CN105646713A - Monoclonal antibody and application thereof - Google Patents
Monoclonal antibody and application thereof Download PDFInfo
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- CN105646713A CN105646713A CN201610116883.1A CN201610116883A CN105646713A CN 105646713 A CN105646713 A CN 105646713A CN 201610116883 A CN201610116883 A CN 201610116883A CN 105646713 A CN105646713 A CN 105646713A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/303—Liver or Pancreas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
- A61K51/1057—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from liver or pancreas
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
Abstract
The invention discloses a monoclonal antibody bioactive in neutralizing human leukocyte differentiation antigen CD24 and a variable region sequence of this monoclonal antibody. Antigen peptide and hemocyanin conjugate CD24-KLH is used as an immunogen immunize-injected to a mouse BALB/c, spleen cells of the immunized mouse are extracted and fused with myeloma cells to obtain a hybridoma cell strain capable of expressing an anti-CD24 antibody, this cell strain is useful in the preparation of an anti-CD24 monoclonal antibody. A variable region amino acid sequence of the anti-CD24 antibody is also cloned herein. The anti-CD24 antibody may specially combine with CD24, thus the monoclonal antibody can be used to treat diseases associated with the excessive, abnormal and out-of-control expression of CD24.
Description
The present invention is application number 2014100277955, and the applying date is 2014-01-22, and denomination of invention is the divisional application of anti-CD24 monoclonal antibody, its variable region sequences and application thereof.
Technical field
The invention belongs to biological technical field, be specifically related to the monoclonal antibody of the specific binding human leukocyte differentiation antigen CD24 of a kind of energy, its variable region sequences and application thereof.
Background technology
Human leukocyte differentiation antigen CD24 (clusterofdifferentiation24) is a kind of newly discovered and primary study tumor markers.
Current research shows, CD24 molecule is apparent molecular weight high glycosylation sialoglycoprotein between 30��70kDa. CD24 comprises the core protein framing structure of 33 amino acid residue compositions, has 16 potential O-or N-glycosylation sites, with mice heat stable antigen very high homology; Its mature polypeptide can be anchored on the surface of cell membrane by the phosphatidylinositols (glycosyl-phosphatidyl-inositol, GPI) of c-terminus.
Under physiological conditions, CD24 molecule is low expression level on immature B cells, ripe granulocyte and minority epithelial cell and neurocyte only; And when body is in pathological state, the surface of most of malignant tumor cell all can detect the CD24 molecule of significantly high horizontal expression, the height of its expression and the generation of tumor and develop closely related.
P selects element to be expressed in platelet and the endothelial cell surface of activation, and CD24 is one of its part. CD24 molecule high expressed enhances the tumor cell adhesive attraction to platelet and endotheliocyte, has promoted recurrence and the transfer of tumor. Certainly, as surface of cell membrane signal transducers, CD24 can by multiple mechanism of action mediate tumor cell propagation, adhesion, transfer and invasion and attack. Research is it has furthermore been found that the dryness that CD24 expresses with hepatoma carcinoma cell is closely related, and CD24 is likely to become a kind of new liver-cancer stem cell surface marker.
As can be seen here, CD24 is expected to become research tumor mechanism and develop the new target drone of dependent diagnostic reagent.
Summary of the invention
The object of the invention one is in that to provide a kind of anti-CD24 method for preparing monoclonal antibody.
The object of the invention two there are provided a kind of anti-CD24 monoclonal antibody.
The object of the invention three is in that to provide the variable region amino acid sequence of anti-CD24 monoclonal antibody.
The present invention is using CD24 Polypeptides and hemocyanin KLH conjugate (CD24-KLH) as immunogen, inoculation BALB/c mouse, take immune mouse spleen cell and by itself and myeloma cell fusion, obtaining the hybridoma cell strain that can express anti-CD24 antibody, this cell strain can be used for preparing anti-CD24 monoclonal antibody.
The heavy chain of antibody of present invention clone, chain variable region amino acid sequence and hypervariable region thereof are if any shown in SEQIDNo.1��16.
The monoclonal antibody of the present invention can be specific binding with CD24.
