CN104098651B - There is PD-L1 IgV affinity peptide and the application thereof of anti-tumor activity - Google Patents
There is PD-L1 IgV affinity peptide and the application thereof of anti-tumor activity Download PDFInfo
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Abstract
The invention belongs to biological pharmacy technical field, be specifically related to the affinity peptide product of a kind of targeting PD-L1 IgV with anti-tumor activity and preparation thereof and application.This affinity peptide specifically binds to PD-L1 IgV district, and its aminoacid sequence is ANGSRLV, and molecular weight is 715.4.This affinity peptide can be prepared by Fomc solid phase polypeptide synthesis synthetic, can play a role as main active in preparing inhibitor against colon carcinoma cells (CT26) medicine.The present invention by utilizing phage display peptide library triage techniques, carries out high-throughout screening with PD-L1 IgV for target spot, and synthetic has the affinity peptide of anti-tumor activity.Inventor further demonstrates affinity peptide provided by the present invention and has good anti-tumor activity, can substantially suppress the growth of mouse interior tumor, thus providing new thinking and theoretical basis for the drug research and development based on PD-L1.
Description
Technical field
The invention belongs to biological pharmacy technical field, be specifically related to a kind of there is the screening of anti-tumour active polypeptide product, preparation and application, more specifically, the present invention relates to the affinity peptide product of a kind of targeting PD-L1IgV with anti-tumor activity and preparation thereof and application.
Background technology
In recent years, the prevention and control situation of tumor is very severe.Raising along with levels such as clinical diagnosis, operative treatment, chemotherapy and radiations, make a part of patient can obtain early discovery, early treatment, and obtain good prognosis, but, find new treatment means and study hotspot that medicine is always up in global range.Compared with traditional Therapeutic Method, immunotherapy of tumors can activate or induced tumor patient sets up the specific immune response to tumor antigen, removes the tumor cell of former, and sets up immunological memory, stops recurrence and the transfer of tumor.
In immunotherapy of tumors process, negativity costimulatory molecules main mediated immunity tolerance and escaping, and the ultimate challenge that forefathers run in immunotherapy of tumors process is precisely due to tumour immunity tolerance and the unsatisfactory curative effect caused of escaping.Therefore, inquire into by suppressing the immunologic tolerance to tumor cell that the signal path that negativity costimulatory molecules mediates has built up to break body to have important theory significance and using value.
The activation of T cell needs two from extracellular signal stimulus, i.e. the dual signal effect of lymphocyte activation.First signal of cell activation is specific binding essentially from cellular antigens identification receptor (TCR) and MHC molecule antigenic peptide complexes, and this process is antigen recognition.The secondary signal of cell activation, also known as costimulatory signal, is that the adhesion molecule by antigen presenting cell (APC) and cell surface is to offer.These adhesion molecules are referred to as costimulatory molecules, are class cell membrane surface molecules, can provide auxiliary signal for the activation of T, B cell, thus regulating the propagation of cell, activation and differentiation.Different according to the effect produced, costimulatory molecules can be divided into positivity costimulatory molecules and negativity costimulatory molecules.Positivity costimulatory molecules includes CD28, ICOS, 4-1BB equimolecular, and negativity costimulatory molecules then includes CTLA-4, PD-1, TIM-3 equimolecular.
PD-1/PD-L1, as B7/CD28 family member, has been found to by suppressing the activation of T cell and propagation to carry out negative regulation immunne response, and plays a significant role in regulating immunologic tolerance, tumor immune escape.Thus utilize the blocker of PD-L1 to have good application prospect and safety as immunotherapy of tumors medicine or adjuvant, and in prior art, still lack the blocker product of good PD-L1.
Summary of the invention
Present invention aim at providing the affinity peptide product of a kind of targeting PD-L1IgV with better anti-tumor activity, PD-L1 protein I gV district (PD-L1IgV) can be utilized as in conjunction with target spot by specificity, thus finally blocking the activation of PD-1/PD-L1, release the inhibitory action of the activation to T cell and propagation, thus playing antitumor action.
