fusion, and performing intraperitoneal injection of 50 mu g of corresponding antigen;
preparation of feeder layer cells (non-immunized mouse splenocytes): spleen from unimmunized normal BAL b/c or ICR mice was harvested one day prior to fusion, and a suspension of the cells was prepared into a suspension of epithelial cells, plated at 5X104 per well, 100. mu.l per well, labeled and cultured overnight in a cell culture incubator (Thermo Fisher, BB150) at 37 ℃ in 5% CO 2. sp2/0-Ag14 cells, discarding the supernatant culture medium, resuspending sp2/0-Ag14 cells with serum-free RPIM1640 culture medium (CellMax, CGM112.05), centrifuging at 1500rpm × 3min, washing the cells with serum-free RPIM1640 culture medium once, discarding the supernatant, resuspending the cells with serum-free RPIM1640, and counting, wherein the total cell number is generally 0.8-2 × 107And (4) standing by. Collecting blood from the eyeballs of the mice to be fused, centrifuging at 12000rpm multiplied by 5min to collect serum, subpackaging and storing at-80 ℃ for later use, wherein the mouse serum can be used as a control positive antibody in fusion and subsequent screening. The mice were sacrificed by cervical dislocation, soaked in 75% alcohol and transferred to a clean bench. The mouse spleen was removed, ground on a 40 μm cell screen (Guangzhoujiert, CSS010040) and filtered to make a single cell suspension, centrifuged at 2000 rpm. times.5 min, and the supernatant discarded. The spleen cells were centrifuged twice and then counted for viable cells by successive washes with RPIM1640 medium. Fusing: according to the technical result, sp20-Ag14 myeloma cells and spleen cells are mixed in a ratio of 1: 5, thoroughly mixed, centrifuged and supernatant discarded. Gently flicking the tube wall to uniformly mix and suspend the precipitated cells, slowly dripping 1ml of PEG1450 (BF 08003) pre-warmed at 37 ℃ into the cell mixed solution while stirring for 90s, immediately adding 30ml of RPIM1640 culture medium preheated at 37 ℃ into the cells, standing for 5min, and centrifuging. 1200rpm 5min, the cell pellet was resuspended in 20% FBS (11011-. And (3) observing and changing liquid after fusion: cell status was observed periodically after fusion, and on the fourth day after fusion, the medium in 100 μ l 96-well plates (Corning, 3599) was replaced with fresh RPIM1640 medium containing 1xHT 20% FBS; post fusion 7 thDay, all the medium in the 96-well plate was replaced with 20% FBS-RPIM1640 medium containing 1XHT (H-Hypoxanthine Hypoxanthine, T-Thymidine Thymidine), and cultured in a cell incubator at 37 ℃ in 5% CO 2.
2. Positive clone selection and subcloning of fusion cells
And marking clone forming holes in the fusion clone holes by using microscope observation, taking important marked cell holes with the monoclonal forming clusters as the focus of attention of subsequent screening of the monoclonal, and detecting the antibody secretion condition in each hole of the fusion cell plate by using indirect ELISA. HEK293 cells expressing rhECD47 protein at 2.5-3X 104Plating cells/hole, culturing overnight in a cell culture box with 5% CO2 at 37 ℃, fixing for 15 minutes by using 4% paraformaldehyde on the next day, sealing for 1 hour by using 5% skimmed milk powder (BD, 232100) to serve as a screening antigen, using HRP-labeled Goat anti Mouse IgG (Jackson, 115-.
EXAMPLE 3 anti-CD 47 ascites type antibody preparation
1. Ascites type antibody preparation
Female Bal b/c born mice, 10-12 weeks old, were picked and injected intraperitoneally with 500. mu.l sterile paraffin oil (Sigma, M5310). One week later, 2X 106 hybridoma cells with good growth status were inoculated by intraperitoneal injection. Observing the survival state of the mouse after about 7 days of inoculation, generally, after 7-10 days of inoculation, obviously bulging the abdomen of the mouse, collecting ascites at the moment, standing the collected ascites at 4 ℃ overnight, centrifuging at 12000rpm for 5min the next day, removing upper-layer oily paraffin, and taking middle-layer ascites for further affinity chromatography purification.
2. Antibody affinity chromatography purification
Antibody affinity chromatography purification was performed using ProteinA/G (Sozhou Nami Microscience, Inc., H10A11) column. The method comprises the following specific operations: diluting ascites with 1xPBS (5-10 times of ascites volume), centrifuging at 12000rpm/min for 20min, filtering supernatant with 0.22 μm filter membrane (Merck Millipore, SLGP033RB), sampling filtered samples, washing chromatographic columns with 1XPBS (pH7.4) of 20 column volumes after all samples flow to chromatographic columns, removing non-specifically bound impurities, eluting with eluent, adding 5-10ml eluent to elute antibodies, collecting eluent, adding corresponding volume of neutralization buffer solution into eluent in advance to quickly restore the pH value to neutrality, regenerating the column packing after eluting the antibodies according to the operation instructions of a production company, finally storing in 20% ethanol, and storing at 4 ℃.
3. Antibody purity and molecular weight characterization
The collected antibody eluate is concentrated by a concentration tube, the buffer solution is replaced by 1XPBS, and then a denatured-reduced sample and a denatured-non-reduced sample are respectively prepared, and the antibody is preliminarily identified by SDS-PAGE electrophoresis, as shown in figure 2.
EXAMPLE 4 monoclonal antibody identification
1. Identification of antibody subtypes
The subtype of the obtained monoclonal antibody is identified by adopting a mouse monoclonal antibody subtype identification kit (SEK 003, Beijing-sense Qianzhou biotechnology, Inc.), according to the use specification of the kit, rabbit anti-mouse specific antibodies of anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM and the like are coated on an enzyme label plate firstly, coated overnight at 4 ℃, sealed by 5 percent skim milk powder for 1h, the antibody to be detected is added into corresponding holes at the concentration of 1mg/ml, each hole is incubated for 1h at the temperature of 100 mu l and 37 ℃, after washing for three times by 1XTBST, the goat anti-mouse antibody marked by enzyme HRP is added, and finally, a color developing solution is added for color development, a stopping solution is added, and reading is carried out. As shown in FIG. 3, the heavy chain subtype of the anti-CD 47 monoclonal antibody was IgG1 and the light chain subtype was Kappa.
The antibody sequence of the invention can also realize antibody subtype conversion by a genetic engineering method.
2. Flow cytometry for detecting binding of CD47 antibody and K562 cells
According to relevant literature reports and control antibody pre-experiments, K562 (human chronic myelogenous leukemia cells) expressing human CD47 on the cell surface is selected, and the specificity of the anti-CD 47 monoclonal antibody is detected by flow cytometry. K562 (human chronic myelogenous leukemia cells) is suspension cells, RPIM-1640 culture medium is firstly discarded, the suspension cells are resuspended by precooled PBS and counted, finally the cell density is adjusted to 1.5 multiplied by 106/ml, then the suspension cells are subpackaged into 1.5ml EP tubes, each tube is 100 mul, the centrifugation is carried out at 1500rpm/min for 5min, supernatant PBS is discarded, primary anti-incubation is carried out, 250 mul of diluted CD47 monoclonal antibody is respectively added into the cell tubes, the antibody concentration is 2.5mg/ml, and the incubation is carried out for 30min at 4 ℃. Washing, centrifuging at 1500rpm/min for 5min, discarding supernatant antibody, adding 500 μ l of precooled PBS into cell tube, gently pipetting and beating resuspended cells, and washing three times. And (3) performing secondary antibody incubation, centrifuging after the third washing to remove the PBS of the supernatant, adding an AF488 goat anti-mouse fluorescent secondary antibody (Jackson,115-546-068), incubating for 30min at 4 ℃ in a dark place, washing, centrifuging to remove PBS of the supernatant, adding 250 mu l of precooled PBS into each tube, re-suspending cell precipitates, and detecting by using a flow cytometer. Binding of mouse IgG, anti-CD 47 positive control antibody, and anti-CD 47 candidate antibody to H1975 cells, respectively, was detected, indicating specific binding to CD 47.
From the flow detection results (fig. 4-8), the anti-CD 47 positive control antibody and the candidate anti-CD 47 monoclonal antibody can be well combined with CD47 on the surface of human cells, and the combination rate can reach more than 95%. Mouse IgG did not bind to K562 cells, indicating that the candidate anti-CD 47 monoclonal antibody specifically binds to CD47 of native structure.
3. Candidate anti-CD 47 antibodies are effective in blocking the binding of CD47 to SIRPa
Published studies have shown that sirpa is the primary ligand of CD47, and either sirpa protein or CD47 protein is capable of binding to CD47 protein or sirpa on the cell surface. In view of the above, the laboratory establishes the stable cell strains SIRP alpha-EGFP/HEK 293 and CD47-RFP/HEK293 expressed by fusion expression of the fluorescent protein and CD47-RFP, and correspondingly purifies the stable cell strains SIRP alpha-EGFP/HEK 293 and CD47-RFP/HEK293 to obtain the SIRP alpha-EGFP and CD47-RFP fusion protein, and the binding rate of the SIRP alpha-1-EGFP fusion protein and the CD47-RFP/HEK293 cell is more than 95% by flow detection. Based on the detection, the flow cytometry method is used for detecting the binding of the candidate anti-CD 47 monoclonal antibody to SIRP alpha-EGFP and CD47-RFP/HEK293The blocking effect of (1). Good growth CD47-RFP/HEK293 was first trypsinized and resuspended in pre-cooled PBS before counting, and finally cell density was adjusted to 1.5X 106And each sample is divided into 1.5ml EP tubes, each tube is centrifuged at 1500rpm for 5min at 100 mu l, supernatant PBS is discarded, 2.5 mu g of SIRP alpha-EGFP protein is added into each group, meanwhile, the candidate anti-CD 47 monoclonal antibody is sequentially diluted by 12 gradients according to the concentration multiple ratio of 20 mu g/ml and added into the corresponding CD47-RFP/HEK293 cell tube for co-incubation, the binding condition of the SIRP alpha-EGFP protein and the CD47-RFP/HEK293 cell is detected by flow cytometry, and the IC50 of the candidate CD47 antibody is obtained by curve fitting according to the average fluorescence intensity detected in the tubes with different concentrations, and the result is shown in figures 9-13.
4. Indirect ELISA method for detecting CD47 monoclonal antibody EC50
Firstly, recombinant human CD47 extracellular region protein is taken as antigen, 100 mul of the antigen is coated overnight at 4 ℃ in each well at 100ng/ml, 5% skimmed milk is used for sealing at 37 ℃ for 1 hour the next day, 1XTBST is washed once, then diluted candidate anti-CD 47 monoclonal antibody and control antibody with different concentrations are added into an enzyme label plate, after 1 hour of reaction at 37 ℃, 1XTBST is washed for 4 times, HPR labeled goat anti-mouse secondary antibody is added, incubation is carried out for 45min at 37 ℃, 1XTBST is washed for 4 times, then chromogenic reaction is carried out for 10min at room temperature in a developing solution, after termination, an enzyme reader (Thermo, Multiskan) reads OD450 value, GraphPad Prism 7 software is used for data processing and mapping analysis, a binding curve of the candidate anti-CD 47 monoclonal antibody to the recombinant human CD47 protein and an EC50 value are obtained through curve fitting, as shown in figure 14-18, the detection result shows that the candidate anti-CD 47 monoclonal antibody can be specifically bound with the recombinant human CD47 protein, and EC50 is 0.10 nmol/L.