Accompanying drawing explanation
Fig. 1 is that SDS-PAGE detects ProteinG affinity column purified product result. Swimming lane M is standard molecular weight albumen; Swimming lane 1 is ascites sample; Swimming lane 2 collects liquid for loading; Swimming lane 3 is washed post for 20mM sodium phosphate buffer and is collected liquid; Swimming lane 4��6 is that 100mM glycine buffer (pH3.5) eluting collects liquid (collection 1ml per minute, collect 3ml altogether), swimming lane 7��10 is that 100mM glycine buffer (pH2.7) eluting collects liquid (collection 1ml per minute, collect 4ml altogether).
Fig. 2 is the interphase interaction of antibody and CD24 after WesternBlot purification Identification. Swimming lane M is standard molecular weight albumen; The commercially available CD24-Fc of swimming lane 1 (purchased from Yi Qiao Divine Land, Beijing); Swimming lane 2 is commercially available Fc (purchased from Yi Qiao Divine Land, Beijing).
Fig. 3 is pcr amplification antibody heavy and light chain variable region gene sepharose electrophoresis result. Swimming lane M is standard molecular weight DNA; Swimming lane 1 is pcr amplification antibody heavy chain variable region gene product; Swimming lane 2 is pcr amplification antibody chain variable region gene product.
Fig. 4 is that competitive ELISA measures monoclonal antibody G7, A7, E4, G5, B4 affinity.
Detailed description of the invention
The preparation of the anti-CD24 monoclonal antibody hybridoma cell of embodiment 1
1. immunogen
CD24 core space is made up of 33 amino acid residues, and its molecular weight is about 3.2kD, less immunogenic. The present invention, by antigenic peptides CD24 and hemocyanin KLH coupling, using conjugate CD24-KLH as immunogen, enhances the immunogenicity of CD24 Polypeptides. CD24-KLH used by the present invention is by giving birth to the synthesis of work biology (Shanghai) Co., Ltd., purity > 90%.
2. immune animal
Immune animal used by the present invention is BALB/c mouse, for SPF rank, comparative medicine institute of Yangzhou University provides. Immunological adjuvant is QuickAntibody, purchased from Beijing Kang Biquan company. Immunity flow process carries out with reference to QuickAntibody description,
Concrete as shown in table 1.
Table 1. immunization route and cycle
3. cell fusion
1) cell prepares
A. feeder layer cells
Take ICR mice (SPF level, purchased from comparative medicine institute of Yangzhou University) peritoneal macrophage the previous day in merging, be resuspended in HAT culture fluid (purchased from GIBCO company, containing xanthine, aminopterin and thymidine). It is inoculated in 96 orifice plates, puts 37 DEG C, 5%CO2 incubator is cultivated 24h.
B. myeloma cell
Murine myeloma cell strain SP20-Ag14 (preservation of this laboratory) does not synthesize and secretes any mouse immunoglobulin heavy and light chain, and guanozola is had resistance by this cell, and can not grow at HAT selective medium. Merge previous week, cultivate hybridoma by common DMEM complete medium (containing 15%FBS, purchased from GIBCO company) sub-bottle, so that cell energy well-grown. Each merges and prepares 250ml stand density is 2 �� l06Cells/m1 is in the healthy cell of mid log phase.
C. immune mouse spleen cell
Put to death the mice of immunity, after infiltrating with ethanol, take out spleen. Be prepared in advance 10cm2Sterile petri dish, puts into cell screen cloth, and adds 10ml plasma-free DMEM medium. Spleen is placed on cell screen cloth, and cuts with aseptic operation spleen is cut into fragment. After plunger crush and grind spleen, rinse by the incomplete culture medium of 5mlDMEM, make cell pass through screen cloth and enter in 50ml centrifuge tube.Collect spleen cell suspension, be used for merging.
2) merge
A. the DMEM culture medium and the 1mlPEG1450 (available from Sigma) that prepare 20ml serum-free are put in 37 DEG C of insulations.
B. collecting myeloma cell and spleen cell, under room temperature, the centrifugal 5min of 1500rpm, washes twice with serum-free medium, is then suspended in 10ml serum-free medium. By 5 �� 108Splenocyte and 1 �� 108Myeloma cell is sufficiently mixed, and 1500rpm is centrifuged 5min, abandons supernatant. Knocking tube bottom gently makes cell loose.