Specifically, the technical solution used in the present invention is as follows:
One has anti-tumor activity targeting PD-L1IgV affinity peptide, specifically binds to PD-L1IgV district, and its aminoacid sequence is: Ala-Asn-Gly-Ser-Arg-Leu-Val, i.e. A-N-G-S-R-L-V, and molecular weight is 715.4.
Described have anti-tumor activity targeting PD-L1IgV affinity peptide, adds pharmaceutically acceptable carrier or/and after additive, play a role as main active in preparing inhibitor against colon carcinoma cells (CT26) medicine.
Described have anti-tumor activity targeting PD-L1IgV affinity peptide, is prepared by Fomc solid phase polypeptide synthesis synthetic.
Target spot being carried out in high flux screening affinity peptide process, the phage display peptide library technology that inventor have employed storage capacity big, easy and simple to handle has been screened.Phage display peptide library technology is that foreign protein or peptide sequence are inserted in the N-terminal of M13 phage capsid protein p III, by the amalgamation and expression of albumen, the random polypeptide sequences of insertion is illustrated in phage surface.Owing to displayed polypeptides is positioned at the N-terminal of p III, these little peptides usually keep comparatively independent space structure such that it is able to simulation part interacts with specific receptor target.
The present invention by utilizing phage display peptide library triage techniques, carries out high-throughout screening with PD-L1IgV for target spot, and synthetic has the affinity peptide of anti-tumor activity.Inventor is further by lotus tumor experiment in Mice Body, demonstrate targeting PD-L1IgV affinity peptide provided by the present invention and there is good anti-tumor activity, can substantially suppress the growth of mouse interior tumor, thus providing new thinking and theoretical basis for the drug research and development based on PD-L1.
Accompanying drawing explanation
Fig. 1 is the ELISA affinity result figure identifying affinity peptide phage monoclonal and PD-L1；
Fig. 2 is the Mass Spectrometric Identification figure of affinity peptide；
Fig. 3 is that affinity peptide affects result figure to what lotus CT26 colon cancer Mouse Weight changed；
Fig. 4 is that lotus CT26 colon cancer mouse tumor change in volume is affected result figure by affinity peptide；
Fig. 5 is the affinity peptide tumor multigraph to lotus CT26 colon cancer mice-transplanted tumor.
Detailed description of the invention
Below in conjunction with embodiment the present invention will be further explained explanation.
Embodiment 1
Provided by the present invention have anti-tumor activity targeting PD-L1IgV affinity peptide, specifically binding to PD-L1IgV district, utilize phage display peptide library technology screening to obtain, its aminoacid sequence is: Ala-Asn-Gly-Ser-Arg-Leu-Val, i.e. A-N-G-S-R-L-V, molecular weight is 715.4.
With high costs due to monoclonal antibody medicine, and can not large-scale production, therefore, we select by the PD-L1IgV district albumen of Prokaryotic expression, purification as target spot, screened by display technique of bacteriophage obtain can be specific binding with PD-L1IgV polypeptide, in order to block PD-1/PD-L1 signal path.The present invention is implemented for ease of those skilled in the art, its screening process brief description is as follows:
In screening process, the preparation of used medium and main solution illustrates as follows:
LB fluid medium: weigh tryptone 10g, yeast powder 5g, sodium chloride 5g, add ultra-pure water and be settled to 1L, 121 DEG C of high pressure steam sterilization 20min, after cooling, room temperature storage is standby.
LB solid medium: adding 15g agar powder in 1LLB fluid medium, heating makes agar powder fully dissolve, and stirs and is packed as 100mL/ bottle, and 121 DEG C of sterilizing 20min store standby after being cooled to room temperature.
LB/IPTG/Xgal flat board: 100mLLB solid medium microwave-oven-heating is dissolved, when being cooled to about 60 DEG C, adds 75 μ LIPTG/Xgal, carefully mix, it is to avoid produce bubble, pour into six orifice plates.Flat board is standby in 4 DEG C of refrigerator stored protected from light.IPTG/Xgal is prepared by formula as below: weighs 0.5gIPTG and 0.2gXgal and is dissolved in 5mLDMF(N, dinethylformamide) in, it is packed as 300 μ L aliquots after mixing, in-20 DEG C of stored protected from light.