4. Cell ELISA method for measuring concentration dependence of monoclonal antibody combined with CD47/HEK293F cell
HEK293F is human embryonic kidney cell, the cell surface does not express human CD47 antigen, we through the transfection method to make it transiently express human CD47, transfected cells inoculated into 96-well plates, each well 2.5X104Culturing individual cell in a 37 deg.C 5% CO2 incubator for 48h, fixing 4% paraformaldehyde for 15min, washing with PBS 3 times, sealing 5% skimmed milk at 37 deg.C for 1h, washing with PBS 1 time, and spin-dryingThe application is as follows. Diluting antibodies of different clones in a gradient manner, adding the diluted monoclonal antibodies into a cell plate, incubating for 1.5h at 37 ℃, washing the cell plate for 4 times by TBST, adding diluted secondary antibodies after spin-drying, incubating for 1h, washing the cell plate for 4 times by TBST, adding TMB color development liquid after spin-drying, stopping color development, reading light absorption value OD450 by an enzyme labeling instrument (Thermo, MultiskGO), and performing data processing and mapping analysis by using GraphPad Prism 7 software to reflect the concentration dependence of the antibody combined with the cells.
From the results of the experiments in FIGS. 19-23, it can be seen that the CD47 monoclonal antibody binds to CD47/HEK293F cells in a very concentration-dependent manner.
EXAMPLE 5 sequencing of heavy and light chain variable region genes of candidate anti-CD 47 monoclonal antibody hybridoma cell lines
1. Acquisition and identification of heavy-light chain variable region gene of anti-CD 47 monoclonal antibody
According to the results of the previous examples, the corresponding CD47, preferably antibody 2D1,2D14,2D27,2D47,2D51, was picked up and its hybridoma cells were expanded, and after the cells had grown to logarithmic phase, the corresponding positive hybridoma cells were counted and collected at about 5X106Total RNA (RNAfast200,220011) is rapidly extracted by using an RNA extraction kit of Shanghai Feijie company, and the corresponding operation is described in the product specification. Add 40. mu.l RNase-free water to dissolve RNA, take 10. mu.l for nucleic acid electrophoresis at 175V/15 min. And reverse transcription of the rest total RNA, and storing at-80 deg.C.
According to a reverse transcription kit (FSQ-101) of TOYOBO company, 10 mu l of RNA is taken as a template, a corresponding random primer, enzyme and buffer are added, and the first cDNA is synthesized by reverse transcription, wherein the specific operation process is described in the specification, and the first cDNA is synthesized by reverse transcription according to the reaction conditions of the specification. The obtained cDNA product is immediately subjected to PCR amplification reaction or temporarily stored at-20 ℃.
PCR method for extracting heavy and light chain variable region gene of antibody
Degenerate primers were designed upstream and downstream of the heavy and light chains of hybridomas according to the reference, and the primer sequences were as follows:
MHV1:5’-ATGAAATGCAGCTGGGGCATSTTCTTC-3’
MHV2:5’-ATGGGATGGAGCTRTATCATSYTCTT-3’
MHV3:5’-ATGAAGWTGTGGTTAAACTGGGTTTTT-3’
MHV4:5’-ATGRACTTTGGGYTCAGCTTGRTTT-3’
MHV5:5’-ATGGGACTCCAGGCTTCAATTTAGTTTTCCTT-3’
MHV6:5’-ATGGCTTGTCYTTRGSGCTRCTCTTCTGC-3’
MHV7:5’-ATGGRATGGAGCKGGRGTCTTTMTCTT-3’
MHV8:5’-AGTAGAGTGCTGATTCTTTTGTG-3’
MHV9:5’-ATGGMTTGGGTGTGGAMCTTGCTTATTCCTG-3’
MHV10:5’-ATGGGCAGACTTACCATTCTCATTCCTG-3’
MHV11:5’-ATGGATTTTGGGCTGATTTTTTTTATTG-3’
MHV12:5’-ATGATGGTGTTAAGTCTTCTGTACCTG-3’
MHCG1:5’-CAGTGGATAGACAGATGGGGG-3’
MHCG2a:5’-CAGTGGATAGACCGATGGGGG-3’
MHCG2b:5’-CAGTGGATGAGCTGATGGGGG-3’
MHCG3:5’-CAAGGGATAGACAGATGGGGC-3’
MKV1：5’-ATGAAGATTGCCTGTTAGGCTGTTGGTGCTG-3’
MKV2：5’-ATGGAGWCAGACACACTCCTGYTATGGGTG-3’
MKV3：5’-ATGAGTGTGCTCACTCAGGTCCTGGSGTTG-3’
MKV4：5’-ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG-3’
MKV5：5’-ATGGATTTWCAGGTGCAGATTWTCAGCTTC-3’
MKV6：5’-ATGAGGTKCYYTGYTSAYCTYCTCTGRGG-3’
MKV7：5’-ATGGGCWTCAAAGATGGAGTCACAKWYYCWGG-3’
MKV8：5’-ATGTGGGGAYCTKTTTYCMMTTTTTCAATG-3’
MKV9：5’-ATGGTRTCCWCASCTCAGTTCCTTG-3’
MKV10：5’-ATGTATATATGTTTGTTGTCTATTTCT-3’
MKV11：5’-ATGGAAGCCCCAGCTCAGCTTCTCTTCC-3’
MKC：5’-ACTGGATGGTGGGAAGATGG-3’
CL12A：5’-ATGRAGTYWCAGACCCAGGTCTTYRT-3’
CL12B：5’-ATGGAGACACATTCTCAGGTCTTTGT-3’
CL13：5’-ATGGATTCACAGGCCCAGGTTCTTAT-3’
CL14：5’-ATGATGAGTCCTGCCCAGTTCCTGTT-3’
CL15：5’-ATGAATTTGCCTGGTCATCTCTTGGTGCT-3’
CL16：5’-ATGGATTTTCAATTGGTCCTCTTGGTGCT-3’
CL17A：5’-ATGAGGTGCCTARCTSAGTTCCTGRG-3’
CL17B：5’-ATGAAGTACTCTGCTCAGTTTCTAGG-3’
CL17C：5’-ATGAGGCATTCTCTTCAATTCTTGGG-3’
(degenerate codon specification: R ═ A, G; Y ═ C, T; M ═ A, C; S ═ C, G; W ═ A, T).
The PCR reaction system is 50 mu l, and the reaction conditions are as follows: 3min at 95 ℃; 10s at 95 ℃; 30s at 55 ℃; 30s at 72 ℃, 30 cycles, and finally 10min extension at 72 ℃. The PCR reaction product is subjected to nucleic acid gel electrophoresis and a band of about 350-400bp is recovered, as shown in FIGS. 24-25, the PCR product recovery uses a gel product recovery kit of Shanghai Jieli bioengineering, Inc., and the specific operation steps are described in the kit specification.
Because the enzyme used in PCR is Taq enzyme, adenine (A) is already carried at the 3 'end of the PCR product, and the PCR product can be directly connected with a T carrier without carrying out reaction of adding A at the 3' end. Connecting the PCR product to a PGM-T carrier (purchased from Beijing Tiangen organisms) by using T4 ligase, taking 0.5 mu l T carrier and 3 mu l of PCR product, adding 0.5 mu l T4 ligase and 4 mu l of ligase buffer, connecting for 5min in water bath at 25 ℃, wherein the kit used in the connection reaction is Beijing kang, a century fast connection kit, and the operation process is detailed in the kit specification.
Transforming the ligation product into competent DH5 alpha by chemical transformation, operating procedure: putting 100 mul of competent cells on ice for min, adding 8 mul of corresponding ligation products into DH5 alpha, stirring while adding, fully mixing uniformly, putting in ice water bath, standing for 30min, preparing a 42 ℃ water bath kettle, thermally shocking for 90s at 42 ℃ in a competent state, quickly taking out, putting on ice for 1min, adding 700 mul of nonresistant LB culture medium into each tube, and carrying out shake culture at 37 ℃ and 240rpm for 40 min. After being taken out, the mixture is centrifuged at 4000rpm multiplied by 3min, and the sediment is coated on a flat plate containing ampicillin resistance, X-gal and IPTG after being resuspended by 100-one 200 mu l LB culture medium and is placed in a constant temperature incubator at 37 ℃ for being cultivated overnight in an inverted way.
Colony PCR preliminary identification, picking white clone on the plate, and culturing in 100 mu lLB culture medium containing benzyl resistance at 37 ℃ for 2h with 240rpm shaking. Adding 3 mu l of bacterial liquid into a pcr reaction system, wherein the reaction condition is 95 ℃ for 3 min; 10s at 95 ℃; 30s at 55 ℃; 30s at 72 ℃, 30 cycles, and finally 10min extension at 72 ℃. Taking 10 mu l of colony PCR product to carry out nucleic acid electrophoresis verification, selecting the clone with the band about 350-400bp for amplification culture, selecting colony identification positive clone, inoculating to 5ml LB culture medium at 37 ℃, carrying out shake culture at 240rpm overnight, taking 500 mu l of bacterial liquid from the clone for sequencing, preserving the quality-improved particles of the other bacterial liquid, and freezing and storing the corresponding bacterial liquid.
2. Analysis of Gene sequencing results for heavy and light chain variable Domain of monoclonal antibodies
According to the sequencing result, the variable region gene of the heavy and light chain of the anti-CD 47 monoclonal antibody is analyzed through a database (https:// www.ncbi.nlm.nih.gov/igblast /) on an NCBI website, and the variable domain gene of the heavy and light chain of the anti-hybridoma cell strain is successfully obtained. We successfully obtained the heavy-light chain variable domain gene of the hybridoma cell strain. The heavy chain variable region comprises hypervariable regions HCDR1, HCDR2 and HCDR3, and the light chain variable region sequence comprises hypervariable regions LCDR1, LCDR2 and LCDR 3.
The sequence information of murine antibody 2D1 is as follows: the heavy chain variable region gene has a total length of 348bp, 116 encoded amino acid residues, a nucleotide sequence shown as SEQ ID NO 38, an amino acid sequence shown as SEQ ID NO 39, a light chain variable region gene has a total length of 312bp, 104 encoded amino acid residues, a nucleotide sequence shown as SEQ ID NO 40 and an amino acid sequence shown as SEQ ID NO 41.
