CN114478693B

CN114478693B - Polypeptide molecule with tumor metastasis inhibiting activity, preparation method and application thereof

Info

Publication number: CN114478693B
Application number: CN202011252220.5A
Authority: CN
Inventors: 段友容; 廖洪泽; 王丽婷; 沈庆; 王琪; 余坚
Original assignee: Shanghai Cancer Institute
Current assignee: Shanghai Cancer Institute
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2024-03-19
Anticipated expiration: 2040-11-11
Also published as: CN114478693A

Abstract

The invention relates to a polypeptide molecule with tumor metastasis inhibiting activity, a preparation method and application thereof. The polypeptide molecule has DR6 death receptor binding activity, can inhibit tumor cell from inducing vascular endothelial cell necrosis, and can prevent tumor cell from permeating vascular wall through blood flow, thus realizing tumor metastasis process. These polypeptide molecules have potential tumor metastasis inhibiting effect. The molecular structural formula is:

Description

Polypeptide molecule with tumor metastasis inhibiting activity, preparation method and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a polypeptide molecule for inhibiting tumor metastasis activity, which is suitable for anti-metastasis treatment of various tumors.

Background

Inhibition of tumor metastasis is critical for tumor treatment. At present, most of the anti-tumor drug action mechanisms are concentrated in the aspects of killing tumor cells, inhibiting tumor growth, reversing tumor drug resistance and the like, and the drugs aiming at tumor metastasis are relatively few, so that the existing drugs for inhibiting the metastasis have unsatisfactory effects. The development of drugs against tumor metastasis remains an urgent need for a solution.

Metastasis from blood, lymphatic metastasis and local invasion are the major pathways for tumor metastasis. Hematogenous metastasis (Hematogenous metastasis) is the primary metastasis of most sarcomas, liver cancer, lung cancer, kidney cancer, follicular thyroid cancer, malignant melanoma, and choriocarcinoma. Tumor cells that pass through the lymphatic pathway are also generally able to enter the blood circulation. Thus, inhibiting tumor hematogenous metastasis pathway is of great importance for tumor metastasis therapy.

The blood-borne pathway is an important tumor metastasis pathway, a complex multi-factor intervention process, and a bidirectional interaction between the tumor and the surrounding environment, involving a great number of cell-to-molecule interactions. The blood transfer process is divided into three phases: in the early stage, primary foci induce angiogenesis and tumor cells break through the stroma layer and enter the blood circulation. Tumor cells migrate with the blood circulation in the metaphase. Later, tumor cells penetrate the vessel wall to the site of metastasis. Later stage of blood metastasis, tumor cells cross vascular endothelium to reach the metastasis target site to realize metastasis. Tumor cells that enter the circulation can be cleared for the most part, and their metastatic efficiency is largely dependent on the rapid and efficient escape from the blood stream through the vascular endothelium. Studies have shown that this extravasation of tumor cells is similar to the transendothelial migration of leukocytes. However, it has not been clear heretofore how tumor cells interact with endothelial cells and the mechanism of molecular action in the process. The interaction of tumor cell surface-expressed Amyloid Precursor Protein (APP) with vascular endothelial cell surface death receptor (DR 6) was reported by Strilic B et al, max-Planck, nature journal, 2016, to induce endothelial cell programmed necrosis (necroptosis) across the vessel wall to target organ. This study result demonstrates a new mechanism for tumor cells to break through the vessel wall.

The existing therapeutic mechanisms acting on blood-circulation pathway drugs mainly aim at the early stage of blood-circulation transfer, namely, the aspects of inhibiting the growth of new blood vessels at the primary tumor part, reducing the movement capacity of tumor cells and the like, and can be divided into the following two types:

