CN104262459B - Acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide, and vaccine and pharmaceutical application of acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide - Google Patents
Acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide, and vaccine and pharmaceutical application of acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide Download PDFInfo
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Abstract
The invention discloses an acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide and an application of the acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide, and belongs to the technical field of biology. The acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide disclosed by the invention can induce specific CTL, and simultaneously can express tumor cells of MLAA-34 and HLA-A*0201, and T2 cells of loaded peptide in vitro to generate the determined specific killing effect; the epitope polypeptide CP701 is applied to preparation of a polypeptide vaccine aiming at acute monocytic leukemia; and the polypeptide vaccine composed of an MLAA-34 epitope peptide CP701 (236ILDRHNFAI244), a T auxiliary epitope and an incomplete Freund adjuvant can inhibit hu-PBL-SCID amplification of tumors in mice loading human monocytic leukemia cells THP-1, prolongs the survival life, and can induce the specific CTL killing activity and NK cytotoxic activity in vivo.
Description
Technical field
The invention belongs to biological technical field is and in particular to a kind of acute monocytic leukemia related antigen mlaa-34
Epitope polypeptide and its vaccine and pharmacy application.
Background technology
Acute myeloid leukemia (acute myeloid leukemia, aml) prognosis is poor, particularly with bad dyeing
Body or molecule abnormality person are higher mainly due to relapse rate after chemotherapy inducer remission.Microresidual disease is eradicated after alleviation
The treatment of (microresidual disease, mrd) still has huge challenge.Allogeneic Hematopoietic Stem Cell Transplantation
(hematopoietic stem cell transplantation, hsct) shows effective leukemia resisting action, but because it is joined
Type is difficult, somewhat expensive is it is often more important that itself have the complication of some threat to life, such as graft versus host disease (graft
Versus host disease, gvhd), only it is suitable to the young suitable patient of small part.Therefore, need development badly based on different anti-
The new Therapeutic Method of leukemia mechanism.Immune system has the exquisite ability that specific killing expresses special cell antigen, this
Species specificity is that immunization therapy can be removed tumor cell and not injured the core of normal cell.Immunology recent advances and have valency
The identification of the LAA of value enables antigen specific immunotherapy to eradicate leukemia mrd after chemotherapy, is expected to change
Total existence of kind aml patient.
Acute monocytic leukemia (acute monocytic leukemia, m5) is more common type in aml,
In China aml, m5 sickness rate is only second to m2, accounts for the 23.2%-26.9% of aml hence it is evident that reporting higher than western countries beutler e etc.
8% leading.Most of m5 patients with clinical manifestations are hypercytosis, easily occur outer marrow infiltration (to include liver, spleen, lymph node, tooth
Gum, skin, eyes, larynx, lung, bladder, meningess, central nervous system), bad karyotype ratio is high, complete incidence graph
(complete remission, cr) rate is low, easy recurrence, prognosis be not good.The heterogeneity of m5 and complexity determine that it still lacks point
Son diagnosis and the specific tumour Research of predicting markers of targeted therapy.
Acute monocytic leukemia related antigen -34 (monocytic leukemia associatedantigen-
34, mlaa-34) it is the seminar of inventor using " restructuring cdna expression library serological analysis method (serological
Identification of antigens by recombinant expression cloning, serex) " to people's monokaryon
The representative neoantigen of cell leukemia cell line screening.Utilize 5 ' and 3 '-cdna end rapid amplifying (rapid afterwards
Amplication of cdna ends, race) technology amplification obtain mlaa-34 total length cdna sequence, sequence number is:
ay288977.2.With the opening code-reading frame finder software analysis of ncbi website (www.ncbi.nlm.nih.gov), find
Mlaa-34 gene can be located at human chromosome 13q14.2；It is the calbindin sample 39 (calcium of Unknown Function
Binging protein 39-like, cab39l) gene new transcript splice variants.Application rnai technical research is sent out
Showing mlaa-34 gene is, with acute monocytic leukemia, related new anti-apoptotic molecule, the table excessively of lentivirus mediated occur
The u937 cell reaching mlaa-34 can significantly inhibit apoptosis, and increases potential oncogenicity.Co-immunoprecipitation, shot gun method is surveyed
Sequence and bioinformatic analysis show that 71 kinds of protein are related to bioprocess and the signal path of apoptosis or propagation.It is up till now
Only, we have applied the real time fluorescent quantitative RT -PCR (reverse of sybr green fluorescent dye determination
Transcription polymerase chain reaction, rt-pcr) and western blot technology white to m5, non-m5
PERIPHERAL BLOOD MONONUCLEAR CELL (the peripheral blood mononuclear of disorders of blood bone marrow blast and normal health
Cells, pbmcs) in expression detected, find: the mrna of mlaa-34 gene and protein level are white in aml-m5 type
It is all specificity overexpression in disorders of blood cell, and low table table or do not express in non-m5 patient and normal healthy controls.