SEQ ID NO:38
CAGGTCCAACTGCAGCAGTCTGGGACTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAATGGATTGGTATGATTGATCCTTCAGACAGTGAAACTCACTACAATCAAATGTTCAAGGACAAGGCCACATTGACTGTAGACGAATCCTCCAGCACAGGCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGCTGGGAAGTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA
SEQ ID NO:39
QVQLQQSGTELVRPGASVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSETHYNQMFKDKATLTVDESSSTGYMQLSSLTSEDSAVYYCARSWEVFDYWGQGTTLTVSS
SEQ ID NO:40
GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCACAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGA
SEQ ID NO:41
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTHNIHPVEEEDAATYYCQHIRELTRSEGG
The sequence information of murine antibody 2D14 is as follows: the heavy chain variable region gene has a total length of 354bp, 118 coded amino acid residues, a nucleotide sequence shown as SEQ ID NO of 5, an amino acid sequence shown as SEQ ID NO of 6, a light chain variable region gene has a total length of 339bp, 113 coded amino acid residues, a nucleotide sequence shown as SEQ ID NO of 7 and an amino acid sequence shown as SEQ ID NO of 8.
SEQ ID NO:42
CAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGCTACACCTTCACTGACTACTATATAAGCTGGATGAAGCAGAAGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAGCGGTAATACTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTTCATGCAGCTCAGCAGCCTGACATCTGAGGACACTGCTGTCTATTTCTTTGCAAGACCCCTAGGCCACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA
SEQ ID NO:43
QIQLQQSGPELVKPGASVKISCKTSGYTFTDYYISWMKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFFARPLGHWYFDVWGAGTTVTVSS
SEQ ID NO:44
GACATTGTGATGTCACAGTCTCCATCCTCCCTACCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGTAATCAAAAGAACCACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCCCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGTTTTACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA
SEQ ID NO:45
DIVMSQSPSSLPVSVGEKVTMSCKSSQSLLYSSNQKNHLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK
The sequence information of murine antibody 2D27 is as follows: the heavy chain variable region gene has a total length of 360bp, 120 coded amino acid residues, a nucleotide sequence shown as SEQ ID NO of 9, an amino acid sequence shown as SEQ ID NO of 10, a light chain variable region gene has a total length of 312bp, 104 coded amino acid residues, a nucleotide sequence shown as SEQ ID NO of 11 and an amino acid sequence shown as SEQ ID NO of 12.
SEQ ID NO:46
GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTCAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGGTGAAGCAGAGGCCTGGGCAGGGCCTTGACTGGGTTGGATATATTAATCCTTACAATGATAATACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGACGCAATAGGTACGACGCCTGGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA
SEQ ID NO:47
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQRPGQGLDWVGYINPYNDNTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGRNRYDAWFPYWGQGTLVTVSA
SEQ ID NO:48
GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGACAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCCATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGA
SEQ ID NO:49
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSDSGSGTDFTLNIHPVEEEDAATHYCQHIRELTRSEGG
The sequence information of murine antibody 2D47 is as follows: the heavy chain variable region gene has a total length of 348bp, 116 encoded amino acid residues, a nucleotide sequence shown as SEQ ID NO 13, an amino acid sequence shown as SEQ ID NO 14, a light chain variable region gene has a total length of 312bp, 104 encoded amino acid residues, a nucleotide sequence shown as SEQ ID NO 15 and an amino acid sequence shown as SEQ ID NO 16.
SEQ ID NO:50
CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAATTACTATATTTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGGAGATTAATCCTAACAATGGTGATACTAACTTCAATGAGAAGTTCAAGACCCAGGCCACACTGACTGTAGACAAATCCTCCACCACAGCCTACATGCAACTCAGCAGCCTGACATCTGACGACTCTGCGGTCTATTACTGTGCAAGAGGGGGCCGGACTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA
SEQ ID NO:51
QVQLQQPGAELVKPGASVKLSCKASGYTFTNYYIYWVKQRPGQGLEWIGEINPNNGDTNFNEKFKTQATLTVDKSSTTAYMQLSSLTSDDSAVYYCARGGRTFDYWGQGTTLTVSS
SEQ ID NO:52
GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGA
SEQ ID NO:53
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGG
The sequence information of murine antibody 2D51 is as follows: the heavy chain variable region gene has a total length of 345bp, 115 coding amino acid residues, a nucleotide sequence shown as SEQ ID NO:17, an amino acid sequence shown as SEQ ID NO:18, a light chain variable region gene has a total length of 312bp, 104 coding amino acid residues, a nucleotide sequence shown as SEQ ID NO:19 and an amino acid sequence shown as SEQ ID NO: 20.
SEQ ID NO:54
GAGGTCCAACTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTTCTCATTCACTGACTACTACGTGCACTGGGTGAAACAAAGTCCTGAAAATAGTCTTGAGTGGATTGGAGAGATTAATCCTCTCTCTGGGGGTACTAGCTACAACCAGAGGTTCAAGGGCAAGGCCACATTAACTGTAGATATATCCTCCAGCACAGCCTACATGCAGCTCAAGAGCCTGACATCTGAAGAGTCTGCAGTCTATTACTGTATCGGTGGTTACTACGGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA
SEQ ID NO:55
EVQLQQSGPELVKPGASMKISCKASGFSFTDYYVHWVKQSPENSLEWIGEINPLSGGTSYNQRFKGKATLTVDISSSTAYMQLKSLTSEESAVYYCIGGYYGAYWGQGTLVTVSA
SEQ ID NO:56
GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGATCATTGTTCATAGTAATGGAAACACCTATTTAGCATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCACCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
SEQ ID NO:57
DVLMTQTPLSLPVSLGDQASISCRSSQIIVHSNGNTYLAWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYYCFQGSHAPYTFGGGTKLEIK
Through the sequence alignment of IgBLSAT, the heavy chain sequences of 2D1,2D14,2D27,2D47 and 2D51 antibodies all accord with the gene characteristics of mouse IgG variable regions, and the light sequences of 2D1,2D14,2D27,2D47 and 2D51 antibodies all accord with the gene characteristics of mouse IgG variable regions. The 3 Complementarity-determining regions (CDRs) and 4 framework region sequences (Frame regions) of the antibody were determined according to the kabat rule, the heavy chain Complementarity-determining region sequences including hypervariable region sequences H-CDR1, H-CDR2 and H-CDR3, and the light chain Complementarity-determining region sequences including hypervariable region sequences L-CDR1, L-CDR2 and L-CDR 3.
Wherein the 2D1 heavy chain complementarity determining region amino acid sequence hypervariable region sequence H-CDR1 is shown as SEQ ID NO. 21, H-CDR2 is shown as SEQ ID NO. 22, and H-CDR3 is shown as SEQ ID NO. 23.
SEQ ID NO:58
GYTFTSYW
SEQ ID NO:59
IDPSDSET
SEQ ID NO:60
ARSWEVFDY
The 2D1 light chain complementarity determining region sequence includes hypervariable region sequence L-CDR1 as shown in SEQ ID NO:24, L-CDR2 as shown in SEQ ID NO:25, L-CDR3 as shown in SEQ ID NO: 26.
SEQ ID NO:61
KSVSTSGYSY
SEQ ID NO:62
LVS
SEQ ID NO:63
QHIRELTR
Wherein the 2D14 heavy chain complementarity determining region amino acid sequence hypervariable region sequence H-CDR1 is shown in SEQ ID NO 27 and H-CDR2Such as SEQ ID NO 28 and H-CDR3 such as SEQ ID NO 29.
SEQ ID NO:64
GYTFTDYY
SEQ ID NO:65
IYPGSGNT
SEQ ID NO:66
ARPLGHWYFDV
The 2D14 light chain complementarity determining region sequence includes hypervariable region sequence L-CDR1 as shown in SEQ ID NO:30, L-CDR2 as shown in SEQ ID NO:31, L-CDR3 as shown in SEQ ID NO: 32.
SEQ ID NO:67
QSLLYSSNQKNH
SEQ ID NO:68
WAS
SEQ ID NO:69
QQYYSYPLT
Wherein the 2D27 heavy chain complementarity determining region amino acid sequence hypervariable region sequence H-CDR1 is shown as SEQ ID NO. 33, H-CDR2 is shown as SEQ ID NO. 34, and H-CDR3 is shown as SEQ ID NO. 35.
SEQ ID NO:70
GYTFTSYV
SEQ ID NO:71
INPYNDNT
SEQ ID NO:72
ARGRNRYDAWFPY
The 2D27 light chain complementarity determining region sequence includes hypervariable region sequence L-CDR1 as shown in SEQ ID NO:24, L-CDR2 as shown in SEQ ID NO:25, L-CDR3 as shown in SEQ ID NO: 26.
SEQ ID NO:61
KSVSTSGYSY
SEQ ID NO:62
LVS
SEQ ID NO:63
QHIRELTR
Wherein the 2D47 heavy chain complementarity determining region amino acid sequence hypervariable region sequence H-CDR1 is shown as SEQ ID NO:36, H-CDR2 is shown as SEQ ID NO:37 and H-CDR3 is shown as SEQ ID NO: 38.
SEQ ID NO:73
GYTFTNYY
SEQ ID NO:74
INPNNGDT
SEQ ID NO:75
ARGGRTFDY
The 2D47 light chain complementarity determining region sequence includes hypervariable region sequence L-CDR1 as shown in SEQ ID NO:24, L-CDR2 as shown in SEQ ID NO:25, L-CDR3 as shown in SEQ ID NO: 26.
SEQ ID NO:61
KSVSTSGYSY
SEQ ID NO:62
LVS
SEQ ID NO:63
QHIRELTR
Wherein the amino acid sequence hypervariable region sequence H-CDR1 of the 2D51 heavy chain complementarity determining region is shown as SEQ ID NO. 39, H-CDR2 is shown as SEQ ID NO. 40, and H-CDR3 is shown as SEQ ID NO. 41.
SEQ ID NO:76
GFSFTDYY
SEQ ID NO:77
INPLSGGT
SEQ ID NO:78
IGGYYGAY
The 2D51 light chain complementarity determining region sequence includes hypervariable region sequence L-CDR1 as shown in SEQ ID NO:43, L-CDR2 as shown in SEQ ID NO:44, L-CDR3 as shown in SEQ ID NO: 45.
SEQ ID NO:79
QIIVHSNGNTY
SEQ ID NO:80
KVS
SEQ ID NO:81
FQGSHAPYT
EXAMPLE 6 hemagglutination assay for CD47 monoclonal antibody
Published studies indicate that some CD47 antibodies can cause hemagglutination, and therefore, hemagglutination characterization of candidate antibodies is particularly important in screening candidate molecules. Collecting whole blood of a healthy volunteer, centrifuging at 1000rpm/min for 5 minutes, removing supernatant serum, re-suspending precipitated red blood cells with PBS, centrifuging at 1000rpm/min for 5 minutes, discarding PBS, washing for three times, centrifuging, discarding PBS to obtain red blood cells, and diluting the red blood cells to 2% to prepare red blood cell suspension. The test antibody and the control antibody are diluted in a multiple ratio, the dilution is started from 20 mu g/ml (666.67nM), 50 mu l of erythrocyte suspension and 50 mu l of different antibodies are added into a U-shaped 96-well plate, the mixture is incubated for 2h in an incubator at 37 ℃, and pictures are taken from the top of the plate by observation, and the result shows that the erythrocytes in the negative control well without the added antibodies are all small and clear circles. The positive control antibody wells showed that the erythrocytes were uniformly dispersed in the form of a cloudy erythrocyte aggregation pad, and the results are shown in FIG. 26.