(1) Tumor angiogenesis inhibitor

Preventing tumor angiogenesis can cut off the supply of nutrients to tumor tissue, effectively inhibit the growth of solid tumor, and cut off the passage of tumor cells from primary foci to remote tissue. For this mechanism, many drug targets and marketed drugs have been developed, such as VEGFR2 inhibitors, VEGF antibodies, integrin receptors αvβ3 and αvβ5 inhibitors, EGFR inhibitors, EGF antibodies, and the like. The number of such inhibitors is huge and the structure and function are various, and many of them become marketed drugs, as shown in the following table. However, paez-Ribes and Ebos et al report in the journal of cancer cells that treatment with different antiangiogenic treatments in preclinical experimental studies increases the likelihood of tumor invasion and spread depending on the treatment situation. Upon inhibition of VEGF activity, the compensatory alternative pathway is activated. Elevated levels of plasma proteins G-CSF, plGF and SDF1 promote infiltration and metastasis of tumor cells. Thus, preventing metastasis of tumor blood by inhibiting the growth of new blood vessels has not been able to effectively block tumor metastasis.

The following table represents a drug for inhibiting tumor neovascularization:

(2) Metalloprotease inhibitors

Extracellular matrix (cell-extracellular matrix, ECM) is a diverse protein and non-protein component that exists from cell to cell, is the microenvironment upon which cells survive, and degradation of ECM is thought to be the onset of tumor angiogenesis and cancer cell infiltration, metastasis. Tumor cells can produce large amounts of lytic enzymes (metalloproteinases MMPs, cathepsins, plasminogen activators, plasmin, etc.) which degrade them, forming local channels that promote migration. Matrix metalloproteinase inhibitors are currently more studied. Based on the classification of structural features, 7 general classes of inhibitors, hydroxamate polypeptide analogs (pepmidoethmates-based inhibitors), new generation Hydroxamates (hydrabamates-based inhibitors), thiols (thio-based inhibitors), pyrimidines (pyrimides-based inhibitors), hydroxypyrones (hydracrylates-based inhibitors), bisphosphonates (phosphoruses-based inhibitors), and Tetracycline derivatives (tetracyclines-based inhibitors have been developed. MMP inhibitors are a class of anti-tumor metastasis drugs which are widely and deeply studied, however, the compounds have some defects such as low selectivity, large toxic and side effects and the like. This has led to MMP inhibitors that have not achieved ideal results in clinical trials. The search for highly selective and well-formed MMP inhibitors is also continuing. The matrix metalloproteinase inhibitors are as follows:

disclosure of Invention

The invention provides an active molecule capable of blocking tumor cells from escaping from blood circulation, which has long half-life and extremely low toxicity, and provides a new scheme for inhibiting tumor metastasis treatment.

In order to solve the technical problems, the invention provides the following technical scheme:

a polypeptide molecule with tumor metastasis inhibiting activity, which has the molecular structural formula:

furthermore, the invention also provides a preparation method of the polypeptide molecule with tumor metastasis inhibiting activity, which comprises the following steps:

firstly, phage display technology obtains 24 leader polypeptides with a certain binding force with DR 6. Performing biological activity verification on the lead polypeptide by adopting a micro thermophoresis experiment, a cell adhesion experiment and a co-culture experiment, and screening to obtain 4 DR6 polypeptides with strong binding force: AHP-03, AHP-10, AHP-12, AHP-13. Its binding constant (Kd) to DR6 protein is: 688.5 + -11.20 nM, 527.8+ -12.09 nM, 321.8 + -11.24 nM, 457.3+ -11.56 nM.

The leader polypeptide sequence is composed of L-type amino acids, the L-type amino acids are easily recognized and degraded by protease in the body, and the metabolic stability is poor. To improve polypeptide stability, all L-form amino acids are replaced with D-form amino acids. Thus, 8D-type polypeptides were obtained: AHP-D-03, AHP-D-10, AHP-D-12, AHP-D-13, AHP-DRI-03, AHP-DRI-10, AHP-DRI-12, AHP-DRI-13. Screening by the same method to obtain the D-type DR6 polypeptide with the strongest binding force and higher stability: AHP-DRI-12. Its binding constant (Kd) to DR6 protein: 225.19 + -4.30 nM.

