CN105646712B - Monoclonal antibody and its application - Google Patents

Monoclonal antibody and its application Download PDF

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CN105646712B
CN105646712B CN201610114516.8A CN201610114516A CN105646712B CN 105646712 B CN105646712 B CN 105646712B CN 201610114516 A CN201610114516 A CN 201610114516A CN 105646712 B CN105646712 B CN 105646712B
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antibody
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CN105646712A (en
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张娟
王旻
罗晨
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China Pharmaceutical University
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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]

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Abstract

The invention discloses a kind of variable region sequences with the monoclonal antibody and the monoclonal antibody that neutralize human leukocytes differentiation antigen CD24 bioactivity.The present invention is using Antigenic Peptide and limpet hemocyanin conjugate CD24-KLH as immunogene, inoculation BALB/c mouse, it takes immune Mouse spleen cells and merges it with myeloma cell, the hybridoma cell strain that can express anti-CD24 antibody is obtained, this cell strain can be used for preparing anti-CD24 monoclonal antibody.The present invention has cloned anti-CD24 antibody variable region amino acid sequence simultaneously.Anti- CD24 antibody of the invention can be specifically bound with CD24, therefore be can be used for treating and expressed excessive, abnormal, relevant disease out of control to CD24.

Description

Monoclonal antibody and its application
The present invention is application number 2014100277955, applying date 2014-01-22, entitled anti-CD24 monoclonal The divisional application of antibody, its variable region sequences and its application.
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of to specifically bind human leukocytes differentiation antigen CD24's Monoclonal antibody, its variable region sequences and its application.
Background technique
Human leukocytes differentiation antigen CD24 (cluster of differentiation 24), which is that one kind is newly discovered, to lay equal stress on The tumor markers of point research.
Current research shows that CD24 molecule is high glycosylation sialic acid of the apparent molecular weight between 30~70kDa Glycoprotein.CD24 includes the core protein skeleton structure of 33 amino acid residues composition, has 16 potential O- or N- glycosyls Change site, with mouse heat stable antigen very high homology；Its mature polypeptide can pass through the phosphatidylinositols of c-terminus (glycosyl-phosphatidyl-inositol, GPI) is anchored on the surface of cell membrane.
Under physiological conditions, CD24 molecule is only in immature B cells, mature granulocyte and a small number of epithelial cells and nerve Low expression level on cell；And when body is in pathological state, the surface of most of malignant tumour cell can detect significantly The CD24 molecule of high level expression, the height of expression and the occurrence and development of tumour are closely related.
P selectin is expressed in the blood platelet and endothelial cell surface of activation, and CD24 is one of its ligand.The high table of CD24 molecule Up to tumour cell is enhanced to the adhesive attraction of blood platelet and endothelial cell, the recurrence and transfer of tumour are promoted.Certainly, as Cell membrane surface signal transducers, CD24 being capable of proliferation by a variety of mechanism of action mediate tumor cells, adherency, transfer And invasion.It has furthermore been found that CD24 expression and the stemness of liver cancer cells are closely related, CD24 is likely to become a kind of new liver for research Cancer stem cell surface marker.
It can be seen that CD24 is expected to become research elaboration of tumour mechanism and develops the new target drone of dependent diagnostic reagent.
Summary of the invention
The object of the invention one is to provide a kind of anti-CD24 method for preparing monoclonal antibody.
The object of the invention two is the provision of a kind of anti-CD24 monoclonal antibody.
The object of the invention three is to provide the variable region amino acid sequence of anti-CD24 monoclonal antibody.
Note is immunized using CD24 Polypeptides and hemocyanin KLH conjugate (CD24-KLH) as immunogene in the present invention BALB/c mouse is penetrated, immune Mouse spleen cells are taken and merges it with myeloma cell, anti-CD24 antibody can be expressed by obtaining Hybridoma cell strain, this cell strain can be used for preparing anti-CD24 monoclonal antibody.
Heavy chain of antibody, chain variable region amino acid sequence and its hypervariable region that the present invention clones if any SEQ ID No.1~ Shown in 16.
Monoclonal antibody of the invention can be specifically bound with CD24.