Example 7 construction and expression of chimeric antibodies
According to the CD47 action mechanism, the constant region of IgG4(S228P) is used as the heavy chain constant region of the antibody, and the mutation of the core hinge region (S228P) of IgG4 can enhance the disulfide bond connection of the core hinge region, reduce the exchange of Fab arms of IgG4 and greatly reduce the formation of half molecules. The human light chain K chain constant region was used as the antibody light chain constant region. Heavy chain and light chain constant region gene cross is synthesized by Nanjing Kisrei, and the heavy chain and the light chain are homologous recombined into a vector through EcoRI and BamHI double enzyme digestion vector PTT 5. After the sequencing is correct, the heavy chain and the light chain of the antibody are co-transfected into the HEK293 cell according to the molar ratio of 1.5: 1. Culturing for 120h, centrifuging, collecting supernatant, and purifying to obtain the chimeric antibody. The chimeric antibodies purified according to this example were designated 2D-1,2D14-1,2D27-1,2D47-1,2D51-1, respectively.
Note that: the gene sequence and amino acid sequence of the chimeric antibody are as follows, respectively. Wherein the italic part represents the gene sequence and amino acid sequence of the antibody signal peptide, the underlined part represents the gene sequence and amino acid sequence of the light and heavy chains of the antibody, and the remaining underline represents the nucleotide sequences of the constant regions of the heavy chain and light chain of the antibody and the amino acid sequences encoded thereby, respectively.
2D1-1 antibody heavy chain encoding nucleotide sequence SEQ ID NO 82
2D1-1 antibody heavy chain amino acid sequence SEQ ID NO 83
2D1-1 light chain sequence encoding nucleotide sequence SEQ ID NO 84
2D1-1 light chain amino acid sequence SEQ ID NO 85
2D14-1 antibody heavy chain encoding nucleotide sequence SEQ ID NO 86
2D14-1 heavy chain amino acid sequence SEQ ID NO:87
2D14-1 light chain encoding nucleotide sequence SEQ ID NO:88
2D14-1 light chain amino acid sequence SEQ ID NO 89
2D27-1 antibody heavy chain encoding nucleotide sequence SEQ ID NO 90
2D27-1 heavy chain amino acid sequence SEQ ID NO 91
2D27-1 light chain encoding nucleotide sequence SEQ ID NO 92
2D27-1 light chain amino acid sequence SEQ ID NO 93
2D47-1 antibody heavy chain encoding nucleotide sequence SEQ ID NO 94
2D47-1 heavy chain amino acid sequence SEQ ID NO 95
2D47-1 light chain encoding nucleotide sequence SEQ ID NO 96
2D47-1 light chain amino acid sequence SEQ ID NO 97
2D51 antibody heavy chain encoding nucleotide sequence SEQ ID NO 98
ATGGAAACCGATACACTGCTGCTGTGGGTCCTGCTGCTGTGGGTCCCTGGGTCAACCGGCGAGGTCCAA CTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTTCTCATT CACTGACTACTACGTGCACTGGGTGAAACAAAGTCCTGAAAATAGTCTTGAGTGGATTGGAGAGATTAATCCTCTCT CTGGGGGTACTAGCTACAACCAGAGGTTCAAGGGCAAGGCCACATTAACTGTAGATATATCCTCCAGCACAGCCTAC ATGCAGCTCAAGAGCCTGACATCTGAAGAGTCTGCAGTCTATTACTGTATCGGTGGTTACTACGGGGCTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCAGCCTCCACAAAGGGACCTAGCGTGTTCCCACTGGCACCTTGCTCTCGGAGCACATCCGAGTCTACCGCCGCCCTGGGATGTCTGGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCTTGGAACAGCGGCGCCCTGACAAGCGGAGTGCACACCTTTCCAGCCGTGCTGCAGAGCTCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCCTCCTCTAGCCTGGGCACCAAGACATATACCTGCAACGTGGACCACAAGCCTTCCAATACCAAGGTGGATAAGAGGGTGGAGTCTAAGTACGGACCACCTTGCCCACCATGTCCAGCACCAGAATTCCTGGGAGGACCTAGCGTGTTCCTGTTTCCTCCAAAGCCAAAGGACACACTGATGATCTCTCGCACACCAGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAGCCAAGGGAGGAGCAGTTTAATAGCACATACCGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGAGCAATAAGGGCCTGCCCTCCTCTATCGAGAAGACAATCTCCAAGGCCAAGGGCCAGCCAAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAGGAGGAGATGACAAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTATCCCTCTGACATCGCCGTGGAGTGGGAGAGCAATGGCCAGCCTGAGAACAATTACAAGACCACACCACCCGTGCTGGACTCCGATGGCTCTTTCTTTCTGTATTCCCGGCTGACCGTGGATAAGAGCCGGTGGCAGGAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCTCTGAGCCTGTCCCTGGGCAAG
2D51-1 antibody heavy chain amino acid sequence SEQ ID NO 99
2D51-1 light chain encoding nucleotide sequence SEQ ID NO 100
ATGGAGACAGATACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCAGCACAGGAGATGTTTTG ATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGATCAT TGTTCATAGTAATGGAAACACCTATTTAGCATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACA AAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATC ACCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGCTCCGTACACGTTCGGAGGGGG GACCAAGCTGGAAATAAAACGGACCGTGGCCGCCCCAAGCGTGTTCATCTTTCCTCCATCCGATGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAATTCCCAGGAGTCTGTGACAGAGCAGGACAGCAAGGATTCCACCTATTCTCTGAGCTCCACACTGACCCTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAGGTCACCCATCAGGGGCTGTCATCACCAGTCACCAAGTCTTTCAACCGAGGGGAATGCTAA
2D51-1 light chain amino acid sequence SEQ ID NO 101
Chimeric antibody EC50 was detected according to the method for determining antibody EC50 in example 4, and the results are shown in Table 1:
TABLE 1 measurement of chimeric antibody EC50
Example 8 phagocytosis of chimeric antibodies
In vitro phagocytosis experiments demonstrated whether chimeric antibodies enhanced phagocytosis of CD 47-expressing target cells by human macrophages. PBMC were isolated from fresh human blood and cultured in AIM-V medium (Life technologies) for 7 days, macrophages from monocyte differentiation were allowed to grow adherently, other cells were removed by washing, and macrophages were re-seeded into 12-well plates and cultured adherently for 24 hours. K562 cells were labeled with 0.3 μ M CFSE for 10min, washed 4 times with pre-chilled PBS, and then treated as target cells: macrophages were added at a ratio of 4:1, followed by incubation in a 37 ℃ incubator for 3h with various concentrations of the CD47 chimeric antibody. Subsequently, the target cells that were not phagocytized were washed away with PBS, and then macrophages were addedCells were digested, washed 3 times with PBS, and macrophages were subsequently labeled with APC-labeled anti-human CD14 antibody. Total macrophages can be expressed by CD14+Detection, phagocytized cells can pass CD14++CFSE+And (6) detecting.
Phagocytosis rate (CD 14)++CFSE+Cell number/CD 14+Cell number) 100%
As shown in FIG. 27, 5 chimeric antibodies showed a better concentration-dependent macrophage phagocytosis of tumor cells, demonstrating that 5 chimeric antibodies all had a certain anti-tumor effect. Among them, 2D14-1 and 2D47-1 showed more excellent macrophage phagocytosis-promoting rate than the control antibody.