The adoption of multi-arm PEG chains to connect different numbers of drug molecules can form a multivalent effect to enhance the binding capacity of the drug to the target. This approach has been widely used in drug research, where more 4-arm and 8-arm PEG chains have been studied and used. Based on these ideas, 4-arm PEG is used for modifying AHP-DRI-12, and activity is further enhanced while the half life of DR6 polypeptide inhibitor is prolonged. After modification of sulfhydryl group by AHP-DRI-12, the modified sulfhydryl group is connected with 4-arm PEG to obtain PEG-tAHP-DRI. The biological activity was verified by the above method, and the binding force to DR6 was 51.12.+ -. 11.56nM, respectively.

Furthermore, the application of the polypeptide molecule with tumor metastasis inhibiting activity provided by the invention is the application of inhibiting tumor cells (gall bladder cancer, melanoma and lung cancer) to induce vascular endothelial cell necrosis and prevent tumor cells from metastasis through vascular walls through blood flow.

The invention has the following technical effects:

1. the invention provides a series of polypeptide molecules with brand new sequence structure and inhibiting effect on DR6 death receptor, and the polypeptide is modified by structure and PEG, so as to protect the polypeptide molecules from being deleted, inserted or substituted by one or more amino acids and the polypeptide molecules have the same biological functions as the polypeptide molecules.

2. The polypeptide molecule has DR6 death receptor binding activity, can inhibit tumor cells (gall bladder cancer, melanoma and lung cancer) from inducing vascular endothelial cell necrosis, prevent the tumor cells from penetrating through the vascular wall through blood flow, and realize the tumor metastasis process. These polypeptide molecules have potential tumor metastasis inhibiting effect.

Drawings

FIG. 1 shows the results of the biological activity verification of 24 leader polypeptides;

FIG. 2 shows the results of the bioactivity verification of 8D peptides;

FIG. 3 shows a nuclear magnetic resonance ¹ H-profile verifies successful attachment of four-arm PEG to DR6 polypeptide;

FIG. 4 shows the in vitro tumor metastasis inhibiting effect;

FIG. 5 shows anti-metastatic effects in vivo.

Detailed Description

1. Obtaining lead polypeptide and polypeptide synthesis by phage display

1) Determination of phage titers

(1) Inoculating ER2738 single colony in 5-10ml LB culture medium, shake culturing to mid-logarithmic phase (OD 600-0.5)

(2) When colonies grew, the microwave oven thaws the upper agar, dividing into 3ml aliquots into sterile test tubes, one tube for each phage dilution. Stored at 45℃for further use.

(3) The LB/IPTG/Xgal plates were pre-warmed at 37℃and one plate was taken for each phage dilution. Phage were prepared in LB 10-fold serial dilutions.

(4) When the cell culture reached mid-log phase, 200. Mu.l aliquots were separated into microcentrifuge tubes, one tube per phage dilution. Mu.l of phage of different dilutions was added to each tube, mixed by rapid shaking, and incubated for 1-5min at room temperature.

(5) The above solution was added to the upper agar culture tubes pre-warmed at 45℃and mixed rapidly one tube at a time, and immediately poured onto LB/IPTG/Xgal plates pre-warmed at 37 ℃. The upper agar was spread evenly by tilting the plate appropriately. After the plates were cooled for 5min, they were incubated overnight at 37℃upside down.

(6) Checking the flat plate, counting 10 ² Number of plaques on plates of each plaque. Then, this number was multiplied by the dilution factor to give a plaque forming unit (pfu) titer per 10. Mu.l phage.

Only when the multiplicity of infection MOI (multiplicity of infection) of the phage was well below 1 (i.e. when the cells were excessive) will the number of plaques increase linearly with the amount of phage added. As such, it is suggested that when detecting the titer of phage stock, dilution is performed prior to infection, rather than dilution of infected cells at high MOI values. The low MOI value helps ensure that each plaque contains only one DNA sequence.

2) Panning:

(1) 100. Mu.g/ml of DR6 protein solution (NaHCO in 0.1M pH 8.6 was prepared ₃ ). 96. 1.5ml of the above solution was added to each plate of the well plate and the rotation was repeated until the surface was completely wetted. Incubate overnight with gentle shaking at 4deg.C in a humidified container (e.g., sealable plastic box lined with wet tissues). The plate can be stored in this container at 4℃for further use.