C. taking out at 37 DEG C of culture medium preheated and PEG, cell is placed in 37 DEG C of water-baths, in 1min, uniform speed slow dropwise adds 1mlPEG, and after adding, the culture medium of timely serum-free terminates. It is centrifuged and abandons supernatant and with 50mlHAT culture fluid re-suspended cell, mix gently.
D. cell is added in 5 pieces of 96 orifice plates being covered with trophocyte, every hole 100 �� l. Put 37 DEG C, 5%CO2Incubator is cultivated 4 days. According to cell growth state, before screening, partly change liquid 1��3 time with HAT culture fluid, it is to avoid in hole, culture medium turns yellow.
E., after merging latter 7 days, use instead and entirely change liquid containing HT culture fluid. After 24h, draw cells and supernatant, indirect ELISA detection screening positive clone.
3) monoclonal of hybridoma
Selecting the clone hole that positive value is higher, adopt limiting dilution assay the cell in hole to be diluted to every milliliter respectively containing 5,10 and 50 cells, be then inoculated in 96 orifice plates, 100 �� l are inoculated in every hole. 37 DEG C, 5%CO2Moistening cultivation 7��10 days, occurs that macroscopic clone can detect cells and supernatant. Observe under inverted microscope, mark the hole of only single clonal growth, take supernatant and do antibody test. The cell expansion taking antibody test positive hole is cultivated, and frozen.
The preparation and purification of embodiment 2CD24 specific antibody
1. ascites is collected
Carry and inject mouse peritoneal with 0.5ml paraffin oil (available from Sigma) the last week. After sensitization one week, discard culture medium by centrifugal for eugonic hybridoma, resuspended with PBS or incomplete culture medium, adjust cell concentration to 2 �� 106Cells/ml, injection 0.5ml cell suspension is to mouse peritoneal. The obvious enlargement of mouse peritoneal after 7��10 days, now gathers ascites. 4 DEG C, 5000g is centrifuged 20min, removes cell debris and oils and fats in ascites, is subsequently adding equal-volume glycerol and is stored in 20 DEG C.
The purification of 2.CD24 specific antibody
Adopt ProteinG affinity column antibody purification. Specifically comprise the following steps that A. cleans ProteinG affinity column with 10 times of cylinder hydrops. B. ProteinG affinity column is rinsed with 10 times of column volumes 20mM sodium phosphate buffer (pH7.0). C. required purification of samples is pumped into chromatographic column. D. with 100mM glycine buffer (pH3.5, pH2.7) eluting, eluting peak is collected; And neutralize gleanings with 1MTris buffer (pH9.0). E.10mMpH7.2 buffer dialysis desalting. F. with SDS-PGAGE electrophoresis Preliminary Identification antibody, there is purpose band in Fig. 1 result display 50kD and 25kD place.
The feature analysis of 3.CD24 specific antibody
1) immunoglobulin hypotype is identified
Adopt the mouse monoclonal antibody hypotype identification kit of Wuhan Sanying Bio-Technology Co., Ltd., identification of cell strain G7, A7, E4, G5, B4 secretory antibody hypotype, result is: the heavy chain immunoglobulin hypotype of this 5 strain cell strain secretion is IgG1, and light chain subtype is Kappa.
2) WesternBlot identifies the specificity of monoclonal antibody
Using antibody purification as primary antibodie, identifying the reaction of itself and CD24, Fig. 2 shows that gained antibody can be specific binding with CD24 part in commercially available CD24-Fc fusion protein.