Sequence listing
<110> Hangzhou Koxing Biotechnology Co., Ltd
<120> anti-CD 47 monoclonal antibody, fragment and medical application thereof
<130> 1
<160> 101
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgaaatgca gctggggcat sttcttc 27
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgggatgga gctrtatcat sytctt 26
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaagwtgt ggttaaactg ggttttt 27
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgractttg ggytcagctt grttt 25
<210> 5
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgggactcc aggcttcaat ttagttttcc tt 32
<210> 6
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atggcttgtc yttrgsgctr ctcttctgc 29
<210> 7
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atggratgga gckggrgtct ttmtctt 27
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
agtagagtgc tgattctttt gtg 23
<210> 9
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atggmttggg tgtggamctt gcttattcct g 31
<210> 10
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgggcagac ttaccattct cattcctg 28
<210> 11
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atggattttg ggctgatttt ttttattg 28
<210> 12
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgatggtgt taagtcttct gtacctg 27
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cagtggatag acagatgggg g 21
<210> 14
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cagtggatag accgatgggg g 21
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cagtggatga gctgatgggg g 21
<210> 16
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
caagggatag acagatgggg c 21
<210> 17
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atgaagattg cctgttaggc tgttggtgct g 31
<210> 18
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atggagwcag acacactcct gytatgggtg 30
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
atgagtgtgc tcactcaggt cctggsgttg 30
<210> 20
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atgaggrccc ctgctcagwt tyttggmwtc ttg 33
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
atggatttwc aggtgcagat twtcagcttc 30
<210> 22
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atgaggtkcy ytgytsayct yctctgrgg 29
<210> 23
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
atgggcwtca aagatggagt cacakwyycw gg 32
<210> 24
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
atgtggggay ctktttycmm tttttcaatg 30
<210> 25
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
atggtrtccw casctcagtt ccttg 25
<210> 26
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
atgtatatat gtttgttgtc tatttct 27
<210> 27
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
atggaagccc cagctcagct tctcttcc 28
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
<210> 29
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
atgragtywc agacccaggt cttyrt 26
<210> 30
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
atggagacac attctcaggt ctttgt 26
<210> 31
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
atggattcac aggcccaggt tcttat 26
<210> 32
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
atgatgagtc ctgcccagtt cctgtt 26
<210> 33
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
atgaatttgc ctggtcatct cttggtgct 29
<210> 34
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
atggattttc aattggtcct cttggtgct 29
<210> 35
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
atgaggtgcc tarctsagtt cctgrg 26
<210> 36
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
atgaagtact ctgctcagtt tctagg 26
<210> 37
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
atgaggcatt ctcttcaatt cttggg 26
<210> 38
<211> 348
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
caggtccaac tgcagcagtc tgggactgag ctggtgaggc ctggggcttc agtgaagctg 60
tcctgcaagg cttctggcta caccttcacc agctactgga tgaactgggt gaagcagagg 120
cctggacaag gccttgaatg gattggtatg attgatcctt cagacagtga aactcactac 180
aatcaaatgt tcaaggacaa ggccacattg actgtagacg aatcctccag cacaggctac 240
atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagaagctgg 300
gaagtctttg actactgggg ccaaggcacc actctcacag tctcctca 348
<210> 39
<211> 116
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Met Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Gly Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Trp Glu Val Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 40
<211> 312
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60
atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120
caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180
ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccca caacatccat 240
cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300
tcggaggggg ga 312
<210> 41
<211> 104
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr His Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg
85 90 95
Glu Leu Thr Arg Ser Glu Gly Gly
100
<210> 42
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60
tcctgcaaga cttctggcta caccttcact gactactata taagctggat gaagcagaag 120
cctggacagg gacttgagtg gattggatgg atttatcctg gaagcggtaa tactaagtac 180
aatgagaagt tcaagggcaa ggccacattg actgtagaca catcctccag cacagccttc 240
atgcagctca gcagcctgac atctgaggac actgctgtct atttctttgc aagaccccta 300
ggccactggt acttcgatgt ctggggcgca gggaccacgg tcaccgtctc ctca 354
<210> 43
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Gln Ile Gln 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 Asp Tyr
20 25 30
Tyr Ile Ser Trp Met Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Phe
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Phe
85 90 95
Ala Arg Pro Leu Gly His Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 44
<211> 339
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gacattgtga tgtcacagtc tccatcctcc ctacctgtgt cagttggaga gaaggttact 60
atgagctgca agtccagtca gagcctttta tatagtagta atcaaaagaa ccacttggcc 120
tggtaccagc agaaaccagg gcagtcccct aaactgctga tttactgggc atccactagg 180
gaatctgggg tccctgatcg ttttacaggc agtggatctg ggacagattt cactctcacc 240
atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagttat 300
ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339
<210> 45
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Pro Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn His Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 46
<211> 360
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gaggtccagc tgcagcagtc tggacctgag ctggtcaagc ctggggcttc agtgaagatg 60
tcctgcaagg cttctggata cacattcact agctatgtta tgcactgggt gaagcagagg 120
cctgggcagg gccttgactg ggttggatat attaatcctt acaatgataa tactaagtac 180
aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240
atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagaggacgc 300
aataggtacg acgcctggtt tccttactgg ggccaaggga ctctggtcac tgtctctgca 360
<210> 47
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Val Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Val
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asp Asn Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Arg Asn Arg Tyr Asp Ala Trp Phe Pro Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 48
<211> 312
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60
atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120
caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180
ggggtccctg ccaggttcag tgacagtggg tctgggacag acttcaccct caacatccat 240
cctgtggagg aggaggatgc tgcaacccat tactgtcagc acattaggga gcttacacgt 300
tcggaggggg ga 312
<210> 49
<211> 104
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Asp Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr His Tyr Cys Gln His Ile Arg
85 90 95
Glu Leu Thr Arg Ser Glu Gly Gly
100
<210> 50
<211> 348
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
caggtccaac tgcagcagcc tggggctgaa ctggtgaagc ctggggcttc agtgaagttg 60
tcctgcaagg cttctggcta caccttcacc aattactata tttactgggt gaagcagagg 120
cctggacaag gccttgagtg gattggggag attaatccta acaatggtga tactaacttc 180
aatgagaagt tcaagaccca ggccacactg actgtagaca aatcctccac cacagcctac 240
atgcaactca gcagcctgac atctgacgac tctgcggtct attactgtgc aagagggggc 300
cggacttttg actactgggg ccaaggcacc actctcacag tctcctca 348
<210> 51
<211> 116
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Tyr Ile Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Asn Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Thr Gln Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Arg Thr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 52
<211> 312
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60
atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120
caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180
ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240
cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300
tcggaggggg ga 312
<210> 53
<211> 104
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg
85 90 95
Glu Leu Thr Arg Ser Glu Gly Gly
100
<210> 54
<211> 345
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gaggtccaac tgcagcagtc tggacctgag ctggtgaagc ctggggcttc aatgaagata 60
tcctgcaagg cttctggttt ctcattcact gactactacg tgcactgggt gaaacaaagt 120
cctgaaaata gtcttgagtg gattggagag attaatcctc tctctggggg tactagctac 180
aaccagaggt tcaagggcaa ggccacatta actgtagata tatcctccag cacagcctac 240
atgcagctca agagcctgac atctgaagag tctgcagtct attactgtat cggtggttac 300
tacggggctt actggggcca agggactctg gtcactgtct ctgca 345
<210> 55
<211> 115
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Phe Ser Phe Thr Asp Tyr
20 25 30
Tyr Val His Trp Val Lys Gln Ser Pro Glu Asn Ser Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Leu Ser Gly Gly Thr Ser Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Ile Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Lys Ser Leu Thr Ser Glu Glu Ser Ala Val Tyr Tyr Cys
85 90 95
Ile Gly Gly Tyr Tyr Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
115
<210> 56
<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctcttgca gatctagtca gatcattgtt catagtaatg gaaacaccta tttagcatgg 120
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
accagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgctccg 300
tacacgttcg gaggggggac caagctggaa ataaaa 336
<210> 57
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Thr Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Ala Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 58
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Gly Tyr Thr Phe Thr Ser Tyr Trp
1 5
<210> 59
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Ile Asp Pro Ser Asp Ser Glu Thr
1 5
<210> 60
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Ala Arg Ser Trp Glu Val Phe Asp Tyr
1 5
<210> 61
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr
1 5 10
<210> 62
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Leu Val Ser
1
<210> 63
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
Gln His Ile Arg Glu Leu Thr Arg
1 5
<210> 64
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
Gly Tyr Thr Phe Thr Asp Tyr Tyr
1 5
<210> 65
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
Ile Tyr Pro Gly Ser Gly Asn Thr
1 5
<210> 66
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 66
Ala Arg Pro Leu Gly His Trp Tyr Phe Asp Val
1 5 10
<210> 67
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn His
1 5 10
<210> 68
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Trp Ala Ser
1
<210> 69
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 69
Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr
1 5
<210> 70
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Gly Tyr Thr Phe Thr Ser Tyr Val
1 5
<210> 71
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Ile Asn Pro Tyr Asn Asp Asn Thr
1 5
<210> 72