Top-layer agar: weigh tryptone 10g, yeast powder 5g, sodium chloride 5g, MgCl2·6H2O1g, agarose 7g, add ultra-pure water and be settled to 1L, and microwave oven boils 3 times, makes agarose fully dissolve, and is cooled to about 60 DEG C, is distributed into the aliquot of 60mL, 121 DEG C of high pressure steam sterilization 20min, cooling rear chamber gentle put standby.
LB-Tet(tetracycline) flat board: microwave-oven-heating dissolves 100mLLB solid medium, when being cooled to about 60 DEG C, adds 100 μ LTet and stores liquid, topple over six flat boards after mixing, and the rear 4 DEG C of stored protected from light of solidification to be cooled made to be finished in one week.Tetracycline (Tet) is store liquid and is prepared by formula as below: weigh 200mg quadracycline, is dissolved in the dehydrated alcohol of 10mL, is packed as 300 μ L aliquots, in-20 DEG C of stored protected from light.
It is coated liquid-0.1MNaHCO3Buffer (pH8.6): weigh NaHCO30.84g adjusts PH to 8.6 with NaOH after dissolving with 80mL tri-distilled water, is settled to 100mL, and 121 DEG C of high pressure steam sterilization 20min cool down latter 4 DEG C and deposit standby.
Confining liquid-0.1MNaHCO3(pH8.6), 5mg/mLBSA: weigh NaHCO30.84g, BSA0.5g adjust pH to 8.6 with NaOH after dissolving with 80mL tri-distilled water, are settled to 100mL, filtration sterilization, are packed as 5mL aliquot, and 4 DEG C of storages are standby.
Eluent-0.2M Glycine-HCl buffer (pH2.2), 1mg/mLBSA: measure 50mL0.2M glycine solution and 44mL0.2MHCl solution, add 200mgBSA, add water and be settled to 200mL, and filtration sterilization, 4 DEG C standby.
Neutralizer-1MTris-HCl buffer (pH9.1): weigh 12.114gTris alkali, appropriate ultra-pure water dissolves, and HCl adjusts pH to 9.1, and constant volume, filtration sterilization, subpackage is standby.
TBS buffer-50mMTris-HCL(PH7.5), 150mMNaCl: prepare according to the following steps, the first step first weighs 6.055gTrisbase, dissolve with a small amount of distilled water (300 ~ 500ml), with dense HCl, pH is adjusted to 7.5 again, finally add distilled water to 1000ml, this step is prepared into Tris buffer (50mM, PH7.5)；Second step weighs NaCl8.766g, first dissolves NaCl with a small amount of distilled water, adds Tris buffer (50mM, the pH7.5) 100ml prepared by the first step, finally add distilled water to 1000ml, fully shake up, autoclaving and get final product.
Cleaning mixture (TBST): 0.1%(v/v) Tween-20TBS, TBS and aforementioned TBS buffer.
PEG/NaCl: weigh PEG-800020g, NaCl14.61g, adds ultra-pure water and is settled to 100mL, 121 DEG C of high pressure steam sterilization 30min, is cooled to room temperature, and 4 DEG C of storages are standby.
TMB stores liquid: weighs 10mgTMB and is dissolved in 1mLDMSO, and 4 DEG C keep in Dark Place standby.
Tmb substrate buffer-phosphoric acid-citrate buffer solution (pH5.0): prepare according to the following steps: first prepare 0.1M/L citric acid solution, namely weigh citric acid (C6H8O7·H2O) 19.2g, adds water to 1000mL, for solution A；Then prepare 0.2M/L disodium hydrogen phosphate liquid, namely weigh disodium hydrogen phosphate (Na2HPO4) 71.7g, add water to 1000mL, for second liquid；Finally take solution A 24.3mL, second liquid 25.7mL, add water and be settled to 100mL, be tmb substrate buffer-phosphoric acid-citrate buffer solution (pH5.0).
Tmb substrate nitrite ion: taking 100 μ LTMB stock solutions in being dissolved in above-mentioned 10mLTMB substrate buffer solution-phosphoric acid-citrate buffer solution (pH5.0), adding volume fraction is 30%H20210 μ L, mix, it should be noted that tmb substrate nitrite ion needs now with the current.