In recent years, the polypeptide vaccine from LAA has been carried out clinical experiment, and aml polypeptide vaccine has
Wt1 polypeptide vaccine, rhamm polypeptide vaccine etc., these polypeptide vaccines have certain curative effect to aml active immunity treatment, but m5 is suffered from
Whether person is specifically effective vaccine, and row is individually studied.Have not yet to see application mlaa-34 polypeptide vaccine immunization therapy anxious
The report of property monocytic leukemia.
Polypeptide vaccine is the aminoacid sequence according to certain section of epitope that is known in pathogen/tumor antigen gene or predicting
Row, the vaccine prepared by chemical synthesising technology.Tumour polypeptide vaccine has the advantage that antigenic peptides such as directly stimulate generation special
Specific immunological reaction is without induction autoimmune response or immunosuppressant；Production process is simple and quick cheap, relatively easily
In large-scale production；Non-carcinogenesis, safety.The greatest weakness of single polypeptide vaccine is that immunogenicity is poor, polypeptide easy quilt in vivo
Peptide enzymatic degradation, the ctl being induced is difficult to break through the threshold value making needed for tumor regression.If although wt1 epi-position peptide vaccine is in clinical examination
Win initial success in testing, but due to current researcher often using the peptide of single ctl epi-position, molecular weight is little is easy to by albumen water
Solution enzymatic degradation, can only excite low-level immunne response, by the multiple ctl epi-positions of joint, increase th epi-position, use immunity assistant
Agent, the structure method such as multi-epitope peptide and fusogenic peptide can improve the immunogenicity of polypeptide.
Th epi-position can activate cd4 in vivo+T cell, cd4+T cell is exciting and to maintain immunization therapy to play important
Effect, therefore add th epi-position in epi-position peptide vaccine and become improve epiposition vaccine immunogenicity with strengthening t cell effect one
Some approach.Th epi-position also can activate cd4+Ctls and directly kill tumor cell.Though adjuvant itself can not effectively cause
Anti tumor immune response, but can enhancing non-specific immunity response, participate in lymphopoiesis and differentiation, and table can be protected
Position peptide is difficult to be easily degraded by proteases.Conventional adjuvant has Granulocyte Colony-stimulating (gm-csf), il-2, heat shock protein
White and incomplete Freund's adjuvant (incomplete freund's adjuvant, ifa) etc..
Content of the invention
It is an object of the invention to provide a kind of acute monocytic leukemia related antigen mlaa-34 epitope polypeptide and its
Through zoopery, vaccine and pharmacy application, verify that this polypeptide vaccine has the effect of anti-acute monocytic leukemia.
The present invention is to be achieved through the following technical solutions:
A kind of acute monocytic leukemia related antigen mlaa-34 epitope polypeptide, this epitope polypeptide is cp701, its ammonia
Base acid sequence is as shown in seq.id.no.1.
A kind of acute monocytic leukemia related antigen mlaa-34 epitope polypeptide vaccine, this polypeptide vaccine contains one
Cp701 epitope polypeptide and a t cell auxiliary epitope polypeptide being used for strengthening vaccine immunogenicity.
It is tetanus toxoid tt830-843 that the described t cell for strengthening vaccine immunogenicity assists epitope polypeptide,
Its aminoacid sequence is as shown in seq.id.no.2.
It is also added with the incomplete Freund's adjuvant for strengthening vaccine immunogenicity in described polypeptide vaccine.
Acute monocytic leukemia related antigen mlaa-34 epitope polypeptide is directed to acute monocytic leukemia in preparation
Medicine in application.
Described medicine is the medicine of suppression people acute monocytic leukemia thp-1 propagation.
Described medicine is the medicine of inducing specific ctl killing activity and nk cytotoxic activity.
Described medicine is to stimulate cd3+cd8+The lymphopoietic medicine of t.
Described medicine is the medicine stimulating t cell to secrete ifn- γ and il-2.
Compared with prior art, the present invention has a following beneficial technique effect:
The acute monocytic leukemia related antigen mlaa-34 epitope polypeptide cp701 that the present invention provides, can induce spy
Different in nature ctl, the t2 cell to the tumor cell expressing mlaa-34 and hla-a*0201 and load cp701 polypeptide simultaneously in vitro
Produce the specific killing action determining, be the restricted ctl epi-position of effective mlaa-34 antigen hla-a*0201.
