CN103833853B - Novel death receptor excitability multivalent antibody and its application in preparation of anti-tumor drugs - Google Patents
Novel death receptor excitability multivalent antibody and its application in preparation of anti-tumor drugs Download PDFInfo
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Abstract
The present invention provides the receptor DR(death receptor of a kind of novel human tumor's necrosin relative death inducing ligand) antibody of extracellular region.Such death receptor excitability multivalent antibody can identify the same or similar epitope of DR4, DR5, form multivalent antibody, and apoptosis occurs for the tumour cell of inducing expression DR4 and/or DR5.It is connected by the p53 structural domain of people, forms tetravalent antibody, which includes heavy chain variable region and light chain variable region, and heavy chain variable amino acid sequence is as shown in SEQ ID NO.1, and chain variable region amino acid sequence is as shown in SEQ ID NO.2.The ability that such agonistic antibody induced while expressing the tumor death of DR4, DR5 is significantly stronger than the agonistic antibody just for DR4 or DR5.The present invention also provides such application of death receptor agonistic antibody in terms of preparing anti-tumor drug.
Description
Technical field
The present invention relates to the excitability multivalent antibody of a kind of novel death receptor, such antibody can with inducing expression DR4 and/
Or apoptosis occurs for the tumour cell of DR5, can be used for preparing anti-tumor drug.
Background technique
TRAIL mRNA (Tumor necrosis factor-related
Apoptosis inducing ligand, TRAIL) it is the TNF superfamily newcomer that willy is equal to nineteen ninety-five discovery.Experiment
Confirm that TRAIL has lethal effect to the most cells system of nearly all System Malignant Tumor, and normal tissue is without killing
It acts on (Nat Med, 1999,5:157-63), therefore has become research by the oncotherapy of target spot of TRAIL receptor
Hot spot.
The biological effect of the inducing apoptosis of tumour cell of TRAIL mainly leads to and the corresponding receptor on cell membrane: DR4
(Death receptor-4), DR5 (Death receptor-5) in conjunction with and generate.When TRAIL is in conjunction with DR4 or DR5
Afterwards, DR4 and DR5 can form homologous/heterotrimer, the death domain intracellular that is contained by it (death domain,
DD it) conducts in apoptosis information to cytoplasm, activation caspase system finally causes Apoptosis (Science. 1997;276:
111–113. Immunity. 2000;12:611–620. Cell Signal. 2004;16:139-144.).
Some research groups develop death receptor agonistic antibody, after these antibody and DR4 or DR5 specific binding,
Simulate the effect of TRAIL, inducing apoptosis of tumour cell (Nat Med. 2001;7:954–960. J Clin Oncol 25:
1390-1395.).Since the molecular weight of antibody is larger, half-life period ratio TRAILL long in vivo, therefore develop swashing for death receptor
Dynamic property antibody replaces TRAIL, becomes the Main way of exploitation drug.
Theoretically, the agonistic antibody of DR4 and DR5 is activated to have the advantage that for while expressing the swollen of DR4 and DR5
Tumor, the ability of inducing apoptosis of tumour cell are significantly stronger than the agonistic antibody just for DR4 or DR5;For only expressing DR4
Or the tumour of DR5, a kind of antibody drug is also only needed, clinical application is convenient for.Although however having multiple laboratory reports at present and obtaining
The agonistic antibody of DR4 or DR5 were obtained, but preparing without laboratory report has while activating DR4 and DR5 ability
Agonistic antibody.This phenomenon explanation prepares according to conventional thought while activating the agonistic antibody feasibility of DR4 and DR5 not
By force.
Summary of the invention
It is an object of that present invention to provide a kind of novel death receptor excitability multivalence that can activate DR4 and DR5 simultaneously is anti-
Body, and its application in terms of preparing anti-tumor drug.
Novel death receptor excitability multivalent antibody identifies the same or similar epitope of DR4, DR5, and it is anti-to form multivalence
With the tumour cell of inducing expression DR4 and/or DR5 apoptosis can occur for body.
Novel death receptor excitability multivalent antibody identifies the same or similar epitope of DR4, DR5, passes through genetic engineering
Method formed multivalent antibody, so as to inducing expression DR4 and/or DR5 tumour cell occur apoptosis.