Stop buffer: for the sulphuric acid liquid that concentration is 2M/L.
Screening step:
(1) it is coated: by the IgV district of the PD-L1 albumen of prokaryotic expression purification, after urea concentration gradients renaturation, obtains destination protein PD-L1IgV(be dissolved in the NaHCO of 0.1MpH8.63Solution).It is coated 96 orifice plates with destination protein, 15 μ g/ holes (150 μ L, 100 μ g/mL), airtight 4 DEG C of overnight incubation of wet box；
(2) closing: discard and be coated liquid, remain residual liquid with rifle head sucking-off and fill it up with rapidly confining liquid, 4 DEG C of airtight wet box hatches 3h；
(3) washing: TBST washs 6 times, every time no less than 2min, notes sterile working, and action wants fast in order to avoid microwell plate dries；
(4) combine: rapidly joining and being diluted to titre with TBST in advance is 2 × 1011The phage 100 μ L/ hole in library, room temperature gentleness shake 1h；In this process, the phage with target protein with affinity can be combined on albumen；
(5) washing: TBST washs 10 times, washes away unconjugated phage；
(6) eluting: every hole adds 100 μ L eluents, room temperature gentleness shake 20min；
(7) neutralize: by the phage of the careful sucking-off eluting of liquid-transfering gun, be transferred in the sterile centrifugation tube having added 15 μ L neutralizers in advance, gentle piping and druming mixing；
(8) amplification: the phage that first round screening is obtained adds LB fluid medium (Tet with escherichia coli ER2738+) in co-cultivation, carry out expanding and purification, obtain secondary peptide storehouse；
(9) titer determination: respectively each secondary peptide storehouse taken turns after screening the phage obtained and amplification is carried out phage titre mensuration on LB/IPTG/Xgal flat board, and carry out response rate calculating, the phage response rate=[wash-out bacteriophage number/input phage number] × 100%；
(10) screening is repeated: phage amplification obtained puts into next round screening, repeats above-mentioned screening process.Take turns affine screening through 5, the phage containing desired polypeptides can be made to obtain highly enriched；
(11) order-checking: picking the 5th take turns screening after titer determination flat board on plaque expand, taking the phage stock solution of the 200 fresh amplifications of μ L to be sent to gold only intelligence order-checking company and automatically check order, sequencing primer is-96g III sequencing primer: 5 '-CCCTCATAGTTAGCGTAACG-3 '.
Peptide sequence is analyzed: derives its coded aminoacid sequence according to DNA sequencing result, obtains the aminoacid sequence of antagonistic peptide.
The selection result:
(1) respectively each bacteriophage elution liquid of obtaining of screening of taking turns being carried out titer determination, and calculate the input phage response rate, result is shown in following table.
(2) we select seven peptide storehouses that PD-L1IgV albumen is screened, and result is shown in following table.Fourth round altogether screen order-checking 18 phage clones, and wherein 11 be all P1 peptide sequence, the affinity peptide that namely present invention is claimed, account for order-checking clone more than 60%.
ELISA identifies the affinity of phage monoclonal and target protein PD-L1IgV
In order to identify the affinity of phage monoclonal and target protein PD-L1IgV, inventor has done further ELISA identification experiment, and authentication step is summarized as follows:
1, it is coated: (be dissolved in the NaHCO of 0.1M with the PD-L1IgV protein solution that concentration is 100 μ g/mL3, pH8.6) coated elisa plate, 100uL/ hole.
Negative control: each clone to be identified is all coated one and corresponding is coated liquid (0.1MNaHCO3, pH8.6) and hole is as negative control.
Blank: set and be coated target molecule and add wild type M13 phage monoclonal sample as blank.
Two multiple holes, airtight 4 DEG C of overnight incubation of wet box are set after being coated.
2, close: discarding and be coated liquid, the filter paper arsis that ELISA Plate is inverted in sterilizing gets rid of to remove residual liquid, and fills it up with rapidly confining liquid.4 DEG C of airtight wet box hatches 3h.
3, washing: TBST washs 6 times, every time no less than 2min.Noting sterile working, action wants fast in order to avoid microwell plate dries.
