CN103936835A - PD-L1IgV-targeting affinity peptide D1 with anti-tumor activity - Google Patents
PD-L1IgV-targeting affinity peptide D1 with anti-tumor activity Download PDFInfo
- Publication number
- CN103936835A CN103936835A CN201410176396.5A CN201410176396A CN103936835A CN 103936835 A CN103936835 A CN 103936835A CN 201410176396 A CN201410176396 A CN 201410176396A CN 103936835 A CN103936835 A CN 103936835A
- Authority
- CN
- China
- Prior art keywords
- affinity peptide
- peptide
- l1igv
- tumor activity
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of bio-pharmacy and specifically relates to a PD-L1IgV-targeting D-configuration affinity peptide D1 product with anti-tumor activity, as well as preparation and application thereof. The affinity peptide D1 is specifically bound to a PD-L1IgV region, the amino acid sequence is NYSKPTDRQYHF, and the molecular weight is 1554.7. The affinity peptide D1 is prepared through a Fomc solid-phase polypeptide synthesis method, and plays a role in preparation of anti-colon cancer medicaments as a main active ingredient. The affinity peptide D1 provided by the invention is obtained by utilizing a mirroring bacteriophage display peptide library screening technology and performing high-throughput screening by taking PD-L1IgV as a target point. Through tumor-bearing experiments in mouse bodies, the inventor proves that the affinity peptide D1 has better anti-tumor activity and can obviously inhibit the growth of tumors in the mouse bodies, so that a new idea and theoretical basis are provided for researching and developing PD-L1-based medicaments.
Description
Technical field
The invention belongs to biological pharmacy technical field, be specifically related to a kind of screening, preparation and application with anti-tumour active polypeptide product, more specifically, the present invention relates to a kind of D-form affinity peptide D1 product and preparation and application with the target PD-L1IgV of anti-tumor activity.
Background technology
In recent years, the prevention and control situation of tumour is very severe.Raising along with levels such as clinical diagnosis, operative treatment, chemotherapy and radiations, make a part of patient can access early discovery, early treatment, and obtain good prognosis, still, find new treatment means and medicine is the study hotspot in global range always.Compare with traditional methods for the treatment of, immunotherapy of tumors can activate or induced tumor patient sets up the specific immune response to tumour antigen, removes the tumour cell of former, and sets up immunological memory, stops recurrence and the transfer of tumour.
In immunotherapy of tumors process, the main mediated immunity tolerance of negativity costimulatory molecules and escape, and the ultimate challenge that forefathers run in immunotherapy of tumors process is exactly the unsatisfactory curative effect that tolerates and escape and cause due to tumour immunity.Therefore, inquire into by suppressing signal path that negativity costimulatory molecules mediates and there is important theory significance and using value with the immunological tolerance to tumour cell of breaking body and having set up.
The activation of T cell needs two from extracellular token stimulus, i.e. the dual signal effect of lymphocyte activation.The first signal of cell activation is the specific binding with MHC molecular antigen peptide complex from cell antigen identification receptor (TCR) mainly, and this process is antigen recognition.The second signal of cell activation claims again costimulatory signal, is that the adhesion molecule by antigen presenting cell (APC) and cell surface provides.These adhesion molecules are called as costimulatory molecules, are class surface of cell membrane molecules, and the activation that can be T, B cell provides subsidiary signal, thereby regulate propagation, activation and the differentiation of cell.Different according to the effect producing, costimulatory molecules can be divided into positivity costimulatory molecules and negativity costimulatory molecules.Positivity costimulatory molecules comprises CD28, ICOS, 4-1BB equimolecular, and negativity costimulatory molecules comprises CTLA-4, PD-1, TIM-3 equimolecular.
PD-1/PD-L1, as B7/CD28 family member, has been proved by suppressing activation and the propagation of T cell and has carried out negative regulation immunne response, and regulated immunological tolerance, tumour immunity to play a significant role in escaping.Thereby utilize the blocker of PD-L1 to there is good application prospect and security as immunotherapy of tumors medicine or adjuvant, and in prior art, still lack the blocker product of good PD-L1.
Summary of the invention
The object of the invention is to provide a kind of D-form affinity peptide D1 product with the target PD-L1IgV of better anti-tumor activity, can specificity utilize PD-L1 protein I gV district (D-PD-L1IgV) conduct in conjunction with target spot, thereby blocking-up PD-1/PD-L1 signal path, the inhibition of releasing to the activation of T cell and propagation, performance antitumor action.
Particularly, the technical solution used in the present invention is as follows:
A kind of have an anti-tumor activity target PD-L1 IgV affinity peptide D1, belong to D-form, specific binding is in PD-L1 IgV district, its aminoacid sequence is: Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe, be N-Y-S-K-P-T-D-R-Q-Y-H-F, molecular weight is 1554.7.
Described have an anti-tumor activity target PD-L1IgV affinity peptide D1, adds pharmaceutically acceptable carrier or/and after additive, play a role in preparing inhibitor against colon carcinoma cells (CT26) medicine as main active ingredient.
Described have an anti-tumor activity target PD-L1IgV affinity peptide D1, by Fomc solid-phase polypeptide synthesis method synthetic, prepares.