Detailed description of the invention
Fig. 1 is that SDS-PAGE detects Protein G affinity column purified product result.Swimming lane M is standard molecular weight egg It is white；Swimming lane 1 is ascites sample；Swimming lane 2 is loading collection liquid；Swimming lane 3 is that 20mM sodium phosphate buffer washes column collection liquid；Swimming lane 4 ~6 elute collection liquid (collecting 1ml per minute, collect 3ml altogether) for 100mM glycine buffer (pH 3.5), and swimming lane 7~10 is 100mM glycine buffer (pH 2.7) elutes collection liquid (collecting 1ml per minute, collect 4ml altogether).
Fig. 2 be Western Blot purification Identification after interact between antibody and CD24.Swimming lane M is standard molecular weight egg It is white；The commercially available CD24-Fc of swimming lane 1 (is purchased from the Divine Land Beijing Yi Qiao)；Swimming lane 2 is commercially available Fc (being purchased from the Divine Land Beijing Yi Qiao).
Fig. 3 is PCR amplification antibody heavy and light chain variable region gene agarose 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.
Specific embodiment
The preparation of the anti-CD24 monoclonal antibody hybridoma cell of embodiment 1
1. immunogene
CD24 core space is made of 33 amino acid residues, and molecular weight is about 3.2kD, less immunogenic.The present invention Antigenic Peptide CD24 and hemocyanin KLH is coupled, using conjugate CD24-KLH as immunogene, enhances CD24 Polypeptides Immunogenicity.CD24-KLH used in the present invention is synthesized by giving birth to work biology (Shanghai) Co., Ltd., purity > 90%.
2. immune animal
It is BALB/c mouse that animal is immunized used in the present invention, is SPF rank, is provided by comparative medicine institute, Yangzhou University.Exempt from Epidemic disease adjuvant is QuickAntibody, is purchased from Beijing Kang Biquan company.Immune process is carried out referring to QuickAntibody specification,
It is specific as shown in table 1.
1. immunization route of table and period
3. cell fusion
1) cell prepares
A. feeder cells
ICR mouse (SPF grades, be purchased from Yangzhou University's comparative medicine institute) peritoneal macrophage is taken in fusion the previous day, is resuspended In HAT culture solution (being purchased from GIBCO company, contain xanthine, aminopterin and thymidine).It is inoculated in 96 holes Plate sets 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator.
B. myeloma cell
Murine myeloma cell strain SP20-Ag14 (preservation of this laboratory) does not synthesize and secretes any mouse immune ball Ferritin heavy chain and light chain, this cell is resistant to guanozola, and cannot grow in HAT selective medium.Fusion Previous week, with common DMEM complete medium (containing 15%FBS, be purchased from GIBCO company) sub-bottle culture hybridoma, So that cell energy well-grown.It is 2 × l0 that each fusion, which prepares 250ml stand density,6Cells/m1 is in logarithmic growth The healthy cell of phase.
C. Mouse spleen cells are immunized
Immune mouse is put to death, takes out spleen after being infiltrated with alcohol.Be prepared in advance 10cm2Sterile petri dish is put into thin Born of the same parents' sieve, and 10ml plasma-free DMEM medium is added.Spleen is placed in cell screen clothes, and is cut with aseptic operation and cuts spleen It fragmentates.After plunger crush and grind spleen, with 5ml DMEM incomplete culture medium rinse, make cell passed through sieve into Enter in 50ml centrifuge tube.Spleen cell suspension is collected, for merging.
2) it merges
A. the DMEM culture medium and 1ml PEG1450 (being purchased from Sigma company) for preparing 20ml serum-free are put in 37 DEG C of heat preservations.
B. myeloma cell and spleen cell are collected, 1500rpm is centrifuged 5min under room temperature, washes two with serum free medium It is secondary, it is then suspended in 10ml serum free medium.By 5 × 108Splenocyte and 1 × 108Myeloma cell is sufficiently mixed, 1500rpm is centrifuged 5min, abandons supernatant.Gently tapping tube bottom keeps cell loose.
C. the culture medium and PEG preheated at 37 DEG C is taken out, cell is placed in 37 DEG C of water-baths, uniform speed slow is dropwise in 1min Add 1ml PEG, adds rear in time with the culture medium termination of serum-free.Centrifugation is abandoned supernatant and is resuspended with 50ml HAT culture solution thin Born of the same parents mix gently.
