CN104127871B - CXCL4 monoclonal antibodies treat tumour after tumour and chemotherapy and accelerate amplified gene screening method again - Google Patents
CXCL4 monoclonal antibodies treat tumour after tumour and chemotherapy and accelerate amplified gene screening method again Download PDFInfo
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Abstract
Accelerate amplified gene screening method again the invention discloses tumour after a kind of CXCL4 monoclonal antibodies treatment tumour and chemotherapy；The purposes is the purposes in medicine for suppressing tumor proliferation is prepared；The tumour is the tumour of CXCL4 height expression caused by the tumour or chemotherapy of CXCL4 height expression；The chemotherapy drugs in combination medication used when the anti-CXCL4 monoclonal antibodies are with chemotherapy, the tumor proliferation for suppressing CXCL4 height expression caused by chemotherapy.The present invention is by applying the Fu chemotherapy models of colon cancer tumor-bearing mice 5, relation between " accelerating to breed again " is disclosed after CXCL4 expression change and the Fu chemotherapy of colon cancer 5, illustrate the molecular mechanism during tumour " accelerating to breed again " after CXCL4 participation chemotherapy, completing antagonism CXCL4 cooperates with 5 Fu to suppress the pharmacological effect research that colon cancer cell is bred again, and foundation is provided successfully to develop CXCL4 antibody drugs.
Description
Technical field
The invention belongs to biological technical field, and in particular to tumour accelerates after a kind of CXCL4 monoclonal antibodies treatment tumour and chemotherapy
Amplified gene screening method again.
Background technology
Tumour " acceleration is bred again " phenomenon that tumor chemoradiotherapy induces：Chemicotherapy is still the most common treatment hand of tumour
Section, its main anti-tumor mechanism is to produce CDCC, causes massive tumor cell death.It has been generally acknowledged that being on the point of during this
The scavenger-cells such as cell or macrophage that dead or dead cell can be survived by surrounding are digested and assimilated, and if few
The tumor cell survival of amount gets off, and estimation slowly can breed, gradually cause tumor recurrence.Document report was begun to before 40 years
Road tumour opens the process for being referred to as " accelerating again to breed (accelerated repopulation) ", this mistake after radiotherapy
Cheng Zhong, under radiotherapy strike under a small amount of tumor cell survival for escaping, and can be rebuild with the growth rate more accelerated it is serious by
Tumor tissues (Hermens, A.F.＆Barendsen, G.W.Changes of the cell proliferation of damage
characteristics in a rat rhabdomyosarcoma before and after x-
irradiation.Eur.J.Cancer,5:173-189,1969.), (Stephens, T.C., Currie, G.A.＆Peacock,
J.H.Repopulation of gamma-irradiated Lewis lung carcinoma by malignant cells
and host macrophage progenitors.Br.J.Cancer,38:573–582,1978.).Recently, inventor's problem
Group also observed after chemotherapy in bearing mouse model, the phenomenon of residual tumor cells " accelerating to breed again ".This this area
Technical staff plays mainly in " acceleration is bred again " phenomenon that molecular level is known little about it during current chemotherapy and radiation
Effect.
Tumour " accelerates to breed again " the oncology important scientific problems that molecule mechanism is urgent need to resolve after treatment：Researcher
The molecule mechanism bred again is accelerated to do substantial amounts of research after tumor chemoradiotherapy in order to illustrate.Recently, China scientist is first
It is secondary to be reported in Dead tumor cell under the conditions of radiotherapy and produce growth stimulation signal in apoptotic process to stimulate tumors remaining thin
Born of the same parents accelerate to breed (Huang Q, Li F, Liu X, et al.Caspase3-mediated stimula tion of tumor again
cell repopulation during during cancer radiotherapy.Nat Me d,17:860-6,2011.)。
They further study the growth stimulation for showing that this apoptotic process key protein of caspase3 take part in tumour cell.
The downstream signaling proteins prostaglandin E2 (PGE2) of caspase3 regulations can stimulate the propagation of viable tumor cell.It is thin in tumour
Antagonism caspase3 can improve the sensitiveness of xenograft tumours or mouse tumor to radiotherapy in born of the same parents or mesenchyma stroma of tumors cell.Face
The caspase3 of the high-level state of activation is deposited with patient's tumour high relapse rate and high mortality in cancer patient's tumor tissues on bed
In significant correlation.Their research discloses caspase3 tumours after radiotherapy and accelerates to breed again to play angled key in path
Color (Huang Q, Li F, Liu X, et al.Caspas e3-mediated stimulation of tumor cell
repopulation during during cance r radiotherapy.Nat Med,17:860-6,2011.).Disclose
Radiotherapy has cell toxicant to tumour growth and accelerates the double action and the mechanism of action of regrowth.Particularly " acceleration is bred again "
Effect, is not the direct effect of radiotherapy, but by having raised caspase3 realizations.Therefore, targeting " acceleration is bred again "
The key molecule caspase3 of path provides the effect for suppressing after radiotherapy " accelerating to breed again ", so as to also improve tumour to putting
The sensitiveness for the treatment of.Targeting caspase3 is likely to become new antineoplastic target spot, and with society and economic value.