Crystalline structure research to PD-1/PD-L1 mixture in prior art is thought, mainly the extracellular fragment IgV district of PD-L1 with the position that acceptor PD-1 is combined, therefore contriver is by building the prokaryotic expression carrier in human PD-L 1 IgV district, obtain human PD-L 1 extracellular fragment IgV district albumen, and take that it screens affinity peptide as target spot, and then the possibility as immunotherapy of tumors medicine and adjuvant is studied to it.
Target spot is being carried out in high flux screening affinity peptide process, contriver has adopted storage capacity phage display peptide library technology large, easy and simple to handle to screen.Phage display peptide library technology is foreign protein or peptide sequence to be inserted in to the N-terminal of M13 phage capsid protein p III, the amalgamation and expression by albumen by the rondom polypeptide sequence shows of inserting at phage surface.Because displayed polypeptides is positioned at the N-terminal of p III, these little peptides can keep comparatively independently space structure conventionally, thereby can simulate part and specific receptors target interacts.Owing to screening resulting polypeptide by phage display peptide library, be natural amino acid residue and form, easily degraded by enzymes and Half-life in vivo is shorter.Mirror image phage selection technology take screening target protein D-form mirror image molecule be target, by phage display peptide library technology screening can with the polypeptide aglucon of its combination, mirror image molecule-D-form polypeptide aglucon of these polypeptide aglucons of resynthesis.The natural polypeptides aglucon energy specific binding D-form target protein obtaining due to screening, according to mirror image symmetric relation, the target protein that D configuration polypeptide aglucon also can specific binding native configurations, so this D-form polypeptide is the mirror image aglucon of natural target protein.Therefore, contriver selects PD-L1IgV district albumen by D-form amino acid synthetic as target spot, obtain the affinity peptide of D-form, thereby the ability that increases substantially its opposing enzyme liberating is to act on the transformation period in extension body by mirror image phage selection technology.
The present invention is by utilizing mirror image phage display peptide library triage techniques, and the D-PD-L1IgV of take carries out high-throughout screening as target spot, synthetic there is the D-form affinity peptide D1 of anti-tumor activity.Contriver further tests by lotus knurl in Mice Body, proved that target PD-L1IgV affinity peptide D1 provided by the present invention has good anti-tumor activity, can obviously suppress the growth of mouse interior tumor, thereby provide new thinking and theoretical basis for the drug research and development based on PD-L1.
Accompanying drawing explanation
Fig. 1 is the Mass Spectrometric Identification figure of affinity peptide D1;
Fig. 2 be the other administration of the knurl of affinity peptide D1 lotus CT26 colorectal carcinoma Mouse Weight is changed affect result figure;
What Fig. 3 was the other administration of the knurl of affinity peptide D1 on lotus CT26 colorectal carcinoma mouse tumor volume change affects result figure;
Fig. 4 is that the other administration of the knurl of affinity peptide D1 is to lotus CT26 colorectal carcinoma mouse tumor multigraph;
Fig. 5 be the intraperitoneal administration of affinity peptide D1 lotus CT26 colorectal carcinoma Mouse Weight is changed affect result figure;
Fig. 6 is that the intraperitoneal administration of affinity peptide D1 affects result figure to lotus CT26 colorectal carcinoma mouse tumor volume change;
Fig. 7 is that the intraperitoneal administration of affinity peptide D1 affects result figure to lotus CT26 colorectal carcinoma survival time of mice;
Fig. 8 is affinity peptide D1 enzyme liberating stability result figure.
Embodiment
Below in conjunction with embodiment the present invention will be further explained explanation.
embodiment 1
The D-form affinity peptide D1 with the target PD-L1 IgV of anti-tumor activity provided by the present invention, specific binding is in PD-L1IgV district, utilize mirror image phage display peptide library technology screening to obtain, its aminoacid sequence is: Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe, be N-Y-S-K-P-T-D-R-Q-Y-H-F, molecular weight is 1554.7.
Owing to screening resulting polypeptide by conventional phage display peptide library, be natural amino acid residue and form, easily degraded by enzymes and Half-life in vivo is shorter.Therefore, we select PD-L1 IgV district albumen by D-form amino acid synthetic as target spot, by mirror image display technique of bacteriophage, screen the antagonistic peptide that obtains D-form, thereby the ability that increases substantially its opposing enzyme liberating is to act on the transformation period in extension body.For ease of those skilled in the art, implement the present invention, as follows to its screening process brief description:
in screening process, the preparation of used medium and main solution is described as follows:
LB liquid nutrient medium: take Tryptones 10 g, yeast powder 5 g, sodium-chlor 5 g, add ultrapure water and are settled to 1 L, 121 ℃ of high pressure steam sterilization 20 min, cooling rear room temperature storage is standby.
LB solid medium: add 15 g agar powders in 1 L LB liquid nutrient medium, heating is fully dissolved agar powder, stirs and is packed as 100 mL/ bottles, 121 ℃ of sterilizing 20 min, be cooled to store after room temperature standby.