A kind of acute monocytic leukemia related antigen mlaa-34 epitope polypeptide vaccine, containing mlaa-34 epitope peptide
cp701(236ildrhnfai244), the polypeptide vaccine of t helper epitope and incomplete Freund's adjuvant composition lotus people can be suppressed single
Human peripheral lymphocyte immunologic reconstitution severe combined immunodeficiency (hu-pbl-scid) of monocytic leukaemia cells thp-1
Mouse tumor body is bred, and extends its existence, can induce specificity ctl killing activity and nk cytotoxic activity in vivo.By a series of
Interior animal experiment confirm its anti-acute monocytic leukemia effect possessing:
1、mlaa-34 cp701(236ildrhnfai244) polypeptide vaccine has obvious suppression to tumor-bearing mice tumour growth
Effect, makes tumor thp-1 cell significantly reduce, a large amount of degeneration necrosis of leukaemia；
2nd, mlaa-34 polypeptide vaccine can extend the existence of lotus thp-1 leukaemia hu-pbl-scid Mus.Mlaa-34 polypeptide
The mean survival time of vaccine group tumor-bearing mice is considerably longer than wt1 polypeptide vaccine group, the thp-1 Cell vaccine group of inactivation, t auxiliary
Polypeptide vaccine group and the mean survival time of matched group tumor-bearing mice；
3rd, mlaa-34 polypeptide vaccine treatment group splenocyte compares thp-1 cell ctl specificity and nk cell in different effect targets
Nonspecific killing-efficiency is obviously higher than blank control group, wt1 polypeptide vaccine group, t Adjuvant Polypeptide vaccine group and inactivation
Thp-1 tumor vaccine group, increases with effect target ratio, killing-efficiency substantially increases.Mlaa-34 polypeptide vaccine treatment group splenocyte is not to
Compare thp-1 cell-specific ctl with effect target and the nonspecific killing-efficiency of nk cell is white obviously higher than person monocytic cell
Disorders of blood cell u937, human breast cancer cell mcf-7 and human lung adenocarcinoma cell a549；
4th, mlaa-34 polypeptide vaccine treatment group cd3+cd8+The ratio of t cell is significantly higher than other groups, and treg cell
Ratio be substantially less than other groups.The t cell secretion ifn- that mlaa-34 polypeptide vaccine treatment group mlaa-34 synthetic peptide stimulates
γ and il-2 substantially increases, and there were significant differences compared with matched group, and remaining each group and matched group have different than also, but ifn-
γ and il-2 secretory volume is significantly lower than mlaa-34 polypeptide vaccine treatment group.Detection il-10 and il-4 result shows, mlaa-34 is many
T cell secretion il-10 and il-4 that peptide vaccine treatment group mlaa-34 synthetic peptide stimulates is substantially less than other each groups.
Shown by study on mechanism, the epitope polypeptide of the present invention can be efficiently applied to preparation for anxious for cp701
The medicine of property monocytic leukemia, is that the treatment of m5 type acute myeloid leukemia opens new research direction.
Brief description
Fig. 1 is the he coloring pathological section photo of each experimental group and matched group；Wherein, (a) transplants tumor tissue for matched group
Pathological section；B () is t auxiliary peptide vaccine treatment group transplanted tumor tissue pathological slice；C () is the thp-1 Cell vaccine group of inactivation
Transplanted tumor tissue pathological slice；D () is wt1 polypeptide vaccine treatment group transplanted tumor tissue pathological slice；E () is mlaa-34 polypeptide
Vaccine therapy group transplanted tumor tissue pathological slice；
Fig. 2 is the life span comparison diagram of different vaccine therapy group mices；
Fig. 3 is the kaplan-meier survival analysises figure of different vaccine therapy group mices；
Fig. 4 is different vaccines to thp-1 cell (hla-a*0201+mlaa34+) ctl killing activity measure figure；
Fig. 5 is the ctl toxicity test figure to different cells for the cp701 polypeptide vaccine；
Fig. 6 is different vaccines to thp-1 cell (hla-a*0201+mlaa34+) nk determination of cytotoxic activity figure；
Fig. 7 is the nk determination of cytotoxic activity figure to different cells for the cp701 polypeptide vaccine；
Fig. 8 is cd3 in different immunization therapy group lotus knurl hu-pbl-scid Mus peripheral bloods+cd4+T raji cell assay Raji figure；Wherein,
A () is matched group；B () is wt1 polypeptide vaccine group；C () assists peptide vaccine group for t；D () is mlaa-34 polypeptide vaccine group；(e)
Thp-1 Cell vaccine group for inactivation；
Fig. 9 is cd3 in different immunization therapy group lotus knurl hu-pbl-scid Mus peripheral bloods+cd8+T raji cell assay Raji figure；Wherein,
A () is matched group；B () is wt1 polypeptide vaccine group；C () assists peptide vaccine group for t；D () is mlaa-34 polypeptide vaccine group；(e)
Thp-1 Cell vaccine group for inactivation；
Figure 10 is treg raji cell assay Raji figure in different immunization therapy group lotus knurl hu-pbl-scid Mus peripheral bloods；Wherein, (a)
For matched group；B () is wt1 polypeptide vaccine group；C () assists peptide vaccine group for t；D () is mlaa-34 polypeptide vaccine group；E () is to go out
The thp-1 Cell vaccine group lived；
Figure 11 is the detection figure of ifn- γ, il-2, il-10, il-4 in peripheral blood；Wherein, (a) is ifn- γ；B () is
il-2；C () is il-10；D () is il-4；
Figure 12 is the gross specimen photo of different vaccine therapy group tumor bodies.
Specific embodiment
With reference to specific embodiment, the present invention is described in further detail, described be explanation of the invention and
It is not to limit.
The invention provides a kind of acute monocytic leukemia related antigen mlaa-34 epitope polypeptide, it is cp701 ctl
Epitope peptide nonapeptide, its aminoacid sequence is236ildrhnfai244(as shown in seq.id.no.1), molecular weight is 1098.8da.