Novel death receptor excitability multivalent antibody identifies the same or similar epitope of DR4, DR5, passes through the p53 of people
Structural domain connection, forms tetravalent antibody, and apoptosis occurs for the tumour cell so as to inducing expression DR4 and/or DR5 simultaneously, should
Tetravalent antibody includes heavy chain variable region and light chain variable region, and for heavy chain variable amino acid sequence as shown in SEQ ID NO.1, light chain can
Become area's amino acid sequence as shown in SEQ ID NO.2.
Above-mentioned novel death receptor excitability multivalent antibody application in preparation of anti-tumor drugs.
In the present invention, using modern biotechnologies such as genetic engineerings, using the B cell of people, using phage display skill
Art obtains the single-chain antibody of the same or similar epitope of one plant of identification people DR4 and DR5, by its multimerization, forms multivalent antibody.
Such multivalent antibody can induced strong apoptosis of tumor cells in vitro.Therefore such antibody can be used for new type antineoplastic medicine
Research and development.Existing death receptor agonistic antibody is only to activate DR4 or DR5(such as Chinese Patent Application No.:
200680009970.1；200410070093.1；200580018426.9).
The invention further relates to the preparation methods of novel death receptor excitability multivalent antibody, it mainly includes following step
It is rapid: to establish antibody library using the B cell of Healthy People, it is the same or similar to wash in a pan sieve identification DR4, DR5 using display technique of bacteriophage
The antibody of epitope, obtains single-chain antibody.It is configured to multivalent antibody.It expresses, after purification, finds one of multivalent antibody tool
There is the ability for the apoptosis of tumor cells for making to express DR4 and/or DR5.
The present invention with both at home and abroad just for the similar patent of DR4 or DR5 compared with, such agonistic antibody identify DR4 and DR5
Common or similar epitope, be made into multivalent antibody, can make express DR4 and/or DR5 apoptosis of tumor cells.Such swashs
Dynamic property antibody promotes the ability of apoptosis of tumor cells to be better than the agonistic antibody that DR4 or DR5 can only be activated traditional；These results are equal
It is unimaginable for those skilled in the art.
Detailed description of the invention
Fig. 1 expresses DR4, DR5 situation before and after showing cancer cell Jurkat transfection DR4: DR4 low expression, DR5 high before transfecting
Expression；DR4 is significantly raised after transfection, and DR5 expression is basically unchanged；
Fig. 2 shows that 2.5mg/L death receptor excitability tetravalent antibody acts on the forward and backward Jurkat cell 20h of transfection DR4 and makees
With result: the antibody can make the Jurkat esophageal cancer cell generation apoptosis of main expression DR5 before transfection, can also make to transfect
The further raising of the Jurkat esophageal cancer cell of high expression DR4 and DR5 simultaneously afterwards；
Fig. 3 expresses DR4, DR5 situation before and after showing cell EC970 transfection DR5: DR5 low expression, DR4 high table before transfecting
It reaches；DR5 is significantly raised after transfection, and DR4 expression is basically unchanged；
Fig. 4 shows that 2.5mg/L death receptor excitability tetravalent antibody acts on the forward and backward EC970 cell 20h of transfection DR4 and makees
With result: the antibody can make the EC970 esophageal cancer cell generation apoptosis of main expression DR4 before transfection, after can also making transfection
The further raising of the EC970 esophageal cancer cell of high expression DR4 and DR5 simultaneously.
Specific embodiment
The present invention is further illustrated by the following examples, but scope of protection of the present invention is not limited thereto.
