CN102952191B - Fully human CD33 single-chain antibody ZJL101 and applications thereof - Google Patents
Fully human CD33 single-chain antibody ZJL101 and applications thereof Download PDFInfo
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Abstract
The invention provides a fully human CD33 single-chain antibody ZJL101 and applications thereof. The fully human CD33 single-chain antibody ZJL101 is prepared through the following steps: through a phage display technology and by taking human CD33-ECD protein as target antigen, repeatedly screening from a high-capacity fully human leukemia phage antibody library, carrying out induction expression of single-chain antibody ZJL101 from coliform bacteria, and conducting affinity purification through Ni column. The obtained fully human CD33 single-chain antibody ZJL101 can specifically inhibit the growth of CD33 positive leukemia cell lines HL-60 and Kasumi-1, after being transformed into other antibody forms through genetic engineering, the single-chain antibody and the variable region sequence thereof can be used as therapeutic drugs or drug carriers for targeted therapy of CD33 positive leukemia. The single-chain antibody has the advantages of being simple in structure, low in immunogenicity, strong in specificity, small in relative molecular weight, strong in penetrability and the like, thereby being a desirable targeted drug and drug carrier.
Description
Technical field
The invention belongs to genetically engineered, relate to screening, evaluation and the prokaryotic expression of total man source anti-CD 33 single-chain antibody, and application in the positive leukemia targeted drug preparation of CD33.
Background technology
Phage displaying antibody technology can be prepared the antibody in total man source, be by the gene clone of protein molecular or peptide section in the genomic dna of filobactivirus, form fusion rotein with the coat protein of phage, thereby make heterologous molecule be showed in phage surface.Principal feature is that the genotype of specific molecule and expression type are appeared in a phage particle simultaneously, can obtain the sequence information of particular display albumen by DNA sequencing.
Phage antibody library technique has been simulated the differentiation and maturation process of mankind B cell, first by molecular biology method, the heavy chain of human antibodies and chain variable region gene are coupled together and form single-chain antibody (Single-chain variable fragment, scFv), insert on phage vector, make single-chain antibody scFv be illustrated in phage surface, go out specific phage antibody with specific antigen selection again, and can express a large amount of single-chain antibodies with escherichia coli expression.
Phage can be expressed antibodies fragment (Fragment antigen binding, Fab), single-chain antibody (scFv), double-chain antibody (Diabody) and bi-specific antibody (Bispecific scFv, BsAb) etc.Wherein single-chain antibody (scFv) is to report to be at present made up of maximum a kind of small molecular antibodies the variable region Fv section of antibody recognition antigen.Fv is by variable region of heavy chain V
hconnect and compose scFv with variable region of light chain VL by connection peptides (linker).Added linker is very important, must not affect the conformation of Fv, the sequence (GGGGS) that the at present the most frequently used repetition being made up of four glycine and Serine is 3 times
3.By scFv gene clone between filobactivirus carrier pIII gene leader and pIII gene, be imported into bacterial film gap with the form of fusion rotein, and be assembled into scFv, and adding after helper phage M13K07, scFv expresses at phage surface with the form of fusion rotein.The feature of pCANTAB-5E carrier is, after scFv gene, contain the sequence of one section of coding Tag tail peptide (E-Tag), after E-Tag, there is an amber (Amber) termination codon, between scFv gene and pIII gene, in inhibition bacterium TG1, it is 20% effective that this amber codon only has, and forms scFv-pIII fusion rotein so can be readed in protein translation process; And in non-inhibity bacterial strain, as HB2151, this terminator is identified, scFv gene stops before pIII gene simultaneously, forms independently antibody protein, is stranded in cytolemma gap, and is released in nutrient solution after long-time cultivation, forms solubility expression.In addition, by the method for molecular cloning, also scFv transgenosis can be entered to pET expression system, and in intestinal bacteria, realize high expression level and obtain single-chain antibody.