4, application of sample: rapidly joining and being diluted to titre with TBST in advance is 2 × 107P1 phage monoclonal sample, 100 μ L/ holes, room temperature gentleness shake 2h.
5, washing: TBST washs 6 times, washes away unconjugated phage.
6, two resist: by confining liquid 1:4000(volume ratio) dilute the anti-M13 antibody of HRP labelling.Every hole 100 μ L, 1h is hatched in room temperature concussion.
7, washing: TBST washs 6 times.
8, colour developing: every hole 100 μ LTMB substrate nitrite ion, room temperature about lucifuge 30min.
9, terminate: the μ L stop buffer color development stopping reaction of every hole 50.
10, plate is read: survey OD at 450nm wavelength place.With OD450Value is positive colony higher than more than 2.1 times persons of negative control, has good affinity with PD-L1IgV.
Qualification result:
As shown in Figure 1, ELISA experiment detects that specific P1 phage monoclonal and target protein have better affinity, its affinity with target molecule compared with the ratio of negative control more than 2.1, illustrate that the phage clone containing P1 seven peptide sequence is exactly the positive peptide clone that screening obtains, it is possible to specific in conjunction with target protein PD-L1IgV.
Embodiment 2
The affinity peptide of the described PD-L1IgV with anti-tumor activity adopts Fomc solid phase polypeptide synthesis to synthesize, and synthesis step is briefly described below:
Main agents used in building-up process has:
End socket liquid: acetic anhydride/pyridine solution (1:1, v/v)；
Indenes check reagent: A. 1,2,3-indantrione monohydrate/alcoholic solution (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: by volume content meter, TFA(82.5%), H2O(5%), phenol (5%), thioanisole (5%), dithioglycol (2.5%).
Synthesis step is briefly described below:
(1) swellable resins, add first aminoacid
A. swellable resins: take 0.3 ~ 0.5gRink resin (the C-terminal aminoacid of the peptide being connected with resin is amide) and be placed in wash clean and dry Peptide synthesizer, add appropriate DMF, soaking about 30min makes resin fully swelling, and vacuum pump extracts 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 2min totally 3 times；Add appropriate DMF, then concussion washs 2min totally 2 times.
C. add first aminoacid: by formula calculate add first aminoacid, HoBt and DIC amount, calculate publicity 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 mass=resin quality × 2.5 times equivalent × HOBT,
The relative molecular mass of DIC consumption=resin quality × 2.5 times equivalent × DIC,
Balance weighs Fmoc-aminoacid, HOBT in 50mL beaker, adds in synthesizer after dissolving with a small amount of DMF, then draws DIC addition synthesizer with liquid-transfering gun, is placed in shaking table by synthesizer, reacts 3h, make aminoacid and resin generation coupled reaction under room temperature.
D. washing: with step B.
E. survey substitution value: owing to, under 290nm, fluorenylmethyloxycarbonyl has very strong uv absorption, therefore by measuring its light absorption value, the situation of first aminoacid and resin-bonded can be measured by colorimetry.In picking step D, resin 1 ~ 1.5mg after reaction, dries, with electronic analytical balance precise resin quality；Then resin is loaded in the centrifuge tube of 5mL, add the deprotection liquid concussion 10min of 3mL；Transition into subsequently in cuvette and measure sample OD value in 290nm；The deprotection liquid adding 3mL in another cuvette is blank, calculates substitution value by formula, calculates publicity as follows:
Substitution value=sample OD value/1.65 × resin quality.
Substitution value is best when 0.4 ~ 0.6, if substitution value is less than 0.4, then shows that aminoacid is very few with the amount of resin-bonded, it should repeat said process and add first aminoacid.
F. end socket: add appropriate end socket liquid to synthesizer and jolt 20min twice totally.
G. washing: with step B.
(2) second aminoacid and follow-up peptide chain extension are added
H. deprotection: add appropriate deprotection liquid in synthesizer, shaking table reaction 20min totally 2 times.
I. washing: with step B.
J. indenes inspection: a small amount of resin in picking step I puts into indenes inspection pipe, dropping indenes check reagent A liquid 1, B liquid 2, C liquid 1 respectively, builds indenes inspection pipe, and boiling water bath 2min observes color of resin；Blue if (after Pro, His, Ser deprotection, indenes inspection color is rufous), then deprotection is normal；Otherwise must repeat deprotection process.