LB/IPTG/Xgal is dull and stereotyped: 100 mL LB solid mediums are dissolved with microwave-oven-heating, while being cooled to 60 ℃ of left and right, add 75 μ L IPTG/Xgal, carefully mix, avoid producing bubble, pour into six orifice plates.Dull and stereotyped standby in 4 ℃ of refrigerator lucifuges storages.IPTG/Xgal is prepared by following formula: take 0.5 g IPTG and 0.2g Xgal and be dissolved in 5 mL DMF(N, dinethylformamide) in, after mixing, be packed as 300 μ L aliquots, in-20 ℃ of lucifuges, store.
Top-layer agar: take Tryptones 10 g, yeast powder 5 g, sodium-chlor 5 g, MgCl
26H
2o 1 g, agarose 7 g, add ultrapure water and are settled to 1L, and microwave oven boils 3 times, and agarose is fully dissolved, and is cooled to 60 ℃ of left and right, is distributed into the aliquot of 60 mL, 121 ℃ of high pressure steam sterilization 20 min, cooling rear room temperature is deposited standby.
LB-Tet(tsiklomitsin) flat board: microwave-oven-heating dissolves 100mL LB solid medium, while being cooled to 60 ℃ of left and right, adds 100 μ L Tet storage liquid, topples over six flat boards after mixing, and to be cooledly solidifies rear 4 ℃ of lucifuges and stores, and in one week, uses.Tsiklomitsin (Tet) storage liquid is prepared by following formula: take 200 mg tetracycline hydrochloride, be dissolved in the dehydrated alcohol of 10 mL, be packed as 300 μ L aliquots, in-20 ℃ of lucifuges, store.
Coating buffer-0.1 M NaHCO
3damping fluid (pH 8.6): take NaHCO
30.84g adjusts PH to 8.6 with NaOH after dissolving with 80mL tri-distilled water, is settled to 100mL, and 121 ℃ of high pressure steam sterilization 20 min, deposit standby for cooling latter 4 ℃.
Confining liquid-0.1M NaHCO
3(pH8.6), 5 mg/mL BSA: take NaHCO
30.84 g, BSA 0.5 g adjust pH to 8.6 with NaOH after dissolving with 80mL tri-distilled water, are settled to 100mL, and filtration sterilization, is packed as 5 mL aliquots, and 4 ℃ of storages are standby.
Elutriant-0.2 M glycine-HCl damping fluid (pH 2.2), 1 mg/mL BSA: measure 50mL 0.2M glycine solution and 44 mL 0.2M HCl solution, add 200 mg BSA, add water and be settled to 200 mL, filtration sterilization, 4 ℃ are standby.
Neutralizer-1M Tris-HCl damping fluid (pH 9.1): take 12.114 g Tris alkali, appropriate ultrapure water dissolves, and HCl adjusts pH to 9.1, constant volume, filtration sterilization, packing is standby.
TBS damping fluid-50 mM Tris-HCL(PH7.5), 150mM NaCl: preparation according to the following steps, the first step first takes 6.055 g Trisbase, with a small amount of distilled water (300 ~ 500ml), dissolve, with dense HCl, pH is adjusted to 7.5 again, finally add distilled water to 1000 ml, this step is prepared into Tris damping fluid (50 mM, PH7.5); Second step takes NaCl 8.766g, first with a small amount of distilled water, dissolves NaCl, then adds the prepared Tris damping fluid of the first step (50 mM, pH7.5) 100ml, finally adds distilled water to 1000ml, fully shakes up autoclaving and get final product.
Washings (TBST): 0.1%(v/v) Tween-20 TBS, TBS is aforementioned TBS damping fluid.
PEG/NaCl: take PEG-8000 20 g, NaCl 14.61 g, add ultrapure water and be settled to 100 mL, and 121 ℃ of high pressure steam sterilization 30 min, are cooled to room temperature, and 4 ℃ of storages are standby.
screening step:
(1) coated: to adopt the synthetic PD-L1 albumen IgV of the chemistry of amino acids district of D-form for fragment assembly technology, after urea concentration gradient renaturation, obtain the NaHCO that target protein D-PD-L1IgV(is dissolved in 0.1M pH 8.6
3solution).By coated 96 orifice plates of target protein, 15 μ g/ holes (150 μ L, 100 μ g/mL), 4 ℃ of overnight incubation of airtight wet box;
(2) sealing: discard coating buffer, also fill it up with rapidly confining liquid with rifle head sucking-off residue raffinate, 4 ℃, airtight wet box is hatched 3 h;
(3) washing: TBST washing 6 times, be no less than 2 min at every turn, note aseptic technique, action wants fast in order to avoid microwell plate is dry;
(4) combination: add fast that to be diluted to titre with TBST be in advance 2 * 10
11the phage 100 μ L/ holes in library, room temperature gentleness is shaken 1h; In this process, the phage with target protein with avidity can be combined on albumen;
(5) washing: TBST washing 10 times, washes away unconjugated phage;
(6) wash-out: every hole adds 100 μ L elutriants, and room temperature gentleness is shaken 20min;
(7) neutralization: with the phage of the careful sucking-off wash-out of liquid-transfering gun, be transferred in the sterilizing centrifuge tube that has added in advance 15 μ L neutralizers, gentle piping and druming mixes;
(8) amplification: phage and intestinal bacteria ER2738 that first round screening is obtained add LB liquid nutrient medium (Tet
+) middle co-cultivation, increase and purifying, obtain secondary peptide storehouse;
(9) titer determination: phage titre mensuration is carried out in the secondary peptide storehouse of respectively each being taken turns after the phage that obtains of screening and amplification on LB/IPTG/Xgal flat board, and carry out rate of recovery calculating, the phage rate of recovery=[wash-out bacteriophage number/input phage number] * 100%;
(10) repeat screening: the phage that amplification is obtained is dropped into next round screening, repeats above-mentioned screening process.Take turns affine screening through 5, can make to obtain highly enriched containing the phage of desired polypeptides;
(11) order-checking: the plaque that picking the 5th is taken turns on the titer determination flat board after screening increases, the phage stock solution of getting the fresh amplification of 200 μ L is sent to Jin Weizhi order-checking company and automatically checks order, and sequencing primer is-96 g III sequencing primers: 5 '-CCC TCA TAG TTA GCG TAA CG-3 '.
