CN101245108B - Antihuman CD34 antibody, producing process and uses thereof - Google Patents
Antihuman CD34 antibody, producing process and uses thereof Download PDFInfo
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Abstract
The invention pertains to the field of biological technology. Particularly, the invention discloses antibodies of 5B12, 4C8 and 2E10 of a mouse anti-human CD34 antibody and chimeric antibodies of c5B12, c4C8 and c2E10, a preparation method of the antibodies and a usage in the separation of bone marrow stems/progenitor cells.
Description
Technical field
The invention belongs to biological technical field, more specifically, the invention discloses a kind of antibody, Preparation Method And The Use.
Background technology
On the clinical treatment of some malignant hematologic disease and solid tumor, hematopoietic stem/progenitor (hematopoietic stem/progenitor cells, HSC) and transplanting have become the unique effective methods for the treatment of of generally acknowledging at present.But cause because containing a large amount of T lymphocytes in the graft that acute, severe graft versus host disease (GVH disease) (graft-versus-host disease GVHD) occurs after transplanting is to affect the one of the main reasons that can Allogeneic Hematopoietic Stem Cell Transplantation obtain long-term survival.It is the hemopoietic stem cell that how to obtain large-scale purification that hematopoietic stem/progenitor is transplanted the matter of utmost importance face, to remove the interference of impurity cell to transplanting, therefore, remove or the enrichment graft in specific cell be current fields of implantation institute problems of concern.Because HSCs does not have clear and definite morphological feature, main manifestations is the single core parent cell of lymphocyte sample, so can only identify with some albumen of cell surface.CD34 be a molecular weight approximately 110Kd wear membrane glycoprotein, be that the knowable expression of institute very early time on human HSC also is antigen the most widely at present, be the preferably sign of weighing the HSC quality and quantity, along with the development of selecting technology based on the positive of CD34 antibody, CD34
+Hemopoietic stem cell is in experimental animal models or all demonstrates function [the Andrews RG of reconstitute hematopoiesis on the person, Bryant EM, BartelmezSH, Muirhead DT, Knitter GH, Bensinger W, Strong DM, Bernstien ID (1992) CD34marrow cells, devoid of T and B Lymphocytes, reconstitute stablelymphopoiesis and myelopoiesis in lethally irradiated baboons.Blood80:1693] [Shpall EJ, Jones RB, Franklin W, Bearman S, Stemmer S, HamiL, Petsche D, Taffs S, Myers S, Purdy M, Heimfeld S, Halligan J, BerensonRJ (1994) Transplantation of autologous CD34
+Hematopoietic progenitorcells into breast cancer patient following high-dose chemotherapy.J ClinOncol 12:28], so they are just becoming the optimal target cell of hematopoietic stem cell transplantation with its unique biological characteristics and function.
The different epi-positions of CD34 monoclonal antibody identification CD34 antigen, to neuraminidase, the sensitivity of papoid and glucoproteinase is different, can be divided three classes according to them for these epi-positions: I class (to three kinds of enzyme sensitivities); II class (to the neuraminidase opposing, responsive to papoid and glucoproteinase); III class (to three kinds of enzyme opposings) [Greaves, MF, Titley I, Colman SM et al.CD34 cluster workshopreport.In:Schlossman S et al (eds) .Leucocyte Typing V.Oxford UniversityPress:Oxford, 1995, pp840-846.].On normal hemopoietic stem cell, the distribution of these epi-positions is different, this may with the differentiation of hemopoietic stem cell, ripe relevant with function.Therefore, just can detect the CD34 of different quantities for the monoclonal antibody of different epi-positions
+Hemopoietic stem cell [Rita Steen, Geir E.Tjqnnfjord, Gustav Gaudernack, Lorentz Brinch, TorsteinEgeland.Differences in the distribution pf CD34 epitopes on normalhaemopoietic progenitor cells and leukaemic blast cells.British Journalof Haematology 1996,94:597-605.].