Chemotherapeutics is in addition to direct CDCC, and may also act to the immune system of body influences it to play anti-swell
Tumor activity.Current research reports (Bruchard M, Mignot G, Ghiringhelli F, et al.Chemothera py-
triggered cathepsin Brelease in myeloid-derived suppressorcells act ivates
the Nlrp3inflammasome and promotes tumor growth.Nat Med,19:57-64,2013.), it is clinical
Two conventional chemotherapeutics gemcitabines and 5-FU, can activate NOD sample receptor families, i.e. marrow source property and suppress cell
What the pyrin domain proteins 3 (Nlrp3) in (myeloid-derived suppressor cell, MDSC) were relied on
Caspase-1 activation compound (also referred to as inflammation body-inflammasome), causes M DSC height expression interleukin 1s
(IL-1).IL-1 can further induce CD4+T cells to discharge Interleukin-17 (IL-17), and IL-17 is by promoting tumor group
Knitting angiogenesis causes tumour after chemotherapy " acceleration is bred again ".Correspondingly, when tumour set up Nlrp3-/-, Caspase1-/-
When on mouse or the wild-type mice handled with interleukin-1 receptor antagonist (IL-1Ra, antagonism IL-1), swell after chemotherapy
Knurl " acceleration is bred again " effect is suppressed so that chemotherapy can play stronger antitumor action (Bruchard M, Mignot
G,Ghiringhelli F,et al.Ch emotherapy-triggered cathepsin Brelease in myeloid-
derived suppresso r cells activates the Nlrp3inflammasome and promotes tumor
growth.Nat Med,19:57-64,2013.).Result of study discloses chemotherapy confrontation tumor cytotoxicity and " acceleration is bred again "
Double action, it is not effect of the chemotherapeutics directly to tumour that it is confirmed to the molecular mechanism research of the latter, but is passed through
The MDSC acted in tumor tissues improves tumor angiogenesis, the blood supply of increase tumor tissues and realized indirectly.Therefore, change
The key molecule that tumour " accelerates to breed again " in path after treatment provides novel targets and new approaches to develop new antineoplastic,
Application such as the IL-1Ra mentioned in article can suppress tumour after chemotherapy " acceleration is bred again " to provide chemotherapy antitumor effect
Really.
These researchs disclose some important mechanisms of tumour after chemicotherapy " accelerating to breed again ".However, research recently is also
Show some chemotherapeutics on the contrary can by remove immunosuppressant cell strengthen anti-tumor immune response (Mattarollo,
S.R.et al.Pivotal role of innate and adaptive immunity in anthracyclinech
emotherapy of established tumors.Cancer Res,71:4809–4820,2011.).Chemotherapeutics 5-Fu
MDSC (Vincent, J.et al.5-Fluorouracil selectively ki lls tumor- can optionally be removed
associated myeloid-derived suppressor cells resulting in enhanced T cell–
dependent antitumor immunity.Cancer Res,70:3052-3061,2010.), and MDSCs be a class not into
Ripe bone marrow cell, can suppress the mankind and mouse T cell activation (Gabrilovich, D.I.＆Nagaraj,
S.Myeloid-derived suppressor cells as regulators of theimmun e
system.Nat.Rev.Immunol,9:162–174,2009.).The MDSC of 5-FU mediations, which is removed, to be also not enough to avoid after chemotherapy
Tumour " acceleration is bred again ".Thus, disclosing causes the other molecular pathway of tumour cell " accelerating to breed again " very necessary.
CXCL4 (PF-4) is the earliest heparin-binding protein for finding to be present in platelet alpha-particle, while other a variety of
Cell can express CXCL4 (Kasper B, Petersen F.Molecular pathways of platelet
factor4/CXCL4signaling.Eur J Cell Biol,90:521-26,2011.).CXCL4 acceptor is CXC chemotactics
Factor acceptor 3 (CXCR3), CXCR3 is the G-protein coupling receptor superfamily member of seven cross-films.People CXCR3 acceptors can be divided into two
Plant subtype C XCR3-A and CXCR3-B.The current expression that CXCR3-B has been found that in T cell and endothelial cell, and CXCL4
Specific receptor be CXCR3-B.Chemokines CC XCL4 acceptors CXCR3 is wide in lymphocyte, endothelial cell and epithelial cell
General expression, the propagation of cell, migration and apoptosis can be adjusted by activating many signal paths.Multinomial research confirmation,
The infiltration and transfer of CXCL4 and its acceptor to colorectal cancer cell have significant impact, and CXCL4 is multiple as colorectal cancer
Biomarker (Pilatova K, Greplova K, Zdrazilova-Dubska L, the et al.Role of of hair
platelet chemokines,PF-4 and CTAP-III,in cancer biology.J Hematol Oncol,6:42,
2013.)。
The content of the invention
It is an object of the invention in view of the deficienciess of the prior art, providing a kind of CXCL4 monoclonal antibodies treatment tumour and change
Tumour accelerates amplified gene screening method again after treatment；Accelerate after the purposes and chemotherapy of tumors of specifically related to anti-CXCL4 monoclonal antibodies
The screening technique of amplified gene cluster again；The tumour that the anti-CXCL4 monoclonal antibodies are used to suppress high expression CXCL4 accelerates to increase again
Grow.
The purpose of the present invention is achieved through the following technical solutions：
In a first aspect, the use the present invention relates to a kind of anti-CXCL4 monoclonal antibodies in medicine for suppressing tumor proliferation is prepared
On the way.
It is preferred that, the purposes is specially in the tumor proliferation medicine for suppressing the high expression of CXCL4 caused by chemotherapy is prepared
Purposes.
It is preferred that, the chemotherapy drugs in combination medication used when the anti-CXCL4 monoclonal antibodies are with chemotherapy.
It is preferred that, anti-CXCL4 monoclonal antibodies CXCL4 expressions after chemotherapy reach to be applied before highest.