The advantage such as that single-chain antibody has is simple in structure, relative molecular weight is little, penetrance is strong, immunogenicity is low is a kind of desirable carrier.Build human antibody storehouse object and be find one can with the single-chain antibody scFv of specific leukemia-associated antigen (as CD33 etc.) specific binding, the single-chain antibody itself of this class specially recognizing tumor cells or after other antibody formation of genetic engineering modified one-tenth, can be used as antitumor drug and develops; Next can be used as pharmaceutical carrier, as coupling drug, toxin or radio isotope, carries out leukemic targeted therapy.
Summary of the invention
The object of this invention is to provide a kind of total man source anti-CD 33 single-chain antibody ZJL101, have the advantages such as simple in structure, relative molecular weight is little, penetrance is strong, immunogenicity is low, is a kind of desirable medicament transport carrier.SEQ ID NO 1 is the DNA sequence dna of this single-chain antibody:
atggcccagg tccagcttgt gcagtctggg actgtagtga agaagcctgg gtcctcggtg
aaggtctcct gcaagacttc tggaggcacc ctccccaacc atgccatcaa ctgggtgcga
caggcctctg gacaagggct tgagtggatg ggatggatga gccctaacac tggtgacaca
ggctatgcac ggaagttcca gggcagagtc accatgacca gggacacctc cataagcaca
gcctacatgg aactgagcag cctgagatct gacgacacgg ccatatatta ctgtgcgacg
ggcgaccccc gggttggtca ttcgtggggc cagggcaccc tggtcactgt ctcctcaggt
ggtggtggtt ctggcggcgg cggctccggt ggtggtggat ctgacatcca gatgacccag
tctccaccct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt
caaaccattg gcagtcattt aaattggtat cagcagaaac cagggaaagc ccctaaactc
ctgatctatg gtgcatccag tttgcaaagt ggggtccccg caaggttcac tggcagtgga
tttgggacag atttcactct caccatcagc agtctacaag ttgaagactt ggcaacttac
tcctgtcaac agacttacaa taccccccgg acgttcggcc aagggaccaa ggtggaaatc
aaa
Total length is 723 bp;
Met Ala Gln Val Gln Leu Val Gln Ser Gly Thr Val Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Leu Pro Asn His Ala Ile Asn Trp Val Arg Gln Ala Ser Gly Gln Gly Leu Glu Trp Met Gly Trp Met Ser Pro Asn Thr Gly Asp Thr Gly Tyr Ala Arg Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Thr Gly Asp Pro Arg Val Gly His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly Ser His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Thr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ala Arg Phe Thr Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Val Glu Asp Leu Ala Thr Tyr Ser Cys Gln Gln Thr Tyr Asn Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Its molecular weight is 25212, contains complete antibody heavy chain variable region V
hwith variable region of light chain V
l, middle connection peptides is (Gly Gly Gly Gly Ser)
3;
Variable region of heavy chain (the V of described single-chain antibody
h) have SEQ ID NO 3 aminoacid sequence:
Met Ala Gln Val Gln Leu Val Gln Ser Gly Thr Val Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Leu Pro Asn His Ala Ile Asn Trp Val Arg Gln Ala Ser Gly Gln Gly Leu Glu Trp Met Gly Trp Met Ser Pro Asn Thr Gly Asp Thr Gly Tyr Ala Arg Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Thr Gly Asp Pro Arg Val Gly His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Containing 119 amino acid, its molecular weight is 12764;
Variable region of light chain (the V of described single-chain antibody
l) have SEQ ID NO 4 aminoacid sequence:
Asp Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly Ser His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Thr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ala Arg Phe Thr Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Val Glu Asp Leu Ala Thr Tyr Ser Cys Gln Gln Thr Tyr Asn Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Containing 107 amino acid, its molecular weight is 11519.
Another object of the present invention is to provide the application of total man source anti-CD 33 single-chain antibody ZJL101 in preparation leukemia targeted drug.Single-chain antibody ZJL101 of the present invention can, to the leukemia cell of the CD33 positive, as marrow series leukemia HL-60 and Kasumi-1 cell, produce growth-inhibiting effect.