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Ala Arg Gly Arg Asn Arg Tyr Asp Ala Trp Phe Pro Tyr
1 5 10
<210> 73
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 73
Gly Tyr Thr Phe Thr Asn Tyr Tyr
1 5
<210> 74
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 74
Ile Asn Pro Asn Asn Gly Asp Thr
1 5
<210> 75
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 75
Ala Arg Gly Gly Arg Thr Phe Asp Tyr
1 5
<210> 76
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 76
Gly Phe Ser Phe Thr Asp Tyr Tyr
1 5
<210> 77
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 77
Ile Asn Pro Leu Ser Gly Gly Thr
1 5
<210> 78
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 78
Ile Gly Gly Tyr Tyr Gly Ala Tyr
1 5
<210> 79
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 79
Gln Ile Ile Val His Ser Asn Gly Asn Thr Tyr
1 5 10
<210> 80
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 80
Lys Val Ser
1
<210> 81
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 81
Phe Gln Gly Ser His Ala Pro Tyr Thr
1 5
<210> 82
<211> 1389
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
atggaaaccg atacactgct gctgtgggtc ctgctgctgt gggtccctgg gtcaaccggc 60
caggtccaac tgcagcagtc tgggactgag ctggtgaggc ctggggcttc agtgaagctg 120
tcctgcaagg cttctggcta caccttcacc agctactgga tgaactgggt gaagcagagg 180
cctggacaag gccttgaatg gattggtatg attgatcctt cagacagtga aactcactac 240
aatcaaatgt tcaaggacaa ggccacattg actgtagacg aatcctccag cacaggctac 300
atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagaagctgg 360
gaagtctttg actactgggg ccaaggcacc actctcacag tctcctcagc ctccacaaag 420
ggacctagcg tgttcccact ggcaccttgc tctcggagca catccgagtc taccgccgcc 480
ctgggatgtc tggtgaagga ctacttcccc gagcctgtga ccgtgtcttg gaacagcggc 540
gccctgacaa gcggagtgca cacctttcca gccgtgctgc agagctccgg cctgtactcc 600
ctgtctagcg tggtgacagt gccctcctct agcctgggca ccaagacata tacctgcaac 660
gtggaccaca agccttccaa taccaaggtg gataagaggg tggagtctaa gtacggacca 720
ccttgcccac catgtccagc accagaattc ctgggaggac ctagcgtgtt cctgtttcct 780
ccaaagccaa aggacacact gatgatctct cgcacaccag aggtgacctg cgtggtggtg 840
gacgtgtccc aggaggaccc cgaggtgcag ttcaactggt acgtggatgg cgtggaggtg 900
cacaatgcca agaccaagcc aagggaggag cagtttaata gcacataccg cgtggtgtcc 960
gtgctgaccg tgctgcacca ggattggctg aacggcaagg agtataagtg caaggtgagc 1020
aataagggcc tgccctcctc tatcgagaag acaatctcca aggccaaggg ccagccaaga 1080
gagccccagg tgtacaccct gccccctagc caggaggaga tgacaaagaa ccaggtgtcc 1140
ctgacctgtc tggtgaaggg cttctatccc tctgacatcg ccgtggagtg ggagagcaat 1200
ggccagcctg agaacaatta caagaccaca ccacccgtgc tggactccga tggctctttc 1260
tttctgtatt cccggctgac cgtggataag agccggtggc aggagggcaa cgtgtttagc 1320
tgctccgtga tgcacgaggc cctgcacaat cactacacac agaagtctct gagcctgtcc 1380
ctgggcaag 1389
<210> 83
<211> 463
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 83
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Val
20 25 30
Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr
35 40 45
Phe Thr Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly
50 55 60
Leu Glu Trp Ile Gly Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr
65 70 75 80
Asn Gln Met Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Glu Ser Ser
85 90 95
Ser Thr Gly Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
100 105 110
Val Tyr Tyr Cys Ala Arg Ser Trp Glu Val Phe Asp Tyr Trp Gly Gln
115 120 125
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
130 135 140
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
145 150 155 160
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
165 170 175
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
180 185 190
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
195 200 205
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
210 215 220
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
225 230 235 240
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
245 250 255
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
260 265 270
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
275 280 285
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
290 295 300
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
305 310 315 320
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
325 330 335
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
340 345 350
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
355 360 365
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
370 375 380
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
385 390 395 400
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
405 410 415
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
420 425 430
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
435 440 445
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
450 455 460
<210> 84
<211> 696
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
atggagacag ataccctgct gctgtgggtg ctgctgctgt gggtgcctgg cagcacagga 60
gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 120
atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 180
caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 240
ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccca caacatccat 300
cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 360
tcggaggggg gacggaccgt ggccgcccca agcgtgttca tctttcctcc atccgatgag 420
cagctgaagt ctggcaccgc cagcgtggtg tgcctgctga acaacttcta ccccagagag 480
gccaaggtgc agtggaaggt ggacaacgcc ctgcagagcg gcaattccca ggagtctgtg 540
acagagcagg acagcaagga ttccacctat tctctgagct ccacactgac cctgagcaag 600
gccgattacg agaagcacaa ggtgtatgcc tgtgaggtca cccatcaggg gctgtcatca 660
ccagtcacca agtctttcaa ccgaggggaa tgctaa 696
<210> 85
<211> 231
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 85
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
20 25 30
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser
35 40 45
Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser
65 70 75 80
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
His Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys
100 105 110
Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly Arg Thr Val Ala
115 120 125
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
130 135 140
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
145 150 155 160
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
165 170 175
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
180 185 190
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
195 200 205
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
210 215 220
Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 86
<211> 1395
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
atggaaaccg atacactgct gctgtgggtc ctgctgctgt gggtccctgg gtcaaccggc 60
cagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 120
tcctgcaaga cttctggcta caccttcact gactactata taagctggat gaagcagaag 180
cctggacagg gacttgagtg gattggatgg atttatcctg gaagcggtaa tactaagtac 240
aatgagaagt tcaagggcaa ggccacattg actgtagaca catcctccag cacagccttc 300
atgcagctca gcagcctgac atctgaggac actgctgtct atttctttgc aagaccccta 360
ggccactggt acttcgatgt ctggggcgca gggaccacgg tcaccgtctc ctcagcctcc 420
acaaagggac ctagcgtgtt cccactggca ccttgctctc ggagcacatc cgagtctacc 480
gccgccctgg gatgtctggt gaaggactac ttccccgagc ctgtgaccgt gtcttggaac 540
agcggcgccc tgacaagcgg agtgcacacc tttccagccg tgctgcagag ctccggcctg 600
tactccctgt ctagcgtggt gacagtgccc tcctctagcc tgggcaccaa gacatatacc 660
tgcaacgtgg accacaagcc ttccaatacc aaggtggata agagggtgga gtctaagtac 720
ggaccacctt gcccaccatg tccagcacca gaattcctgg gaggacctag cgtgttcctg 780
tttcctccaa agccaaagga cacactgatg atctctcgca caccagaggt gacctgcgtg 840
gtggtggacg tgtcccagga ggaccccgag gtgcagttca actggtacgt ggatggcgtg 900
gaggtgcaca atgccaagac caagccaagg gaggagcagt ttaatagcac ataccgcgtg 960
gtgtccgtgc tgaccgtgct gcaccaggat tggctgaacg gcaaggagta taagtgcaag 1020
gtgagcaata agggcctgcc ctcctctatc gagaagacaa tctccaaggc caagggccag 1080
ccaagagagc cccaggtgta caccctgccc cctagccagg aggagatgac aaagaaccag 1140
gtgtccctga cctgtctggt gaagggcttc tatccctctg acatcgccgt ggagtgggag 1200
agcaatggcc agcctgagaa caattacaag accacaccac ccgtgctgga ctccgatggc 1260
tctttctttc tgtattcccg gctgaccgtg gataagagcc ggtggcagga gggcaacgtg 1320
tttagctgct ccgtgatgca cgaggccctg cacaatcact acacacagaa gtctctgagc 1380
ctgtccctgg gcaag 1395
<210> 87
<211> 465
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 87
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
20 25 30
Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr
35 40 45
Phe Thr Asp Tyr Tyr Ile Ser Trp Met Lys Gln Lys Pro Gly Gln Gly
50 55 60
Leu Glu Trp Ile Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr
65 70 75 80
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser
85 90 95
Ser Thr Ala Phe Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
100 105 110
Val Tyr Phe Phe Ala Arg Pro Leu Gly His Trp Tyr Phe Asp Val Trp
115 120 125
Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
225 230 235 240
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
245 250 255
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
260 265 270
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
275 280 285
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
290 295 300
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
305 310 315 320
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
325 330 335
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
340 345 350
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
355 360 365
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
370 375 380
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
385 390 395 400
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
405 410 415
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
420 425 430
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
435 440 445
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
450 455 460
Lys
465
<210> 88
<211> 723
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
atggagacag ataccctgct gctgtgggtg ctgctgctgt gggtgcctgg cagcacagga 60
gacattgtga tgtcacagtc tccatcctcc ctacctgtgt cagttggaga gaaggttact 120
atgagctgca agtccagtca gagcctttta tatagtagta atcaaaagaa ccacttggcc 180
tggtaccagc agaaaccagg gcagtcccct aaactgctga tttactgggc atccactagg 240
gaatctgggg tccctgatcg ttttacaggc agtggatctg ggacagattt cactctcacc 300
atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagttat 360
ccgctcacgt tcggtgctgg gaccaagctg gagctgaaac ggaccgtggc cgccccaagc 420
gtgttcatct ttcctccatc cgatgagcag ctgaagtctg gcaccgccag cgtggtgtgc 480
ctgctgaaca acttctaccc cagagaggcc aaggtgcagt ggaaggtgga caacgccctg 540
cagagcggca attcccagga gtctgtgaca gagcaggaca gcaaggattc cacctattct 600
ctgagctcca cactgaccct gagcaaggcc gattacgaga agcacaaggt gtatgcctgt 660
gaggtcaccc atcaggggct gtcatcacca gtcaccaagt ctttcaaccg aggggaatgc 720
taa 723
<210> 89
<211> 240
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 89
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Pro
20 25 30
Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
35 40 45
Leu Leu Tyr Ser Ser Asn Gln Lys Asn His Leu Ala Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
65 70 75 80
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr
100 105 110
Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr
115 120 125
Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
130 135 140
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
145 150 155 160
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
165 170 175
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
180 185 190
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
195 200 205
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
210 215 220
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235 240
<210> 90
<211> 1401
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
atggaaaccg atacactgct gctgtgggtc ctgctgctgt gggtccctgg gtcaaccggc 60
gaggtccagc tgcagcagtc tggacctgag ctggtcaagc ctggggcttc agtgaagatg 120
tcctgcaagg cttctggata cacattcact agctatgtta tgcactgggt gaagcagagg 180
cctgggcagg gccttgactg ggttggatat attaatcctt acaatgataa tactaagtac 240
aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 300
atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagaggacgc 360
aataggtacg acgcctggtt tccttactgg ggccaaggga ctctggtcac tgtctctgca 420
gcctccacaa agggacctag cgtgttccca ctggcacctt gctctcggag cacatccgag 480
tctaccgccg ccctgggatg tctggtgaag gactacttcc ccgagcctgt gaccgtgtct 540
tggaacagcg gcgccctgac aagcggagtg cacacctttc cagccgtgct gcagagctcc 600
ggcctgtact ccctgtctag cgtggtgaca gtgccctcct ctagcctggg caccaagaca 660
tatacctgca acgtggacca caagccttcc aataccaagg tggataagag ggtggagtct 720
aagtacggac caccttgccc accatgtcca gcaccagaat tcctgggagg acctagcgtg 780
ttcctgtttc ctccaaagcc aaaggacaca ctgatgatct ctcgcacacc agaggtgacc 840
tgcgtggtgg tggacgtgtc ccaggaggac cccgaggtgc agttcaactg gtacgtggat 900
ggcgtggagg tgcacaatgc caagaccaag ccaagggagg agcagtttaa tagcacatac 960
cgcgtggtgt ccgtgctgac cgtgctgcac caggattggc tgaacggcaa ggagtataag 1020
tgcaaggtga gcaataaggg cctgccctcc tctatcgaga agacaatctc caaggccaag 1080
ggccagccaa gagagcccca ggtgtacacc ctgcccccta gccaggagga gatgacaaag 1140
aaccaggtgt ccctgacctg tctggtgaag ggcttctatc cctctgacat cgccgtggag 1200
tgggagagca atggccagcc tgagaacaat tacaagacca caccacccgt gctggactcc 1260
gatggctctt tctttctgta ttcccggctg accgtggata agagccggtg gcaggagggc 1320
aacgtgttta gctgctccgt gatgcacgag gccctgcaca atcactacac acagaagtct 1380
ctgagcctgt ccctgggcaa g 1401
<210> 91
<211> 467
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 91
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
20 25 30
Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr
35 40 45
Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Arg Pro Gly Gln Gly
50 55 60
Leu Asp Trp Val Gly Tyr Ile Asn Pro Tyr Asn Asp Asn Thr Lys Tyr
65 70 75 80
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser
85 90 95
Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
100 105 110
Val Tyr Tyr Cys Ala Arg Gly Arg Asn Arg Tyr Asp Ala Trp Phe Pro
115 120 125
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys
130 135 140
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
145 150 155 160
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
165 170 175
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
180 185 190
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
195 200 205
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
210 215 220
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
225 230 235 240
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
245 250 255
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
275 280 285
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
290 295 300
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
305 310 315 320
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
325 330 335
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
340 345 350
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
355 360 365
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
370 375 380
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
385 390 395 400
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
420 425 430
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
435 440 445
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
450 455 460
Leu Gly Lys
465
<210> 92
<211> 696
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
atggagacag ataccctgct gctgtgggtg ctgctgctgt gggtgcctgg cagcacagga 60
gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 120
atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 180
caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 240
ggggtccctg ccaggttcag tgacagtggg tctgggacag acttcaccct caacatccat 300
cctgtggagg aggaggatgc tgcaacccat tactgtcagc acattaggga gcttacacgt 360
tcggaggggg gacggaccgt ggccgcccca agcgtgttca tctttcctcc atccgatgag 420
cagctgaagt ctggcaccgc cagcgtggtg tgcctgctga acaacttcta ccccagagag 480
gccaaggtgc agtggaaggt ggacaacgcc ctgcagagcg gcaattccca ggagtctgtg 540
acagagcagg acagcaagga ttccacctat tctctgagct ccacactgac cctgagcaag 600
gccgattacg agaagcacaa ggtgtatgcc tgtgaggtca cccatcaggg gctgtcatca 660
ccagtcacca agtctttcaa ccgaggggaa tgctaa 696
<210> 93
<211> 231
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 93
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
20 25 30
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser
35 40 45
Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser
65 70 75 80
Gly Val Pro Ala Arg Phe Ser Asp Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr His Tyr Cys
100 105 110
Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly Arg Thr Val Ala
115 120 125
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
130 135 140
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
145 150 155 160
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
165 170 175
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
180 185 190
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
195 200 205
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
210 215 220
Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 94
<211> 1389
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
atggaaaccg atacactgct gctgtgggtc ctgctgctgt gggtccctgg gtcaaccggc 60
caggtccaac tgcagcagcc tggggctgaa ctggtgaagc ctggggcttc agtgaagttg 120
tcctgcaagg cttctggcta caccttcacc aattactata tttactgggt gaagcagagg 180
cctggacaag gccttgagtg gattggggag attaatccta acaatggtga tactaacttc 240
aatgagaagt tcaagaccca ggccacactg actgtagaca aatcctccac cacagcctac 300
atgcaactca gcagcctgac atctgacgac tctgcggtct attactgtgc aagagggggc 360
cggacttttg actactgggg ccaaggcacc actctcacag tctcctcagc ctccacaaag 420
ggacctagcg tgttcccact ggcaccttgc tctcggagca catccgagtc taccgccgcc 480
ctgggatgtc tggtgaagga ctacttcccc gagcctgtga ccgtgtcttg gaacagcggc 540
gccctgacaa gcggagtgca cacctttcca gccgtgctgc agagctccgg cctgtactcc 600
ctgtctagcg tggtgacagt gccctcctct agcctgggca ccaagacata tacctgcaac 660
gtggaccaca agccttccaa taccaaggtg gataagaggg tggagtctaa gtacggacca 720
ccttgcccac catgtccagc accagaattc ctgggaggac ctagcgtgtt cctgtttcct 780
ccaaagccaa aggacacact gatgatctct cgcacaccag aggtgacctg cgtggtggtg 840
gacgtgtccc aggaggaccc cgaggtgcag ttcaactggt acgtggatgg cgtggaggtg 900
cacaatgcca agaccaagcc aagggaggag cagtttaata gcacataccg cgtggtgtcc 960
gtgctgaccg tgctgcacca ggattggctg aacggcaagg agtataagtg caaggtgagc 1020
aataagggcc tgccctcctc tatcgagaag acaatctcca aggccaaggg ccagccaaga 1080
gagccccagg tgtacaccct gccccctagc caggaggaga tgacaaagaa ccaggtgtcc 1140
ctgacctgtc tggtgaaggg cttctatccc tctgacatcg ccgtggagtg ggagagcaat 1200
ggccagcctg agaacaatta caagaccaca ccacccgtgc tggactccga tggctctttc 1260
tttctgtatt cccggctgac cgtggataag agccggtggc aggagggcaa cgtgtttagc 1320
tgctccgtga tgcacgaggc cctgcacaat cactacacac agaagtctct gagcctgtcc 1380
ctgggcaag 1389
<210> 95
<211> 1389
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 95
Ala Thr Gly Gly Ala Ala Ala Cys Cys Gly Ala Thr Ala Cys Ala Cys
1 5 10 15
Thr Gly Cys Thr Gly Cys Thr Gly Thr Gly Gly Gly Thr Cys Cys Thr
20 25 30
Gly Cys Thr Gly Cys Thr Gly Thr Gly Gly Gly Thr Cys Cys Cys Thr
35 40 45
Gly Gly Gly Thr Cys Ala Ala Cys Cys Gly Gly Cys Cys Ala Gly Gly
50 55 60
Thr Cys Cys Ala Ala Cys Thr Gly Cys Ala Gly Cys Ala Gly Cys Cys
65 70 75 80
Thr Gly Gly Gly Gly Cys Thr Gly Ala Ala Cys Thr Gly Gly Thr Gly
85 90 95
Ala Ala Gly Cys Cys Thr Gly Gly Gly Gly Cys Thr Thr Cys Ala Gly
100 105 110
Thr Gly Ala Ala Gly Thr Thr Gly Thr Cys Cys Thr Gly Cys Ala Ala
115 120 125
Gly Gly Cys Thr Thr Cys Thr Gly Gly Cys Thr Ala Cys Ala Cys Cys
130 135 140
Thr Thr Cys Ala Cys Cys Ala Ala Thr Thr Ala Cys Thr Ala Thr Ala
145 150 155 160
Thr Thr Thr Ala Cys Thr Gly Gly Gly Thr Gly Ala Ala Gly Cys Ala
165 170 175
Gly Ala Gly Gly Cys Cys Thr Gly Gly Ala Cys Ala Ala Gly Gly Cys
180 185 190
Cys Thr Thr Gly Ala Gly Thr Gly Gly Ala Thr Thr Gly Gly Gly Gly
195 200 205
Ala Gly Ala Thr Thr Ala Ala Thr Cys Cys Thr Ala Ala Cys Ala Ala
210 215 220
Thr Gly Gly Thr Gly Ala Thr Ala Cys Thr Ala Ala Cys Thr Thr Cys
225 230 235 240
Ala Ala Thr Gly Ala Gly Ala Ala Gly Thr Thr Cys Ala Ala Gly Ala
245 250 255
Cys Cys Cys Ala Gly Gly Cys Cys Ala Cys Ala Cys Thr Gly Ala Cys
260 265 270
Thr Gly Thr Ala Gly Ala Cys Ala Ala Ala Thr Cys Cys Thr Cys Cys
275 280 285
Ala Cys Cys Ala Cys Ala Gly Cys Cys Thr Ala Cys Ala Thr Gly Cys
290 295 300
Ala Ala Cys Thr Cys Ala Gly Cys Ala Gly Cys Cys Thr Gly Ala Cys
305 310 315 320
Ala Thr Cys Thr Gly Ala Cys Gly Ala Cys Thr Cys Thr Gly Cys Gly
325 330 335
Gly Thr Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly Cys Ala Ala
340 345 350
Gly Ala Gly Gly Gly Gly Gly Cys Cys Gly Gly Ala Cys Thr Thr Thr
355 360 365
Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Ala
370 375 380
Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr Cys Ala Cys Ala Gly
385 390 395 400
Thr Cys Thr Cys Cys Thr Cys Ala Gly Cys Cys Thr Cys Cys Ala Cys
405 410 415
Ala Ala Ala Gly Gly Gly Ala Cys Cys Thr Ala Gly Cys Gly Thr Gly
420 425 430
Thr Thr Cys Cys Cys Ala Cys Thr Gly Gly Cys Ala Cys Cys Thr Thr
435 440 445
Gly Cys Thr Cys Thr Cys Gly Gly Ala Gly Cys Ala Cys Ala Thr Cys
450 455 460
Cys Gly Ala Gly Thr Cys Thr Ala Cys Cys Gly Cys Cys Gly Cys Cys
465 470 475 480
Cys Thr Gly Gly Gly Ala Thr Gly Thr Cys Thr Gly Gly Thr Gly Ala
485 490 495
Ala Gly Gly Ala Cys Thr Ala Cys Thr Thr Cys Cys Cys Cys Gly Ala
500 505 510
Gly Cys Cys Thr Gly Thr Gly Ala Cys Cys Gly Thr Gly Thr Cys Thr
515 520 525
Thr Gly Gly Ala Ala Cys Ala Gly Cys Gly Gly Cys Gly Cys Cys Cys
530 535 540
Thr Gly Ala Cys Ala Ala Gly Cys Gly Gly Ala Gly Thr Gly Cys Ala
545 550 555 560
Cys Ala Cys Cys Thr Thr Thr Cys Cys Ala Gly Cys Cys Gly Thr Gly
565 570 575
Cys Thr Gly Cys Ala Gly Ala Gly Cys Thr Cys Cys Gly Gly Cys Cys
580 585 590
Thr Gly Thr Ala Cys Thr Cys Cys Cys Thr Gly Thr Cys Thr Ala Gly
595 600 605
Cys Gly Thr Gly Gly Thr Gly Ala Cys Ala Gly Thr Gly Cys Cys Cys
610 615 620
Thr Cys Cys Thr Cys Thr Ala Gly Cys Cys Thr Gly Gly Gly Cys Ala
625 630 635 640
Cys Cys Ala Ala Gly Ala Cys Ala Thr Ala Thr Ala Cys Cys Thr Gly
645 650 655
Cys Ala Ala Cys Gly Thr Gly Gly Ala Cys Cys Ala Cys Ala Ala Gly
660 665 670
Cys Cys Thr Thr Cys Cys Ala Ala Thr Ala Cys Cys Ala Ala Gly Gly
675 680 685
Thr Gly Gly Ala Thr Ala Ala Gly Ala Gly Gly Gly Thr Gly Gly Ala
690 695 700
Gly Thr Cys Thr Ala Ala Gly Thr Ala Cys Gly Gly Ala Cys Cys Ala
705 710 715 720
Cys Cys Thr Thr Gly Cys Cys Cys Ala Cys Cys Ala Thr Gly Thr Cys
725 730 735
Cys Ala Gly Cys Ala Cys Cys Ala Gly Ala Ala Thr Thr Cys Cys Thr
740 745 750
Gly Gly Gly Ala Gly Gly Ala Cys Cys Thr Ala Gly Cys Gly Thr Gly
755 760 765
Thr Thr Cys Cys Thr Gly Thr Thr Thr Cys Cys Thr Cys Cys Ala Ala
770 775 780
Ala Gly Cys Cys Ala Ala Ala Gly Gly Ala Cys Ala Cys Ala Cys Thr
785 790 795 800
Gly Ala Thr Gly Ala Thr Cys Thr Cys Thr Cys Gly Cys Ala Cys Ala
805 810 815
Cys Cys Ala Gly Ala Gly Gly Thr Gly Ala Cys Cys Thr Gly Cys Gly
820 825 830
Thr Gly Gly Thr Gly Gly Thr Gly Gly Ala Cys Gly Thr Gly Thr Cys
835 840 845
Cys Cys Ala Gly Gly Ala Gly Gly Ala Cys Cys Cys Cys Gly Ala Gly
850 855 860
Gly Thr Gly Cys Ala Gly Thr Thr Cys Ala Ala Cys Thr Gly Gly Thr
865 870 875 880
Ala Cys Gly Thr Gly Gly Ala Thr Gly Gly Cys Gly Thr Gly Gly Ala
885 890 895
Gly Gly Thr Gly Cys Ala Cys Ala Ala Thr Gly Cys Cys Ala Ala Gly
900 905 910
Ala Cys Cys Ala Ala Gly Cys Cys Ala Ala Gly Gly Gly Ala Gly Gly
915 920 925