The preparation of 1 death receptor single-chain antibody of embodiment
The single nuclear blood cell in periphery of 50 normal adults is acquired, total serum IgE is extracted.According to " recombinant antibodies " (Shen is put forth energy again,
Science Press) scheme, using cDNA as template, an amplification full set VH and VL gene, the weight that amplification is obtained, light chain product are carried out
Overlap extension PCR amplifies ScFv segment.ScFv segment and carrier pcantab-5E are after Sfi I, Not I digestion, T4 connection
Enzyme connection, electrotransformation enter E. coli XL1-Blue competent cell, prepare phage antibody library.With death receptor antigen packet
By 96 orifice plates (the 1st wheel each 100 μ g/ml of DR4, DR5;2nd wheel 30 μ g/ml of elder generation DR4, then 30 μ g/ml of DR5；3rd wheel elder generation DR4
10 μ g/ml, then 10 μ g/ml of DR5；4th wheel 1 μ g/ml of elder generation DR4, then 1 μ g/ml of DR5.), every hole adds 50 μ after 1%BSA closing
L phage antibody library, 37 DEG C of 2 h of placement wash (the 1st wheel 10 times, later several rounds 20 times) with TBST.The phasmid that will be obtained
Serve the sequencing of Hai Yingjun biotech firm.
CDNA the first chain people's heavy chain constant region primer
IgM(F): TGG AAG AGG CAC GTT CTT TTC TTT
(1) heavy chain forward primer
H-1(F) : CAG CCG GCC ATG GCC SAG GTC CAG CTG GTR CAG TCT GG
H-2b(F) : CAG CCG GCC ATG GCC CAG RTC ACC TTG AAG GAG TCT GG
H-3b(F) : CAG CCG GCC ATG GCC SAG GTG CAG CTG GTG GAG TCT GG
H-3c(F) : CAG CCG GCC ATG GCC GAG GTG CAG CTG GTG GAG WCY GG
H-4b(F) : CAG CCG GCC ATG GCC CAG GTG CAG CTA CAG CAG TGG GG
H-4c(F) : CAG CCG GCC ATG GCC CAG STG CAG CTG CAG GAG TCS GG
H-5(F) : CAG CCG GCC ATG GCC GAR GTG CAG CTG GTG CAG TCT GG
H-6(F) : CAG CCG GCC ATG GCC CAG GTA CAG CTG CAG CAG TCA GG
H-7(F) : CAG CCG GCC ATG GCC CAG RTG CAG CTG GTG CAR TCT GG
HuVH1: GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC SAG GTC CAG CTG GTR
CAG TCT GG(Sfi I)
HuVH2b: GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG RTC ACC TTG AAG
GAG TCT GG(Sfi I)
HuVH3b: GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC SAG GTG CAG CTG GTG
GAG TCT GG(Sfi I)
HuVH3c: GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC GAG GTG CAG CTG GTG
GAG WCY GG(Sfi I)
HuVH4b: GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTG CAG CTA CAG
CAG TGG GG(Sfi I)
HuVH4c: GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG STG CAG CTG CAG
GAG TCS GG(Sfi I)
HuVH5: GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC GAR GTG CAG CTG GTG
CAG TCT GG(Sfi I)
HuVH6: GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTA CAG CTG CAG
CAG TCA GG(Sfi I)
HuVH7: GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG RTG CAG CTG GTG
CAR TCT GG(Sfi I)
(2) heavy chain reverse primer
H-8(R) : TGA GGA GAC GGT GAC CAG GGT GCC
H-9(R) : TGA AGA GAC GGT GAC CAT TGT CCC
H-10(R): TGA GGA GAC GGT GAC CAG GGT TCC
H-extension(R)1\2: ggagccgccgccgcc(agaaccaccaccacc)2 TGA GGA GAC GGT
GAC CAG GGT GCC