The invention provides a kind of new anti-acute monocytic leukemia polypeptide vaccine, give the system of this polypeptide vaccine
Preparation Method, and confirm the curative effect of the anti-acute monocytic leukemia of this polypeptide vaccine by zoopery.The group of this polypeptide vaccine
+ Freund is not to become mlaa-34 cp701 polypeptide (50 μ g)+versatility t helper epitopes (t) (tetanus endotoxin) (50 μ g)
Freund's complete adjuvant (incomplete freund ' s adjuvant, ifa) (1ml).
Wherein, acute monocytic leukemia antigen mlaa-34 cp701 ctl epitope peptide is nonapeptide, aminoacid sequence
For236ildrhnfai244, molecular weight is 1098.8da, and t helper epitopes are tetanus toxoid (tt830-843), amino
Acid sequence is qyikanskfigite.Sequence according to design, manually synthesizing, specific entrusts Shanghai biotechnology of shining by force to have
Limit company synthesizes this 2 polypeptides using Peptide synthesizer, using the synthesis of semi-automatic solid-phase polypeptide method, the purification of polypeptide and purity
Analysis adopts rp-hplc (waters company of the U.S.) method, with Electrospray Mass Spectrometry (electrospray ionization mass
Spectrometers, esi-ms) tandem mass spectrum (pe company of the U.S.) measures its molecular weight and identified.mlaa-34 cp701
Polypeptide and t helper epitopes peptide purity all reach more than 95%.The polypeptide lyophilized powder of synthesis, is dissolved in the concentration of 10 μm of ol/l
In dmso, standby under the conditions of being placed in -20 DEG C.
Early stage adopts Bioinformatics Prediction, semi-automatic solid-phase polypeptide method to synthesize mlaa-34 epitope peptide, classical peptide-mhc
Adhesion experiment display mlaa-34 epitope peptide cp701 (236ildrhnfai244) with hla-a*0201, there is very strong affinity,
And experiment in vitro display mlaa-34 epitope peptide cp701 (236ildrhnfai244) can be with inducing specific ctl, to expressing simultaneously
The t2 cell of the tumor cell of mlaa-34 and hla-a*0201 and load cp701 polypeptide produces the specific killing action determining,
These results prompting cp701 (236ildrhnfai244) it is the restricted ctl epi-position of effective mlaa-34 antigen hla-a*0201.
The invention discloses acute monocytic leukemia related antigen mlaa-34 epitope polypeptide is directed to acute list in preparation
Application in the medicine of monocytic leukaemia, this medicine is biological genetic engineering antibody medicine.
The mensure of leukemia curative effect in the mlaa-34 polypeptide vaccine body of the present invention:
First, prepare vaccine
1st, polypeptide vaccine: prepared before use.
Polypeptide therapeutic group vaccine consists of polypeptide (50 μ g)+versatility t helper epitopes (t) (tetanus endotoxin)
(50 μ g)+incomplete Freund's adjuvant (incomplete freund ' s adjuvant, ifa)；
Wt1 polypeptide vaccine group as wt1 polypeptide (126rmfpnapyl134, 50 μ g) and+t (50 μ g)+ifa, t Adjuvant Polypeptide epidemic disease
Seedling group is t (50 μ g)+ifa；
Blank group is iaf.
Antigen and ifa adjuvant are administered to mice by after the complete emulsifying of 100 μ g:1ml.
2nd, the thp-1 Cell vaccine preparation of inactivation: well-grown thp-1 cell of taking the logarithm, add mitomycin c dense eventually
Spend and inactivate thp-1 cell for 20mg/ml, after 37 DEG C of effect 30min, with 2000 revs/min of centrifugation 5min, abandon supernatant, pbs washes 2
Time, adjust cell number 1 × 106/ ml, in pbs, adds ifa, the volume ratio of the two is 1:1.
2nd, the foundation of animal model
Rebuild after scid mouse immune system 24h with hla-a*0201 positive human peripheral lymphocyte, subcutaneous vaccination
Thp-1 cell, sets up the lotus knurl scid mouse model of people's immunologic reconstitution；
1st, human peripheral lymphocyte separates
(1) the White Blood Cells Concentrate suspension of Healthy People is purchased from Xi'an City Central Blood Supply Station, adopts after hla-a*0201 antibody mark
Flow cytomery, using hla-a*0201 positive as object of study.
(2) add 5ml lymphocyte separation medium in 10ml centrifuge tube, take healthy women volunteer's anticoagulant heparin venous blood
5ml, is slowly superimposed on lymphocyte separation medium liquid level along tube wall with dropper, keeps clearly interface, 2000rpm horizontal centrifugal
20min.
(3) it is divided into three layers in pipe after being centrifuged, upper strata is serum, lower floor is mainly erythrocyte and granulocyte.Middle level is lymph
Cell separation liquid, has the white based on mononuclearcell (including lymphocyte and mononuclear cell) in upper, middle level interface
Cloud and mist layer is tunica albuginea layer.
(4) it is inserted into cloud and mist layer with capillary pipette, draw mononuclearcell.Insert in another 1.5ml centrifuge tube, add pbs
Liquid, 1200rpm is centrifuged 5min, and washed cell is twice.