Advantage of the present invention is: the full human single chain variable fragments antibody ZJL101 that (1) provides is simple in structure, is antibody heavy chain variable region V
hwith variable region of light chain V
lby connection peptides (GGGGS)
3be formed by connecting, MW is 25.212 kD, and contains complete antigen-binding site; (2) single-chain antibody ZJL101 of the present invention can, to the leukemia cell of the CD33 positive, as marrow series leukemia HL-60 and Kasumi-1 cell, produce growth-inhibiting effect; (3) single-chain antibody ZJL101 molecular weight of the present invention is little, penetrance is strong, immunogenicity is low, can be used as medicine or pharmaceutical carrier transportation medicine, isotropic substance or lps molecule, can treat the leukemia of the CD33 positive.
Accompanying drawing explanation
Fig. 1 is the combination activity that ELISA detects phage single-chain antibody.
Fig. 2 A is phage single-chain antibody ZJL101 structure V
hfragment sequence analysis chart.
Fig. 2 B is phage single-chain antibody ZJL101 structure V
lfragment sequence analysis chart.
Fig. 3 is the Specific Inhibitory Effect of positive bacteriophage (showing single-chain antibody ZJL101) to leukemia cell line.
Fig. 4 is the expression in intestinal bacteria HB2151 with SDS-PAGE electrophoretic analysis single-chain antibody ZJL101.
Fig. 5 is the Western Blot analysis chart of the single-chain antibody ZJL101 that expresses in intestinal bacteria HB2151.
Fig. 6 is expression and the purge process in e. coli bl21 with SDS-PAGE electrophoretic analysis single-chain antibody ZJL101.
Fig. 7 is that single-chain antibody ZJL101 measures leukemia cell's Specific Inhibitory Effect.
Embodiment
The present invention with the following Examples and accompanying drawing be further described.
Embodiment 1: the screening of phage antibody library
Experimental technique: antigen selection total man source, CD33 extracellular region (CD33-ECD) the leukemia antibody library that uses DNA recombinant expression.Use Na2CO3/NaHCO
3by after antigen diluent, add 96 hole elisa plates, 4 ℃ of coated spending the night.Next day, confining liquid seals 1 h, adds phage antibody library, hatches 2 h for 37 ℃.TBS washing (first round 5 times, second takes turns 10 times, third and fourth is taken turns 10 times).With glycine-hydrochloric acid (pH 2.2) wash-out and collect phage, Tris-HCl is neutralized to pH 7.0, infects logarithmic phase
e. colitG1,37 ℃ leave standstill 20 min, get 10 mL and survey titre.All the other go to 20 mL 2 × YT-A-G, and 37 ℃ of joltings are cultured to OD
600nmreach 0.5, then add helper phage, 37 ℃ of jolting 1 h, centrifugal, precipitation is resuspended in 200 mL 2 × YT-AK, and 30 ℃ of joltings are spent the night, and next day, collect phage.
Experimental result: phage antibody library, after four-wheel screening, obtains the enrichment of 105 times, in table 1.
Presentation of results: the efficiency (2.0 × 10 of fourth round screening
-6) be the efficiency (1.9 × 10 of first round screening
-8) 105 times, show to have obtained effective enrichment with the phage of target antigen CD33-ECD specific combination.
Embodiment 2: antibody library diversity analysis
Experimental technique: 200 mono-clonals that random choose screens send Nanjing Genscript Biotechnology Co., Ltd., the DNA sequence dna that inserts region checks order.The mono-clonal DNA sequence dna of energy correct coding aminoacid sequence is translated into amino acid, carry out sequence alignment analysis (http://www.ncbi.nlm.nih.gov/igblast/) through ClustaIX software.
Experimental result: the sequencing results shows, there is the DNA in the monoclonal insertion of 8 strain phage region can correct coding amino acid, aminoacid sequence comparison result is in table 2, and the DNA sequence dna that wherein inserts regions with No. 4 two strain phages No. 3 is identical, and its sequence is SEQ ID NO 1.
Presentation of results: in the 8 strain mono-clonal phages that filter out, insert the DNA sequence dna identical (sequence is SEQ ID NO 1) in region except No. 3 with No. 4 two strain phages, all the other all have good diversity.