H-extension(R)3: ggagccgccgccgcc(agaaccaccaccacc)2 TGA AGA GAC GGT
GAC CAT TGT CCC
H-extension(R)4\5: ggagccgccgccgcc(agaaccaccaccacc)2 TGA GGA GAC GGT
GAC CAG GGT TCC
H-extension(R)6: ggagccgccgccgcc(agaaccaccaccacc)2 TGA GGA GAC GGT
GAC CGT GGT CCC
(3) κ chain forward primer
K-1(F) : GGT GGC TCC GGA GGT GGC GGA TCG GAC ATC CAG WTG ACC CAG TCT
CC
K-2(F) : GGT GGC TCC GGA GGT GGC GGA TCG GAT GTT GTG ATG ACT CAG TCT
CC
K-3(F) : GGT GGC TCC GGA GGT GGC GGA TCG GAA ATT GTG WTG ACR CAG TCT
CC
K-4(F) : GGT GGC TCC GGA GGT GGC GGA TCG GAT ATT GTG ATG ACC CAC ACT
CC
K-5(F) : GGT GGC TCC GGA GGT GGC GGA TCG GAA ACG ACA CTC ACG CAG TCT
CC
K-6(F) : GGT GGC TCC GGA GGT GGC GGA TCG GAW RTT GTG MTG ACT CAG TCT
CC
K-extension(F): ggcggcggcggctccggtggtggtggatccGGT GGC TCC GGA GGT GGC
GGA TCG(linker)
(4) κ chain reverse primer
K-7(R) : TCG ACT TGC GGC CGC ACG TTT GAT TTC CAC CTT GGT CCC
K-8(R) : TCG ACT TGC GGC CGC ACG TTT GAT CTC CAG CTT GGT CCC
K-9(R) : TCG ACT TGC GGC CGC ACG TTT GAT ATC CAC TTT GGT CCC
K-10(R) : TCG ACT TGC GGC CGC ACG TTT GAT CTC CAC CTT GGT CCC
K-11(R) : TCG ACT TGC GGC CGC ACG TTT AAT CTC CAG TCG TGT CCC
K-extension(R): ATT TGC GGC CGC ACG TTT(Not Ⅰ)
HuJk1:GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT TTC CAC CTT GGT CCC
(Not Ⅰ)
HuJk2:GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT CTC CAG CTT GGT CCC
(Not Ⅰ)
HuJk3:GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT ATC CAC TTT GGT CCC
(Not Ⅰ)
HuJk4:GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT CTC CAC CTT GGT CCC
(Not Ⅰ)
HuJk5:GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT AAT CTC CAG TCG TGT CCC
(Not Ⅰ)
(5) λ chain forward primer
λ-1(F) : GGT GGC TCC GGA GGT GGC GGA TCG CAG TCT GTS BTG ACK CAG CCR
CC
λ-2(F) : GGT GGC TCC GGA GGT GGC GGA TCG CAR TCT GCC CTG ACT CAG CCT
λ-3a(F) : GGT GGC TCC GGA GGT GGC GGA TCG TCC TAT GWG CTG ACT CAG CCA
CC
λ-3b(F) : GGT GGC TCC GGA GGT GGC GGA TCG TCT TCT GAG CTG ACT CAG GAC
CC
λ-4(F) : GGT GGC TCC GGA GGT GGC GGA TCG CAC GTT ATA CTG ACT CAA CCG
CC
λ-5(F) : GGT GGC TCC GGA GGT GGC GGA TCG CAG GCT GTG CTG ACT CAG CCG
TC
λ-6(F) : GGT GGC TCC GGA GGT GGC GGA TCG AAT TTT ATG CTG ACT CAG CCC
CA
λ-7/8(F) : GGT GGC TCC GGA GGT GGC GGA TCG CAG RCT GTG GTG ACY CAG
GAG CC
λ-9(F) : GGT GGC TCC GGA GGT GGC GGA TCG CWG CCT GTG CTG ACT CAG CCM
CC
λ-extension(F): ggcggcggcggctccggtggtggtggatccGGT GGC TCC GGA GGT GGC
GGA TCG(linker)
(6) λ chain reverse primer
λ-10(R): TTC TCG ACT TGC GGC CGC ACC TAG GAC GGT GAC CTT GGT CCC
λ-11(R): TTC TCG ACT TGC GGC CGC ACC TAG GAC GGT CAG CTT GGT CCC
λ-12(R): TTC TCG ACT TGC GGC CGC ACT TAA AAC GGT GAG CTG GGT CCC
λ-extension(R): ATT TGC GGC CGC ACC(Not Ⅰ)
HuJλ1:GAG TCA TTC TCG ACT TGC GGC CGC ACC TAG GAC GGT GAC CTT GGT CCC
(NotⅠ)
HuJλ2:GAG TCA TTC TCG ACT TGC GGC CGC ACC TAG GAC GGT CAG CTT GGT CCC
(NotⅠ)
HuJλ3:GAG TCA TTC TCG ACT TGC GGC CGC ACT TAA AAC GGT GAG CTG GGT CCC
(NotⅠ)
Note: Y=C T；W=A\T；B=C\G\T；M=A\C；R=A\G；K=G\T；S=G\C.The above primer sequence left side is 5 '
End, the right are 3 ' ends.