(5) after final centrifugation, abandon supernatant, add 1mlpbs liquid re-suspended cell.Take a cell suspension in cell counting count board
On, count the total cellular score in four block plaid.
Mononuclearcell concentration (cell number/1ml cell suspension)=4 block plaid inner cell sum/4 × 104
(6) with pbs re-suspended cell and adjust cell density to be 8 × 10 after being separated with lymphocyte separation medium is conventional7/ml.
2nd, the qualified c.b17/scid mouse inbred liness of selection (having used elisa method to measure Mus igg ＜ 1 μ g/ml), 6-8 week old,
Female, closes the immunologic reconstitution of nk cytoactive laggard pedestrian peripheral blood lymphocyte with anti-asialo gm1.
μ l anti-asialo gm1 is to close nk cytoactive, parallel x for -1d: every scid mouse peritoneal injection 20
Line sublethal irradiation (3gy, x line ionization chamber m-150we).
0d: immunologic reconstitution: (concentration is 8 × 10 to lumbar injection 0.5ml peripheral blood lymphocyte7/ml).
1d: inoculate the exponential phase thp-1 of In vitro culture under every hu-pbl-scid Corium Mus after immunologic reconstitution 24h
(concentration is 1 × 10 to cell7Individual/mlpbs), every about axillary fossa and totally 3 points of back, every inoculum concentration is 0.2ml.
3rd, experiment packet, selects no immune seepage sciid Mus 45, is randomly divided into 5 groups.
First group 9, blank group: vaccine composition is only incomplete Freund's adjuvant；
Second group 9, wt1 polypeptide vaccine group: vaccine composition is wt1 polypeptide+versatility t helper epitopes (tetanus
Endotoxin)+incomplete Freund's adjuvant；
3rd group 9, t Adjuvant Polypeptide vaccine group: vaccine composition is versatility t helper epitopes (tetanus endotoxin)
+ incomplete Freund's adjuvant；
4th group 9, mlaa-34 polypeptide vaccine treatment group: vaccine composition is above preferred mlaa-34 polypeptide
(cp701,236ildrhnfai244)+versatility t helper epitopes (tetanus endotoxin)+incomplete Freund's adjuvant；
5th group: 9, the thp-1 Cell vaccine group of inactivation: vaccine composition is the thp-1 Cell vaccine of inactivation.
4. vaccination
When gross tumor volume is about 100-120mm3When, three are inferior to dorsal sc vaccination, every time inoculation interval one week.Exempt from
Epidemic disease treatment end 14d, puts to death scid Mus, and each group stays 6 observation of nature life spans respectively at random.
5.hla-a*0201 detects
Scid mice hla-a*0201 positive human peripheral lymphocyte immunologic reconstitution gathered mouse peripheral blood after 2 weeks, drenched
Lymphocyte, after employment hla-a*0201 labeling of monoclonal antibody, flow cytometer is obtained after the conventional separation of bar cell separation liquid
Measure the positive lymphocyte of people hla-a*0201 in display Mouse Peripheral Blood Lymphocyte and account for 95%, point out scid mouse immune
Rebuild successfully, and the hla type of the human lymphocyte in mice body is hla-a*0201.
6. in serum people igg mensure
Before putting to death scid Mus, gather non-anticoagulated blood through eyeball, measure people igg with elisa method.
(1) blood specimen dress centrifuge tube, centrifugation, 37 DEG C, 1500rpm × 20min, separate and obtain serum, -80 DEG C of preservations, institute
There is specimen to detect simultaneously；
(2) take out ELISA Plate, according to the corresponding standard substance adding 100 μ l respectively of order and quality-control product in blank micropore；
(3) difference labelling sample number into spectrum, plus 100 μ l samples are in blank micropore；
(4) every hole adds the enzyme label solution of 50 μ l, 18-25 DEG C of incubation reaction 90min；
(5) wash trigger to clean 5 times, stand 10-20s every time；
(6) every hole adds each 50 μ l of substrate a, b liquid, 18-25 DEG C of lucifuge incubation reaction 15min；
(7) every hole adds 50 μ l terminate liquids, terminating reaction；
(8) the od value in each hole is read on microplate reader, wavelength is 450nm.
After 2 weeks, elisa method just can detect people in mice serum to human peripheral lymphocyte (pbl) lumbar injection
igg.4th week different group mice serum people's igg level: blank control group (3.84 ± 0.20mg/ml), wt1 polypeptide vaccine group
(8.42 ± 0.28mg/ml), t auxiliary peptide vaccine group (6.36 ± 0.35mg/ml), mlaa-34 polypeptide vaccine group (17.20 ±
2.21mg/ml), thp-1 Cell vaccine group (4.66 ± 0.30mg/ml) of inactivation.Transplant the 6th week blank control group mice serum
People's igg level is 6.18 ± 0.36mg/ml, and mlaa-34 polypeptide vaccine group is 37.31 ± 4.68mg/ml, significantly increase (p <
0.01).8th week mlaa-34 polypeptide vaccine group mice serum people's igg level is 76.18 ± 8.36mg/ml, in gradually rising
Gesture.