D. cell is added 5 pieces to be covered in 96 orifice plates of trophocyte, every 100 μ l of hole.37 DEG C are set, 5%CO2Incubator Middle culture 4 days.It according to cell growth state, is partly changed liquid 1~3 time before screening with HAT culture solution, culture medium in hole is avoided to become It is yellow.
E. it after merging after 7 days, uses instead and changes liquid entirely containing HT culture solution.After for 24 hours, cells and supernatant, indirect ELISA are drawn Detect screening positive clone.
3) monoclonal of hybridoma
Select the positive higher clone hole of value, using limiting dilution assay by the cell in hole be diluted to respectively every milliliter contain 5, 10 and 50 cells, are then inoculated in 96 orifice plates, and every hole is inoculated with 100 μ l.37 DEG C, 5%CO2Wet culture 7~10 days, occurs Macroscopic clone can be detected cells and supernatant.It is observed under inverted microscope, marks only single clonal growth Hole takes supernatant to make antibody test.The cell expansion culture in antibody test positive hole is taken, and is frozen.
The preparation and purification of embodiment 2CD24 specific antibody
1. ascites is collected
The last week is mentioned with 0.5ml paraffin oil (purchased from Sigma company) injection mouse peritoneal.Sensitization after a week, will be grown prosperous The hybridoma centrifugation of Sheng discards culture medium, is resuspended with PBS or incomplete culture medium, and adjustment cell concentration to 2 × 106Cells/ml injects 0.5ml cell suspension into mouse peritoneal.The obvious enlargement of mouse peritoneal, acquires at this time after 7~10 days Ascites.4 DEG C, 5000g is centrifuged 20min, removes cell fragment and grease in ascites, isometric glycerol is then added and is stored in ﹣ 20 ℃。
2.CD24 the purifying of specific antibody
Using Protein G affinity column antibody purification.Specific step is as follows: A. is cleaned with 10 times of cylinder ponding ProteinG affinity column.B. ProteinG affinity chromatography is rinsed with 10 times of column volume 20mM sodium phosphate buffers (pH 7.0) Column.C. required purification of samples is pumped into chromatographic column.D. it is eluted with 100mM glycine buffer (pH 3.5, pH 2.7), collection is washed De- peak；And gleanings are neutralized with 1M Tris buffer (pH 9.0).E.10mM 7.2 buffer dialysis desalting of pH.F. SDS- is used There is purpose band at 50kD and 25kD as the result is shown in PGAGE electrophoresis Preliminary Identification antibody, Fig. 1.
3.CD24 the signature analysis of specific antibody
1) immunoglobulin subtype identification
Using the mouse monoclonal antibody subtype identification kit of Wuhan Sanying Bio-Technology Co., Ltd., identification of cell strain G7, A7, E4, G5, B4 secretory antibody hypotype, as a result are as follows: this 5 plants of cell strains secretion heavy chain immunoglobulin hypotype be IgG1, light chain subtype are Kappa.
2) Western Blot identifies the specificity of monoclonal antibody
Using antibody purification as primary antibody, its reacting with CD24 is identified, Fig. 2 display gained antibody can be with commercially available CD24-Fc The part CD24 is specifically bound in fusion protein.