Embodiment 3:ELISA identifies the antigen-binding activity of single-chain antibody
Experimental technique: select the phage mono-clonal obtaining after screening four-wheel, send order-checking.By can read over the mono-clonal of sequence containing order-checking, prepare mono-clonal recombinant phage.Dilute antigen (CD33-ECD) with PBS, join in 96 well culture plates, 4 ℃ of coated spending the night.After taking-up, with 37 ℃ of sealing 1h of confining liquid, mono-clonal recombinant phage to be measured is added in 96 orifice plates to 37 ℃ of incubation 2 h.Using PBS as negative control, add HRP/anti-M13 antibody, 37 ℃ of incubation 1 h, add substrate TMB-H
2o
2colour developing, microplate reader is measured OD
450nmvalue.
Experimental result: do ELSIA checking with mono-clonal phage, result is referring to Fig. 1, wherein 9 and 10, be respectively phage M13 negative control and PBS control group, OD
450nmvalue <0.2; The complete phase of sequence of No. 3 and No. 4 phages, OD
450nmvalue is about 1.6; The OD of No. 1, No. 2 and No. 5 phage
450nmvalue is between 0.8-1.2; The OD of No. 7 phages
450nmbe 0.5, No. 6 and No. 8 OD
450nmvalue <0.2.
Presentation of results: No. 3 and No. 4 phage antigens are in conjunction with being strong positive, No. 1, No. 2 and No. 5 phages positive, No. 7 phages are the weak positive, No. 6 and No. 8 phages negative.
Embodiment 4: positive bacteriophage single-chain antibody determined dna sequence and structural analysis
Experimental technique: No. 3 and No. 4 phages insert regions (single-chain antibody) and carry out DNA sequence analysis; According to DNA sequence analysis result, get coding region sequence, input VBASE2 database (http://www.vbase2.org/), analyzes and obtains single-chain antibody structural analysis figure.
Experimental result: No. 3 identical with the DNA sequence dna in No. 4 phage insertion regions (single-chain antibody), and its sequence is SEQ ID NO 1, and analytical results is referring to Fig. 2 A and Fig. 2 B, and wherein Fig. 2 A is V
hfragment sequence (comprising linker), Fig. 2 B are V
lfragment sequence figure, in figure, visible heavy chain and light chain have respectively FR1, FR2, FR3, FR4, CDR1, CDR2 and CDR3 structural domain.
Presentation of results: this positive strain single-chain antibody has complete antigen binding domain is correct single-chain antibody structure, called after ZJL101.
Embodiment 5: the single-chain antibody ZJL101 of phage display measures leukemia cell's Specific Inhibitory Effect
Experimental technique: select the marrow series leukemia cell strain HL-60 of the CD33 positive and the leukemia cell Jurkat strain of Kasumi-1 and CD33 feminine gender, at 37 ℃ and 5%CO
2condition under be cultured to logarithmic phase; By cell cultures suspension, centrifugal, abandon supernatant, collecting cell; Use cell culture fluid suspension cell, and with 5.0 × 10
3-1.0 × 10
4the density in cells/ hole is inoculated in 96 porocyte culture plates, at 37 ℃ and 5% CO
2condition under cultivate 24 h; Above-mentioned ELISA is identified to No. 3 positive phage amplifications, and obtaining phage concentration is 7.5x 10
11cfu/mL; Four concentration gradients are set, add respectively phage to each cell hole, make phage final concentration be respectively 0,7.5x10
7cfu/mL, 7.5x10
8cfu/mL, 7.5x10
9cfu/mL, 7.5x10
10cfu/mL; Then, cell is hatched to 72 h, 4 multiple holes are established in every hole, do negative control with the M13 phage of PBS dilution; Add cck-8 solution, hatch 1 h for 37 ℃; Under 470 nm wavelength, measure absorbance, and calculate the growth inhibition ratio of cell under phage (displaying the has single-chain antibody ZJL101 sequence) effect of different concns.
Experimental result: referring to Fig. 3, the single-chain antibody ZJL101 of this phage display, in each dosage group, all can produce restraining effect to the Kasumi-1 of the CD33 positive and HL-60 cell, wherein the restraining effect of Kasumi-1 cell is reached as high as to 50%, and can reach 30% to HL-60 cell.Do negative control with Jurkat cell, only have slight restraining effect, and be not dose-dependence, have significant difference (P< 0.05) with above-mentioned two kinds of marrow series leukemia cells.