3rd, observation of curative effect
Scid mice tumor volume after measurement immunization therapy in every 3 days after subcutaneous vaccination thp-1 cell, tumor volume (mm3)
=tumor body length × (tumor body width)2×π/6.14d after immunization therapy, each group takes scid mice three at random, and place after death, takes
Tumor body tissue carries out conventional fixing, paraffin embedding and section, observes cancer pathology and changes (he dyeing).Observing and nursing mouse survival
Phase, determine the internal action effect of polypeptide vaccine.
Referring to Figure 12, after immunization therapy 2 weeks, tumorous size is respectively as follows: matched group (11.06 ± 5.64cm3), t assists peptide epidemic disease
Seedling group (7.28 ± 2.21cm3), the thp-1 Cell vaccine group (6.82 ± 1.70cm of inactivation3), wt1 polypeptide vaccine group (3.03 ±
0.33cm3), mlaa-34 polypeptide vaccine group (1.39 ± 0.36cm3).As can be seen here, mlaa-34 polypeptide vaccine swells to tumor-bearing mice
Tumor has obvious growth inhibition effect (p=0.002), and tumour growth is slow, and matched group and simple t assist peptide vaccine group, go out
The thp-1 Cell vaccine group tumor lived is in that progressive grows.Compared with matched group, wt1 polypeptide vaccine also has significantly to tumor
Inhibitory action (p=0.006) is it can be seen that mlaa-34 polypeptide vaccine can suppress lotus thp-1 leukaemia hu-pbl-scid
Mus tumor bulk-growth.
Referring to Fig. 1, the pathological section of each experiment packet he dyeing of (a)～(e), visible in matched group he pathological section
A large amount of leukaemias, growth substantially enlivens, uniform in size, and form is consistent, clear-cut, and cell membrane is complete, and karyon is larger,
Dyeing is dark blue.Compared with matched group, t auxiliary peptide vaccine treatment group thp-1 cellular morphology is unchanged；The thp-1 glucagonoma of inactivation
Seedling group visible fraction thp-1 cell death, after birth is damaged, not of uniform size, soft edge；Wt1 polypeptide vaccine group majority cell
Form is irregular, and after birth is damaged, nucleus shrinkage, most leukaemia's degeneration necrosis；Mlaa-34 polypeptide vaccine group tumor is cut
In piece, thp-1 cell significantly reduces, and cell outline obscures, and after birth is damaged, karyopycnosis fragmentation, and a large amount of degeneration of leukaemia is bad
Extremely.
Referring to Fig. 2, observe the life cycle of mice, result show, the matched group tumor-bearing mice mean survival time for 48.50 ±
4.89d；mlaa-34 cp701(236ildrhnfai244) polypeptide vaccine treatment group and wt1 polypeptide vaccine group tumor-bearing mice averagely raw
Time of depositing is respectively 88.33 ± 1.63d and 66.17 ± 3.06d, the thp-1 Cell vaccine group of t Adjuvant Polypeptide vaccine group and inactivation
The tumor-bearing mice mean survival time is respectively 52.50 ± 3.02d and 54.34 ± 3.14d.mlaa-34 cp701
(236ildrhnfai244) polypeptide vaccine treatment group, wt1 polypeptide vaccine group and inactivation thp-1 Cell vaccine group tumor-bearing mice
Mean survival time is considerably longer than matched group (p < 0.01, p < 0.01, p=0.034), and t Adjuvant Polypeptide vaccine group tumor-bearing mice
Mean survival time compared with matched group, there was no significant difference (p=0.119).T Adjuvant Polypeptide vaccine group and the thp- of inactivation
The mean survival time of 1 Cell vaccine group tumor-bearing mice is compared, and also there was no significant difference (p=0.334), and this two groups with
mlaa-34 cp701(236ildrhnfai244) polypeptide vaccine group compares with wt1 polypeptide vaccine group, have significant difference (p <
0.01, p < 0.01, p < 0.01, p < 0.01).mlaa-34 cp701(236ildrhnfai244) polypeptide vaccine group tumor-bearing mice flat
All life spans are longer than wt1 polypeptide vaccine group (p < 0.01).
Referring to Fig. 3, survival analysises are carried out using kaplan-meier, result shows compared with matched group, mlaa-34
cp701(236ildrhnfai244) polypeptide vaccine can significantly improve the existence (p < 0.01) of thp-1 tumor-bearing mice.Wt1 polypeptide vaccine
Also improve the existence (p=0.01, p=0.007) of thp-1 tumor-bearing mice with the thp-1 Cell vaccine of inactivation, and simple t is auxiliary
Help polypeptide vaccine fail to be obviously improved thp-1 tumor-bearing mice long term survival (p=0.055) it can be seen that, mlaa-34 polypeptide
Vaccine can extend the existence of lotus thp-1 leukaemia hu-pbl-scid Mus.
4th, study on mechanism
After vaccine injection, detect leukemia mouse ctl activity and nk cytotoxic activity, Peripheral T Lymphocyte Subsets and treg
The change of ifn- γ, il-2, il-10, il-4 in cell, peripheral blood, to determine action effect and the mechanism of action of different vaccines.