Over nearly 20 years along with the appearance of immunomagnetic beads, preparing corresponding immunomagnetic beads by CD34 monoclonal antibody coupling magnetic Nano microsphere comes the sorting marrow hemopoietic stem cells to be widely used in clinical hematopoietic stem cell transplantation, since with its sorting cells have easy and simple to handle, separate rapidly fully, the cell purity advantages of higher, become some malignant hematologic disease, severe immune deficiency and tumor chemoradiotherapy hemopoietic function extremely hang down the first-selected therapy of inferior disease.
In the process of hemopoietic stem cell sorting, these antibody will be accompanied by inevitably hemopoietic stem cell and enter human body.After entering human body, the mouse resource monoclonal antibody that hybridoma technology is produced to cause human antimouse antibody immunne response human antimouse antibody response (HAMA) [Winter G, Harris WJ.Humanized antibodies.Immunol Today.1993 Jun; 14 (6): 243-6].Therefore, how to make up the anti-immunogenic antibody that reduces mouse source antibody by genetic engineering technique, the incidence that effectively reduces the HAMA reaction is the problem that those skilled in the art is eager to solve.
Summary of the invention
In order to address the above problem, the present inventor has at first obtained the mouse source CD34 monoclonal antibody for different epi-positions, and its transformation is obtained chimeric antibody, has reached to reduce its immunogenicity, reduce the incidence of HAMA reaction, improve the purpose of the security of hematopoietic stem/progenitor transplanting.
Further, the invention discloses:
1, antihuman CD 34 antibody, its weight chain variable region amino acid sequence are one of SEQ ID NO:2, SEQ IDNO:6, SEQ ID NO:10, and its light chain variable region amino acid sequence is one of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12.
2, the encode nucleotide sequence of above-mentioned antibody, wherein the nucleotides sequence of encoding heavy chain variable region is classified one of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9 as, and the nucleotides sequence of encoded light chain variable region is classified one of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11 as.
3, chimeric antihuman CD 34 antibody, its weight chain variable region amino acid sequence is one of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, its light chain variable region amino acid sequence is one of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, constant region behaviour antibody constant region.
4, chimeric antihuman CD 34 antibody, its weight chain variable region amino acid sequence are SEQ ID NO:2, and the light chain variable region amino acid sequence is SEQ ID NO:4, and the constant region aminoacid sequence is SEQID NO:16, SEQ ID NO:14.
5, chimeric antihuman CD 34 antibody c4C8, the aminoacid sequence of its heavy chain are SEQ ID NO:18, and light-chain amino acid sequence is SEQ ID NO:20.
6, chimeric antihuman CD 34 antibody, its weight chain variable region amino acid sequence are SEQ ID NO:6, and the light chain variable region amino acid sequence is SEQ ID NO:8, and the constant region aminoacid sequence is SEQID NO:16, SEQ ID NO:14.
7, chimeric antihuman CD 34 antibody c5B12, the aminoacid sequence of its heavy chain are SEQ ID NO:22, and light-chain amino acid sequence is SEQ ID NO:24.
8, chimeric antihuman CD 34 antibody, its weight chain variable region amino acid sequence are SEQ ID NO:9, and the light chain variable region amino acid sequence is SEQ ID NO:11, and the constant region aminoacid sequence is SEQID NO:16, SEQ ID NO:14.
9, chimeric antihuman CD 34 antibody c2E10, the aminoacid sequence of its heavy chain are SEQ ID NO:26, and light-chain amino acid sequence is SEQ ID NO:28.
10, the immunomagnetic beads of above-mentioned antibody preparation is used for the sorting hematopoietic progenitor.
Particularly, the invention discloses three kinds for the antibody (5B12 of the antihuman CD 34 of different epi-positions, 4C8 and 2E10) sequence, the invention also discloses the chimeric antibody (c5B12 that utilizes above-mentioned antibody to obtain, c4C8 and c2E10) and prepare these chimeric antibodies method, particularly: at first adopt 5 ' RACE technology from 4C8 hybridoma genome (IgG1, separate κ) and identify functional murine antibody variable region gene, splice mutually by certain way through gene recombination and people's antibody constant region gene, be cloned in the carrier for expression of eukaryon, make up respectively the light of chimeric antibody, the heavy chain expression carrier, then will be light, heavy chain expression carrier liposome method cotransfection Chinese hamster ovary celI, with the screening of medicaments screening of pressurizeing, carry out subclone with limiting dilution assay, with the high-expression clone serum free medium enlarged culturing that screening obtains, use at last Protein A affinity column separation and purification chimeric antibody c4C8, the same c4C8 of the structure of c5B12 and c2E10 and purification process.