(7) primer pairing (referring to table one)
Table one: design of primers matching table
The building of 2 tetravalent antibody expression vector of embodiment
Using death receptor single-chain antibody gene as template, using L1, H2 as primer, PCR amplification obtains ScFv, reaction condition:
95 DEG C of 3min, 95 DEG C of 30 sec, 60 DEG C of 40 sec, 72 DEG C of 30 sec, 30 circulations.Using P53a, P53b as primer, PCR
Amplification obtains P53 segment, reaction condition: 95 DEG C of 30 sec, 60 DEG C of 40 sec, 72 DEG C of 30 sec, 30 circulations.With above-mentioned
Product is template, using L1, P532 as primer, Overlap extension PCR, and reaction condition: 95 DEG C of 3min, 95 DEG C of 30 sec, 60 DEG C 40
Sec, 72 DEG C of 30 sec, 30 circulations.Target gene recovery product VL+VH2+P53 is taken, I He of restriction enzyme Xho is utilized
Hind III, as segment double digestion system.Empty carrier plasmid pcDNA3.1 is taken, using restriction enzyme Xho I and Hind III,
Double digestion.Gel extraction target gene, the connection of T4 ligase, electrotransformation enter E. coli DH5 ɑ competent cell, screen, picking
Positive colony sequencing.
L1:ATGAAGCTTATGGCCGAGGTGCAGCTGGTGGAGT；
H2:GTTGTGTCACCAAGTGGGGTTGGTCCCTG
P53a:ACCCCACTTGGTGACACAACTCACACATCCGGAAAACCACTGGATGGAGAA TATTTCACCCTT
CA GATCCGTGGGCGTGAGCGCTTCG；
P53b:TGGCTCCTTCCCAGCCTGGGCATCCTTGAGTTCCAAGGCCTCATTCAGCTC TCGGAACATCTC
GA AGCGCTCACGCCCACGGATCT；
P532:CGCCTCGAGTGGCTCCTTCCCAGCCT.
The preparation of 3 excitability tetravalent antibody of embodiment
Tetravalent antibody plasmid is transiently transfected to Chinese hamster ovary celI, trypsin digestion and cell is used after 48 h, harvests supernatant.It will
Supernatant is collected with the flow velocity of 1 ml/min by nickel column, it is steady to baseline with 20 mM imidazoles balance pillar after upper complete sample,
Then unbonded albumen and foreign protein are eluted with 50 mM imidazoles.Then destination protein is eluted with 250 mM imidazoles, be collected simultaneously
Eluent.30 μ l are taken to add 30 μ l 2 × Loading Buffer the eluent of above-mentioned collection, be suspended cracking, incubates on ice
20 min are educated, 10 min are then boiled in boiling water, 4 DEG C, 12 000 g, 5 min is centrifuged, supernatant is taken to be transferred in new EP pipe, to
After SDS-PAGE analysis, merge all eluents containing destination protein, measures protein concentration with BCA method, and use western
The destination protein of blot verifying purifying.
Embodiment 4 transfects DR4 stable cell line and establishes
DR4 plasmid is transiently transfected to Jurkat cell, trypsin digestion and cell is used after 48 h, 2 ml DMEM are added
Cell is resuspended complete culture solution, in two holes of average mark to 6 orifice plates, while using the Chinese hamster ovary celI of untransfected as negative right
According to each Zeocin that 400 μ g/ml are added, which pressurizes, to be screened.After a week, it can see under inverted microscope, untransfected pressurization hole
Complete cell death, transfection hole cell still have cell survival, continue pressurization culture.When cell covers with substantially in hole, it will transfect
It is cells trypsinised, cell is resuspended in DMEM complete culture solution containing 400 μ g/ml Zeocin, counts, according to 5 holes 3
The principle of a cell, 10 cells of ml culture solution 60 spread 96 orifice plates, every 100 μ l of hole.It is during the cultivation process it can be observed that thin
The formational situation of born of the same parents' monoclonal, cell-free hole and many cells clone hole discard during culture, simply continue to culture and form list
Clone the cell in hole.And cell culture is gradually from 96 orifice plates to 24 orifice plates then to 6 orifice plates transition.Continue in incubation
The Zeocin of 200 μ g/ml is maintained to pressurize.