1st, ctl activity and nk determination of cytotoxic activity
1), when Leukemia Model mice gives vaccine injection and terminates rear 14d, detect that its specificity ctl killing activity and nk are thin
Born of the same parents' cytotoxic activity；
2) ctl determination of activity
Prepared by effector lymphocyte: take each experiment and the splenocyte of control group mice to prepare single cell suspension under aseptic condition, use
Rpmi RPMI-1640 (containing 10%fcs) adjustment cell concentration is to 5 × 106/ ml, 3ml/ hole is seeded to 6 orifice plate cultures.Add
Mlaa-34 polypeptide cp701 (236ildrhnfai244), 50 μ g/ holes.Recombined human il-2 20u/ml is added (every three days in culture medium
Supplement once).After culture 5d, 1000rpm is centrifuged 5min, collects cell, and counts, as effector lymphocyte.
Prepared by target cell: collect exponential phase thp-1 cell (hla-a2+mlaa-34+), human monocyte cell line u937
(hla-a2-mlaa-34+), human breast cancer cell mcf-7 (hla-a2+mlaa-34-) and human lung adenocarcinoma cell a549 (hla-a2-
mlaa-34-), with the rpmi RPMI-1640 adjustment target cell concentration containing 5%fcs to 1 × 105/ml.
Effect/target cell co-cultures: in different effects target ratio (50:1,25:1,12.5:1 and 6.25:1) plus effector lymphocyte and
The each 50 μ l of target cell, in 96 well culture plates (3 holes are repeated), cultivate 4h, measure ctl activity with lactic dehydrogenase enzyme process.
3) nk determination of cytotoxic activity
Take mouse spleen under aseptic condition, prepare single cell suspension action effect cell.Collect exponential phase thp-1 thin
Born of the same parents (hla-a2+mlaa-34+), human monocyte cell line u937 (hla-a2-mlaa-34+), human breast cancer cell mcf-7 (hla-a2+
mlaa-34-) and human lung adenocarcinoma cell a549 (hla-a2-mlaa-34-), with the rpmi RPMI-1640 adjustment containing 5%fcs
Cell concentration is to 4 × 105/ ml, as target cell.Remaining operating procedure is with ctl determination of activity.
Referring to Fig. 4～7, external ctl killing activity result: mlaa-34 cp701 (236ildrhnfai244) polypeptide vaccine controls
Treatment group splenocyte compares thp-1 cell (hla-a2 in different effect targets+mlaa34+) specificity ctl and nk cell killing-efficiency equal
Apparently higher than blank control group, wt1 polypeptide vaccine group, t Adjuvant Polypeptide vaccine group and inactivation thp-1 Cell vaccine group (p <
0.05), increase with effect target ratio, killing-efficiency substantially increases.mlaa-34 cp701(236ildrhnfai244) polypeptide vaccine controls
Treatment group splenocyte compares thp-1 cell (hla-a2 in different effect targets+mlaa34+) specificity ctl and nk cell killing-efficiency
Obviously higher than human monocyte cell line u937 (hla-a2-mlaa-34+), human breast cancer cell mcf-7 (hla-a2+mlaa-34-)
With human lung adenocarcinoma cell a549 (hla-a2-mlaa-34-)(p<0.05).These experiment in vivo results point out mlaa-34 cp701
(236ildrhnfai244) polypeptide vaccine induction ctl specific cytotoxicity there is mhc (hla-a*0201) and mlaa-34 epi-position
Dual restricted.
2nd, in peripheral blood t cell subsets and treg cell detection
Leukemia Model mice gives vaccine injection when terminating rear 14d, and eyeball takes blood.The mensure of t cell subsets, taking heparin
Sodium anticoagulated whole blood 50 μ l adds 10 μ l percp-cd3/fitc-cd4/pe-cd8 tri- len antibody, lucifuge incubation 30min at 4 DEG C,
Haemolysis and after washing with facs detection, and adopt bdfacsdiva software analysis data, to analyze cd4+、cd8+The change of t cell
Change.With cd4, cd25 monoclonal antibody labelling cell, detect the change of treg cell with facs.
Referring to Fig. 8～10, cd3 in the different immunization therapy group of detection+cd4+T cell proportion: matched group (13.2%), wt1
Polypeptide vaccine group (21.7%)；T auxiliary peptide vaccine group (23.8%), mlaa-34 polypeptide vaccine treatment group (25.4%), inactivation
Thp-1 Cell vaccine group (16.3%), mlaa-34 polypeptide vaccine treatment group cd3+cd4+T cell proportion is significantly higher than matched group (p
< 0.05), raise than other groups, no significant difference；cd3+cd8+T cell proportion: matched group (15.7%), wt1 is many
Peptide vaccine group (18.3%)；T auxiliary peptide vaccine group (16.5%), mlaa-34 polypeptide vaccine treatment group (58.7%), inactivation
Thp-1 Cell vaccine group (17.1%).Mlaa-34 polypeptide vaccine treatment group cd3+cd8+The ratio of t cell is significantly higher than other groups
Not (p < 0.05).The ratio of treg cell: matched group (45.2%), wt1 polypeptide vaccine group (37.7%)；T assists peptide vaccine group
(40.6%), mlaa-34 polypeptide vaccine treatment group (16.4%), thp-1 Cell vaccine group (41.7%) of inactivation.mlaa-34
The ratio of polypeptide vaccine treatment group treg cell is substantially less than other groups (p < 0.05).