It is preferred that, the chemotherapeutics used during the chemotherapy is cell cycle dependant chemotherapeutic.
It is preferred that, the chemotherapeutics is 5-FU.
It is preferred that, the anti-CXCL4 monoclonal antibodies and 5-FU drug combinations.
It is preferred that, after the 5-FU medications within 4 days, carry out anti-CXCL4 monoclonal antibodies administration.
It is preferred that, the tumour is the tumour of CXCL4 height expression caused by the tumour or chemotherapy of CXCL4 height expression.
It is preferred that, the tumour is 5-FU responsive type tumours.
It is preferred that, the tumour is tumor in digestive tract.
It is preferred that, the tumour is stomach cancer.
It is preferred that, the tumour is colon cancer tumours.
Second aspect, the invention further relates to accelerate the screening technique of amplified gene cluster again after a kind of chemotherapy of tumors, including such as
Lower step：
A, set up tumor-bearing model or tumor cell in vitro culture systems；
B, the combination for giving different chemotherapeutics or chemotherapeutics；
After C, chemotherapy, tumor sample is taken in different time points；
D, with gene expression chip examination obtain tumor tissues gene expression profile；
E, bioinformatic analysis, which are obtained, has the gene of expression rule as accelerating to breed candidate gene again after chemotherapy of tumors；
F, the pharmacological effect research for carrying out system to the candidate gene, accelerate what is bred again after confirming influence chemotherapy of tumors
Key gene.
The third aspect, the invention further relates to a kind of purposes of CXCL4 in medicine for suppressing tumor proliferation is prepared.
Preferably, the purposes is specially：CXCL4 is as the target spot of tumor proliferation in medicine for suppressing tumor proliferation is prepared
Purposes.
Preferably, the purposes comprises the following steps：Obtain can and CXCL4 or CXCL4 downstream signals neutralize combination
Thing, using in the conjugate and CXCL4 target spots or downstream signal, realizes and suppresses tumor proliferation.
Preferably, the conjugate is the antibody that is prepared by antigen of CXCL4, or the antibody fragment, or described resist
The fusion protein of body.
Preferably, the conjugate is the antibody that is prepared by antigen of CXCL4 acceptors, or the antibody fragment, or institute
State the fusion protein of antibody.
Preferably, the CXCL4 acceptors are CXCR3, or CXCR3A, or CXCR3B.
Preferably, the conjugate be CXCL4 acceptors, or CXCL4 acceptors soluble recepter, or CXCL4 acceptors by
Body fusion protein；
The CXCL4 acceptors are CXCR3, CXCR3A, or CXCR3B.
Preferably, the conjugate be CXCL4 mutant, the CXCL4 mutant can competition binding CXCL4 acceptors,
Do not cause receptor activation simultaneously.
Preferably, the conjugate is targeting CXCL4, or targets CXCL4 acceptors, or targets downstream signal member's
mRNA。
Preferably, the conjugate is with CXCL4, or with CXCL4 acceptor, or using CXCL4 downstream passages member as target
The antagonist that mark screening is obtained.
Tumour can open the process for being referred to as " accelerating to breed again " after chemotherapy, i.e., the tumour escaped under chemotherapy strike is thin
Born of the same parents survive, and can be bred with the speed more accelerated.Genotype determines phenotype, accelerates again after tumour cell or tissue chemotherapy
During propagation, certainly exist chemotherapy induced activation plays the interior acceleration in regulation and control driving effect amplified gene cluster again.Accelerate
Amplified gene cluster may play positive regulation or negative regulation again, thus tumour cell or be organized in different chemotherapeutics or
In the presence of the combination of chemotherapeutics, amplified gene cluster is accelerated again to occur to give birth to tumour cell or tissue at different time points
Long speed is consistent or antipodal regular fluctuation, and the rule of this gene expression dose is sexually revised can be in full-length genome table
Shown on up to chip.According to gene after gene expression chip examination chemotherapy in expression of tumor tissue difference, through extensive raw
The data analysis of thing informatics and judgement, expression confirmation is carried out to candidate gene, so as to obtain after participation chemotherapy " acceleration is bred again "
Candidate gene.
Amplified gene cluster screening technique is accelerated again to filter out difference on a large scale after the chemotherapy of tumors being related in the present invention
Tumour breeds relevant gene cluster again under the compound action of different chemotherapeutics or chemotherapeutics with accelerating after chemotherapy of tumors, from
And provide combination chemotherapy new departure for the different chemotherapy means of different tumours for clinic.
The purposes for the anti-CXCL4 monoclonal antibodies being related in the present invention is to accelerate after the chemotherapy of tumors being related in the present invention
The application example of amplified gene cluster screening technique again, the present invention uses mouse junction cancer model, to may participate in tumour after chemotherapy " plus
Speed is bred again " gene cluster carried out system examination.Take the tumour of different time points after subcutaneous mouse CT26 colon cancer 5-Fu chemotherapy
Tissue, prepares full-length genome chip of expression spectrum, according to gene after gene expression chip examination chemotherapy in expression of tumor tissue difference,
Analyze and judge through large-scale data, expression confirmation is carried out to candidate gene, so as to obtain after participation chemotherapy " acceleration is bred again "
Candidate gene.Most research emphasis focuses on chemotactic factor (CF) family at last, has filtered out Chemokines CC XCL4.CXCL4 is in 5-Fuization
Show significantly first to rise the expression characteristic declined afterwards after treatment, point out it to participate in after colon cancer chemotherapy " acceleration is bred again "
Process.The present invention illustrates the molecule mechanism that CXCL4 " accelerates to breed again " after chemotherapy of tumors, and is carried by intervening this mechanism
For combination chemotherapy colon cancer new departure.