The present inventor has carried out the epi-position identification experiment to above-mentioned antibody, and the epi-position qualification result shows 5B12, and 4C8 and 2E10 identify respectively the I of CD34 molecule, II and III class epi-position.No matter exo-antigen shows it is mouse source antibody 5B12 in conjunction with the determination of activity result, 4C8 and 2E10, or chimeric antibody c5B12, c4C8 and c2E10 can both be well and people's marrow leukaemia cell KG-1a specific binding of high expression level CD34.Competition suppresses experimental result and shows that three kinds of chimeric antibodies have all kept avidity and the specificity of each self-corresponding mouse source antibody, can be with them and magnetic Nano material coupling, the preparation immunomagnetic beads, come the sorting marrow hemopoietic stem cells, can effectively reduce the incidence of HAMA, improve the success ratio of clinical hematopoietic stem cell transplantation, be used for the treatment of some malignant hematologic disease and solid tumor.
Description of drawings
Fig. 1. antihuman CD 34 mouse source antibody 5B12, the qualification result of 4C8 and 2E10, the antigen-binding activity result of Fig. 1-1 antihuman CD 34 mouse source antibody, membranin and the 5B12 of Fig. 1-2 KG-1a cell, the western blotting analytical results of 4C8 and 2E10, the mouse source antibody 581 of commercial antihuman CD 34 in contrast.
Fig. 2. the epi-position qualification result of antihuman CD 34 mouse source antibody 5B12,4C8 and 2E10, the dyeing of hollow figure representative cell after enzyme is processed, solid figure representative is without the dyeing of the cell of enzyme processing.
Fig. 3. the qualification result of antihuman CD 34 chimeric antibody c5B12, c4C8 and c2E10, the antigen-binding activity result of Fig. 3-1 antihuman CD 34 chimeric antibody, Fig. 3-2, Fig. 3-3, Fig. 3-4 suppress experimental result for competition, wherein Fig. 3-2 is FITC-5B12, Fig. 3-3 is FITC-4C8, and Fig. 3-4 is FITC-2E10.
Embodiment
Following examples, experimental example only are further detailed the present invention, should not be construed as limiting the invention.
The preparation of embodiment 1. antihuman CD 34 monoclonal antibody 5B12,4C8 and 2E10-cell fusion and hybridization knurl prepares monoclonal antibody
KG-1a cell (KG-1a cell ATCC CCL-246.1) immune BALB/c mouse (available from available from the Shanghai Experimental Animal Center) with high expression level CD34, make bone-marrow-derived lymphocyte in its spleen can produce the antibody of antihuman CD 34, the splenocyte and the NS-1 (BALB/c mouse myeloma cell) that get the rear mouse of immunity merge, cultivate through the HAT selectivity, through cultivating, filter out the antihuman CD 34 positive colony, after cloning, filter out again subclone, to guarantee that antibody is to be produced by single clone cell, then collect single clone cell culture supernatant behind Protein G column purification, just obtain the monoclonal antibody 5B12 of antihuman CD 34,4C8 and 2E10.
The clone of embodiment 2. antihuman CD 34 monoclonal antibody 5B12,4C8 and 2E10 variable region gene
Extract 2 * 10 by " Trizol Reagent " test kit (U.S. Gibco BRL company product) specification sheets
6Total RNA of the hybridoma 4C8 of secretion antihuman CD 34 monoclonal antibody.Select antibody (IgG1, κ) 3 gene-specific primer GSP1, GSP2, GSP3 are designed respectively in the appropriate location of heavy chain and constant region of light chain, and wherein GSP1 distance variable district gene is used for reverse transcription reaction farthest, GSP2 is used for first run pcr amplification, and GSP3 is used for the nido amplification.Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized, and sequence is as follows: GSP1-H, 5 '-GTA GAG GTC AGACTG CAG GAC-3 '; GSP2-H, 5 '-CTC AGG GAA ATA GCC CTT GAC-3 '; GSP3-H, 5 '-AGA TCC AGG GGC CAG TGG ATA GAC-3 ' .GSP1-L, 5 '-TTG CTG TCC TGA TCAGTC CAA CT-3 '; GSP2-L, 5 '-TGT CGT TCA CTG CCA TCA ATC TT-3 '; GSP3-L, 5 '-TTG TTC AAG AAG CAC ACG ACT GA-3 '.