(2) The following day, ER2738 monoclonal (phage titer plate plated) was picked in 10ml LB liquid medium. And (3) carrying out severe shake culture at 37 ℃. The coating liquid in each plate was poured off, each well was topped up with a blocking liquid and the reaction was carried out at 4℃for at least 1 hour. Removing the blocking liquid. Further using TBST (TBS+0.1% [ v/v)]Tween-20) buffer was used to wash the plates 6 times. Each time rotated so that the bottom and edges of the plate or well were washed, the buffer was decanted. Dilution with 1ml of TBST buffer 4X 10 ¹⁰ I.e.10. Mu.l of the original library) and then added to the coated plate, gently shaken at room temperature for 10-60min. Depending on the intermolecular interactions studied, the bound phage was eluted with 1ml of an appropriate elution buffer. The eluate was aspirated into another clean microcentrifuge tube by gentle shaking at room temperature for 10-60min. The titer of a small (. About.1. Mu.l) eluate was determined by the procedure described above. The remaining eluate should be amplified: the eluate was added to 20ml ER2738 culture (the cells should be in the log phase) and cultured with vigorous shaking at 37℃for 4.5 hours. The culture was transferred to a centrifuge tube, and then centrifuged at 10,000rpm at 4℃for 10min. The supernatant was transferred to another centrifuge tube and centrifuged again. The upper 80% of the supernatant was transferred to a fresh tube and 1/6 volume of PEG/NaCl was added. Phage were allowed to pellet at 4℃for at least 60min overnight.

(3) The next day. PEG precipitation was centrifuged at 10,000rpm at 4℃for 15min. The supernatant was decanted, centrifuged briefly and the residual supernatant was aspirated. The pellet was resuspended in 1ml TBS and the suspension transferred to a microcentrifuge tube and centrifuged at 4℃for 5min to pellet. The supernatant was transferred to another fresh microcentrifuge tube and re-precipitated with 1/6 volume of PEG/NaCl. Incubating on ice for 15-60min. Centrifuging at 4deg.C for 10min, discarding supernatant, centrifuging briefly, and removing residual supernatant. The pellet was resuspended in 200. Mu.l TBS,0.02% NaN 3. Centrifuging for 1min, and precipitating any residual insoluble material. The supernatant was transferred to a fresh tube. This is the eluate after amplification. The eluate after amplification was titrated with LB/IPTG/Xgal plates according to the method described above. Stored at 4 ℃.

(4) A plate was coated again in preparation for the second round of panning.

(5) The next day. The number of blue spots on the plate was counted to determine the titer. This value is used to calculate the corresponding 1-2X 10 ¹¹ The amount of pfu added. If the titer is too low, the next rounds of panning can be as low as 10 ⁹ phage addition to pfu was tested. A second round of panning was performed: 1-2×10 of the eluate amplified by the first round of panning ¹¹ The phage amount of pfu was repeated in step (2) (3), and the concentration of Tween was increased to 0.5% (v/v) in the washing step. Titer after amplification of the eluate from the second round of panning was determined on LB/IPTG/Xgal plates.

(6) A plate was coated again in preparation for a third panning.

(7) A third round of panning was performed: 2X 10 of the eluate amplified by the second round of panning ¹¹ phage amount of pfu step (2) was repeated, again with 0.5% (v/v) Tween in the wash step. Titer was determined on LB/IPTG/Xgal plates when the eluate from the third round of panning was not amplified. The third round of eluate was not amplified. Plaques obtained at titer determination can be used for sequencing: as long as the plate culture time is not more than 18 hours, the deletion is liable to occur when the culture time is too long. The remaining eluate was stored at 4 ℃. ER2738 was selected and grown overnight in LB-Tet medium (without plating the diluted plating).

3) Amplification of plaques

(1) ER2738 overnight cultures were inoculated in LB medium at a 1:100 dilution and split into 1ml into culture tubes. One tube per clone to be identified. In general, 10 clones from the third round of panning were sufficient to detect consensus sequences in the binding peptides.