Compared with prior art, the device have the advantages that being：The present invention is by applying colon cancer tumor-bearing mice 5-
Fu chemotherapy models, disclose after CXCL4 expression change and colon cancer 5-Fu chemotherapy the relation between " accelerating to breed again ", illustrate
CXCL4 participates in the molecular mechanism during tumour after chemotherapy " accelerating to breed again ", completes antagonism CXCL4 (using independent research
CXCL4 monoclonal antibodies) cooperate with 5-Fu to suppress the pharmacological effect research that colon cancer cell is bred again, resist successfully to develop CXCL4
Body medicine provides foundation.
Meanwhile, present invention discover that the molecular mechanisms that participate in after chemotherapy during tumour " accelerate breed again " of CXCL4 have
Meaning is started, CXCL4 can play a part of tumor proliferation target spot, and then the correlation of tumour can be carried out by acting on the target spot
Treat work；The mechanism acted on target spot has many kinds, and these mechanism can apply to the present invention, realizes and suppresses tumor proliferation
Effect；These mechanism include but is not limited to following：
The enough conjugates neutralized with CXCL4 or CXCL4 downstream signals of capacitation, using in the conjugate with CXCL4 target spots
Or downstream signal, realize and suppress tumor proliferation；
Further, with reference to common sense in the field understand, can action target spot and realize suppress tumour mechanism also include：
The conjugate that can be neutralized with CXCL4 or CXCL4 downstream signals is obtained, using in the conjugate and CXCL4 targets
Point or downstream signal, realize and suppress tumor proliferation, the conjugate can be：
The conjugate is the antibody that is prepared by antigen of CXCL4, or the antibody fragment, or the antibody fusion
Albumen.
The conjugate is the antibody that is prepared by antigen of CXCL4 acceptors, or the antibody fragment, or the antibody
Fusion protein.
The CXCL4 acceptors are CXCR3, or CXCR3A, or CXCR3B.
The conjugate be CXCL4 acceptors, or CXCL4 acceptors soluble recepter, or CXCL4 acceptors receptors fusion egg
In vain；The CXCL4 acceptors are CXCR3, CXCR3A, or CXCR3B.
The conjugate be CXCL4 mutant, the CXCL4 mutant can competition binding CXCL4 acceptors, while not drawing
Play receptor activation.
The conjugate is targeting CXCL4, or targets CXCL4 acceptors, or targets the mRNA of downstream signal member.
The conjugate is, with CXCL4, or with CXCL4 acceptor, or to be obtained as target sieving using CXCL4 downstream passages members
The antagonist obtained.
Obviously, the mechanism that can be acted on claimed CXCL4 target spots, related substances all should be the present invention's
Within protection domain, however it is not limited to content listed above, this be those skilled in the art it is to be understood that.
The successful implementation of the present invention, provides novel targets and new antibodies medicine for colorectal cancer new drug development, is colorectal cancer
Drug therapy provides new approaches；Thus, with great social effect and economic value.
Brief description of the drawings
By reading the detailed description made with reference to the following drawings to non-limiting example, further feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is disposably injects the 0th after 5-FU (200mg/kg), chemotherapy to CT26 tumor-bearing mices, subcutaneously swell within 2,4,6,8 days
The measurement result figure of knurl volume；
Fig. 2 is the 0th after 5-FU (200mg/kg) chemotherapy, 2,4,6, CT26 tumor tissue sections HE coloration results signals in 8 days
Figure；
Fig. 3 is the detection that hypodermic tumour tissue after 5-FU (200mg/kg), chemotherapy is disposably injected to CT26 tumor-bearing mices
Result schematic diagram；Wherein, A is CXCL4mRNA levels becoming after 5-Fu chemotherapy in the tumor tissues that gene chip results are shown
Gesture figure, B be RT-PCR detect CT26 chemotherapy of tumors after the 4th day tissue in CXCL4 gene level expression change schematic diagram (n
=3) (* * p<0.01vs the 0th day)；
After Fig. 4 is is incubated in vitro culture CT26 cells and MFC cells 5-FU (200mg/kg) or L-OHP (30ug/ml),
RT-PCR detection CXCL4mRNA expression quantity change schematic diagrams (n=3, * p<0.05, * * p<0.01)；Wherein, A.5-FU it is incubated
CT26 cells, B.L-OHP is incubated CT26 cells, is C.5-FU incubated MFC cells, and D.L-OHP is incubated MFC cells；
Fig. 5 is that mtt assay detects proliferation function schematic diagram (* * ps of the rhCXCL4 to CT26 cells and IEC-6 cells<
0.01,***p<0.001)；Wherein, A illustrates for the rhCXCL4 (0-3ug/ml) of various concentrations to the proliferation function of IEC-6 cells
Figure, B is proliferation function schematic diagrames of the rhCXCL4 (0-3ug/ml) to CT26 cells of various concentrations；
Fig. 6 is the identification schematic diagram of Anti-CXCL4mAb characteristics；Wherein, A is SDS/PAGE (8%) (a) and Western
The anti-CXCL4mAb of blotting (b) methods analysis purifying, B are that BIAcore methods analyze the affine of anti-CXCL4mAb
Power, C is growth inhibition effect schematic diagrames of the rhCXCL4 to ACHN cells, and D is Anti-CXCL4mAb antagonisms rhCXCL4
The inhibitory action schematic diagram of (72.6ug/ml) to ACHN cells；
Fig. 7 is that anti-CXCL4 monoclonal antibodies combine inhibitory action schematic diagrames of the 5-FU to tumour growth；Wherein, A is swollen for each group mouse
Knurl volume size variation schematic diagram, B is each group dead mouse situation of change schematic diagram, and C is that chemotherapeutical control group adds monoclonal antibody with chemotherapy
4th day and the 6th day tumor tissue section BrdU dyeing schematic diagram after administration group mouse chemotherapy, D is that the section of BrdU SABCs is swollen
Tumor cell proliferation index counts schematic diagram, (n=3), * * p ＜ 0.01；
Fig. 8 is that anti-CXCL4 monoclonal antibodies combine the inhibitory action signal that 5-FU transplants nude inoculation tumour growth to T lymphocytes
Figure；Wherein, A is the BALB/C nu/nu nude mouses of subcutaneous vaccination CT26 tumours, after 5-FU (150mg/kg) chemotherapy plus monoclonal antibody is given
Medicine group is subcutaneously injected anti-CXCL4 monoclonal antibodies (1mg/kg), and gross tumor volume changes with time schematic diagram, and B is that tail vein transplants health
Monoclonal antibody is added to give after the BALB/C nu/nu CT26 lotus knurl nude mouses of BALB/C mice lymphocyte, 5-FU (150mg/kg) chemotherapy
Medicine group is subcutaneously injected anti-CXCL4 monoclonal antibodies (1mg/kg), and gross tumor volume changes with time schematic diagram.