Take GSP1 as primer total RNA reverse transcription is become cDNA according to 5 ' RACE test kit (U.S. Gibco BRL company product) specification sheets, add poly (C) tail then for the 3 ' end of the first chain cDNA, be that primer carries out pcr amplification with GSP2 and AAP behind the tailing, 100 times of amplified production dilutions are carried out the nest-type PRC amplification take AUAP and GSP3 as primer again.Warm start, reaction conditions are all adopted in twice PCR reaction: 94 ℃ 5 minutes; 94 ℃ 45 seconds, 60 ℃ 45 seconds, 72 ℃ 1 minute 10 seconds, 30 circulations; 72 ℃ 7 minutes.The nest-type PRC product reclaims purifying purpose segment and is cloned in the pGEM-Teasy carrier after 1% agarose gel electrophoresis separates, the screening positive clone order-checking is analyzed sequencing result.Then with the correct pGEM-T/V that checks order
HBe template, design primer H has adopted AAG CTT GCC GCC ACC ATG GAT TGG CTG TGG AAC TTG and H antisense GCT AGC TGC AGA GAC AGT GAC CAG, adopt Onestep RT-PCR reaction amplification VH chain variable region gene and make its 5 ' end contain restriction enzyme sites HindIII, 3 ' end contain restriction enzyme sites NheI, reaction conditions is: 50 ℃ 30 minutes; 95 ℃ 15 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 10 minutes.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) through the agarose gel electrophoresis purifying, screening positive clone sequence verification, result prove that the sequence of this sequence and 5 ' RACE is in full accord.SEQ ID NO:1 and SEQ ID NO:2 have shown respectively nucleotide sequence and the aminoacid sequence of 4C8 variable region of heavy chain.Correct clone in this example is denoted as pGEM-T/VH.
With the correct pGEM-T/V that checks order
LBe template, design primer L has adopted AAG CTT GCC GCC ACC ATGAAG TTG CCT GTT AGG CTG and L antisense GAC AGA TGG TGC AGC CAC AGT CCG TTT GATTTC CAG CTT G, adopt Onestep RT-PCR reaction amplification VL gene and make its 5 ' end contain restriction enzyme sites HindIII, 3 ' end contains the complementary sequence of human antibody light chain constant region 5 ' end, and reaction conditions is: 94 ℃ 5 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 10 minutes.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) through the agarose gel electrophoresis purifying, screening positive clone sequence verification, result prove that the sequence of this sequence and 5 ' RACE is in full accord.SEQID NO:3 and SEQ ID NO:4 have shown respectively nucleotide sequence and the aminoacid sequence of 4C8 variable region of light chain.Correct clone in this example is denoted as pGEM-T/VL.
The hypotype of 5B12 is (IgG2a, κ), select the appropriate location of heavy chain of antibody and constant region of light chain to design respectively 3 gene-specific primer: GSP1-H, 5 '-AGC TGG GAA GGT GTG CAC ACC ACT-3 ': GSP2-H, 5 '-CAG AGT TCC AGG TCA AGG TCA-3 '; GSP3-H, 5 '-CTT GAC CAG GCATCC TAG AGT-3 '.GSP1-L, 5 '-TTG CTG TCC TGA TCA GTC CAA CT-3 '; GSP2-L, 5 '-TGT CGT TCA CTG CCA TCA ATC TT-3 '; GSP3-L, 5 '-TTG TTC AAG AAG CACACG ACT GA-3 ', the same 4C8 of the cloning process of 5B12 variable region gene, SEQ ID NO:5 and SEQ ID NO:6 have shown respectively nucleotide sequence and the aminoacid sequence of 5B12 variable region of heavy chain; SEQ ID NO:7 and SEQ IDNO:8 have shown respectively nucleotide sequence and the aminoacid sequence of 5B12 variable region of light chain.