Peptide sequence is analyzed: according to DNA sequencing result its coded aminoacid sequence of deriving, obtain the aminoacid sequence of antagonistic peptide.
the selection result:
(1) respectively each is taken turns to the phage elutriant that obtains of screening and carry out titer determination, and calculate and drop into the phage rate of recovery, the results are shown in following table.
(2) the non-specific sequence that we obtain for screening and eliminating with the not high sequence of target protein D-PD-L1IgV avidity, select dodecapeptide storehouse to carry out mirror image screening to D-PD-L1IgV albumen, obtain two D peptide sequences with repeated cloning, comprising D1 peptide, concrete outcome is as following table.
embodiment 2
The described D-form affinity peptide D1 with the PD-L1 IgV of anti-tumor activity adopts Fomc solid-phase polypeptide synthesis method to synthesize.
In building-up process, main agents used has:
End socket liquid: diacetyl oxide/pyridine solution (1:1 v/v);
Indenes check reagent: A. triketohydrindene hydrate/ethanolic soln (5% w/v), B. phenol/ethanol (4:1 w/v), C. potassium cyanide/pyridine (2% v/v);
Deprotection liquid: piperidines/DMF solution (20% v/v);
Cutting reagent: content meter by volume, TFA(82.5%), H
2o(5%), phenol (5%), thioanisole (5%), dithioglycol (2.5%).
Synthesis step is briefly described below:
(1) swelling resin, add first amino acid
A. swelling resin: get 0.3 ~ 0.5 g Rink resin (the C-terminal amino acid of the peptide being connected with resin is acid amides) and be placed in wash clean and dry Peptide synthesizer, add appropriate DMF, soak 30 min left and right and make the abundant swelling of resin, vacuum pump is extracted the DMF in synthesizer out.
B. washing: step is as follows, adds appropriate DMF, concussion washing 2min totally 2 times; Add appropriate MeOH, concussion washing 2min totally 3 times; Add appropriate DCM, concussion washing 2 min totally 3 times; Add appropriate DMF, then concussion is washed 2min totally 2 times.
C. add first amino acid: by formula, calculate the amount of first amino acid, HoBt and the DIC that add, calculating publicity is as follows:
Amino acid masses=resin quality * 2.5 times equivalent * with the amino acid whose relative molecular mass of blocking group,
The relative molecular mass of HOBT quality=resin quality * 2.5 times equivalent * HOBT,
The relative molecular mass of DIC consumption=resin quality * 2.5 times equivalent * DIC,
Balance weighs D-Fmoc-amino acid, HOBT in 50 mL beakers, adds in synthesizer, then add synthesizer with liquid-transfering gun absorption DIC with a small amount of DMF after dissolving, and synthesizer is placed in to shaking table, under room temperature, reacts 3h, makes amino acid and resin generation coupled reaction.
D. washing: with step B.
E. survey substitution value: due under 290nm, fluorenylmethyloxycarbonyl has very strong uv-absorbing, therefore, by measuring its light absorption value, by colorimetry, can measure the situation of first amino acid and resin-bonded.In picking step D, resin 1 ~ 1.5 mg after reaction, dries, and with electronic analytical balance, accurately weighs resin quality; Then resin is packed in the centrifuge tube of 5mL, add the deprotection liquid concussion 10min of 3mL; Shift and enter in cuvette in 290nm working sample OD value subsequently; The deprotection liquid that adds 3mL in another cuvette is blank, by formula, calculates substitution value, and calculating publicity is as follows:
Substitution value=sample OD value/1.65 * resin quality.
Substitution value is best at 0.4 ~ 0.6 o'clock, if substitution value is less than 0.4, shows that the amount of amino acid and resin-bonded is very few, should repeat said process and add first amino acid.