Presentation of results: the single-chain antibody ZJL101 of this phage display can produce obvious growth-inhibiting effect to the HL-60 of the CD33 positive and Kasumi-1 cell, and to the Jurkat cell of CD33 feminine gender without obvious restraining effect.
Embodiment 6: the solubility expression of single-chain antibody ZJL101 in intestinal bacteria HB2151
Experimental technique: the recombinant phage of getting positive colony infects the intestinal bacteria HB215l of logarithmic phase, is inoculated on SOBAG-pyrimidine acid plate 30 ℃ of overnight incubation; Clone of picking, in 2 × YT-AG, 30 ℃ of overnight incubation; The bacterium liquid 1:10 that will spend the night next day adds in 50 mL 2 × YT-AG, cultivate l h for 30 ℃, centrifugal, supernatant discarded, the resuspended precipitation of 50 mL 2 × YT-Amp-IPTG, 30 ℃, cultivate after 6 ~ 7 h, centrifugal, precipitation is resuspended containing the 25 mM Tris-HCl (pH 7.3) of 20% sucrose with 2.5 mL, fully suspend, ice bath 30 min; Again centrifugal collection thalline, adds 5 mL distilled waters resuspended, ice bath 30 min, centrifugal after, get supernatant and be periplasm protein and carry out 12% conventional SDS-PAGE electrophoretic analysis.
Experimental result: through IPTG induction, SDS-PAGE electrophoresis detection, referring to Fig. 4: M, is molecular weight standard; 1, for not inducing full cell; 2, be induction group periplasm protein; 3, for full cell is organized in induction, 4, be culture supernatant.No. 2 and No. 3 are visible molecular weight 27 kD left and right object bands (band His label) all, in No. 1 and No. 4 without this band.
Presentation of results: through IPTG induction, single-chain antibody ZJL101 can be at bacterium periplasm protein and full cells, and has no expression in culture supernatant.
Embodiment 7:Western blot identifies single-chain antibody ZJL101
Experimental technique: the protein sample of above-mentioned expression, carries out, after SDS-PAGE electrophoresis, taking off the acrylamide gel after electrophoresis transferring film, 100V constant voltage, transferring film 90 min; Take off pvdf membrane, confining liquid (TBST-5% skim-milk) sealing pvdf membrane, slowly shakes 1 h; Dilution primary antibodie Rabbit Anti-6 × His tag antibody, takes out pvdf membrane in confining liquid, slowly shakes 1 h, washs 5 min × 5 time with TBST again; Confining liquid dilutes two anti-Goat anti-rabbit IgG-FIFC conjugate, slowly shakes 1h, washs 5 min × 5 time with TBST; Chemoluminescence ECL colour developing, Taking Pictures recording result.
Experimental result: Western blot detected result is referring to Fig. 5, No. 1 is induction group cell periplasm protein, is for No. 2 culture supernatant, organizes full cell for induction No. 3.In the cell periplasm protein of visible induction group, full enchylema and culture supernatant, all there is the band (band His label) of 27 kD.
Presentation of results: through IPTG induction, single-chain antibody ZJL101 has expressed in cell periplasm protein and full enchylema, also has a small amount of expression in culture supernatant.
Embodiment 8: expression and the purifying of single-chain antibody ZJL101 in e. coli bl21
Experimental technique: get ELISA and detect positive No. 3 the highest phages and carry out pcr amplification target DNA fragment, enter expression vector pET30a (+) with Xho I and Nde I double digestion rear clone, express in e. coli bl21.Add IPTG to final concentration be 0.5 mM, abduction delivering 5 h under 30 ℃ of conditions, after ultrasonication, centrifugal through 12000 rpm/10 min, getting supernatant crosses Ni post and carries out purifying, with the level pad wash-out target protein containing 100 mM imidazoles, with 12% SDS-PAGE electrophoretic analysis expression and purified product.