The hypotype of 2E10 is (IgG3, κ), selects the appropriate location of heavy chain of antibody and constant region of light chain to design respectively 3 gene-specific primer: GSP1-H, 5 '-CTA CGT TGC AGA TGA CAG TC-3 '; GSP2-H, 5 '-ACA GTC ACC AAG CTG CTG AG-3 '; GSP3-H, 5 '-ATG AGA CTG TGC GCA CACC-3 ' .GSP1-L, 5 '-TTG CTG TCC TGA TCA GTC CAA CT-3 '; GSP2-L, 5 '-TGTCGT TCA CTG CCA TCA ATC TT-3 '; GSP3-L, the same 4C8 of cloning process of 5 '-TTG TTC AAG AAG CAC ACG ACTGA-3 ' .2E10 variable region gene, SEQ ID NO:9 and SEQ ID NO:10 have shown respectively nucleotide sequence and the aminoacid sequence of 2E10 variable region of heavy chain: SEQ ID NO:11 and SEQ IDNO:12 have shown respectively nucleotide sequence and the aminoacid sequence of 2E10 variable region of light chain.
Embodiment 3. people's antibody are light, the clone of weight chain constant area gene
With lymphocyte separation medium (ancient cooking vessel state biotech development company product) separating health human lymphocyte, extract total RNA with Trizol reagent (Invitrogen company product), according to document (Cell, 1980,22:197-207) and document (Nucleic Acids Research, 1982,10:4071-4079) sequence of report designs respectively primer and adopts RT-PCR reaction amplification heavy chain of antibody and constant region of light chain gene.The PCR product reclaims and is cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, confirms to have obtained correct clone after the sequence verification.SEQ ID NO:13 and SEQ ID NO:14 have shown respectively nucleotide sequence and the aminoacid sequence of CH (CH).SEQ ID NO:15 and SEQ ID NO:16 have shown respectively nucleotide sequence and the aminoacid sequence of constant region of light chain (CL).Correct clone in this example is denoted as pGEM-T/CH and pGEM-T/CL.
Embodiment 4. chimeric antibody c4C8, the structure of c5B12 and c2E10
The plasmid pGEM that above-mentioned Onestep RT-PCR order-checking is correct-T/VH HindIII and NheI double digestion, obtaining the approximately enzyme section of 430bp through the recovery of agarose gel electrophoresis purifying breaks, be connected with the plasmid pGEM-T/CH that cuts with enzyme, cut with HindIII and EcoRI enzyme after selecting correct clone, reclaim the purpose fragment through the agarose gel electrophoresis purifying, be connected with T4DNA ligase enzyme (Invitrogen company product) with the plasmid pcDNA3.1 (+) that cuts with enzyme (American I nvitrogen company product), be built into carrier for expression of eukaryon pcDNA3.1 (+) (VHCH).SEQ ID NO:17 and SEQ ID NO:18 have shown respectively nucleotide sequence and the aminoacid sequence of chimeric antibody c4C8H chain.
Adopt the Overlapping PCR clone pGEM-T/VL that above-mentioned Onestep RT-PCR order-checking is correct directly with the correct clone pGEM-T/CL fusion of constant region of light chain, reaction conditions is: 50 ℃ 30 minutes; 95 ℃ 15 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 10 minutes, obtain PCR product VLCL, its 5 ' end contains restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoRI.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) the screening positive clone order-checking through the agarose gel electrophoresis purifying.The VLCL gene that order-checking is correct downcuts from the pGEM-T carrier with HindIII and the two enzymic digestions of EcoRI, be cloned in pcDNA3.1/ZEO (+) carrier (American I nvitrogen company product), be built into carrier for expression of eukaryon pcDNA3.1/ZEO (+) (VLCL).SEQ ID NO:19 and SEQ ID NO:20 have shown respectively nucleotide sequence and the aminoacid sequence of chimeric antibody c4C8 L chain.