Fig. 9 is IFN-r and GRAN-b mrna expression amount change schematic diagrams in RT-PCR detection CT26 tumor tissues；Wherein,
A is the expression quantity that RT-PCR detects IFN-γ mRNA, and B is the expression quantity that RT-PCR detects GRAN-b mRNA；(n=3) (* p<
0.05vs PBS control groups).
Embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that to the ordinary skill of this area
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
The hybridoma 16D6-3 being related in embodiment submits Chinese Typical Representative culture guarantor on November 29th, 2012
Tibetan center preservation, preservation address is Wuhan Wuhan Universitys of China, and deposit number is CCTCC NO:C2012186.
Embodiment
The foundation of 1.1 colon cancer tumor-bearing mice chemotherapy models
The chemotherapy regimen of clinical treatment colorectal cancer is single medicine chemotherapy or the Combination chemotherapy (5-FU based on 5-FU
+ L-OHP or CPT-11).For approach clinic, selection 5-FU single dose of chemotherapies build the subcutaneous tumor-bearing mice chemotherapy model of colon cancer.Close
Suitable chemotherapy doses is also significant for the stability of model.If chemotherapeutic dosage is too low, killing of the chemotherapy to tumour
Effect is not obvious；If chemotherapeutic dosage is too high, too high mouse death rate can be caused.Grope through overtreatment, moved in follow-up
In thing experimental study, 5-FU single medicine chemotherapy doses is controlled in 150-200mg/kg.
In Balb/c right side of mice oxter subcutaneous vaccination mouse colorectal cancer tumour cell CT261 × 106It is individual, treat tumour body
Product is long to 400-500mm3When, disposable tail vein injection chemotherapeutics 5-FU (200mg/kg) uses vernier in every two days after chemotherapy
Slide calliper rule measure gross tumor volume size, this results in mouse Colon tumor after 5-FU chemotherapy its growth change situation；Fig. 1 is
CT26 tumor-bearing mices are disposably injected the 0th after 5-FU (200mg/kg), chemotherapy, the measurement of 2,4,6,8 days subcutaneous tumor volumes
Result schematic diagram, as shown in Figure 1, CT26 tumours are after 5-FU chemotherapy, and gross tumor volume first reduces, and touch the bottom within the 4th day, then
Bounce-back increase, shows as typical " acceleration is bred again " phenomenon.This shows that single dose of chemotherapy is made to the growth inhibition of colon cancer tumours
With limited, tumour there is a situation where to recur propagation after chemotherapy, consistent with clinical observation phenomenon.
To the 0th after 5-FU chemotherapy, the CT26 tumor tissues of 2,4,6,8 days carry out H＆E stained slices, Fig. 2 is 5-FU
The 0th after (200mg/kg) chemotherapy, 2,4,6,8 days CT26 tumor tissue sections HE coloration result schematic diagrames, as shown in Figure 2, as a result
There is large area necrosis after chemotherapy in display tumor tissues, the most serious with the 4th day, but downright bad drastically reduce is after the 6th day
It is consistent with situation of change of the tumour growth volume after chemotherapy to disappearing.
1.25-FU induction colon cancer cell expression CXCL4
Take 0 after 5 FU 5 fluorouracil (5-FU) chemotherapy, the fresh CT26 mouse junction cancers group at 2,4,6,8 days totally 5 time points
Knit, extract RNA, sample presentation makes high density oligonucleotide microarray, obtained after colon cancer tumours chemotherapy in resistance growth course
Gene expression dynamic collection of illustrative plates.By the expression for analyzing mRNA in high density oligonucleotide microarray, it is found that chemotactic factor (CF)
The expression quantity up-regulation of CXCL4 CT26 tumor tissues after 5-FU chemotherapy, and in single dose of chemotherapy advanced stage tumours restoration ecosystem period table
Decline up to amount.From Fig. 3 A, compared with before chemotherapy, CXCL4 expression reached peak at the 2nd day after 5-FU chemotherapy, with
After begin to decline.For further proofing chip result, CT26 have detected using real-time PCR (RT-PCT) method and swell
CXCL4 expression of the 4th day after 5-FU chemotherapy changes in tumor tissue, such as Fig. 3 B (n=3, * * p<0.01) shown in, result verification
Compared with the 0th day control group of non-chemotherapy, CXCL4 gene expressions are significantly raised.