(2) A blue plaque was picked up into the 1ml culture tube using a sterilized toothpick or tip. Note that: plates with a total of less than 100 plaques were selected to ensure that each plaque picked contained only one DNA sequence.

(3) Shaking culture at 37deg.C for 4.5-5hrs (without excessive length).

(4) The culture was transferred to a microcentrifuge tube and centrifuged for 30sec. The supernatant was transferred to fresh tubes and then centrifuged. 80% of the supernatant was transferred to fresh centrifuge tubes using a pipette, which is a stock of amplified phage that can be stored for several weeks at 4℃with little impact on titer. Long term storage after 1:1 dilution with sterilized glycerol, storage at-20 ℃.

4) Sequencing. Sequences in each plaque were obtained.

5) Polypeptide synthesis

(1) The synthesis method adopts a Boc protection solid-phase polypeptide synthesis method. The PAM-amino acid-Boc resin (or MBHA resin) was swollen with DMF for 15min, deprotected with trifluoroacetic acid (TFA) twice for 1min each, the Boc-protected amino acid was dissolved in 0.5M HBTU (solvent DMF) and reacted for 15min at room temperature, the DMF was washed, TFA removed the Boc protection, and the reaction was sequentially carried out according to the amino acid sequence, after completion of the reaction CHO (W-containing amino acid sequence) protecting group was removed with 20% piperidine DMF solution twice for 15min each. After TFA is deprotected by Boc, the polypeptide is cut off from the resin by hydrogen fluoride, and a crude polypeptide is obtained by suction filtration of a sand core funnel after precipitation of glacial ethyl ether. After dissolution with 50% acetonitrile (containing 0.1% tfa), the polypeptide was prepared as a liquid phase purification and lyophilized.

(2) And detecting purity by HPLC and identifying by MS mass spectrum.

The polypeptide sequences obtained by screening are shown in Table 1. The purity of the synthesized polypeptide is over 95 percent through HPLC detection. Table 1 shows 24 leader polypeptide sequences and binding to DR 6.

TABLE 1

2. Identification of biological Activity

1) Binding force constant of polypeptide and DR6 protein by micro thermophoresis

(1) DR6 protein was labeled with Red-tris-NTA 2nd generation dye. An equal volume (200 nM) of DR6 protein was mixed with (100 nM) dye and incubated at room temperature for 30min before centrifugation at 15000g at 4 ℃.

(2) 12 PCR tubes were prepared and 10ul of PBS-T was added to each of the tubes 2-12.

(3) Into tube 1, 20uL of the polypeptide solution (20 uM) was added, and 10uL of the solution in tube 1 was transferred to tube 2 and thoroughly mixed. Transfer the solution in 10uL of tube 2 to tube 3 and mix well. This operation is repeated. Excess 10uL of solution in tube 12 is discarded.

(4) 10uL of the labeled DR6 protein was added to each well and mixed well. The final target protein concentration was 50nM. This concentration was used to calculate the Kd value.

(5) Kd values were determined using capillary loading.

As shown in table one. Kd values of peptide-3, peptide-10, peptide-12, and peptide-13 were in nM scale, respectively.

2) HUVEC and tumor cell co-culture to observe that tumor cells induce endothelial cell programmed necrosis

(1) HUVEC cells at 1X 10 ⁵ Cells/well were seeded in 6-well plates at 37℃with 5% CO ₂ The cell incubator was incubated for 24h.

(2) GFP-labeled B16F10, SGC-996, LLC1 cells at 2X 10 ⁴ Cells/well were added to the 6-well plate described above. 37 ℃,5% CO ₂ The cell culture was co-cultured in an incubator for 24 hours.

(3) Staining with Hoechst33342, ethD-III staining kit, and observing with fluorescence microscope.

3) Cell adhesion experiments

(1) DR6 protein (dissolved in PBS,50 nM) was coated in 96-well plates at 50uL per well, incubated at 37 ℃ for 2h, and allowed to stand overnight at 4 ℃.

(2) The next day PBS was lightly washed and blocked with 1% BSA at 37℃for 2h. The PBS was lightly washed.