3) competitive ELISA measures affinity of antibody
According to the method measuring affinity such as Friguet, designing antigenic competition binding antibody measuring affinity costant, sessile antibody concentration is 10pmol L-1, change antigen concentration and form series reaction system. 50 �� L antigens (0,1.95 �� 10 of 7 variable concentrations-11��3.91��10-11��7.81��10-11��1.56��10-10��3.13��10-10��6.25��10-10mol��L-1) mix with 50 �� L monoclonal antibody G7, A7, E4, G5, B4 respectively, 4 DEG C of reaction overnight. CD24 polypeptide is dissolved in 0.05mol L-1In carbonic acid buffer (pH9.6), whole mass concentration is 10 �� g mL-1, it is added in 96 orifice plates, every hole 100 �� L, 4 DEG C are coated 15h. 5% defatted milk powder closes 2h. It is separately added into the reactant liquor of reaction overnight, hatches 90min, PBST and PBS for 37 DEG C and wash 3 times respectively. Add the Goat anti-mouse antibodies of 1: 5000 dilution HRP labelling. TMB nitrite ion develops the color, and every hole adds 1mol L-1H2SO450 �� L terminate reaction, adopt double-wavelength method to read data, each monoclonal antibody " A450nm-A630nm " value in microplate reader. Antigen initial concentration is a0, antibody initial concentration is b0, A0And AiThe respectively absorption angle value of the antibody of initial antibodies and conjugated antigen. B=(A0-Ai)/A0, B is antibodies rate. KD=(1-B) (a0-b0B)/B. Measure the B value of each response system, with B/ (1-B) for abscissa, (a0-b0* B) to map (Fig. 4) for vertical coordinate, slope is exactly affinity constant KD. According to linear regression result, monoclonal antibody G7, A7, E4, G5, B4 affinity respectively 0.8nM, 0.72nM, 0.84nM, 0.82nM, 0.76nM.
The clone of embodiment 3 hybridoma cell strain G7, A7, E4, G5, B4 heavy and light chain variable region gene
1. anti-CD24 monoclonal antibody heavy and light chain variable region gene extracts, expands and Preliminary Identification
1) total serum IgE is extracted
Collect logarithmic (log) phase hybridoma, extract test kit (biological purchased from raw work) with RNA and extract total serum IgE, be dissolved in 20��50 �� l without RNase water, 70 DEG C of preservations.
2) RT-PCR synthesizes cDNA the first chain
Synthesizing cDNA the first chain with total serum IgE for template reverse transcription, test kit is purchased from Sheng Gong biotech firm, and reaction is undertaken by product description.
3) pcr amplification weight chain variable region gene
Polymerase is PrimerSTAR exo+ polymerase. The primer is the degenerate primer in document designed by hybridoma upstream, strain antibody weight chain variable region and downstream gene sequence, and wherein primer sequence is by light chain VLF (forward primer) and VLB (downstream primer) and heavy chain VHF1, VHF2 and VHB two:
VLF:gggagctcgayattgtgmtsacmcarwctmca;
VLB:ggtgcatgcggatacagttggtgcagcatc;
VHF1:cttccggaattcsargtnmagctgsagtc
VHF2:cttccggaattcsargtnmagctgsagtcwgg
VHB:ggaagatctatagacagatgggggtgtcgttttggc
(degenerate codon illustrates: r=a, g; Y=c, t; M=a, c; S=c, g; W=a, t)
Reaction volume 50 �� l, reaction condition is: 94 DEG C of 5min; 94 DEG C of 30s, 58 DEG C of 1min, 72 DEG C of 1min, circulate 30 times; 72 DEG C of 10min. Take 5 �� l end-products to identify at 100g/L agarose gel electrophoresis. Fig. 3 shows, pcr amplification obtains about 380bp heavy and light chain gene. Reclaim test kit (biological purchased from raw work) with glue PCR primer is purified.
4) glue recovery DNA product 3 ' end adds adenine (A)
Taking 1 �� l (5U/ �� l) rTaq enzyme, 4 �� ldNTP, 5 �� l10 �� PCRbuffer and 40 �� l purification electrophoresis product, reaction condition is: 94 DEG C, 10min, 70 DEG C of effect 40min, 4 DEG C, 10min.
5) PCR primer is connected to carrier T
Taking 4.5 �� lPCR products, 0.5 �� lpMD18-T carrier, 5 �� lligationbuffersolutionI, 16 DEG C connect 4h.
6) conversion and the Preliminary Identification of product are connected
Adopt CaCl2Conversion method will connect product and convert to competence DH5 ��. Concrete operations are as follows:
A. 200 �� l competent cells and 10 �� lDNA are gently mixed, ice bath 30min.
B. in 42 DEG C of circulator baths, place 90s, be transferred to ice bath 3min with by centrifuge tube.
C. often pipe adds the nonresistant LB culture medium of 1ml, hatches 1h for 37 DEG C.
D. centrifugal 4000rpm, 10min, and be resuspended in the middle of 300 �� lLB culture medium. The 300 �� l bacterial suspensions glass of aseptic process is coated with bacterium rod, is spread evenly across on Amp+ (100 �� g/ml) resistance agar plate.