1.35-FU specificity mediation colon cancer cell CXCL4 activation expression
5-FU induced tumor tissues CXCL4 expression activation, and the heterogeneity of tumor tissues make it that research CXCL4 is by swelling
Which class cell expression in tumor tissue seems extremely necessary.Mouse colonic cell CT26 is cultivated in vitro, with 200ug/ml5-
FU changes liquid after being incubated 3 hours, collect the cell changed 4 hours, 24 hours and 48 hours after liquid, and extracting RNA utilizes RT-PCR technology
Study CXCL4mRNA expression quantity change.By Fig. 4 A (n=3, * p<0.05, * * p<0.01) understand, from 4 hours after 5-FU chemotherapy
Rise, compared with the control without chemotherapy, CT26 cells significantly activate up-regulation CXCL4mRNA.
Next it further study whether tumour cell CXCL4 expression activation is chemotherapeutic specificity or is tumour cell
Specificity.Cultured Mouse colon cancer cell CT26 and Mouse Gastric Cancer cell MFC, it is difficult to understand with 200ug/ml5-FU or 30ug/ml
Husky profit platinum (L-OHP) changes liquid after being incubated 3 hours, collects the cell changed 4 hours, 24 hours and 48 hours after liquid, extracting RNA, profit
Changed with RT-PCR technology research CXCL4mRNA expression quantity.By Fig. 4 C (n=3, p<0.05) understand, as a result show, compared to
CT26 cells, MFC cells are 48 hours after 5-FU, and CXCL4mRNA just has significantly activation up-regulation.And L-OHP can not activate CT26
The up-regulation CXCL4 expression of (Fig. 4 B) or MFC cells (Fig. 4 D).It these results suggest that colon cancer cell CXCL4 activation expression is by 5-
FU specificity mediations.
1.4rhCXCL4 does not work to CT26 Colon Cancer Cells in vitro
Above research shows that 5-FU chemotherapy can specificity induction colon cancer cell activation expression CXCL4, next research
Whether CXCL4 has direct effect to colon cancer cell.In vitro with the rhCXCL4 (0-3ug/ml) of various concentrations to mouse Colon
Cancer cell CT26 and the undifferentiated crypts of small intestine epithelial cell IEC-6 (positive control) of rat use MTT after carrying out cell culture, 48h
Development process detects living cells relative populations, calculates CT26 cells and IEC-6 cells under various concentrations rhCXCL4 condition of culture
Survival rate.Fig. 5 A are proliferation function schematic diagrames of the rhCXCL4 (0-3ug/ml) to IEC-6 cells of various concentrations, and Fig. 5 B is not
RhCXCL4 (0-3ug/ml) with concentration is to the proliferation function schematic diagrames of CT26 cells；Fig. 5 A, 5B result shows, in vitro
Growths of the rhCXCL4 just to IEC-6 cells under 0.33ug/ml concentration has significant inhibitory action, and inhibitory action is presented
Concentration dependent, and the external propagation on CT26 cells of rhCXCL4 is without influence.
The preparation of 1.5 anti-CXCL4 monoclonal antibodies and its identification of physicochemical property
Polyclonal antibody is complex due to its serum origin composition, and the present embodiment has been prepared and purified with neutralization people source
And mouse source CXCL4 monoclonal antibody.Through purity detecting, endotoxin detection and biological activity detection (Fig. 6), it was demonstrated that monoclonal antibody
Quality be reached zoopery standard.
SD rats are immunized with the people CXCL4 recombinant proteins of purifying, one plant has been obtained through cell fusion and the screening of 3 time clonings
The hybridoma cell strain of anti-recombined human and mouse CXC L4 monoclonal antibodies is secreted, 16D6-3 is named as.16D6-3 cell lines are external
Continuous culture 1 month, being detected through ELISA method titre proves, antibody-secreting ability is stable.Nude mice is injected intraperitoneally with the cell line
Collect after ascites, the monoclonal antibody in ascites is purified with affinity column HiTrap protein G HP (GE Healthcare)
Anti-CXCL4mAb, reaches 98% (Fig. 6 A-a), and detected with Western blot methods through SDS-PAGE detection antibody purities
Monoclonal antibody and the binding activity (Fig. 6 A-b) of recombined human CXCL4 albumen.The anti-that purifying is obtained is detected with BIAcore
CXCL4mAb and recombined human CXCL4 affinity, through Biaevaluation analysis software processing datas, using one-to-one
Langmuir binding model calculations incorporateds rate (k on) and dissociation yield (k off), equilibrium dissociation constant (k d) are k on and k
Off ratio；It is respectively 1.79 × 10 to calculate obtained Percentage bound (k on) and dissociation yield (k off)-6(1/s) and 97.8 (1/
Ms), equilibrium dissociation constant (kd) is 1.83 × 10-8M (Fig. 6 B).It further have detected the biological activity of anti-CXCL4 monoclonal antibodies, root
ACHN cells are incubated with 10ug/ml-100ug/ml rhCXCL4 albumen according to the literature, MTT detections are found after 72 hours
RhCXCL4 (IC50=72.6ug/ml) effectively inhibits the propagation of ACHN cells, (Fig. 6 C) consistent with document report, and resists
CXCL4 monoclonal antibodies have effectively blocked CXCL4 (72.6ug/ml) to the growth inhibition effects of ACHN cells, and concentration dependant is presented
Property (Fig. 6 D).