In 3.5cm tissue culture ware, inoculate 3 * 10
5The CHO-K1 cell, cell cultures is carried out transfection when 90%-95% merges: (plasmid pcDNA3.1 (+) is 4 μ g (VHCH) to get plasmid 10 μ g, plasmid pcDNA3.1/ZEO (+) is 6 μ g (VLCL)) and 20 μ l Lipofectamine2000 Reagent (Invitrogen company product) be dissolved in respectively 500 μ l serum-free DMEM substratum, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA liposome complex that forms is joined in the plate CO
2Incubator is cultivated after 4 hours and is added the DMEM perfect medium that 2ml contains 10% serum, places CO
2Continue in the incubator to cultivate.Cell changed the selection Screening of Media resistance clone that contains 600 μ g/ml G418 and 250 μ g/ml Zeocin after 24h was carried out in transfection.Get cells and supernatant and detect the screening high-expression clone with ELISA: goat anti-human igg (Fc) is coated in elisa plate, 4 ℃ are spent the night, seal 2h with 2%BSA-PBS in 37 ℃, add resistance clone culture supernatant to be measured or standard substance (Human myelomaIgG1, κ), 37 ℃ of incubation 2h, add HRP-goat anti-human igg (κ) and carry out association reaction, 37 ℃ of incubation 1h add TMB in 37 ℃ of effect 5min, use at last H
2SO
4Termination reaction is surveyed A
450Value.With the high-expression clone serum free medium enlarged culturing that screening obtains, use Protein A affinity column (GE company product) separation and purification chimeric antibody c4C8.Antibody purification is dialysed with PBS, at last with the uv-absorbing standard measure.
The same c4C8 of the preparation method of chimeric antibody c5B12 and c2E10 and purification process.SFQ ID NO:21 and SEQ ID NO:22 have shown respectively nucleotide sequence and the aminoacid sequence of chimeric antibody c5B12 heavy chain; SEQID NO:23 and SEQ ID NO:24 have shown respectively nucleotide sequence and the aminoacid sequence of chimeric antibody c5B12 light chain.SEQ ID NO:25 and SEQ ID NO:26 have shown respectively nucleotide sequence and the aminoacid sequence of chimeric antibody c2E10 heavy chain; SEQ ID NO:27 and SEQ ID NO:28 have shown respectively nucleotide sequence and the aminoacid sequence of chimeric antibody c2E10 light chain.
Experimental example
The evaluation of experimental example 1. monoclonal antibody 5B12,4C8 and 2E10
With dialysis labelling method traget antibody: with the carbonate buffer solution of 0.025M pH9.5 the antibody (5B12,4C8 and 2E10) of preliminary making is diluted to 1% concentration, in the dialysis tubing of packing into.With same damping fluid the solution that FITC is made into 0.1mg/ml is contained in the small beaker, dialysis tubing is immersed in the FITC solution, stir 24h 4 ℃ of lucifuges.Take out marking fluid in the dialysis tubing, cross post with Sephadex G-50, remove free fluorescein, collect fluorescence antibody FITC-5B12, FITC-4C8 and FITC-2E10 are for subsequent use.
Antigen-binding activity detects: with the KG-1a cell resuspended one-tenth 1 * 10 of 1%FCS-PBS
6Cells/ml, add respectively different dilution monoclonal antibody FITC-5B12, the monoclonal antibody FITC-581 of FITC-4C8 and FITC-2E10 and commercialization antihuman CD 34 in contrast (available from Britain Serotec company), place 4 ℃ to hatch 60min, with analyzing with FCM behind the 1%FCS-PBS Shen cell 2 times, calculate the average fluorescent strength of stained positive cell and the relative affinity of each antibody relatively.Corresponding antibody concentration when the relative affinity percentage ratio that is defined as the stained positive cell reaches 50% here.
Experimental result is seen Fig. 1-1, compares with 581 contrasts (Control) of antihuman CD 34 mouse source antibody, and 5B12,4C8 and 210 can both be combined with the KG-1a cell-specific well.
Western blot analyzes: extracting KG-1a epicyte protein, separate with the SDS electrophoresis, and then electricity forwards on the pvdf membrane.Pvdf membrane is at first with the PBS sealing that contains 5% skim-milk, then with three kinds of monoclonal antibody 5B12,4C8 and 2E10 are hatched 60min at 37 ℃, after PBS washes, add again the HRP-goat anti-mouse igg and hatched 1 hour in 37 ℃, develop the color to show immunoreactive band by DAB after washing cell.