F. end socket: add appropriate end socket liquid to jolt 20min twice totally to synthesizer.
G. washing: with step B.
(2) add second amino acid and follow-up peptide chain extension
H. deprotection: add appropriate deprotection liquid in synthesizer, shaking table reacts 20 min totally 2 times.
I. washing: with step B.
J. indenes inspection: a small amount of resin in picking step I is put into indenes inspection pipe, drips respectively 1 of indenes check reagent A liquid, 2 of B liquid, 1 of C liquid, builds indenes inspection pipe, and boiling water bath 2min observes color of resin; Blue if (after Pro, His, Ser deprotection, indenes inspection color is reddish-brown), deprotection is normal; Otherwise must repeat deprotection process.
K. add amino acid: calculate and by load weighted amino acid and HOBT with adding Peptide synthesizer after a small amount of DMF dissolving, then add DIC with liquid-transfering gun, be placed in shaking table, under room temperature, react 2.5 h.
L. washing: with step B.
M. indenes inspection: process is with step J, if resin is glassy yellow, without locus coeruleus, amino acid condensation is complete, can carry out next step reaction.If blue, amino acid condensation is incomplete, need repeat this amino acid whose condensation reaction, until indenes inspection resin is glassy yellow completely.
N. repeating step H-M is to the condensation reaction that completes whole piece peptide.
(3)
polypeptide cutting
O. deprotection: with step H.
P. indenes inspection: with step J.
Q. washing: with step B.
R. cutting: configuration cuts reagent pour synthesizer in stink cupboard, agitator is put into synthesizer, start cleavage reaction, agitator slowly stirs cutting 2.5 h left and right.After completion of the reaction, with vacuum filtration pump suction filtration, go out cutting reagent, more several all over stirring rake and resin with DCM washing, and the cutting liquid that suction filtration is obtained packs round-bottomed flask into.
S. rotary evaporation: with Rotary Evaporators, the TFA in cutting liquid is removed, Rotary Evaporators temperature is adjusted into 65 ℃, rotary evaporation 10 ~ 20min.Add again afterwards ice ether, continue rotary evaporation, 10 ~ 20min, this step repeats 6 times, owing to containing arginine and Methionin in peptide sequence, need carry out ice and cut, to remove the protection group of side chain.In bottle, add 5mL TFA, be placed in ice, on shaking table, jolt 30min.After ice is cut, continue to be placed in Rotary Evaporators, rotary evaporation 10 ~ 20min.
T. precipitate: after rotary evaporation, cutting liquid is poured in the beaker that 50mL ice ether is housed, the standing 30min of ice bath, makes polypeptide precipitation.
U. centrifugal: suction pipe is drawn beaker bottom ether sedimentation mixed solution, 2000r/min, centrifugal 2min, abandons supernatant collecting precipitation, the resuspended washing precipitation of ice ether 5 times.
V. dry: the polypeptide of collection is deposited in to 37 ℃ of oven for drying, and electronic analytical balance is weighed, and calculates thick peptide productive rate, by its sealing be stored in-20 ℃ standby.
(4)
rP-HPLC analyzes and prepares purified polypeptide
Moving phase solution preparation during RP-HPLC analyzes:
Mobile phase A liquid (1 ‰ TFA solution): transfer pipet measures 1mL TFA, adds ultrapure water to be settled to 1000 mL, the ultrasonic degasification de-soak of ultrasonic instrument;
Mobile phase B liquid (acetonitrile): use chromatographically pure level acetonitrile solution as Mobile phase B liquid, note by the ultrasonic degasification de-soak of ultrasonic instrument.
Peptide purification preparation adopts gradient elution separation purifying:
W. the 30 min preheatings of starting shooting in advance, open software operation interface, parameter setting: wavelength arranges 228 nm, and flow velocity arranges 5 mL/min;
X. balance: balance approximately 30 min;
Y. thick peptide preparation: take thick peptide approximately 20 mg, dissolve sample introduction after 0.45 μ m filtering with microporous membrane with the 0.1% TFA aqueous solution (4 mL) containing certain concentration acetonitrile.Collection main peak product is placed in-80 ℃ and spends the night frozen.
Z. freeze-drying: the main peak product sample having frozen is placed in to Freeze Drying Equipment freeze-drying 24h, collects afterwards and weigh smart peptide ,-20 ℃ of sealings save backup.
(5) Mass Spectrometric Identification
Sperm chromosome peptide, by electrospray ionization mass spectrometry Analysis and Identification peptide molecule quality, as shown in Figure 1, qualification result meets expection to Mass Spectrometric Identification result.
embodiment 3
The D-form affinity peptide D1 of the target PD-L1 IgV with anti-tumor activity prepared by the embodiment 2 of take is example, and contriver has done experiment in further tumor-bearing mice body, and specific experiment process is as follows:
(1) affinity peptide D1 tests lotus CT26 colorectal carcinoma mice-transplanted tumor growth-inhibiting
Select 24 experiments to use Balb/c mouse, mouse source colorectal carcinoma (CT26) physiological saline for cell (NS) is adjusted to 5 * 10 by cell concn
6individual cell/mL, (contains 5 * 10 by 0.1 mL cell suspension after routine disinfection
5individual cell) be inoculated in every Balb/c mouse right fore armpit subcutaneous, continue to observe subcutaneous tumors bulk-growth situation.