1.6 anti-CXCL4 monoclonal antibodies collaboration 5-FU chemotherapy suppress colon cancer tumours cell growth
In vitro on the basis of cell experiment confirmation CXCL4 directly effects no to colon cancer tumours cell, next grind
Study carefully effects of the CXCL4 in vivo to CT26 tumour cells.The anti-CXCL4 monoclonal antibodies prepared and purified using laboratory oneself
(application number：201310066089.7 Chinese invention patent《The anti-monoclonal antibody of chemotactic factor (CF) 4, hybridoma cell line and should
With》) effects of the CXCL4 in CT26 tumour 5-FU chemotherapy is demonstrated from the negative.
In Balb/c right side of mice oxter subcutaneous vaccination mouse colorectal cancer tumour cell CT261 × 106It is individual, treat tumour body
Product is long to 200-300mm3When, packet, including negative control group at random, chemotherapeutical control group, chemotherapy adds monoclonal antibody administration group.Chemotherapy side
Case is disposable tail vein injection chemotherapeutics 5-FU (150mg/kg).Anti- CXCL4 monoclonal antibodies dosage regimen is subcutaneous on the day of being chemotherapy
Administration, dosage 1mg/kg.Same volume PBS, chemotherapeutical control group injection same volume PBS is only subcutaneously injected in PBS control group.Administration
Hypodermic tumour size is measured with slide measure within every 2 days afterwards.Fig. 7 is that anti-CXCL4 monoclonal antibodies combine suppression works of the 5-FU to tumour growth
With schematic diagram, as a result show, compared with independent 5-FU chemotherapy, anti-CXCL4 monoclonal antibodies can cooperate with 5-FU to significantly improve chemotherapy to swollen
Inhibitory action (Fig. 7 A) (n=10, p of knurl growth<0.01).
Fig. 7 B (n=10, p<0.05) it is influence of the anti-CXCL4 monoclonal antibodies to the death rate after tumor-bearing mice chemotherapy, experimental result
Show that anti-CXCL4 monoclonal antibodies also can cooperate with 5-FU to significantly improve the time-to-live after tumor-bearing mice chemotherapy.
The tumor tissues for adding monoclonal antibody administration group to chemotherapeutical control group and chemotherapy are cut into slices, the detection of BrdU immunohistochemical stainings
The proliferative conditions of cell in tumor tissues, such as Fig. 7 C, 7D；As a result show, compared to 5-FU chemotherapeutical control groups, anti-CXCL4 is mono-
Anti- be combined with 5-FU reduces tumour the 2nd after chemotherapy, 4,6 days BrdU positive cell numbers (being in brown colouration in section) (figure
7C), proliferation index statistical discrepancy is notable (p ＜ 0.01) (Fig. 7 D).Proliferation index is counted：Microscope 400 randomly selects 3 under the visual field
The individual visual field, counts BrdU positive cells number (n=3) under each visual field.
Result above shows, compared with independent 5-FU chemotherapy, and anti-CXCL4 monoclonal antibodies are significantly inhibited " to be accelerated to increase again after 5-FU
Grow ", show as collaboration 5-FU and suppress tumor cell proliferation, while monoclonal antibody can also significantly extend tumor-bearing mice chemotherapy with 5-FU combinations
Time-to-live afterwards.This also demonstrates tumour cell after chemotherapy and can obtained by activating expression CXCL4 after chemotherapy from the negative
" acceleration breed again " advantage.
1.7 anti-CXCL4 monoclonal antibodies cooperate with 5-FU chemotherapy suppression colon cancer cell to breed again by acting on T lymphocytes
Anti- CXCL4 monoclonal antibodies collaboration 5-FU improves chemotherapy to the inhibitory action of tumour growth, and this shows tumour cell after chemotherapy
The proliferative advantage of itself can be obtained by activating expression CXCL4.And In vitro cell experiment confirms that CXCL4 does not have to tumour cell
There is direct effect, this indication CXCL4 may help colon cancer cell by interstitial cell of the indirectly-acting in tumor tissues
Obtain proliferative advantage.
First CT26 is set up using the BALB/C nu/nu nude mouses of congenital athymia T lymphocyte development defects as object
Bearing mouse model, oxter subcutaneous vaccination mouse colorectal cancer tumour cell CT261 × 10 on the right side of nude mice6It is individual, treat tumour body
Product is long to 200-300mm3, the random packet afterwards of disposable injection chemotherapeutics 5-FU (150mg/kg), experimental group chemotherapy same day skin
The lower anti-CXCL4 monoclonal antibodies 1mg/kg of injection, control group injection same volume PBS, measures hypodermic tumour in every 2 days after administration with slide measure
Size.Fig. 8 is that anti-CXCL4 monoclonal antibodies combine the inhibitory action schematic diagram that 5-FU transplants nude inoculation tumour growth to T lymphocytes；
As a result show, compared with independent 5-FU chemotherapy, the suppression that the 5-FU chemotherapy that anti-CXCL4 monoclonal antibodies can not cooperate with improves to tumour growth is made
With (Fig. 8 A) (n=6).