3) competitive ELISA measures affinity of antibody
According to the method that Friguet etc. measures affinity, antigenic competition binding antibody measuring affinity costant is designed, Gu Determining antibody concentration is 10pmolL-1, change antigen concentration and form series reaction system.50 μ L antigens of 7 various concentrations (0、1.95×10-11、3.91×10-11、7.81×10-11、1.56×10-10、3.13×10-10、6.25×10-10mol·L-1) It is mixed respectively with 50 μ L monoclonal antibody G7, A7, E4, G5, B4,4 DEG C of reaction overnights.CD24 polypeptide is dissolved in 0.05mol L-1In carbonic acid buffer (pH 9.6), whole mass concentration is 10 μ gmL-1, it is added in 96 orifice plates, every 100 μ L of hole, 4 DEG C of coatings 15h.5% skimmed milk power closes 2h.It is separately added into the reaction solution of reaction overnight, 37 DEG C of incubations 90min, PBST and PBS wash 3 respectively It is secondary.The Goat anti-mouse antibodies of 1: 5000 dilution HRP label are added.1molL is added in the colour developing of TMB developing solution, every hole-1H2SO450μ L terminates reaction, using double-wavelength method in reading data, each monoclonal antibody " A450nm-A630nm " value in microplate reader.At the beginning of antigen Beginning concentration is a0, antibody initial concentration is b0, A0And AiThe respectively absorption angle value of the antibody of initial antibodies and combination antigen.B= (A0-Ai)/A0, B is antibody Percentage bound.KD=(1-B) (a0-b0B)/B.The B value of each reaction system is measured, is with B/ (1-B) Abscissa, (a0-b0* B) it is that ordinate is mapped (Fig. 4), slope is exactly affinity constant KD.According to linear regression as a result, monoclonal Antibody G7, A7, E4, G5, B4 affinity is respectively 0.8nM, 0.72nM, 0.84nM, 0.82nM, 0.76nM.
The clone of 3 hybridoma cell strain G7, A7, E4, G5, B4 heavy and light chain variable region gene of embodiment
1. anti-CD24 monoclonal antibody heavy and light chain variable region gene extraction, amplification and Preliminary Identification
1) total serum IgE is extracted
Logarithmic phase hybridoma is collected, extracts total serum IgE with RNA extracts kit (purchased from raw work biology), it is dissolved in 20~ 50 μ l are without RNA enzyme water, 70 DEG C of ﹣ preservations.
2) RT-PCR synthesizes the first chain of cDNA
The first chain of cDNA is synthesized by template reverse transcription of total serum IgE, kit is purchased from Sheng Gong biotech firm, and reaction is said by product Bright book carries out.
3) PCR amplification weight chain variable region gene
Polymerase is PrimerSTAR exo+ polymerase.The primer is light according to hybridoma strain antibody in document Degenerate primer designed by heavy chain variable region upstream and downstream gene sequence, wherein light chain VL F (upstream primer) and VL B (under Swim primer) and heavy chain VH F1, VH F2 and VH B two to primer sequence are as follows:
VL F:gg gag ctc gay att gtg mts acm car wct mca；
VL B:ggt gca tgc gga tac agt tgg tgc agc atc；
VH F1:ctt ccg gaa ttc sar gtn mag ctg sag tc
VH F2:ctt ccg gaa ttc sar gtn mag ctg sag tcw gg
VH B:gga aga tct ata gac aga tgg ggg tgt cgt ttt ggc
(degenerate codon explanation: r=a, g；Y=c, t；M=a, c；S=c, g；W=a, t)
50 μ l of reaction volume, reaction condition are as follows: 94 DEG C of 5min；94 DEG C of 30s, 58 DEG C of 1min, 72 DEG C of 1min are recycled 30 times； 72℃10min.5 μ l final products are taken to be identified in 100g/L agarose gel electrophoresis.Fig. 3 shows that PCR amplification obtains 380bp Left and right heavy and light chain gene.Purified with plastic recovery kit (purchased from raw work biology) to PCR product.
4) glue recycling DNA product 3 ' end plus adenine (A)
1 μ l (5U/ μ l) rTaq enzyme is taken, 4 μ l dNTP, 5 μ 10 × PCR of l buffer and 40 μ l purify electrophoresis product, reaction Condition are as follows: 94 DEG C, 10min, 70 DEG C effect 40min, 4 DEG C, 10min.
5) PCR product is connected to carrier T
Take 4.5 μ l PCR products, 0.5 μ l pMD 18-T carrier, 5 μ l ligation buffer solution I, 16 DEG C connection 4h.
6) conversion and Preliminary Identification of connection product
Using CaCl2Conversion method converts connection product to competence DH5 α.Concrete operations are as follows:
A. 200 μ l competent cells and 10 μ l DNA are gently mixed, ice bath 30min.
B. 90s is placed in 42 DEG C of circulator baths, with centrifuge tube is transferred to ice bath 3min.
C. the LB culture medium of 1ml non-resistant, 37 DEG C of incubation 1h are added in every pipe.
D. it is centrifuged 4000rpm, 10min, and is resuspended in 300 μ l LB culture mediums.By 300 μ l bacterial suspensions with without The glass of bacterium processing applies bacteria stick, is spread evenly across on Amp+ (100 μ g/ml) resistance agar plate.