Embodiment 5 transfects DR5 stable cell line and establishes
DR5 plasmid is transiently transfected to human esophagus cancer cell EC970, trypsin digestion and cell is used after 48 h, is added 2
Cell is resuspended ml DMEM complete culture solution, in two holes of average mark to 6 orifice plates, while using the Chinese hamster ovary celI of untransfected as
Negative control, each Zeocin that 400 μ g/ml are added, which pressurizes, to be screened.After a week, it can see under inverted microscope, untransfected
Pressurize hole complete cell death, and transfection hole cell still has cell survival, continues pressurization culture.When cell covers with substantially in hole,
By the cells trypsinised of transfection, cell is resuspended in the DMEM complete culture solution containing 400 μ g/ml Zeocin, is counted, is pressed
According to the principle of 53 cells in hole, 10 cells of ml culture solution 60 spread 96 orifice plates, every 100 μ l of hole.It can see during the cultivation process
The formational situation of cell monoclonal is observed, cell-free hole and many cells clone hole discard during culture, simply continue to cultivate
Form the cell in monoclonal hole.And cell culture is gradually from 96 orifice plates to 24 orifice plates then to 6 orifice plates transition.Incubation
In continue to 200 μ g/ml Zeocin pressurization.
The detection of expression of embodiment 6 Jurkat cell surface DR4, DR5
1 by the good Jurkat cell routine culture of growth conditions in containing 10% fetal calf serum RPMI1640 culture solution
In.
Cell is adjusted to 3 × 10 by 26A/ml, every kind of cell take 3 parts, and 200 l/ parts of μ cell suspensions are washed with PBS
Cell 3 times.
3 developmental tubes add concentration to be the 200 μ l of DR4, DR5 monoclonal antibody (RD company) of 5 μ g/ml, and mouse is added in Isotype control group
200 μ l(1 ︰ 200 of normal serum dilution), negative control adds the PBS of equivalent, 45 min of ice bath.
4 are washed cell 3 times so that PBS is pre-chilled, and 3 ml PBS are added in every pipe every time, are discarded supernatant.
5 are added sheep anti-mouse igg (1 ︰ 50 dilution) the 50 μ l mixing of 50 μ l PBS and FITC label, and 40 min of ice bath is used
PBS is washed 2 times, the expression of flow cytomery DR4, DR5, with Cellquest software analysis cell DR4, DR5 expression (knot
Fruit sees Fig. 1).
The detection of expression of embodiment 7 EC970 cell surface DR4, DR5
1 by growth conditions good human esophagus cancer cell EC970 routine cultures in the fetal calf serum containing 10%
In RPMI1640 culture solution.
Cell is adjusted to 3 × 10 by 26A/ml, every kind of cell take 3 parts, and 200 l/ parts of μ cell suspensions are washed with PBS
Cell 3 times.
3 developmental tubes add concentration to be the 200 μ l of DR4, DR5 monoclonal antibody (RD company) of 5 μ g/ml, and mouse is added in Isotype control group
200 μ l(1 ︰ 200 of normal serum dilution), negative control adds the PBS of equivalent, 45 min of ice bath.
4 are washed cell 3 times so that PBS is pre-chilled, and 3 ml PBS are added in every pipe every time, are discarded supernatant.
5 are added sheep anti-mouse igg (1 ︰ 50 dilution) the 50 μ l mixing of 50 μ l PBS and FITC label, and 40 min of ice bath is used
PBS is washed 2 times, the expression of flow cytomery DR4, DR5, with Cellquest software analysis cell DR4, DR5 expression (knot
Fruit sees Fig. 3).