Next it is thin to the BALB/C nu/nu nude mices lymph that the healthy BALB/C mice spleen of tail vein transplanting is separated weekly
Born of the same parents 1 × 107It is individual, subcutaneous vaccination CT26 cells 1 × 10 after lymphocyte is transplanted 3 days first6It is individual, treat gross tumor volume length to 200-
300mm3, disposable injection chemotherapeutics 5-FU (150mg/kg).It is subcutaneously injected on the day of random packet, experimental group chemotherapy anti-
CXCL4 monoclonal antibody 1mg/kg, control group injection same volume PBS, hypodermic tumour size are measured with slide measure in every 2 days after administration.Knot
Fruit shows that anti-CXCL4 monoclonal antibodies can cooperate with 5-FU to significantly improve inhibitory action (Fig. 8 B) (n=8, the p of chemotherapy to tumour growth<
0.05)。
Above experimental result all shows that anti-CXCL4 antibody is to cooperate with 5-FU to suppress by acting on T lymphocytic immunities system
Colon cancer cell is bred again, and in other words 5-FU chemotherapy induced tumor cell-stimulating expression CXCL4 can act on T lymphocytes
Enable tumour cell to escape immunosurveillance and obtain proliferative advantage.
The antitumor action of T lymphocytes in 1.8 anti-CXCL4 monoclonal antibodies enhancing tumor tissues
Lymphocyte infiltration has antitumor action, such as cytotoxic T lymphocyte (CTL) and nature in tumor tissues
Killing cell (NK) can be pressed down by secreting (GRAN-b) such as interferon (IFN-γ), granzyme A (GRAN-a) and granzyme Bs
Tumour growth processed.When existing document shows lymphocyte infiltration and patient's prognosis and survival in clinical cancer patient's tumor tissues
Between have significantly correlated.The verified anti-CXCL4 monoclonal antibodies of previous experiments are to cooperate with 5-FU chemotherapy to press down by acting on T lymphocytes
Colon cancer tumours growth processed, it is assumed that the synergistic mechanism of anti-CXCL4 monoclonal antibodies is that anti-CXCL4 monoclonal antibodies can strengthen tumor infiltrating lymphocyte
Antitumor action.
In Balb/c right side of mice oxter subcutaneous vaccination CT26 cells 1 × 106It is individual, treat gross tumor volume length to 200-300mm3
When, packet, including negative control group at random, chemotherapeutical control group, chemotherapy adds monoclonal antibody administration group.PBS control group is only subcutaneously injected together
Volume PBS, chemotherapeutical control group disposably injects chemotherapeutics 5-FU (150mg/kg).Chemotherapy adds monoclonal antibody administration group in 5-FU chemotherapy
Anti- CXCL4 monoclonal antibodies 1mg/kg is subcutaneously injected in same day neck.Put to death mouse within the 4th day after chemotherapy, strip fresh tumor tissue, extract
RNA, the expression quantity of IFN-γ and GRAN-b mRNA in each group mouse tumor tissue are compared using RT-PCR technology.Fig. 9 is RT-
IFN-r and GRAN-b mrna expression amount change schematic diagrams in PCR detection CT26 tumor tissues；As a result show, 5-FU chemotherapy can press down
The expression of IFN-γ and GRAN-b processed, and anti-CXCL4 monoclonal antibodies can significantly improve IFN-γ in chemotherapy tumor tissues (Fig. 9 A) and
GRAN-b (Fig. 9 B) expression (n=3, p<0.05).Above test result indicates that, anti-CXCL4 monoclonal antibodies can significantly increase tumor group
Knit the antitumor action of middle T lymphocytes.
In summary, the present invention by apply colon cancer tumor-bearing mice 5-Fu chemotherapy models, disclose CXCL4 expression change with
Relation between " accelerating to breed again " after colon cancer 5-Fu chemotherapy, illustrates CXCL4 and participates in tumour after chemotherapy " acceleration is bred again " mistake
Molecular mechanism in journey, completes antagonism CXCL4 collaborations 5-Fu and suppresses the pharmacological effect research that colon cancer cell is bred again, the present invention
Successful implementation, provide novel targets and new antibodies medicine for colorectal cancer new drug development, provided newly for colorectal cancer drug therapy
Thinking；Thus, with great social effect and economic value.
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (4)
1. purposes of a kind of anti-CXCL4 monoclonal antibodies in medicine for suppressing tumor proliferation is prepared, it is characterised in that described anti-
CXCL4 monoclonal antibodies are that preserving number is CCTCC NO：The antibody that C2012186 hybridoma cell strain is produced, the tumour is
The colon cancer tumours of CXCL4 height expression caused by 5-FU chemotherapy.
2. purposes as claimed in claim 1, it is characterised in that the 5- used when the anti-CXCL4 monoclonal antibodies are with chemotherapy
FU drug combinations.
3. purposes as claimed in claim 2, it is characterised in that anti-CXCL4 monoclonal antibodies CXCL4 after chemotherapy is expressed
Level reaches to be applied before highest.
4. purposes as claimed in claim 3, it is characterised in that after the 5-FU medications within 4 days, carries out anti-CXCL4 Dan Ke
Grand antibody administration.
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| CN103173419A (en) * | 2013-03-01 | 2013-06-26 | 上海交通大学 | Chemokine-4 resisting monoclonal antibody, chemokine-4 resisting hybridoma cell line and applications of monoclonal antibody |
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| CN103173419A (en) * | 2013-03-01 | 2013-06-26 | 上海交通大学 | Chemokine-4 resisting monoclonal antibody, chemokine-4 resisting hybridoma cell line and applications of monoclonal antibody |
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