E.37 after DEG C incubator cultivates 12h, observed result.
F. picked clones, adds shaken overnight in the middle of Amp+ (100 �� g/ml) LB culture medium.
G. take 1 �� l bacterium solution as template, carry out bacterium colony PCR checking with former PCR step. Choose positive colony, send by raw work biology order-checking.
2. the order-checking of anti-CD24 monoclonal antibody weight chain variable region gene and analysis
Sequencing result shows, is successfully obtained five strain of hybridoma strain antibody variable region genes. Utilize IMGT-VQUEST data base (http://www.imgt.org/) to analyze anti-CD24 monoclonal antibody weight chain variable region gene, analyze result and show: A7 hybridoma strain antibody heavy and light chain variable region aminoacid sequence and hypervariable region sequence thereof are such as shown in SEQIDNo.1��8; G7 hybridoma strain antibody heavy and light chain variable region aminoacid sequence and hypervariable region sequence thereof are such as shown in SEQIDNo.9��16; E4 hybridoma strain antibody heavy and light chain variable region aminoacid sequence and hypervariable region sequence thereof are such as shown in SEQIDNo.17��24; G5 hybridoma strain antibody heavy and light chain variable region aminoacid sequence and hypervariable region sequence thereof are such as shown in SEQIDNo.25��32; B4 hybridoma strain antibody heavy and light chain variable region aminoacid sequence and hypervariable region sequence thereof are such as shown in SEQIDNo.33��40.
Sequence table
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GlnProAlaSerIleSerCysLysSerSerGlnSerLeuLeuArgSer
202530
AspGlyLysThrTyrLeuAsnTrpLeuLeuGlnArgProGlyGlnSer
354045
ProLysArgLeuIleTyrValLeuSerLysLeuAspSerGlyValPro
505560
AspArgPheThrGlySerGlySerGlyThrAspPheThrLeuLysVal
65707580
SerArgValGluAlaGluAspLeuGlyValTyrTyrCysSerGlnGly
859095
AlaHisPheProTyrThrPheGlyGlyGlyThrLysLeuGluIleLys
100105110
<210>22
<211>11
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(11)
<223>VL3CDR1
<400>6
GlnSerLeuLeuArgSerAspGlyLysThrTyr
1510
<210>23
<211>3
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(3)
<223>VL3CDR2
<400>7
ValLeuSer
1
<210>24
<211>9
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(9)
<223>VL3CDR3
<400>24
SerGlnGlyAlaHisPheProTyrThr
15
<210>25
<211>113
<212>PRT
<213>artificial sequence
<220>
<221>V_region
<222>(1)..(113)
<223>VH4
<400>25
GlnValLysLeuGluGluSerGlyGlyGlyLeuValGlnProGlyGly
151015
SerLeuLysIleSerCysAlaAlaSerAlaPhePheAspSerArgTyr
202530
TrpMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpIle
354045
GlyGluIleMetProAspTyrSerThrIleAsnTyrThrProSerLeu
505560
ArgAspLysPheIleIleSerArgAspAsnAlaLysAsnThrLeuTyr
65707580
LeuGlnMetSerLysValArgTyrGluAspThrSerLeuTyrTyrCys
859095
AlaArgGlnGlyAspTyrTrpGlyGlnGlyThrSerValThrValSer
100105110
Ser
<210>26
<211>8
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(8)
<223>VH4CDR1
<400>26
AlaPhePheAspSerArgTyrTrp
15
<210>27
<211>8
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(8)
<223>VH4CDR2
<400>27
IleMetProAspSerSerThrIle
15
<210>28
<211>6
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(6)