Experimental result: electrophoretic analysis result is referring to Fig. 6, and M, is molecular weight standard; 1, be contrast before induction; 2, be full bacterium after induction; Being for No. 3 ultrasonic rear supernatant, is for 4, No. 5 unconjugated foreign protein after sample upper prop, and No. 6 is the target protein after purifying, is single band, MW 27 kD(band His labels), consistent with expection.
Presentation of results: single-chain antibody ZJL101 can express in intestinal bacteria, can carry out purifying with Ni post, can prepare highly purified single-chain antibody ZJL101.
Embodiment 9: single-chain antibody ZJL101 measures leukemia cell's Specific Inhibitory Effect
Experimental technique: select the marrow series leukemia cell strain HL-60 of the CD33 positive and the leukemia cell Jurkat of Kasumi-1 and CD33 feminine gender, at 37 ℃ and 5%CO
2condition under be cultured to logarithmic phase; By centrifugal cell cultures suspension, abandon supernatant, collecting cell; Use cell culture fluid re-suspended cell, and with 5.0 × 10
3-1.0 × 10
4the density in cells/ hole is inoculated in 96 porocyte culture plates, at 37 ℃ and 5% CO
2condition under cultivate 24 h; Add the single-chain antibody ZJL101 of different concns, hatch 72 h, 3 multiple holes are established in every hole, do negative control with the cell that only adds PBS; Add cck-8 solution, hatch 1 h for 37 ℃; Under 470 nm wavelength, measure absorbance, and calculate the growth inhibition ratio of cell under the single-chain antibody ZJL101 of different concns effect.
Experimental result: referring to Fig. 7, this single-chain antibody ZJL101 can produce restraining effect to HL-60 and Kasumi-1 cell, its middle and high concentration single-chain antibody (0.25 mg/mL) reaches 50.15% to the restraining effect of Kasumi-1 cell, and HL-60 cell is reached to 30.52%; Middle concentration single-chain antibody (0.12 mg/mL) reaches 33.99% to the restraining effect of Kasumi-1 cell, and HL-60 cell is reached to 20.35%; Lower concentration single-chain antibody (0.06 mg/mL) reaches 23.39% to the restraining effect of Kasumi-1 cell, and HL-60 cell is reached to 16.55%; All do not observe the restraining effect to Jurkat cell (negative control) in high, middle concentration group, low dose group has been observed slight restraining effect, belongs to normal experimental error.
Presentation of results: this monoclonal antibody ZJL101 can produce obvious growth-inhibiting effect to the HL-60 of the CD33 positive and Kasumi-1 cell, and Jurkat cell unrestraint effect to CD33 feminine gender; Show that its restraining effect has specificity.
In sum, we screen and have obtained the special phage single-chain antibody ZJL101 of being combined with CD33 molecule of a kind of energy from human antibody storehouse, through determined dna sequence, elisa assay and its structure of electrophoresis assay certificate are correct, and can prepare through escherichia coli expression, in the leukemia cell of the CD33 positive of cultivating, can cause special restraining effect, this single-chain antibody is separately or after the antibody formation that is transformed into other through methods such as genetically engineereds, can be used as antitumor drug or pharmaceutical carrier, as coupling drug, toxin or radio isotope, carry out the positive leukemic targeted therapy of CD33.
Without further elaborating, believe employing disclosed content above, those skilled in the art can apply the present invention to greatest extent.Therefore, preferred specific embodiments is above interpreted as only illustrating, but not limits the scope of the invention by any way.