Experimental result is seen Fig. 1-2, the same with antihuman CD 34 mouse source antibody 581 contrast (Control) (Britain Serotec company products), 5B12,4C8 and 2E10 can both with the approximately specific band reaction of 110kDa of a molecular weight, 5B12 namely, 4C8 and 2E10 can specific recognition CD34 molecules.
Experimental example 2. epi-positions are identified
The epi-position of CD34 molecule can be divided three classes according to different to the sensitivity of enzyme (neuraminidase, papoid and glucoproteinase):
The I class: to Neu, Chy, Gly are all responsive, such as My10, and BI-3C5, ICH3,12.8 etc.
The II class: to Chy, Gly is responsive, to the Neu opposing, such as QBFnd10
The III class: to Neu, Chy, Gly all resists, such as HPCA-2,8G12, TUK3,115.2 etc.
According to this conclusion, we are to 5B12, and the molecule epi-position of 4C8 and 2E10 detects.At first with 1 * 10
6People's acute myeloid leukemia cells in children KG-1a is resuspended in 100 μ lPBS, then respectively with neuraminidase (the VCN) (Sigma of 0.1U/ml, MO, USA), papoid (the Calbiochem of 100U/ml, Cambridge, U.K.) or the pasteurella haemolytica glucoproteinase hatch 30min at 37 ℃, wash cell 2 times with 5%FCS-PBS, to end the reaction of enzyme, three kinds of cells after enzyme is processed add respectively 5B12,4C8,2E10 places 4 ℃ of incubation 45min, uses 1%FCS-PBS Shen cell 2 times, add again FITC-goat anti-mouseIgG (H+L) (U.S. Zymed company) and hatch 45min in 4 ℃, wash behind the cell and analyze with FCM, undressed cell in contrast, to detect losing of enzyme susceptibility epi-position.
Experimental result is seen Fig. 2, and 5B12 is responsive to three fermentoids, so its identification is I class epi-position; 4C8 resists neuraminidase, and is responsive to papoid and glucoproteinase, so its identification is II class epi-position; 2E10 resists three fermentoids, so its identification is III class epi-position.
The evaluation of experimental example 3. chimeric antibody c5B12, c4C8 and c2E10
Antigen-binding activity detects: with the KG-1a cell resuspended one-tenth 1 * 10 of 1%FCS-PBS
6Cells/ml adds respectively different dilution monoclonal antibody c5B12, and c4C8 and c2E10 place 4 ℃ to hatch 60min, wash cell 2 times with 1%FCS-PBS, adds FITC goat anti-human igg (H+L) again and hatches 60min in 4 ℃, analyzes with FCM after washing cell.Each sample is established three multiple pipes.
Experimental result is seen Fig. 3-1, and c5B12, c4C8 and c2E10 can both be combined with the KG-1a cell-specific well.
Competition suppresses experiment: join target cell KG-1a (in 1 * 106/ml) after the unmarked antibody purification of fluorescent-labeled antibody FITC-5B12, the FITC-4C8 of fixing sub-saturated concentration and FITC-2E10 and serial dilution is mixed respectively, hatch 60min for 4 ℃, 1%FCS-PBS washes cell 2 times, and flow cytometer detects and use the Cellquest software analysis.(preparation is referring to Li BH for the monoclonal antibody c12F6 of chimeric anti-CD3, Wang H, Dai JX, Ji JJ, Qian WZ, Zhang DP, Hou S, Guo YJ.Constructionand characterization of a humanized anti-human CD3 monoclonal antibody12F6 with effective immunoregulation functions.Immunology.2005,116 (4): 487-98) in contrast.Each concentration of competition antibody is established 3 multiple pipes, calculation of half inhibitory concentration IC50 value.
Experimental result is seen Fig. 3-2,3-3 and 3-4, chimeric antibody c5B12, c4C8 and c2E10 can both with each self-corresponding mouse source antibody competition, illustrate that three kinds of chimeric antibodies have all kept avidity and the specificity of each self-corresponding mouse source antibody, and their IC
50Value similar (seeing Table 1).