(3) The cells are resuspended in culture medium to prepare single cell suspension, and the concentration is adjusted to 3 multiplied by 10 ⁵ 100uL of seed was inoculated per well. Incubate in cell incubator for 1h. The culture was discarded, and the PBS was gently washed to remove non-adherent cells. Fixing with 4% paraformaldehyde at room temperature for 30min, washing, dyeing with 5% crystal violet 50 uL/well for 10min, light washing with PBS, and decolorizing with 33% acetic acid. BIO-RAD enzyme label instrument, 590nm, measuring absorbance.

The results of the biological activity identification of 24 DR6 polypeptides are shown in FIG. 1. peptide-3,10,12,13 exhibited higher bioactivity.

3. Structural modification of polypeptides

1) And D, synthesizing the peptide. Polypeptides composed of D-amino groups are not readily degraded by proteases. Therefore, in order to improve the stability of DR6 polypeptide, all L-type amino acids in peptide-3,10,12,13 were replaced with D-type amino acids, and the sequences are shown in Table 2. Table 2 shows 8D peptide sequences and binding force with DR 6; the biological activity of the D peptide was identified as described above, and the results are shown in FIG. 2, wherein ^D peptide-12’, ^D The biological activity of peptide-13' is highest.

TABLE 2

2) Four-arm PEG modified polypeptides. A cysteine is connected to the polypeptide chain, and maleimide is modified at the tail end of the four-arm PEG. Four-arm PEG and corresponding ratio of polypeptides (molar ratio = 1:8) were dissolved in an appropriate volume of PH = 8, 0.1M PBS. The reaction was stirred at room temperature for 4 hours, dialyzed in dialysis bags with molecular weight 3500 for 24 hours, and lyophilized. As shown in fig. 3, nuclear magnetism ¹ H-spectra showed that the polypeptide was successfully linked to the four-arm PEG. The results of the biological activity assay are shown in FIG. 4.

4. Experiment of polypeptide anti-transfer function

1) In vitro anti-metastatic assay

The experimental group is as follows: blank, polypeptide treated, zVAD treated, nec-1 treated

(1) Transendothelial migration experiments: 2X 10 ⁵ HUVEC cells 0.1mL were added to a 24-well plate transwell upper chamber, and 1mL medium was added to the lower chamber, 37℃and 5% CO ₂ Culturing, changing culture medium every two days, measuring transendothelial resistance every day by using EVOM resistance tester, and measuring when TEER resistance value is more than 200Ω cm ² When cells grow into endothelial cell monolayers with tight junctions. Adding 2X 10 ⁵ GFP-TCs, treated with different reagents overnight. The cells in the 24-well plate were observed by fluorescent microscopy.

(2) Co-culture experiments: HUVEC cells at 1X 10 ⁵ Cells/well were seeded in 6-well plates at 37℃with 5% CO ₂ The cell incubator was incubated for 24h. GFP-TCs at 2X 10 ⁴ Cells/well were added to the 6-well plate described above. After being treated by different reagents, the temperature is 37 ℃ and CO is 5 percent ₂ The cell culture was co-cultured in an incubator for 24 hours. Staining with Hoechst33342, ethD-III staining kit, and fluorescent microscopy.

As shown in FIG. 5, peptides have better anti-metastatic activity in vitro.

2) In vivo anti-metastasis experiments

The experimental group is as follows: blank control group, polypeptide treatment group

GFP-B16F10 (2X 105) in 100uLPBS was injected into the tail vein of the mice. Mice were intravenously injected with PBS, polypeptide (20 uM) intravenously 24 hours before and after B16F10 injection. The administration was once every 2 days for 7 times. After 15 days, mice were sacrificed and perfused with PBS and 4% paraformaldehyde. The lungs of the mice in the different groups were observed for metastatic nodules. As shown in fig. 5, both the D peptide and the four-arm PEG-modified peptide showed the tumor metastasis inhibiting effect, and the PEG-modified peptide had the strongest inhibiting effect.

Claims

1. A polypeptide molecule with tumor metastasis inhibiting activity, which has the molecular structural formula:

。