The affinity peptide D1 of embodiment 2 preparations is dissolved in physiological saline, makes polypeptide drugs ,-20 ℃ of packing are preserved, standby.Inoculation mouse source colorectal carcinoma (CT26) cell, after 9 days, by the grouping of knurl volume, 6 every group, is divided into negative control group (NS), positive controls (5-FU), polypeptide drugs experimental group by mouse.
For determining the impact of administering mode difference on antitumous effect, twice lotus knurl experiment before and after contriver has carried out, different administering modes is taked in twice experiment.Specific experiment arranges as follows:
Lotus knurl is tested all experimental group and is all adopted the other administration of knurl for the first time.Polypeptide drugs are divided into high dose group (2 mg/kg) and low dose group (0.5 mg/kg) experimental group is administered once for every 3 days, and administration is 7 days altogether; 5-Fu positive controls is administered once every day, and successive administration finishes for four days.Experimental session mouse ad lib and drinking-water, measure length (a) short (b) footpath of Mouse Weight and tumour every day, and press formula knurl volume=(π/6) * a * b
2calculate gross tumor volume and draw tumor growth curve.After administration finishes, mouse is put to death and takes out tumour and weigh.
It is high dose group (5.0 mg/kg) and low dose group (2.0 mg/kg) that intraperitoneal administration mode, administration component are taked in lotus knurl experiment for the second time, and be administered once every day, successive administration 12 days.Experimental session mouse ad lib and drinking-water, measure length (a) short (b) footpath of Mouse Weight and tumour every day, and calculate gross tumor volume and draw tumor growth curve by formula.After administration finishes, mouse is put to death and takes out tumour and weigh.
Experimental result is shown in Fig. 2～Fig. 6.From Fig. 2, Fig. 5, we can find out polypeptide drugs group experiment mice Normal-weight, and positive controls is because the toxic side effect of 5-FU is compared with occurring greatly the situation of becoming thin.As can be seen here, affinity peptide D1 does not have obvious toxic-side effects.From Fig. 3, Fig. 4, we can find out that, while adopting the other administering mode of knurl, medicine group experiment mice gross tumor volume and knurl are heavy all little than negative control group.High dosage and low dosage gap are little, and supposition is because affinity peptide D1 is difficult for degraded, or dosage is relatively higher, thereby does not show dose-dependently.From Fig. 6, we can find out that while adopting intraperitoneal administration mode, medicine group experiment mice gross tumor volume is less than negative control group.Now, low dosage is relatively slower than the experiment mice knurl volume growth of high dosage administration group, illustrates that the D1 of 2mg/kg dosage can better suppress the growth of tumor-bearing mice tumour.
(2) experiment lifetime
Select 24 experiments to use Balb/c mouse, mouse source colorectal carcinoma (CT26) physiological saline for cell (NS) is adjusted to 5 * 10 by cell concn
6individual cell/mL, (contains 5 * 10 by 0.1 mL cell suspension after routine disinfection
5individual cell) be inoculated in every Balb/c mouse right fore armpit subcutaneous, continue to observe subcutaneous tumors bulk-growth situation.
The affinity peptide D1 of embodiment 2 preparations is dissolved in physiological saline, makes polypeptide drugs ,-20 ℃ of packing are preserved, standby.
Inoculation mouse source colorectal carcinoma (CT26) cell, after 9 days, by the grouping of knurl volume, 6 every group, is divided into negative control group (NS), positive controls (5-FU), polypeptide drugs experimental group by mouse.Each group all adopts the other administration of knurl, and be administered once every day, and administration is 12 days altogether, observes mouse survival state, records mouse diing time.
Experimental result is shown in Fig. 7.From Fig. 7, we can find out that D1 medicine group experiment mice lifetime (2mg/kg organizes 37d to 53d, and 5mg/kg organizes 36d to 52d) compares significant prolongation with negative control group (17d to 36d).Supposition is because D1 peptide is difficult for degraded, or dosage is relatively higher, thereby compares not advantage the lifetime of high dose group mouse with low dose group.
embodiment 4
For checking the stability of affinity peptide D1 provided by the present invention, contriver has further done enzyme liberating stability experiment, and specific experiment process is as follows:
By 10uL 10
-2m D1 peptide and contrast L peptide (the affine dodecapeptide of L-configuration that the PD-L1IgV albumen of take obtains as target sieving phage dodecapeptide storehouse) mother liquor add in 190uL serum (with ten times of dilutions of PBS), and concussion immediately mixes, and takes out 20uL mixed solution, add in centrifuge tube, now timing is 0min, and remaining continues to hatch at 37 ℃, and respectively at different time point (0min, 15min, 30min, 60min, 120min, 240min, 480min) taking-up 20uL.
Add 90uL acetonitrile concussion to mix to stop enzymolysis process in the sample of taking-up, sample is placed in 5 min on ice, then the glacial acetic acid that takes out 90 uL 0.5% dilutes to guarantee that enzymolysis process stops.13000 g, 15 min are centrifugal, collect supernatant, and-80 ℃ are frozen, analyze to RP-HPLC.