<223>VH4CDR3
<400>28
AlaArgGlnGlyAspTyr
15
<210>29
<211>112
<212>PRT
<213>artificial sequence
<220>
<221>V_region
<222>(1)..(112)
<223>VL4
<400>29
AspIleValMetThrGlnThrProLeuSerLeuSerValThrIleGly
151015
GlnProAlaSerIleSerCysLysSerSerGlnSerValLeuHisSer
202530
AspGlyLysThrTyrLeuAsnTrpLeuLeuGlnArgProGlyGlnSer
354045
ProLysArgLeuIleTyrValLeuSerLysLeuAspSerGlyValPro
505560
AspArgPheThrGlySerGlySerGlyThrAspPheThrLeuLysVal
65707580
SerArgValGluAlaGluAspLeuGlyValTyrTyrCysTrpGlnGly
859095
GlyHisPheProTyrThrPheGlyGlyGlyThrLysLeuGluIleLys
100105110
<210>30
<211>11
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(11)
<223>VL4CDR1
<400>30
GlnSerValLeuHisSerAspGlyLysThrTyr
1510
<210>31
<211>3
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(3)
<223>VL4CDR2
<400>31
ValLeuSer
1
<210>32
<211>9
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(9)
<223>VL4CDR3
<400>32
TrpGlnGlyGlyHisPheProTyrThr
15
<210>33
<211>113
<212>PRT
<213>artificial sequence
<220>
<221>V_region
<222>(1)..(112)
<223>VH5
<400>33
GlnValLysLeuGluGluSerGlyGlyGlyLeuValGlnProGlyGly
151015
SerLeuLysIleSerCysAlaAlaSerGlyPheAspPheTyrArgTyr
202530
SerMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpIle
354045
GlyGluIleAsnProAspTrpSerThrIleAsnTyrThrProSerLeu
505560
ArgAspLysPheIleIleSerArgAspAsnAlaLysAsnThrLeuTyr
65707580
LeuGlnMetSerLysValArgTyrGluAspThrSerLeuTyrTyrCys
859095
AlaHisGlnGlyAspTyrTrpGlyGlnGlyThrSerValThrValSer
100105110
Ser
<210>34
<211>8
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(8)
<223>VH5CDR1
<400>34
GlyPheAspPheTyrArgTyrSer
15
<210>35
<211>8
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(8)
<223>VH5CDR2
<400>35
IleAsnProAspTrpSerThrIle
15
<210>36
<211>6
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(6)
<223>VH5CDR3
<400>36
AlaHisGlnGlyAspTyr
15
<210>37
<211>112
<212>PRT
<213>artificial sequence
<220>
<221>V_region
<222>(1)..(112)
<223>VL5
<400>37
AspIleValMetThrGlnThrProLeuSerLeuSerValThrIleGly
151015
GlnProAlaSerIleSerCysLysSerSerGlnAsnGluLeuHisSer
202530
AspGlyLysThrTyrLeuAsnTrpLeuLeuGlnArgProGlyGlnSer
354045
ProLysArgLeuIleTyrAlaValAlaLysLeuAspSerGlyValPro
505560
AspArgPheThrGlySerGlySerGlyThrAspPheThrLeuLysVal
65707580
SerArgValGluAlaGluAspLeuGlyValTyrTyrCysTrpGlnLeu
859095
LeuHisPheProTyrThrPheGlyGlyGlyThrLysLeuGluIleLys
100105110
<210>38
<211>11
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(11)
<223>VL5CDR1
<400>38
GlnAsnGluLeuHisSerAspGlyLysThrTyr
1510
<210>39
<211>3
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(3)
<223>VL5CDR2
<400>39
AlaValAla
1
<210>40
<211>9
<212>PRT
<213>artificial sequence
<220>
<221>D-segment
<222>(1)..(9)
<223>VL5CDR3
<400>40
TrpGlnLeuLeuHisPheProTyrThr
15
<210>41
<211>33
<212>PRT
<213>Homosapiens
<400>41
SerGluThrThrThrGlyThrSerSerAsnSerSerGlnSerThrSer
151015
AsnSerGlyLeuAlaProAsnProThrAsnAlaThrThrLysAlaAla
202530
Gly
Claims (7)
1. the variable region sequences of an anti-CD24 monoclonal antibody, it is characterised in that comprise CDR1 ~ 3 region amino acid sequence shown in SEQIDNo.18 ~ 20 and SEQIDNo.22 ~ 24 in its heavy chain, variable region of light chain respectively.
2. the monoclonal antibody of the anti-CD24 containing variable region as claimed in claim 1, it is characterised in that the structure of described monoclonal antibody includes the aminoacid sequence with heavy chain shown in SEQIDNo.17 and 21 and light chain.
3. a genetic engineering antibody, it is characterised in that its contained heavy chain is consistent with light-chain variable sequence with the variable region sequences described in claim 1;This genetic engineering antibody includes but not limited to: Chimeric antibody; Or humanized antibody; Or the functional fragment Fab of antibody; Or single-chain antibody; Or the antibody functional fragment VH-L that variable region of heavy chain and Whole light chains merge; Or the arrangement of one or more CDR of heavy chain and light chain, the antibody functional fragment connecting or combine; Or above-mentioned antibody and antibody functional fragment and other various albumen or polypeptide are attached, splice, merge and functional fusion protein of antibody-like of obtaining.
4. an antibody coupling matter, it is characterised in that using monoclonal antibody described in claim 2 or the genetic engineering antibody described in claim 3 as targeting moiety, with radionuclide or chemicals or toxin conjugated.
5. monoclonal antibody described in claim 2 or the genetic engineering antibody described in claim 3, it is characterised in that be optionally combined with CD24 or suppress the combination of CD24 and CD24 part or neutralize CD24.
6. monoclonal antibody described in claim 2 or right want the genetic engineering antibody described in 3 or the antibody coupling matter described in claim 4, it is characterised in that for preparing treatment and CD24 abnormal expression or medicine excessive, wild phase related disorders.
7. an anti-CD24 method for preparing monoclonal antibody, it is characterised in that using shown in CD24 Polypeptides with hemocyanin KLH conjugate as antigen.
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| CN201610116883.1A CN105646713B (en) | 2014-01-22 | 2014-01-22 | A kind of monoclonal antibody and its application |
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| CN201410027795.5A CN103819561A (en) | 2014-01-22 | 2014-01-22 | Anti-CD24 (Cluster of Differentiation 24) monoclonal antibody, and variable region sequence and application thereof |
| CN201610116883.1A CN105646713B (en) | 2014-01-22 | 2014-01-22 | A kind of monoclonal antibody and its application |
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| CN201410027795.5A Division CN103819561A (en) | 2014-01-22 | 2014-01-22 | Anti-CD24 (Cluster of Differentiation 24) monoclonal antibody, and variable region sequence and application thereof |
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| CN105646713B CN105646713B (en) | 2019-07-12 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114106180A (en) * | 2021-12-13 | 2022-03-01 | 中国药科大学 | Monoclonal antibody and application thereof |
| WO2023232110A1 (en) * | 2022-06-01 | 2023-12-07 | 迈威(上海)生物科技股份有限公司 | Anti-human cd24 antibody and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127523A (en) * | 2010-11-24 | 2011-07-20 | 江苏省农业科学院 | Hybridoma cell line and application thereof |
| CN102250828A (en) * | 2010-11-10 | 2011-11-23 | 北京大学 | CD24 (glycosyl phosphatidyl inositol-linked surface mucin) and new use of CD24 antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250828A (en) * | 2010-11-10 | 2011-11-23 | 北京大学 | CD24 (glycosyl phosphatidyl inositol-linked surface mucin) and new use of CD24 antibody |
| CN102127523A (en) * | 2010-11-24 | 2011-07-20 | 江苏省农业科学院 | Hybridoma cell line and application thereof |
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| Title |
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| E WEBER等: "Antibodies to the protein core of the small cell lung cancer workshop antigen cluster-w4 and to the leucocyte workshop antigen CD24 recognize the same short protein sequence leucine-alanine-proline", 《CLINICAL & EXPERIMENTAL IMMUNOLOGY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114106180A (en) * | 2021-12-13 | 2022-03-01 | 中国药科大学 | Monoclonal antibody and application thereof |
| WO2023232110A1 (en) * | 2022-06-01 | 2023-12-07 | 迈威(上海)生物科技股份有限公司 | Anti-human cd24 antibody and use thereof |
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| Publication number | Publication date |
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| CN105646713B (en) | 2019-07-12 |
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