Ala Gly Cys Ala Gly Thr Thr Thr Ala Ala Thr Ala Gly Cys Ala Cys
930 935 940
Ala Thr Ala Cys Cys Gly Cys Gly Thr Gly Gly Thr Gly Thr Cys Cys
945 950 955 960
Gly Thr Gly Cys Thr Gly Ala Cys Cys Gly Thr Gly Cys Thr Gly Cys
965 970 975
Ala Cys Cys Ala Gly Gly Ala Thr Thr Gly Gly Cys Thr Gly Ala Ala
980 985 990
Cys Gly Gly Cys Ala Ala Gly Gly Ala Gly Thr Ala Thr Ala Ala Gly
995 1000 1005
Thr Gly Cys Ala Ala Gly Gly Thr Gly Ala Gly Cys Ala Ala Thr Ala
1010 1015 1020
Ala Gly Gly Gly Cys Cys Thr Gly Cys Cys Cys Thr Cys Cys Thr Cys
1025 1030 1035 1040
Thr Ala Thr Cys Gly Ala Gly Ala Ala Gly Ala Cys Ala Ala Thr Cys
1045 1050 1055
Thr Cys Cys Ala Ala Gly Gly Cys Cys Ala Ala Gly Gly Gly Cys Cys
1060 1065 1070
Ala Gly Cys Cys Ala Ala Gly Ala Gly Ala Gly Cys Cys Cys Cys Ala
1075 1080 1085
Gly Gly Thr Gly Thr Ala Cys Ala Cys Cys Cys Thr Gly Cys Cys Cys
1090 1095 1100
Cys Cys Thr Ala Gly Cys Cys Ala Gly Gly Ala Gly Gly Ala Gly Ala
1105 1110 1115 1120
Thr Gly Ala Cys Ala Ala Ala Gly Ala Ala Cys Cys Ala Gly Gly Thr
1125 1130 1135
Gly Thr Cys Cys Cys Thr Gly Ala Cys Cys Thr Gly Thr Cys Thr Gly
1140 1145 1150
Gly Thr Gly Ala Ala Gly Gly Gly Cys Thr Thr Cys Thr Ala Thr Cys
1155 1160 1165
Cys Cys Thr Cys Thr Gly Ala Cys Ala Thr Cys Gly Cys Cys Gly Thr
1170 1175 1180
Gly Gly Ala Gly Thr Gly Gly Gly Ala Gly Ala Gly Cys Ala Ala Thr
1185 1190 1195 1200
Gly Gly Cys Cys Ala Gly Cys Cys Thr Gly Ala Gly Ala Ala Cys Ala
1205 1210 1215
Ala Thr Thr Ala Cys Ala Ala Gly Ala Cys Cys Ala Cys Ala Cys Cys
1220 1225 1230
Ala Cys Cys Cys Gly Thr Gly Cys Thr Gly Gly Ala Cys Thr Cys Cys
1235 1240 1245
Gly Ala Thr Gly Gly Cys Thr Cys Thr Thr Thr Cys Thr Thr Thr Cys
1250 1255 1260
Thr Gly Thr Ala Thr Thr Cys Cys Cys Gly Gly Cys Thr Gly Ala Cys
1265 1270 1275 1280
Cys Gly Thr Gly Gly Ala Thr Ala Ala Gly Ala Gly Cys Cys Gly Gly
1285 1290 1295
Thr Gly Gly Cys Ala Gly Gly Ala Gly Gly Gly Cys Ala Ala Cys Gly
1300 1305 1310
Thr Gly Thr Thr Thr Ala Gly Cys Thr Gly Cys Thr Cys Cys Gly Thr
1315 1320 1325
Gly Ala Thr Gly Cys Ala Cys Gly Ala Gly Gly Cys Cys Cys Thr Gly
1330 1335 1340
Cys Ala Cys Ala Ala Thr Cys Ala Cys Thr Ala Cys Ala Cys Ala Cys
1345 1350 1355 1360
Ala Gly Ala Ala Gly Thr Cys Thr Cys Thr Gly Ala Gly Cys Cys Thr
1365 1370 1375
Gly Thr Cys Cys Cys Thr Gly Gly Gly Cys Ala Ala Gly
1380 1385
<210> 96
<211> 696
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
atggagacag ataccctgct gctgtgggtg ctgctgctgt gggtgcctgg cagcacagga 60
gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 120
atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 180
caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 240
ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 300
cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 360
tcggaggggg gacggaccgt ggccgcccca agcgtgttca tctttcctcc atccgatgag 420
cagctgaagt ctggcaccgc cagcgtggtg tgcctgctga acaacttcta ccccagagag 480
gccaaggtgc agtggaaggt ggacaacgcc ctgcagagcg gcaattccca ggagtctgtg 540
acagagcagg acagcaagga ttccacctat tctctgagct ccacactgac cctgagcaag 600
gccgattacg agaagcacaa ggtgtatgcc tgtgaggtca cccatcaggg gctgtcatca 660
ccagtcacca agtctttcaa ccgaggggaa tgctaa 696
<210> 97
<211> 231
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 97
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
20 25 30
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser
35 40 45
Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser
65 70 75 80
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys
100 105 110
Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly Arg Thr Val Ala
115 120 125
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
130 135 140
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
145 150 155 160
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
165 170 175
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
180 185 190
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
195 200 205
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
210 215 220
Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 98
<211> 1386
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
atggaaaccg atacactgct gctgtgggtc ctgctgctgt gggtccctgg gtcaaccggc 60
gaggtccaac tgcagcagtc tggacctgag ctggtgaagc ctggggcttc aatgaagata 120
tcctgcaagg cttctggttt ctcattcact gactactacg tgcactgggt gaaacaaagt 180
cctgaaaata gtcttgagtg gattggagag attaatcctc tctctggggg tactagctac 240
aaccagaggt tcaagggcaa ggccacatta actgtagata tatcctccag cacagcctac 300
atgcagctca agagcctgac atctgaagag tctgcagtct attactgtat cggtggttac 360
tacggggctt actggggcca agggactctg gtcactgtct ctgcagcctc cacaaaggga 420
cctagcgtgt tcccactggc accttgctct cggagcacat ccgagtctac cgccgccctg 480
ggatgtctgg tgaaggacta cttccccgag cctgtgaccg tgtcttggaa cagcggcgcc 540
ctgacaagcg gagtgcacac ctttccagcc gtgctgcaga gctccggcct gtactccctg 600
tctagcgtgg tgacagtgcc ctcctctagc ctgggcacca agacatatac ctgcaacgtg 660
gaccacaagc cttccaatac caaggtggat aagagggtgg agtctaagta cggaccacct 720
tgcccaccat gtccagcacc agaattcctg ggaggaccta gcgtgttcct gtttcctcca 780
aagccaaagg acacactgat gatctctcgc acaccagagg tgacctgcgt ggtggtggac 840
gtgtcccagg aggaccccga ggtgcagttc aactggtacg tggatggcgt ggaggtgcac 900
aatgccaaga ccaagccaag ggaggagcag tttaatagca cataccgcgt ggtgtccgtg 960
ctgaccgtgc tgcaccagga ttggctgaac ggcaaggagt ataagtgcaa ggtgagcaat 1020
aagggcctgc cctcctctat cgagaagaca atctccaagg ccaagggcca gccaagagag 1080
ccccaggtgt acaccctgcc ccctagccag gaggagatga caaagaacca ggtgtccctg 1140
acctgtctgg tgaagggctt ctatccctct gacatcgccg tggagtggga gagcaatggc 1200
cagcctgaga acaattacaa gaccacacca cccgtgctgg actccgatgg ctctttcttt 1260
ctgtattccc ggctgaccgt ggataagagc cggtggcagg agggcaacgt gtttagctgc 1320
tccgtgatgc acgaggccct gcacaatcac tacacacaga agtctctgag cctgtccctg 1380
ggcaag 1386
<210> 99
<211> 462
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 99
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
20 25 30
Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Phe Ser
35 40 45
Phe Thr Asp Tyr Tyr Val His Trp Val Lys Gln Ser Pro Glu Asn Ser
50 55 60
Leu Glu Trp Ile Gly Glu Ile Asn Pro Leu Ser Gly Gly Thr Ser Tyr
65 70 75 80
Asn Gln Arg Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Ile Ser Ser
85 90 95
Ser Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Glu Ser Ala
100 105 110
Val Tyr Tyr Cys Ile Gly Gly Tyr Tyr Gly Ala Tyr Trp Gly Gln Gly
115 120 125
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
130 135 140
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
145 150 155 160
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
165 170 175
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
180 185 190
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
195 200 205
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro
210 215 220
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
225 230 235 240
Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe
245 250 255
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
260 265 270
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
275 280 285
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
290 295 300
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
305 310 315 320
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
325 330 335
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
340 345 350
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
355 360 365
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
370 375 380
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
385 390 395 400
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
405 410 415
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
420 425 430
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
435 440 445
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
450 455 460
<210> 100
<211> 720
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
atggagacag ataccctgct gctgtgggtg ctgctgctgt gggtgcctgg cagcacagga 60
gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 120
atctcttgca gatctagtca gatcattgtt catagtaatg gaaacaccta tttagcatgg 180
tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 240
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 300
accagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgctccg 360
tacacgttcg gaggggggac caagctggaa ataaaacgga ccgtggccgc cccaagcgtg 420
ttcatctttc ctccatccga tgagcagctg aagtctggca ccgccagcgt ggtgtgcctg 480
ctgaacaact tctaccccag agaggccaag gtgcagtgga aggtggacaa cgccctgcag 540
agcggcaatt cccaggagtc tgtgacagag caggacagca aggattccac ctattctctg 600
agctccacac tgaccctgag caaggccgat tacgagaagc acaaggtgta tgcctgtgag 660
gtcacccatc aggggctgtc atcaccagtc accaagtctt tcaaccgagg ggaatgctaa 720
<210> 101
<211> 239
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 101
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro
20 25 30
Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile
35 40 45
Ile Val His Ser Asn Gly Asn Thr Tyr Leu Ala Trp Tyr Leu Gln Lys
50 55 60
Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
65 70 75 80
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
85 90 95
Thr Leu Lys Ile Thr Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
100 105 110
Cys Phe Gln Gly Ser His Ala Pro Tyr Thr Phe Gly Gly Gly Thr Lys
115 120 125
Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
130 135 140
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
145 150 155 160
Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
165 170 175
Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
180 185 190
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
195 200 205
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
210 215 220
Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
Claims (3)
1. A CD47 monoclonal antibody or antigen binding fragment thereof, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the CD47 monoclonal antibody are respectively SEQ ID NO 64, SEQ ID NO 65 and SEQ ID NO 66, and the amino acid sequences of CDR1, CDR2 and CDR3 are respectively SEQ ID NO 67, SEQ ID NO 68 and SEQ ID NO 69.
2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO 43 and the amino acid sequence of the light chain variable region is SEQ ID NO 45.
3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is SEQ ID NO. 42, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO. 44.
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| WO2021032174A1 (en) * | 2019-08-21 | 2021-02-25 | 和铂医药(上海)有限责任公司 | Anti-cd47 antigen binding protein, and application thereof |
| CN111087470B (en) * | 2020-01-19 | 2022-05-10 | 中国人民解放军第四军医大学 | Anti-human CD47 monoclonal antibody 7G4mAb and application thereof |
| WO2021190441A1 (en) * | 2020-03-23 | 2021-09-30 | 倍而达药业（苏州）有限公司 | Cd47/humanized cd47 antibody or antigen binding fragment or immunologically active fragment thereof and use thereof |
| BR112022019795A2 (en) * | 2020-04-02 | 2022-11-16 | Chia Tai Tianqing Pharmaceutical Group Co Ltd | ANTIGEN-BINDING POLYPEPTIDE THAT BINDS CD47 AND USE THEREOF |
| AR121805A1 (en) * | 2020-04-10 | 2022-07-13 | Hutchison Medipharma Ltd | ANTI-CD47 ANTIBODY AND USES OF THE SAME |
| WO2022100694A1 (en) * | 2020-11-12 | 2022-05-19 | 迈威(上海)生物科技股份有限公司 | Antibody and preparation method therefor |
| AR127271A1 (en) * | 2021-10-09 | 2024-01-03 | Hutchmed Ltd | BISPECIFIC ANTIBODIES THAT BIND SPECIFICALLY TO CD47 AND CD20, AND USES THEREOF |
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| CA2303072A1 (en) * | 1997-09-11 | 1999-03-18 | Chugai Seiyaku Kabushiki Kaisha | Monoclonal antibody inducing apoptosis |
| MY169341A (en) * | 2012-02-06 | 2019-03-21 | Inhibrx Inc | Cd47 antibodies and methods of use thereof |
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