K. aminoacid is added: calculate and add Peptide synthesizer after load weighted aminoacid and a small amount of DMF of HOBT being dissolved, then adding DIC with liquid-transfering gun, being placed in shaking table, under room temperature, reacting 2.5h.
L. washing: with step B.
M. indenes inspection: the rapid J of course synchronization, if resin is glassy yellow, without locus coeruleus, then amino acid condensation is complete, can carry out next step reaction.If blue, then amino acid condensation is incomplete, need to repeat this amino acid whose condensation reaction, until indenes inspection resin is completely in glassy yellow.
N. step H-M is repeated to the condensation reaction completing 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 fume hood, puts into synthesizer by agitator, starts cleavage reaction, and agitator is slowly stirred cutting about 2.5h.After completion of the reaction, go out cutting reagent with vacuum filtration pump sucking filtration, more several all over stirring paddle and resin with DCM washing, and the cutting liquid obtained by sucking filtration loads round-bottomed flask.
S. rotary evaporation: being removed by the TFA in cutting liquid with Rotary Evaporators, Rotary Evaporators temperature is adjusted to 65 DEG C, rotary evaporation 10 ~ 20min.Adding ice ether afterwards, continue rotary evaporation, 10 ~ 20min, this step repeats 6 times, owing to containing arginine in peptide sequence, need to carry out ice and cut, to remove the protection group of side chain.In bottle, add 5mLTFA, be placed in ice, shaking table jolts 30min.After ice is cut, continue to be placed in Rotary Evaporators, rotary evaporation 10 ~ 20min.
T. precipitation: after rotary evaporation, poured into by cutting liquid in the beaker equipped with 50mL ice ether, ice bath stands 30min, makes polypeptide precipitate.
U. it is centrifuged: suction pipe draws beaker bottom ether sedimentation mixed liquor, 2000r/min, centrifugal 2min, abandons supernatant and collects precipitation, the resuspended washing precipitation of ice ether 5 times.
V. dry: the polypeptide of collection is deposited in 37 DEG C of oven for drying, and electronic analytical balance is weighed, and calculates thick peptide productivity, be sealed against being stored in-20 DEG C standby.
(4) RP-HPLC analyzes and prepares purified polypeptide
Mobile phase solution preparation in RP-HPLC analysis:
Mobile phase A liquid (1 ‰ TFA solution): pipet measures 1mLTFA, adds ultra-pure water and is settled to 1000mL, 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 purification:
W. start 30min preheating in advance, opens software operation interface, and parameter is arranged: wavelength arranges 228nm, and flow velocity arranges 5mL/min；
X. balance: balance about 30min；
Y. prepared by thick peptide: weighs thick peptide and is about 20mg, dissolves with the 0.1%TFA aqueous solution (4mL) containing certain concentration acetonitrile, sample introduction after 0.45 μm of filtering with microporous membrane.Collect main peak product and be placed in-80 DEG C of frozen overnight.
Z. lyophilizing: be placed in freeze dryer by the main peak Product samples frozen lyophilizing 24h, collects afterwards and weighs fine peptide, and-20 DEG C of sealings save backup.
(5) Mass Spectrometric Identification
Sperm chromosome peptide, by electrospray ionization mass spectrometry Analysis and Identification peptide molecule quality, affinity peptide Mass Spectrometric Identification result is as in figure 2 it is shown, qualification result meets expection.
Embodiment 3
For the affinity peptide of the PD-L1IgV with anti-tumor activity of embodiment 2 preparation, inventor has done further tumor-bearing mice experiment in vivo, and specific experiment process is as follows:
Select 24 experiments to use Balb/c mice, cell concentration is adjusted to 5 × 10 by Mus source colon cancer (CT26) cell normal saline (NS)6Individual cell/mL, by 0.1mL cell suspension (containing 5 × 10 after routine disinfection5Individual cell) to be inoculated in every Balb/c mice right fore axillary fossa subcutaneous, continuous observation subcutaneous tumors bulk-growth situation.