Cytotoxicity of 8 tetravalent antibody of embodiment to tumour cell
1) detection of Jurkat cell apoptosis rate
Jurkat cell suspension is collected and prepared, 24 well culture plates, 2 holes ml/ are added, final concentration of cells is 1 × 105
A/hole sets 37 DEG C, 5% CO212 hr of CMC model is separately added into single-chain antibody, tetravalent antibody makes its final concentration of 2.5 mg/
L；Negative control hole RPMI1640 culture solution.In 37 DEG C and 5% CO2Under conditions of cultivate 20 h, collect all cells in every hole
In corresponding centrifuge tube.After PBS washs cell, every hole cell is suspended in respectively in the AnnExin V combination liquid of 100 μ l, point
Not plus 5 μ l AnnExin V-FITC solution and 5 μ l PI solution, after mixing, in 20 min of room temperature Incubation in dark, with combining liquid
It mends to 400 μ l.Cell is detected using flow cytometer.10,000 cells of each sample detection, with CellQuest software
The apoptosis rate of cell is analyzed, experiment is repeated 3 times (result is shown in Fig. 2).The antibody can make the Jurkat of main expression DR5 before transfection
Apoptosis can be such that the Jurkat cell of the DR4 and DR5 of height expression simultaneously after conversion before transfecting further increases.
2) detection of EC970 apoptosis rate
EC970 cell suspension is collected and prepared, 24 well culture plates, 2 holes ml/ are added, final concentration of cells is 1 × 105A/
Hole sets 37 DEG C, 5% CO212 hr of CMC model is separately added into single-chain antibody, tetravalent antibody makes its final concentration of 2.5 mg/L；
Negative control hole RPMI1640 culture solution.In 37 DEG C and 5% CO2Under conditions of cultivate 20 h, collect all cells in every hole in
In corresponding centrifuge tube.After PBS washs cell, every hole cell is suspended in respectively in the AnnExin V combination liquid of 100 μ l, respectively
Add 5 μ l AnnExin V-FITC solution and 5 μ l PI solution, after mixing, in 20 min of room temperature Incubation in dark, is mended with combination liquid
To 400 μ l.Cell is detected using flow cytometer.10,000 cells of each sample detection, with CellQuest software point
The apoptosis rate of cell is analysed, experiment is repeated 3 times (result is shown in Fig. 4).The antibody can make the EC970 food of main expression DR4 before transfection
Apoptosis occurs for pipe cancer cell, and the EC970 esophageal cancer cell of the DR4 and DR5 of height expression simultaneously after conversion before transfecting can be made further
It increases.
SEQUENCE LISTING
<110>He'nan University
<120>novel death receptor excitability multivalent antibody and its application in preparation of anti-tumor drugs
<130>
<160> 2
<170> PatentIn version 3.4
<210> 1
<211> 116
<212> PRT
<213>artificial sequence
<400> 1
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser
1 5 10 15
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly
20 25 30
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
35 40 45
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Val Glu Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
100 105 110
Thr Val Ser Ser
115
<210> 2
<211> 112
<212> PRT
<213>artificial sequence
<400> 2
Asp Ile Val Met Thr His Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asp Gly Asn Thr Tyr Leu Ser Trp Leu His Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Ile Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met His Gly
85 90 95
Val Gln Leu Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Claims (2)
1. death receptor excitability multivalent antibody, it is characterised in that: the same epitope of identification DR4, DR5 pass through the p53 structure of people
Domain connection, forms tetravalent antibody, so that apoptosis occurs for the tumour cell of inducing expression DR4 and DR5 simultaneously, which includes
Heavy chain variable region and light chain variable region, heavy chain variable amino acid sequence is as shown in SEQ ID NO.1, chain variable region amino acid sequence
As shown in SEQ ID NO.2.
2. death receptor excitability multivalent antibody application in preparation of anti-tumor drugs as described in claim 1, feature
It is, the tumour is tumor type corresponding to Jurkat or EC970 cell.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310725221.0A CN103833853B (en) | 2013-12-25 | 2013-12-25 | Novel death receptor excitability multivalent antibody and its application in preparation of anti-tumor drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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