Table 1
| Antibody types | IC 50(μg/ml) a | SD | n |
| m5B12 c5B12 m4C8 c4C8 m2E10 c2E10 | 1.306 1.238 1.789 1.848 2.060 1.931 | 0.044 0.056 0.048 0.037 0.082 0.085 | 3 3 3 3 3 3 |
SEQUENCE LISTING
Claims (6)
1. antihuman CD 34 antibody, its weight chain variable region amino acid sequence is SEQ ID NO:2, the light chain variable region amino acid sequence is SEQ ID NO:4.
2. the nucleotide sequence of coding claim 1 described antibody, wherein the nucleotides sequence of encoding heavy chain variable region is classified SEQ ID NO:1 as, and the nucleotides sequence of encoded light chain variable region is classified SEQ IDNO:3 as.
3. chimeric antihuman CD 34 antibody, its weight chain variable region amino acid sequence is SEQ ID NO:2, the light chain variable region amino acid sequence is SEQ ID NO:4, constant region behaviour antibody constant region.
4. chimeric antihuman CD 34 antibody claimed in claim 3, its weight chain variable region amino acid sequence is SEQID NO:2, the light chain variable region amino acid sequence is SEQ ID NO:4, and the CH aminoacid sequence is SEQ ID NO:14, and the constant region of light chain aminoacid sequence is SEQID NO:16.
5. chimeric antihuman CD 34 antibody claimed in claim 4, its heavy chain amino acid sequence is SEQIDNO:18, light-chain amino acid sequence is SEQ ID NO:20.
6. the immunomagnetic beads of the arbitrary described antibody preparation of claim 1-5 is used for the sorting hematopoietic progenitor.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710094456 CN101245108B (en) | 2007-12-13 | 2007-12-13 | Antihuman CD34 antibody, producing process and uses thereof |
| CA2708407A CA2708407A1 (en) | 2007-12-13 | 2008-12-03 | Humanized anti-human cd34 antibody, the preparation method and uses thereof |
| EP08865354A EP2233501B1 (en) | 2007-12-13 | 2008-12-03 | Humanized anti-cd34 monoclonal antibody, the preparation and uses thereof |
| PCT/CN2008/001963 WO2009079922A1 (en) | 2007-12-13 | 2008-12-03 | Humanized anti-cd34 monoclonal antibody, the preparation and uses thereof |
| US12/746,990 US20100311955A1 (en) | 2007-12-13 | 2008-12-03 | Humanized anti-human cd34 antibody, the preparation method and uses thereof |
| JP2010537235A JP2011505810A (en) | 2007-12-13 | 2008-12-03 | Humanized anti-human CD34 antibody, its preparation method and use |
| BRPI0821211-2A BRPI0821211A2 (en) | 2007-12-13 | 2008-12-03 | Humanized cd34 anti-human antibody, the method of preparation and uses thereof. |
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| CN 200710094456 CN101245108B (en) | 2007-12-13 | 2007-12-13 | Antihuman CD34 antibody, producing process and uses thereof |
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| JP2011505810A (en) * | 2007-12-13 | 2011-03-03 | シャンハイ グオジアン バイオ−テック インスティチュート | Humanized anti-human CD34 antibody, its preparation method and use |
| CN102127148B (en) * | 2010-01-14 | 2013-04-03 | 中国农业科学院北京畜牧兽医研究所 | Polypeptide in cluster-of-differentiation part on surface of vascular endothelial cell and use thereof |
| CN105254717B (en) * | 2015-08-18 | 2018-08-24 | 中山大学 | The polypeptide combined with CD34 molecular specificities and its application |
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Non-Patent Citations (3)
| Title |
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| 任素萍 等.CD34分子及其单克隆抗体应用.《中国输血杂志》.2003,第16卷(第5期),350-354. * |
| 佘鸣 等.抗CD34 单克隆抗体HI273 的制备和鉴定.《细胞与分子免疫学杂志》.2000,第16卷(第1期),79-80. * |
| 孙玉英 等.人CD34 抗原胞外区cDNA的克隆、真核表达载体的构建.《解放军医学杂志》.2003,第28卷(第12期),1090-1095. * |
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