<110> Zhejiang University
<120> total man source anti-CD 33 single-chain antibody ZJL101 and application thereof
<160> 4
<210> 1
<211> 723
<212> DNA
<213> artificial sequence
<220>
<223> is containing the DNA sequence dna of the anti-CD 33 single-chain antibody ZJL101 of connection peptides
<400>1
atggcccagg tccagcttgt gcagtctggg actgtagtga agaagcctgg gtcctcggtg 60
aaggtctcct gcaagacttc tggaggcacc ctccccaacc atgccatcaa ctgggtgcga 120
caggcctctg gacaagggct tgagtggatg ggatggatga gccctaacac tggtgacaca 180
ggctatgcac ggaagttcca gggcagagtc accatgacca gggacacctc cataagcaca 240
gcctacatgg aactgagcag cctgagatct gacgacacgg ccatatatta ctgtgcgacg 300
ggcgaccccc gggttggtca ttcgtggggc cagggcaccc tggtcactgt ctcctcaggt 360
ggtggtggtt ctggcggcgg cggctccggt ggtggtggat ctgacatcca gatgacccag 420
tctccaccct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt 480
caaaccattg gcagtcattt aaattggtat cagcagaaac cagggaaagc ccctaaactc 540
ctgatctatg gtgcatccag tttgcaaagt ggggtccccg caaggttcac tggcagtgga 600
tttgggacag atttcactct caccatcagc agtctacaag ttgaagactt ggcaacttac 660
tcctgtcaac agacttacaa taccccccgg acgttcggcc aagggaccaa ggtggaaatc 720
aaa 723
<210> 2
<211> 241
<212> PRT
<213> artificial sequence
<223> is containing the polypeptide chain sequence of the anti-CD 33 single-chain antibody ZJL101 of connection peptides
<400>2
Met Ala Gln Val Gln Leu Val Gln Ser Gly Thr Val Val Lys Lys
1 5 10 15
Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr
20 25 30
Leu Pro Asn His Ala Ile Asn Trp Val Arg Gln Ala Ser Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Trp Met Ser Pro Asn Thr Gly Asp Thr
50 55 60
Gly Tyr Ala Arg Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp
65 70 75
Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser
80 85 90
Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Thr Gly Asp Pro Arg Val
95 100 105
Gly His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly
110 115 120
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
125 130 135
Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly
140 145 150
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly Ser
155 160 165
His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
170 175 180
Leu Ile Thr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ala Arg
185 190 195
Phe Thr Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile Ser
200 205 210
Ser Leu Gln Val Glu Asp Leu Ala Thr Tyr Ser Cys Gln Gln Thr
215 220 225
Tyr Asn Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
230 235 240
Lys
241
<210> 3
<211> 119
<212> PRT
<213> artificial sequence
The polypeptide chain sequence of the heavy chain of <223> anti-CD 33 single-chain antibody ZJL101
<400>3
Met Ala Gln Val Gln Leu Val Gln Ser Gly Thr Val Val Lys Lys
1 5 10 15
Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr
20 25 30
Leu Pro Asn His Ala Ile Asn Trp Val Arg Gln Ala Ser Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Trp Met Ser Pro Asn Thr Gly Asp Thr
50 55 60
Gly Tyr Ala Arg Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp
65 70 75
Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser
80 85 90
Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Thr Gly Asp Pro Arg Val
95 100 105
Gly His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
110 115 119
<210> 4
<211> 107
<212> PRT
<213> artificial sequence
The polypeptide chain sequence of the light chain of <223> anti-CD 33 single-chain antibody ZJL101
<400>4
Asp Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly
20 25 30
Ser His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Thr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ala
50 55 60
Arg Phe Thr Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Gln Val Glu Asp Leu Ala Thr Tyr Ser Cys Gln Gln
80 85 90
Thr Tyr Asn Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu
95 100 105
Ile Lys
107
Claims (2)
1. a total man source anti-CD 33 single-chain antibody ZJL101, SEQ ID NO 1 is the DNA sequence dna of this single-chain antibody, and SEQ ID NO 2 is aminoacid sequences of this single-chain antibody, and the molecular weight of this single-chain antibody is 25212, contains complete antibody heavy chain variable region V
hwith variable region of light chain V
l, middle connection peptides is (Gly Gly Gly Gly Ser)
3, its variable region of heavy chain V
haminoacid sequence be SEQ ID NO 3, containing 119 amino acid, its molecular weight is 12764, variable region of light chain V
laminoacid sequence be SEQ ID NO 4, containing 107 amino acid, its molecular weight is 11519.
2. the application of a kind of total man according to claim 1 source anti-CD 33 single-chain antibody ZJL101 in preparation leukemia targeted drug, it is characterized in that, suppress the application in the HL-60 of the CD33 positive and Kasumi-1 leukemia cell's target therapeutic agent in preparation.
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