3rd, in peripheral blood ifn- γ, il-2, il-10, il-4 detection
Leukemia Model mice gives vaccine injection when terminating rear 14d, and eyeball takes blood.Application elisa method, detects respectively
The level of il-2, ifn- γ, il-4, il-10 in serum.
Detection ifn- γ result shows, referring to Figure 11, mlaa-34 cp701 (236ildrhnfai244) polypeptide vaccine treatment
The t cell secretion ifn- γ that group mlaa-34 synthetic peptide stimulates substantially increases, and there were significant differences compared with matched group (p < 0.05),
Remaining each group and matched group have different than also, but ifn- γ secretory volume is significantly lower than mlaa-34 cp701
(236ildrhnfai244) polypeptide vaccine treatment group (p < 0.05).Detection il-2 result shows, mlaa-34 cp701
(236ildrhnfai244) the t cell secretion il-2 that stimulates of polypeptide vaccine treatment group mlaa-34 synthetic peptide is significantly higher than other each groups
(p<0.05).Detection il-10 and il-4 result shows, mlaa-34 cp701 (236ildrhnfai244) polypeptide vaccine treatment group
T cell secretion il-10 and il-4 that mlaa-34 synthetic peptide stimulates is substantially less than other each groups (p < 0.05).The above results are pointed out
Mlaa-34 polypeptide can induce th1 and th2 activation, and th1 cytokines level dramatically increases, under th2 cytokines level
Fall, gradually can be developed to th1 direction with inducing cellular immune response, induce ctl response.
Claims (8)
1. a kind of acute monocytic leukemia related antigen mlaa-34 epitope polypeptide vaccine is it is characterised in that this polypeptide vaccine
It is used for strengthening the t cell auxiliary epitope polypeptide of vaccine immunogenicity, cp701 epi-position containing a cp701 epitope polypeptide and one
The aminoacid sequence of polypeptide is as shown in seq.id.no.1.
2. a kind of acute monocytic leukemia related antigen mlaa-34 epitope polypeptide vaccine as claimed in claim 1, it is special
Levy and be, it is tetanus toxoid tt830-843 that the described t cell for strengthening vaccine immunogenicity assists epitope polypeptide,
Its aminoacid sequence is as shown in seq.id.no.2.
3. a kind of acute monocytic leukemia related antigen mlaa-34 epitope polypeptide vaccine as claimed in claim 1 or 2,
It is characterized in that, it is also added with the incomplete Freund's adjuvant for strengthening vaccine immunogenicity in described polypeptide vaccine.
4. a kind of acute monocytic leukemia related antigen mlaa-34 epitope polypeptide is directed to the white blood of Acute monocytic in preparation
It is characterised in that this epitope polypeptide is cp701, its aminoacid sequence is as shown in seq.id.no.1 for application in the medicine of disease.
5. application as claimed in claim 4 is it is characterised in that described medicine is suppression people's acute monocytic leukemia
The medicine of thp-1 propagation.
6. application as claimed in claim 4 is it is characterised in that described medicine is inducing specific ctl killing activity and nk
The medicine of cytotoxic activity.
7. application as claimed in claim 4 is it is characterised in that described medicine is to stimulate cd3+cd8+T is lymphopoietic
Medicine.
8. application as claimed in claim 4 is it is characterised in that described medicine is to stimulate t cell secretion ifn- γ and il-2
Medicine.
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| CN107217066A (en) * | 2017-05-26 | 2017-09-29 | 吉林医药学院 | The method and its application of DNA fragmentation and the recombinant vector containing the fragment, structure recombinant vector |
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| JP2007319069A (en) * | 2006-05-31 | 2007-12-13 | Aichi Prefecture | LOC284293 VARIANT GENE AND CD8+ CYTOTOXIC T LYMPHOCYTE mHA EPITOPE PEPTIDE ENCODED WITH THE GENE AND APPLICATION THEREOF |
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| CN1793178A (en) * | 2005-11-17 | 2006-06-28 | 中国人民解放军第四军医大学 | Fusion protein of tetanus toxin T cell expressing bit polypeptide and human bata lymph cell stimulating factor and preparing thereof |
| JP2007319069A (en) * | 2006-05-31 | 2007-12-13 | Aichi Prefecture | LOC284293 VARIANT GENE AND CD8+ CYTOTOXIC T LYMPHOCYTE mHA EPITOPE PEPTIDE ENCODED WITH THE GENE AND APPLICATION THEREOF |
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| 白血病相关抗原MLAA-34 HLA-A2+限制性CTL 表位的预测及鉴定;赵建强等;《西安交通大学学报（医学版）》;20110731;第32卷(第4期);摘要和第426页表1-2 * |
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