D1 still can not be degraded after 480min as can be seen from Figure 8, and L peptide in contrast has been degraded half when 120 min.Hence one can see that, and D1 peptide in vivo environment is difficult for being degraded, and can effectively extend its action time, thereby reach better result for the treatment of.
SEQUENCE LISTING
<110> Zhengzhou University
<120> has the target PD-L1IgV affinity peptide D1 of anti-tumor activity
<130> none
<160> 1
<170> PatentIn version 3.4
<210> 1
<211> 12
<212> PRT
<213> target PD-L1IgV affinity peptide D1
<400> 1
Asn Tyr Ser Lys Pro Thr Asp Arg Gln Tyr His Phe
1 5 10
Claims (3)
1. one kind has anti-tumor activity target PD-L1 IgV affinity peptide D1, it is characterized in that, this affinity peptide D1 belongs to D-form, specific binding is in PD-L1 IgV district, its aminoacid sequence is: Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe, be N-Y-S-K-P-T-D-R-Q-Y-H-F, molecular weight is 1554.7.
2. described in claim 1, there is the application of anti-tumor activity target PD-L1 IgV affinity peptide D1 in preparing inhibitor against colon carcinoma cells medicine.
3. the preparation method described in claim 1 with anti-tumor activity target PD-L1 IgV affinity peptide D1, is characterized in that, by Fomc solid-phase polypeptide synthesis method, prepares.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410176396.5A CN103936835B (en) | 2014-04-29 | 2014-04-29 | There is the target PD-L1IgV affinity peptide D1 of anti-tumor activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410176396.5A CN103936835B (en) | 2014-04-29 | 2014-04-29 | There is the target PD-L1IgV affinity peptide D1 of anti-tumor activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103936835A true CN103936835A (en) | 2014-07-23 |
| CN103936835B CN103936835B (en) | 2016-03-30 |
Family
ID=51184738
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410176396.5A Active CN103936835B (en) | 2014-04-29 | 2014-04-29 | There is the target PD-L1IgV affinity peptide D1 of anti-tumor activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103936835B (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017072273A1 (en) * | 2015-10-30 | 2017-05-04 | Affibody Ab | New polypeptide |
| WO2018153208A1 (en) * | 2017-02-25 | 2018-08-30 | 朱乃硕 | Human pd-l1 high-protein-affinity peptide and use thereof |
| CN109053862A (en) * | 2018-08-07 | 2018-12-21 | 复旦大学附属肿瘤医院 | Target PD-L1 polypeptide derivative and its99mThe preparation and application of Tc complex |
| CN110205291A (en) * | 2019-04-28 | 2019-09-06 | 蚌埠医学院第一附属医院 | The preparation method and pharmaceutical composition of a kind of film surface engineering NK cell and application |
| CN110312736A (en) * | 2017-01-23 | 2019-10-08 | 苏州康宁杰瑞生物科技有限公司 | PD-L1 combination polypeptide or compound |
| CN110330550A (en) * | 2019-08-02 | 2019-10-15 | 郑州大学 | The affinity peptide of PD-L1-IgV and its application |
| CN111423497A (en) * | 2020-03-16 | 2020-07-17 | 山东大学 | Antagonistic peptide, copolymer and nano assembly thereof, and preparation method and application thereof |
| CN111650382A (en) * | 2020-06-12 | 2020-09-11 | 上海维洱生物医药科技有限公司 | Targeting peptide-mediated gold nanoparticle biosensor and preparation method and application thereof |
| CN112402622A (en) * | 2020-11-10 | 2021-02-26 | 福建医科大学 | Anti-tumor polypeptide nano-drug carrier targeting PD-L1 and application thereof |
| US11155596B2 (en) | 2015-10-30 | 2021-10-26 | Affibody Ab | Polypeptide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010077634A1 (en) * | 2008-12-09 | 2010-07-08 | Genentech, Inc. | Anti-pd-l1 antibodies and their use to enhance t-cell function |
| CN103304638A (en) * | 2013-07-08 | 2013-09-18 | 郑州大学 | PD-L1 affinity peptide with anti-tumour activity and application for same |
-
2014
- 2014-04-29 CN CN201410176396.5A patent/CN103936835B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010077634A1 (en) * | 2008-12-09 | 2010-07-08 | Genentech, Inc. | Anti-pd-l1 antibodies and their use to enhance t-cell function |
| CN103304638A (en) * | 2013-07-08 | 2013-09-18 | 郑州大学 | PD-L1 affinity peptide with anti-tumour activity and application for same |
Non-Patent Citations (1)
| Title |
|---|
| 张意等: "PD-L1-Ig融合蛋白的酵母表达及其对T细胞的抑制作用", 《中国肿瘤生物治疗杂志》, vol. 16, no. 05, 31 October 2009 (2009-10-31), pages 464 - 468 * |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10364280B2 (en) | 2015-10-30 | 2019-07-30 | Affibody Ab | Polypeptide |
| US11155596B2 (en) | 2015-10-30 | 2021-10-26 | Affibody Ab | Polypeptide |