E.37 after DEG C incubator culture 12h, result is observed.
F. shaken overnight in Amp+ (100 μ g/ml) LB culture medium is added in picked clones.
G. it takes 1 μ l bacterium solution as template, carries out bacterium colony PCR verifying with original PCR step.Positive colony is chosen, is sent by raw work Biological order-checking.
2. anti-CD24 monoclonal antibody weight chain variable region gene sequencing and analysis
Sequencing result is shown, successfully obtains five strain of hybridoma strain antibody variable region genes.Utilize IMGT-VQUEST number Analyze anti-CD24 monoclonal antibody weight chain variable region gene according to library (http://www.imgt.org/), analysis the result shows that: A7 hybridoma strain antibody heavy and light chain variable region amino acid sequence and its hypervariable region sequence are as shown in No.1~8 SEQ ID；G7 Hybridoma strain antibody heavy and light chain variable region amino acid sequence and its hypervariable region sequence are as shown in No.9~16 SEQ ID；E4 Hybridoma strain antibody heavy and light chain variable region amino acid sequence and its hypervariable region sequence are as shown in No.17~24 SEQ ID；G5 Hybridoma strain antibody heavy and light chain variable region amino acid sequence and its hypervariable region sequence are as shown in No.25~32 SEQ ID；B4 Hybridoma strain antibody heavy and light chain variable region amino acid sequence and its hypervariable region sequence are as shown in No.33~40 SEQ ID.
Sequence table
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<223> VH3
<400> 17
Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly Pro Asp Phe Ser Arg Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Asp Tyr Ser Thr Ile Asn Tyr Thr Pro Ser Leu
50 55 60
Arg Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Lys Val Arg Tyr Glu Asp Thr Ser Leu Tyr Tyr Cys
85 90 95
Val Arg Gln Gly Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 18
<211> 8
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(8)
<223> VH3CDR1
<400> 18
Gly Pro Asp Phe Ser Arg Tyr Trp
1 5
<210> 19
<211> 8
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(8)
<223> VH3CDR2
<400> 3
Ile Asn Pro Asp Tyr Ser Thr Ile
1 5
<210> 20
<211> 6
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(6)
<223> VH3CDR3
<400> 20
Val Arg Gln Gly Asp Tyr
1 5
<210> 21
<211> 112
<212> PRT
<213>artificial sequence
<220>
<221> V_region
<222> (1)..(112)
<223> VL3
<400> 21
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Arg Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Val Leu Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Val
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Ser Gln Gly
85 90 95
Ala His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 22
<211> 11
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(11)
<223> VL3CDR1
<400> 6
Gln Ser Leu Leu Arg Ser Asp Gly Lys Thr Tyr
1 5 10
<210> 23
<211> 3
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(3)
<223> VL3CDR2
<400> 7
Val Leu Ser
1
<210> 24
<211> 9
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(9)
<223> VL3CDR3
<400> 24
Ser Gln Gly Ala His Phe Pro Tyr Thr
1 5
<210> 25
<211> 113
<212> PRT
<213>artificial sequence
<220>
<221> V_region
<222> (1)..(113)
<223> VH4
<400> 25
Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Ile Ser Cys Ala Ala Ser Ala Phe Phe Asp Ser Arg Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Met Pro Asp Tyr Ser Thr Ile Asn Tyr Thr Pro Ser Leu
50 55 60
Arg Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Lys Val Arg Tyr Glu Asp Thr Ser Leu Tyr Tyr Cys
85 90 95
Ala Arg Gln Gly Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 26
<211> 8
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(8)
<223> VH4CDR1
<400> 26
Ala Phe Phe Asp Ser Arg Tyr Trp
1 5
<210> 27
<211> 8
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(8)
<223> VH4CDR2
<400> 27
Ile Met Pro Asp Ser Ser Thr Ile
1 5
<210> 28
<211> 6
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(6)
<223> VH4CDR3
<400> 28
Ala Arg Gln Gly Asp Tyr
1 5
<210> 29
<211> 112
<212> PRT
<213>artificial sequence
<220>
<221> V_region
<222> (1)..(112)
<223> VL4
<400> 29
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Val Leu His Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Val Leu Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Val
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly
85 90 95
Gly His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 30
<211> 11
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(11)
<223> VL4CDR1
<400> 30
Gln Ser Val Leu His Ser Asp Gly Lys Thr Tyr
1 5 10
<210> 31
<211> 3
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(3)
<223> VL4CDR2
<400> 31
Val Leu Ser
1
<210> 32
<211> 9
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(9)
<223> VL4CDR3
<400> 32
Trp Gln Gly Gly His Phe Pro Tyr Thr
1 5
<210> 33
<211> 113
<212> PRT
<213>artificial sequence
<220>
<221> V_region
<222> (1)..(112)
<223> VH5
<400> 33
Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly Phe Asp Phe Tyr Arg Tyr
20 25 30
Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Asp Trp Ser Thr Ile Asn Tyr Thr Pro Ser Leu
50 55 60
Arg Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Lys Val Arg Tyr Glu Asp Thr Ser Leu Tyr Tyr Cys
85 90 95
Ala His Gln Gly Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 34
<211> 8
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(8)
<223> VH5CDR1
<400> 34
Gly Phe Asp Phe Tyr Arg Tyr Ser
1 5
<210> 35
<211> 8
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(8)
<223> VH5CDR2
<400> 35
Ile Asn Pro Asp Trp Ser Thr Ile
1 5
<210> 36
<211> 6
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(6)
<223> VH5CDR3
<400> 36
Ala His Gln Gly Asp Tyr
1 5
<210> 37
<211> 112
<212> PRT
<213>artificial sequence
<220>
<221> V_region
<222> (1)..(112)
<223> VL5
<400> 37
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Asn Glu Leu His Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Ala Val Ala Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Val
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Leu
85 90 95
Leu His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 38
<211> 11
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(11)
<223> VL5CDR1
<400> 38
Gln Asn Glu Leu His Ser Asp Gly Lys Thr Tyr
1 5 10
<210> 39
<211> 3
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(3)
<223> VL5CDR2
<400> 39
Ala Val Ala
1
<210> 40
<211> 9
<212> PRT
<213>artificial sequence
<220>
<221> D-segment
<222> (1)..(9)
<223> VL5CDR3
<400> 40
Trp Gln Leu Leu His Phe Pro Tyr Thr
1 5
<210> 41
<211> 33
<212> PRT
<213> Homo sapiens
<400> 41
Ser Glu Thr Thr Thr Gly Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser
1 5 10 15
Asn Ser Gly Leu Ala Pro Asn Pro Thr Asn Ala Thr Thr Lys Ala Ala
20 25 30
Gly

Claims (5)

1. a kind of anti-CD24 monoclonal antibody, which is characterized in that separately include SEQ ID No in its heavy chain, light chain variable region ~ 12 and the region amino acid sequence of CDR1 ~ 3 shown in .14 ~ 16 SEQ ID No .10.
2. a kind of monoclonal antibody of anti-CD24, which is characterized in that include heavy chain variable region in the structure of the monoclonal antibody And light chain variable region, amino acid sequence is respectively as shown in SEQ ID No.9 and 13.
3. a kind of genetic engineering antibody, which is characterized in that heavy chain and light-chain variable sequence contained by it contain claim 1 institute The region amino acid sequence of CDR1 ~ 3 shown in .10 ~ 12 ID No SEQ and .14 ~ 16 SEQ ID No stated；The genetic engineering antibody packet It includes: Chimeric antibody；Either humanized antibody；The either functional fragment Fab of antibody；Either single-chain antibody.
4. a kind of antibody coupling matter, which is characterized in that will be described in monoclonal antibody described in claim 2 or claim 3 Genetic engineering antibody as targeting moiety, be coupled with radionuclide or chemicals.
5. monoclonal antibody or right described in claim 2 want 3 described in genetic engineering antibody or as claimed in claim 4 Antibody coupling matter is used to prepare treatment and CD24 abnormal expression or excessive, related disease out of control drug.
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