Being dissolved in normal saline by affinity peptide prepared by embodiment 2, make polypeptide drugs ,-20 DEG C of subpackages preserve, standby.
Inoculation Mus source colon cancer (CT26) cell is after 9 days, by mice by the packet of tumor volume, often group 6, is divided into negative control group (NS), positive controls (5-FU), high dose group (2mg/kg), middle dosage group (1.0mg/kg) and low dose group (0.5mg/kg).
Each group all adopts the other administration of tumor, and every day is administered once, altogether administration 7 days, mice ad lib and drinking-water during experiment.Measure length (a) short (b) footpath of Mouse Weight and tumor every day, and press and by tumor volume=π/6 × a × b2Formula calculates gross tumor volume and draws tumor growth curve.After 7 days, sacrifice is taken out tumor and weighs.Experimental result is shown in that in Fig. 3 Fig. 5, figure, use P1 is instead of affinity peptide provided by the present invention.
Medicine group experiment mice Normal-weight as can be seen from Figure 3, there is, because the toxic and side effects of 5-FU is relatively big, the situation of becoming thin in positive controls.As can be seen here, affinity peptide does not only have certain antitumous effect, and does not have obvious toxic and side effects.
We can see that medicine group experiment mice gross tumor volume is less than negative control group from Fig. 4.And within the scope of finite concentration, peptide concentration is more big, experiment mice gross tumor volume is more little.
We can see that medicine group experiment mice tumor anharmonic ratio negative control group is little from Fig. 5.Within the scope of finite concentration, affinity peptide concentration is more big, and experiment mice tumor is heavily more little.
SEQUENCELISTING
<110>Zhengzhou University
<120>there is PD-L1IgV affinity peptide and the application thereof of anti-tumor activity
<130>none
<160>1
<170>PatentInversion3.4
<210>1
<211>7
<212>PRT
<213>affinity peptide
<400>1
AlaAsnGlySerArgLeuVal
15
Claims (3)
1. one kind has anti-tumor activity targeting PD-L1IgV affinity peptide, it is characterised in that this affinity peptide specifically binds to PD-L1IgV district, and its aminoacid sequence is: Ala-Asn-Gly-Ser-Arg-Leu-Val, i.e. A-N-G-S-R-L-V, and molecular weight is 715.4Da.
2. there is described in claim 1 anti-tumor activity targeting PD-L1IgV affinity peptide and preparing the application of drugs against colon cancer.
3. the preparation method described in claim 1 with anti-tumor activity targeting PD-L1IgV affinity peptide, it is characterised in that prepared by Fmoc solid phase polypeptide synthesis.
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| CN104387453A (en) * | 2014-12-08 | 2015-03-04 | 深圳市同康生物医药有限公司 | Dendritic cell targeted peptide, coding gene and application |
| CN105504018B (en) * | 2016-01-13 | 2019-01-04 | 郑州大学 | LAG-3 affinity peptide N13, preparation method and applications |
| CN108727470B (en) * | 2017-04-17 | 2020-04-24 | 上海大学 | Polypeptide and application thereof |
| WO2018192443A1 (en) * | 2017-04-17 | 2018-10-25 | 中国科学院苏州纳米技术与纳米仿生研究所 | Polypeptide and application thereof |
| CN109265533B (en) * | 2018-10-30 | 2020-11-17 | 广东赛尔生物科技有限公司 | Improved tumor inhibiting peptide capable of being specifically combined with PD-1 and application thereof |
| CN109456390B (en) * | 2018-12-27 | 2021-11-16 | 西华师范大学 | Artificially synthesized polypeptide H-473 and application thereof |
| CN110330550B (en) * | 2019-08-02 | 2021-04-13 | 郑州大学 | Affinity peptide of PD-L1-IgV and application thereof |
| CN110478472B (en) * | 2019-09-29 | 2020-08-28 | 北京鼎成肽源生物技术有限公司 | PD-1 sealant and application thereof |
| CN110818776B (en) * | 2019-12-04 | 2022-07-12 | 南阳师范学院 | Affinity peptide and application thereof |
| CN112724197A (en) * | 2021-02-03 | 2021-04-30 | 河南大学 | 7 peptide for blocking PDL1 in immunotherapy |
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