| WO2017072273A1 (en) * | 2015-10-30 | 2017-05-04 | Affibody Ab | New polypeptide |
| CN110312736A (en) * | 2017-01-23 | 2019-10-08 | 苏州康宁杰瑞生物科技有限公司 | PD-L1 combination polypeptide or compound |
| JP2020508069A (en) * | 2017-02-25 | 2020-03-19 | 朱乃碩ZHU, Naishuo | Peptide having high affinity for human PD-L1 protein and use thereof |
| CN108503691A (en) * | 2017-02-25 | 2018-09-07 | 复旦大学 | A kind of human PD-L 1 albumen high-affinity peptide and its application |
| RU2725954C1 (en) * | 2017-02-25 | 2020-07-07 | Найшо ЧЖУ | Peptide having high affinity to pd-l1 protein, and use thereof |
| CN108503691B (en) * | 2017-02-25 | 2021-07-23 | 复旦大学 | High-affinity peptide of human PD-L1 protein and application thereof |
| WO2018153208A1 (en) * | 2017-02-25 | 2018-08-30 | 朱乃硕 | Human pd-l1 high-protein-affinity peptide and use thereof |
| CN109053862A (en) * | 2018-08-07 | 2018-12-21 | 复旦大学附属肿瘤医院 | Target PD-L1 polypeptide derivative and its99mThe preparation and application of Tc complex |
| CN110205291A (en) * | 2019-04-28 | 2019-09-06 | 蚌埠医学院第一附属医院 | The preparation method and pharmaceutical composition of a kind of film surface engineering NK cell and application |
| CN110330550A (en) * | 2019-08-02 | 2019-10-15 | 郑州大学 | The affinity peptide of PD-L1-IgV and its application |
| WO2021023140A1 (en) * | 2019-08-02 | 2021-02-11 | 郑州大学 | Affinity peptide of pd-l1-igv and application thereof |
| CN111423497A (en) * | 2020-03-16 | 2020-07-17 | 山东大学 | Antagonistic peptide, copolymer and nano assembly thereof, and preparation method and application thereof |
| CN111650382A (en) * | 2020-06-12 | 2020-09-11 | 上海维洱生物医药科技有限公司 | Targeting peptide-mediated gold nanoparticle biosensor and preparation method and application thereof |
| CN112402622A (en) * | 2020-11-10 | 2021-02-26 | 福建医科大学 | Anti-tumor polypeptide nano-drug carrier targeting PD-L1 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103936835B (en) | 2016-03-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103936835A (en) | PD-L1IgV-targeting affinity peptide D1 with anti-tumor activity | |
| CN104086627B (en) | There is the PD-L1 IgV affinity peptide S10 of anti-tumor activity | |
| CN104098651A (en) | PD-L1 IgV affinity peptide with antineoplastic activity, and preparation method and application of thereof | |
| CN103936836B (en) | There is the target PD-L1IgV affinity peptide D2 of anti-tumor activity | |
| CN105504018B (en) | LAG-3 affinity peptide N13, preparation method and applications | |
| ES2762580T3 (en) | Macrocyclic inhibitors of protein / protein PD-1 / PD-L1 and CD80 (B7-1) / PD-L1 interactions | |
| ES2768610T3 (en) | Macrocyclic inhibitors of protein / protein PD-1 / PD-L1 and CD80 (B7-1) / PD-L1 interactions | |
| CN103304638B (en) | PD-L1 affinity peptide with anti-tumour activity and application for same | |
| CN103897036B (en) | A kind of PD-1 albumen extracellular fragment affinity peptide L8 and application thereof | |
| BR112020025985A2 (en) | bicyclic peptide linkers specific for nectin-4 | |
| CN109311943A (en) | PD-1/PD-L1 and CD80/PD-L1 protein/protein interaction macrocyclic hcv inhibitors | |
| KR20110093832A (en) | Cxcr4 receptor compounds | |
| CN104017049B (en) | A kind of tumor specific target polypeptide and application thereof | |
| BR122019016210B1 (en) | COMPOUND, PHARMACEUTICAL COMPOSITION, USE OF THE COMPOUND AND PROCESS FOR THE MANUFACTURE OF THE SAME | |
| WO2022234853A1 (en) | Cyclic compound having inhibitory effect selective for kras but not for hras and nras | |
| CN101010339A (en) | Human glucagon-like-peptide-1 modulators and their use in treatment of diabetes and related conditions | |
| CN105315350B (en) | Antiangiogenic polypeptide mPEG-Mal-Cys-AS16 | |
| Kuncewicz et al. | BTLA-derived peptides as inhibitors of BTLA/HVEM complex formation–design, synthesis and biological evaluation | |
| CN111956795B (en) | Application of chimeric antigen receptor combined anti-tumor drug taking CD99 as target | |
| US20220267376A1 (en) | P53 activator peptidomimetic macrocycles | |
| CN109157656B (en) | Double-target tumor vaccine and preparation method and application thereof | |
| JP2023029958A (en) | Novel compounds (immunorhelins) | |
| CN111592580A (en) | Polypeptide with immune checkpoint CTLA-4 inhibitory activity and application thereof | |
| Bojko et al. | Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer | |
| CN105017385A (en) | Vaccine based on simulation of histamine acceptor 4 (HR4) epitope and construction method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |