CN106632613B

CN106632613B - Affinity peptides related to coxsackie virus adenovirus receptor

Info

Publication number: CN106632613B
Application number: CN201710037694.XA
Authority: CN
Inventors: 顾月清; 赵梦路; 韩智豪
Original assignee: China Pharmaceutical University
Current assignee: Nanjing Nuoyuan Medical Devices Co Ltd
Priority date: 2017-01-17
Filing date: 2017-01-17
Publication date: 2020-07-31
Anticipated expiration: 2037-01-17
Also published as: CN106632613A

Abstract

The invention discovers a series of affinity polypeptides YQC-1 and YQC-2 related to a coxsackie virus adenovirus receptor, the specific binding property of the polypeptides to the coxsackie virus adenovirus receptor can realize near-infrared imaging of specific malignant tumors, and the affinity polypeptides YQC-1 and YQC-2 can be used for preparation of drugs for inhibiting cell adhesion, diagnosing or tracing tumors, targeted chemotherapy and pharmaceutical excipients or connectors, thereby achieving the purpose of real-time nondestructive in-situ monitoring of early malignant tumors. The invention relates to the field of medicaments related to tumor diagnosis, in particular to a plurality of polypeptides, an affinity test thereof, a pharmaceutical composition taking the polypeptides as active ingredients and application thereof in preparing diagnostic medicaments.

Description

Affinity peptides related to coxsackie virus adenovirus receptor

Technical Field

The invention belongs to the technical field of bioengineering pharmacy and the field of protein polypeptide drugs and biomedical engineering, and particularly relates to a polypeptide with high affinity for a coxsackie virus adenovirus receptor and application thereof, such as cancer diagnosis and treatment.

Background

At present, cancer becomes the biggest culprit to harm the health of residents in China, and seriously threatens the health of human beings. The clinical methods for treating tumors include surgical methods, chemical drug therapy and radiotherapy. Among them, chemotherapy is the most widely used method for tumor therapy at present, and chemotherapy is the main treatment means in patients with advanced tumors who lose surgical chances; postoperative adjuvant chemotherapy has also been shown to reduce the rate of postoperative tumor recurrence after surgery in patients with high risk factors; in various tumors such as breast cancer, preoperative neoadjuvant chemotherapy is also increasingly gaining attention.

CARs are co-receptors for coxsackie group B virus (CBV) and Adenovirus (Adenovirus, Ad) to bind to target cells. In 1997, Bergelson et al first isolated a cDNA clone encoding the co-receptor for CVB and Adv located on human chromosome 21q21-cen, which encodes a 365 amino acid polypeptide belonging to the immunoglobulin superfamily. CARs are widely distributed and different species, tissues are distributed differently. In mice, CAR is distributed in the heart, liver, brain, kidney, lung, etc., and is expressed in the liver in the highest amount. CARs are widely distributed and different species, tissues are distributed differently. In mice, CAR is distributed in the heart, liver, brain, kidney, lung, large intestine, etc., with the highest expression in the liver. In humans, CAR mRNA is detected in many tissues and organs, including heart, liver, brain, kidney, lung, intestine, pancreas, and testis, and is expressed in very abundant amounts in the neointima of heart, pancreas, and arteries. Notably, CARs are highly expressed in certain tumors and cell lines, such as liver cancer cell lines, cervical cancer cell lines, melanoma, and the like, but there is a decrease, loss of CAR expression in glioma, prostate cancer, renal cancer, bladder cancer, human ovarian cancer cell lines, and the like.

As a protein involved in infection with COXSACKIEVIRUS AND the like, a COXSACKIEVIRUS-ADENOVIRUS RECEPTOR (CoXSACKIEVIRUS AND ADENOVIRUS RECEPTOR; CXADR) is known. In addition, it has been reported that the expression of the protein is enhanced in liver cancer and basal cell carcinoma of the skin. On the other hand, in cholangiocarcinoma, the homozygous deletion of CXADR was confirmed, suggesting that CXADR functions as an oncogene. As described above, although CXADR has been reported to be associated with cancer, it is not yet determined whether CXADR contributes to the onset, malignancy, and the like of cancer, or exerts an inhibitory effect on cancer. Therefore, it is also unclear whether antibodies against CXADR have anti-cancer activity.

The role of CARs in tumors has been considered to be a tumor suppressor. In some human malignancies, including bladder, prostate, and malignant gliomas, the expression level of CAR is down-regulated as the malignancy of the tumor progresses. Transfection of prostate and glial tumor cell lines that lack CAR expression of CARs can result in a decrease in cell proliferative activity and tumor formation ability. In vitro and in vivo studies of glioma, CAR plays a role in tumor inhibition, which provides a reasonable explanation for the expression down-regulation of CAR in malignant glioma, and CAR expression is similarly down-regulated in other tumors including ovarian cancer, malignant melanoma, head and neck tumors, musculoskeletal system tumors, immature chondroblastoma and human rhabdomyosarcoma. Antibodies are currently proteins that demonstrate high affinity for the coxsackie virus adenovirus receptor (see for details: anti-CXADR antibodies, application No. 201380024727.7).

The invention discloses polypeptides capable of highly-affinity coxsackie virus adenovirus receptors, which can be efficiently combined with coxsackie virus adenovirus receptors, thereby targeting tumor cells highly expressing the receptors and finally achieving the effect of marking the tumor cells. The polypeptides can be prepared into medicaments for diagnosing cancers.

Disclosure of Invention

The invention aims to provide a targeting polypeptide related to a coxsackie virus adenovirus receptor, so that the targeting polypeptide can be used for in-vivo diagnosis of tumors with high expression of the coxsackie virus adenovirus receptor and can be used for preparing a novel targeting drug carrier.

It is also an object of the present invention to provide a means of action of a molecule comprising or partially comprising a polypeptide or protein of the YQC-1, YQC-2 sequence, which binds to the coxsackie virus adenovirus receptor and induces a decrease in cell adhesion.

Another purpose of the invention is to provide an antibody containing YQC-1 and YQC-2 as an effective component for detecting coxsackie virus adenovirus receptors in vitro and in vivo.

The YQC-1 and YQC-2 peptides are obtained by optimizing the structure of an adenovirus affinity group, have natural amino acid sequences, and have small immunogenicity and good biological activity.

In order to detect the pharmacological activity of the polypeptide, the polypeptide is marked by a fluorescent dye rhodamine B in an in vitro experiment, and the polypeptide is marked by a near infrared dye ICG-Der-02 in an in vivo experiment so as to detect the in vivo and in vitro targeting of the polypeptide to tumors. In-vitro cell affinity experiments and uptake experiments, the YQC-1 and YQC-2 peptides show good targeting capability to tumor cell coxsackie virus adenovirus receptors. In vitro cytotoxicity experiments, the YQC-1 and YQC-2 peptides of the invention do not show toxicity to cells.

The detailed invention content is as follows:

marking in vitro experiment fluorescent dye

The fluorescent dye applied to in vitro cell experiments is Rhodamine B (Rhodamine B), named RhB for short, which is respectively connected with YQC-1 and YQC-2 peptides of the invention through amido bonds, named YQC-1-RhB and YQC-2-RhB for short, and the connection method specifically comprises the steps of taking 1mg of Rhodamine B (for details, see the reference document: Cao, J., et al, Fast cleaning RGD-base near-enriched fluorescent probes for in vivo tissue diagnosis. contrast Media MolImaging, 2012.7 (4): p.390-402), dissolving in 1ml of PBS (pH is 7.4), adding 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride, N-hydroxysuccinimide (EDC/NHS) (the molar ratio of Rhodamine B: NHS is 1: 1.5: EDC, and activating in a dark place. 1mmol of YQC-1 and YQC-2 peptides are respectively weighed and dissolved in 1ml of PBS buffer solution (pH7.4) containing the fluorescent dye rhodamine B, and stirred overnight at room temperature in a dark place. After the reaction, the reaction solution was concentrated and passed through Sephadex G-25 column, and PBS buffer (pH7.4) was used as eluent to obtain purified YQC-1-RhB and YQC-2-RhB, which were stored at-20 ℃ for further use.

Second, in vitro cell affinity experiment

The cultured human hepatoma HepG2 and human brain glioma U87MG cells are eluted from a 12-well plate and then suspended in a PBS solution, and are respectively incubated with rhodamine B labeled YQC-1 and YQC-2 peptides (10 umol/L) for 2 hours, and the average fluorescence intensity is detected by flow cytometry, the stronger the fluorescence intensity is, the stronger the affinity to the cells is, when the affinity of the probes to receptors on the cells is strong, the higher the average fluorescence intensity value of the cells detected by a flow cytometer is, the in vitro affinity experiment result shows that the probes of the YQC-1 and the YQC-2 peptides labeled with RhB have the same concentration are respectively incubated with the HepG2 cells highly expressed by Coxsackie adenovirus receptors, the average fluorescence intensity of the YQC-1 peptide probes in the HepG2 cells is 68, the average fluorescence intensity of the YQC-2 peptide probes is 344, the blank dye average fluorescence intensity is 5, and the result shows that the probes have the highest fluorescence intensity when the affinity of the probes is compared with the HepG2 cells, the invention is very strong, but the maximum fluorescence intensity of the YQC-2 peptide probes is very low, and the average fluorescence intensity of the YQC-2 probes is very low as well as the fluorescence intensity of the fluorescence of the Coxsack.

Third, in vitro cell uptake assay

The cultured human hepatoma cell HepG2 and human brain glioma U87MG cells are transferred to a confocal dish, after overnight incubation, rhodamine B labeled YQC-1 and YQC-2 peptides (40 umol/L) with the same concentration are respectively added, after incubation for 4 hours at 37 ℃, a staining reagent of 12 mu g/m L Hochests 33342 is added for staining, and finally, the uptake condition of the probes in the cells is observed by a laser confocal microscope.

Fourth, in vitro cytotoxicity test

U87MG cells, HepG2 cells and normal liver L02 cells, when they grow to more than 90%, they are digested with 0.25% trypsin solution to prepare single cell suspension (complete growth medium), which is spread on 96-well plate at 3000 cells/well and at 37 deg.C with 5% CO₂The culture medium of (1%) FBS was replaced with a low serum culture medium after 24 hours, and then different concentration gradients of YQC-1 and YQC-2 peptides were added to make their final concentrations 2.5, 5, 10, 20, 40, 80 and 100. mu.M. The results showed (FIG. 3) that both YQC-1 and YQC-2 peptides showed over 80% survival for these cells, indicating that the YQC-1 and YQC-2 peptides are essentially non-toxic for these cells.

Five, living tumor targeting experiment

The fluorescent dye applied to in vivo animal experiments is a near infrared organic dye ICG-Der-02 which is respectively connected with YQC-1 and YQC-2 through amide bonds, is ICG-Der-02-C1 for short and ICG-Der-02-C2, and the specific connection method is that 2mg of ICG-Der-02 (detailed in a reference document) is dissolved in 1ml of PBS (pH is 7.4), 1-ethyl- (3-dimethylaminopropyl) carbonyl di-industrial amine hydrochloride and N-hydroxysuccinimide (EDC/NHS) (molar ratio ICG-Der-02: EDC: NHS is 1: 1.5) are added, and the mixture is reacted for 4 hours in a dark place and activated. 1mmol of YQC-1 and YQC-2 peptides were dissolved in 1ml of PBS buffer (pH7.4) containing the fluorescent dye ICG-Der-02 and stirred overnight at room temperature in the dark. After completion of the reaction, the reaction mixture was concentrated and passed through a Sephadex G-25 column and eluted with PBS buffer (pH7.4) to obtain purified ICG-Der-02-C1, ICG-Der-02-C2, which was stored at-20 ℃ until use. Nude mice were imaged separately by near infrared imaging system at different time points and the difference between different probes was compared 0-24 hours after injection. The target specific Region (ROI) analysis of the tumor site fluorescence signal intensity was calculated as follows: tumor/normal tissue ratio ═ tumor signal intensity-background signal intensity ]/[ normal tissue (muscle) signal intensity-background signal intensity ], to quantify the targeting of different probes in different tumors, a larger value indicating a stronger targeting.

The HepG2 tumor-bearing nude mice are injected with ICG-Der-02 marked YQC-1 and YQC-2 by tail vein. And (3) respectively carrying out imaging detection on the nude mice at different time points by a near-infrared imaging system 0-12h after injection.

HepG2 liver cancer cell is a high expression cell of Coxsackie adenovirus receptor, if the probe is gathered at the tumor position in the nude mouse within a certain time and can last for a certain time, the probe can be targeted to the tumor cell. As shown in figure 4, after injecting several probes labeled with the same amount of ICG-Der-02 and ICG-Der-02 respectively into a HeGp2 tumor-bearing nude mouse, the ICG-Der-02, ICG-Der-02-C1 and ICG-Der-02-C2 are injected for 2h to distribute fluorescence throughout the body, and then are gradually transferred to kidney-bladder metabolism, no obvious fluorescence signal exists at the tumor site, the fluorescence signal at the tumor site is gradually enhanced after injecting probes ICG-Der-02-C1 and C2 for 4h, along with the metabolism of the probes in vivo, the fluorescence intensity of the probes is more and more accumulated at the tumor site, the peak value is reached at 6h after injection, the outline of the tumor edge is clearer, and the fluorescence signal at the tumor part basically disappears after 24 h. The fluorescence intensity of the probe at the tumor site was further quantitatively analyzed by using the ROI values, and the ratios of the tumor fluorescence intensity to the normal tissue after 6h of injecting the ICG-Der-02-C1 and the ICG-Der-02-C2 probes were 15.23 +/-0.53 and 8.41 +/-0.24, respectively. The result shows that the probes ICG-Der-02-C1 and ICG-Der-02-C2 have better targeting property and can be quickly targeted to tumor parts, and the result shows that the affinity peptide can be targeted to cells with high expression of Coxsackie adenovirus receptors on an animal level.

In conclusion, the YQC-1 and YQC-2 peptides can target cells with high expression of a Coxsackie adenovirus receptor, have no targeting effect on the cells with low expression of the Coxsackie adenovirus receptor basically, and have no toxicity on the cells by the YQC-1 and the YQC-2 peptides. Therefore, the YQC-1 and YQC-2 peptides can be used for in vitro diagnosis of high-expression tumors of Coxsackie adenovirus receptors.

The YQC-1 and YQC-2 peptides of the present invention can be preferably prepared by the solid phase synthesis method of examples 1 and 2.

The polypeptide of the invention is a coxsackie adenovirus receptor affinity peptide. While not wishing to be bound by theory, the inventors believe that the affinity action of the polypeptides of the invention is based on their inhibition of the coxsackie adenovirus receptor effect.

The pharmaceutical composition comprises: pharmaceutical compositions based on the YQC-1, YQC-2 peptides, or pharmaceutically acceptable salts or prodrugs, including esters, thereof may be prepared in any therapeutically effective amount for the indications described herein, including inhibition of proliferation of brain glioma cells, breast cancer cells, optionally in combination with pharmaceutically acceptable additives, carriers or excipients. The therapeutically effective amount may vary with the infection or condition to be treated, its severity, the treatment regimen employed, the pharmacokinetic properties of the agent used, and the patient being treated.

The invention also includes pharmaceutical formulations comprising as an active ingredient a peptide YQC-1, YQC-2 or an ester or prodrug thereof or a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, and refers to one or more inert, nontoxic solid or liquid fillers, diluents, adjuvants, etc., which do not adversely affect the active compound or the patient.

The dosage form of the composition can be tablets, capsules, pills, suppositories, soft capsules, oral liquid, suspensions, injection and other pharmaceutically commonly used dosage forms. Tablets and capsules for oral use contain conventional excipients such as fillers, diluents, lubricants, dispersants and binders.

The various dosage forms of the pharmaceutical compositions of the present invention may be prepared according to methods well known in the pharmaceutical art.

The dosage of the above active agents will vary depending on the formulation.

Has the advantages that:

1. the invention discloses a series of novel polypeptides with high affinity to a coxsackie virus adenovirus receptor, and expands the types of molecules which are combined with the coxsackie virus adenovirus receptor.

2. The polypeptides are low molecular weight polypeptides, and the synthesis cost is low.

3. The peptides are reported for the first time, and the acquisition channel is convenient.

4. The product is prepared from ICG-Der-02 cyanine near-infrared fluorescent dye, which has a carboxyl functional group on its side chain and is suitable for biological fluorescent labeling. The polypeptide can be effectively activated by an EDC/NHS catalytic system, and is covalently coupled with amino at the tail end of a polypeptide molecular chain to form an amido bond, so that an ICG-Der-02-peptide molecular probe is constructed, and the polypeptide has obvious near-infrared fluorescence characteristics.

5. The property of the specific combination of the polypeptides to the coxsackie virus adenovirus receptor can realize the near infrared imaging of specific malignant tumors, thereby achieving the purpose of real-time nondestructive on-site monitoring of early malignant tumors. The Coxsackie virus adenovirus receptor has high-level expression on the surfaces of various solid tumor cells such as breast cancer, liver cancer, glioblastoma, prostatic cancer, bladder cancer, osteosarcoma and the like, a HepG2 liver cancer cell is selected and connected with a better target with polypeptide, and the tumor part has an obvious fluorescent signal after administration.

Brief description of the drawings:

FIG. 1 flow cytometry for cell affinity experiment Observation of rhodamine B labeled probe for affinity of different tumor cells (AD for HepG2 cell, BE for U87MG cell)

FIG. 2 laser confocal cell uptake experiment to observe the uptake of rhodamine B labeled probe into different tumor cells (A is HepG2 cell, B is U87MG cell)

FIG. 3 in vitro cytotoxicity test to examine the toxicity of YQC-1 and YQC-2 peptides on different cells

FIG. 4 experiment for examining polypeptide targeting ability in vivo by small animal in vivo imaging technology

Detailed Description

The present invention will be further described with reference to the following examples. It should be noted that the following examples are only for illustration and are not intended to limit the present invention. Variations of the teachings of the present invention may be made by those skilled in the art without departing from the scope of the claims of the present application.

Example 1

Synthesis Process of polypeptide YQC-1 of the present invention

1: coupling of

Fmoc-Arg-Rink amide MBHA resin (fluorenylmethoxycarbonyl-arginine-Rink amino resin) 0.33/2mmol 6.06g is swelled in dimethylformamide, 10ml/g resin is removed, 20% piperidine/dimethylformamide solution is used to remove Fmoc protecting group (hereinafter referred to as decapping), the uncapped resin is washed with dimethylformamide, raw material Fmoc-L ys (Boc) -OH 2.3g is added, condensing agent is HBTU 1.44g, reaction time is 30 minutes, kaiser method is used for detecting if detection result is negative, indicating that coupling reaction is completed, uncapping is performed for 30 minutes, indicating that coupling reaction is completed if detection result is still positive, indicating that coupling reaction is not completed, extension of coupling time or re-feeding of coupling reaction is required until coupling reaction is completed, repeating the above operations for sequential coupling, Fmoc-Ala-OH, Fmoc-Asp- (OtBu) -OH, (Fmoc) -OH, Fmoc-t-OH, (Pro-Pro, Fmoc-t-OH, Trboc-t-Pro, Fmoc-Pro, Fmoc-Arg-Asp-Pro, Fmoc-Arg-.

2: separation and cleavage of resin from peptide

Adding 120ml of lysate (10ml/g peptidyl resin) into the resin, magnetically stirring for 2.5h at 25 ℃, separating the lysate from the resin by using a sand core funnel, discarding the resin, and keeping the filtrate. Slowly dripping the filtrate into 10eq volume of ice anhydrous ether, and naturally settling for 30min after dripping. And centrifuging with a high-speed centrifuge for 10min (4000rpm), discarding supernatant, retaining precipitate, and drying the obtained precipitate in a drying oven for 8-10h to obtain crude dry powder.

3: purification of

Taking 1g of the crude dry powder, dissolving the crude dry powder with 0.1% trifluoroacetic acid/water, filtering, loading the powder to a C18 preparation column, performing gradient elution by using a high performance liquid phase, collecting an elution liquid of a target peptide, detecting the liquid purity, mixing qualified samples, performing rotary evaporation, and finally performing freeze-drying by using a freeze-drying machine to obtain Asp-Pro-Pro-Pro-Asn-Cys-Ser-L eu-Ile-Gln-Glu-L eu-Asp-Ala-L ys-Arg, wherein the purity is more than 98%.

The sequence is Asp-Pro-Pro-Pro-Asn-Cys-Ser-L eu-Ile-Gln-Glu-L eu-Asp-Ala-L ys-Arg

The mass spectrogram shows that the molecular weight of Asp-Pro-Pro-Pro-Asn-Cys-Ser-L eu-Ile-Gln-Glu-L eu-Asp-Ala-L ys-Arg is 599.35 x 3-3 ═ 1795.05

The ultraviolet spectrogram has a peptide absorption peak at 273 nm.

Example 2

Synthesis Process of polypeptide YQC-2 of the present invention

1: coupling of

Fmoc-L ys-Rink amide MBHA resin (fluorenylmethoxycarbonyl-lysine-Rink amino resin) 0.33/2mmol 6.06g is swelled in dimethylformamide, 10ml/g resin is removed, Fmoc protecting group is removed by 20% piperidine/dimethylformamide solution (hereinafter referred to as uncapping), the uncapped resin is washed by dimethylformamide, raw material Fmoc-Cys (Trt) -OH 2.3g is added, condensing agent is 1.44g by HBTU, reaction time is 30 minutes, kaiser method is used for detecting if detection result is negative, indicating that coupling reaction is completed, uncapping is carried out for 30 minutes, if detection result is still positive, indicating that coupling reaction is not complete, coupling time is extended or feeding coupling material again until coupling reaction is completed, the above operations are repeated, coupling is carried out in turn, Fmoc-Cys) -OH, (Fmoc-Asn (Trt) (Pro, Fmoc-Asp-Pro, Trboc-OH, Trboc-t-OH, Fmoc-Ser-Pro, Fmoc-Pro, Trboc-Arg-t-OH, Fmoc-Pro, and Pro, Fmoc-Pro, and Fmoc.

2: separation and cleavage of resin from peptide

3: purification of

Taking 1g of the crude dry powder, dissolving the crude dry powder with 0.1% trifluoroacetic acid/water, filtering, loading the obtained product to a C18 preparation column, performing gradient elution by using a high performance liquid phase, collecting an elution liquid of a target peptide, detecting the liquid purity, mixing qualified samples, performing rotary evaporation, and finally performing freeze-drying by using a freeze-drying machine to obtain Pro-Asp-Pro-Ser-Pro-Asn-Cys-Arg-Ile-His-Ser-Asp-Asn-Asp-Cys-L ys, wherein the purity is more than 98%.

The sequence is Pro-Asp-Pro-Ser-Pro-Asn-Cys-Arg-Ile-His-Ser-Asp-Asn-Asp-Cys-L ys

The mass spectrogram shows that the molecular weight of Pro-Asp-Pro-Ser-Pro-Asn-Cys-Arg-Ile-His-Ser-Asp-Asn-Asp-Cys-L ys is 599.98 x 3-3 ═ 1796.94

The ultraviolet spectrogram has a peptide absorption peak at 273 nm.

Claims

1. The first polypeptide YQC-1 with high affinity to coxsackie virus adenovirus receptor is characterized in that the sequence is SEQ ID NO: 1;

the polypeptide YQC-1 is obtained by structural optimization of adenovirus affinity groups.

2. The second polypeptide YQC-2 with high affinity for coxsackie virus adenovirus receptors is characterized in that the sequence is SEQ ID NO: 2;

the polypeptide YQC-2 is obtained by structural optimization of adenovirus affinity groups.

3. A pharmaceutical agent for examining a disease associated with a Coxsackie virus adenovirus receptor protein, comprising the polypeptide of claim 1 to 2 as an active ingredient.