CN114507270B

CN114507270B - Targeting PD-L1 polypeptide, preparation method, assembled nanotube and application

Info

Publication number: CN114507270B
Application number: CN202210011286.8A
Authority: CN
Inventors: 张黎民; 王蔚芝
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2022-01-06
Filing date: 2022-01-06
Publication date: 2023-07-14
Anticipated expiration: 2042-01-06
Also published as: CN114507270A

Abstract

The invention discloses a targeting PD-L1 polypeptide, a preparation method, a nanotube formed by assembly and application, wherein the general formula of the polypeptide is as follows: X1X2X3X4X5X6X7X8X9X10; wherein X1, X2, X8, X9 and X10 are hydrophilic amino acids, X3 is a basic or acidic amino acid, and X4, X5, X6 and X7 are hydrophobic amino acids; the polypeptide self-assembles to form a nanotube which can be used as a tumor therapeutic drug or an imaging reagent carrier for immune combination therapy and imaging of tumors; the polypeptide obtained by the invention is self-assembled to form a nanotube, and residues with recognition function can be displayed on the surface of the nanotube to show specific binding to PD-L1.

Description

Targeting PD-L1 polypeptide, preparation method, assembled nanotube and application

Technical Field

The invention relates to the technical field of biological materials and medicines, in particular to a targeting PD-L1 polypeptide, a preparation method, a nanotube formed by assembly and application.

Background

Malignant tumor seriously endangers human health, and the global new cancer in 2020 has 1929 ten thousand cases and death has nearly 1000 ten thousand cases. In recent years, with the rapid development of the fields of biological materials, immunology and the like, tumor immunodiagnosis and treatment have obtained a great breakthrough. Unlike traditional chemotherapy, radiotherapy and surgery therapies, tumor immunotherapy kills tumor cells by activating the immune system of the human body, has various advantages of persistence, systemic property, pertinence, safety and the like, is known as the third revolution of cancer treatment, and is also considered as a new hope for overcoming tumors.

Tumor immunotherapy mainly includes tumor vaccine, adoptive immune cell therapy, and immune checkpoint therapy; among these immune checkpoint therapies are a focus of attention. Immune checkpoints are a class of immunosuppressive molecules that can reduce the intensity of immune responses, avoiding hyperimmunization from damaging and destroying normal tissues. However, this mechanism is also used skillfully in the cancer development process, and immune checkpoint molecules are expressed on the surface of tumor cells, thereby causing immune escape. Immune checkpoint therapy is the process of killing tumor cells by using inhibitor molecules to bind to immune checkpoints, thereby blocking tumor cells from binding to immune cells, thereby activating immunity. A number of immune checkpoints have been discovered, such as cytotoxic T cell associated protein-4 (CTLA-4), programmed death receptor (PD-1) and its ligand 1 (PD-L1), T cell immunoglobulins and ITIM domain proteins (TIGIT), among others. The therapy has been developed to date with a variety of inhibitor drugs either on the market or entering the clinical stage and achieving satisfactory results. Such as ipilimumab and tremeliumab for CTLA-4 checkpoints, and pembrolizumab and nivolumab as PD-1 inhibitors, and the like.

The existing immune checkpoint inhibitor mainly comprises antibodies, and the antibodies have the advantages of high inhibition efficiency, capability of remarkably prolonging the survival time of tumor patients, and the like, but have the defects of inherent immunogenicity, weak penetrability, large side effect, low thermal stability, complex preparation, high price and the like, so that the development of the immune checkpoint inhibitor is limited.

Since PD-L1 is involved in the tumor development and progression, tumor cells are assisted in escaping the killing of the immune system of the organism. Unlike conventional target proteins, PD-L1 has been a difficulty in the development of inhibitors due to its specific binding site. The polypeptide has important roles in the development of immunosuppressants based on molecular recognition by virtue of the advantages of good biocompatibility, low immunogenicity, easy excretion and clearance, chemical synthesis and the like. In addition, different amino acids have unique physicochemical properties such as chargeability, hydrophilicity and hydrophobicity, chirality, and the like. The amino acid molecules with different properties can generate polypeptide chains containing abundant structural information and identification information according to the arrangement sequence and the difference of the number of residues in the process of forming the polypeptide through dehydration condensation, which cannot be achieved by other molecules. And the polypeptide shows strong superiority in aspects of tumor immunity, diagnosis and the like by virtue of the advantages. The micromolecular targeting polypeptide has the dilemma of monovalent combination with a target point, instability and easy degradation by enzyme in an in-vivo dynamic environment, and the polypeptide can form a supermolecular nano structure such as a nano tube, a nano fiber, a nano ball and the like through self-assembly. The formation of the nano-assemblies greatly increases the enzymolysis resistance of the nano-assemblies, and can be used as carriers for efficiently carrying anti-tumor drugs and imaging reagents to tumor sites to realize the joint diagnosis and treatment of various methods. However, in the process of polypeptide self-assembly based on β -sheet secondary structure, polypeptide molecules are arranged in a manner perpendicular to the surface of the assembly body in order to form a stable self-assembled structure, resulting in that the side chain active site is completely buried in the assembly body, and structural-functional connection cannot be established. In order to make the assembly body have a targeting function, the current common method is to splice a polypeptide unit with an identification function and a unit with an assembly function, so that the assembly and the identification are realized to a certain extent, but the problem that the units cannot be coordinated exists; the other way is to design the recognition sequence longer than the assembly sequence by adopting a co-assembly way, so that the recognition sequence is exposed outside the assembly body after co-assembly, but the method needs to strictly regulate the assembly density of the two polypeptides.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides a targeting PD-L1 polypeptide which has high affinity to PD-L1 and is formed by self-assembly, wherein residues with an identification function are displayed on the surface of the nanotube, a preparation method of the targeting PD-L1 polypeptide, the assembled nanotube and application of the targeting PD-L1 polypeptide.

The technical scheme adopted by the invention is as follows:

a targeted PD-L1 polypeptide having the general formula:

X1X2X3X4X5X6X7X8X9X10

wherein X1, X2, X8, X9 and X10 are hydrophilic amino acids, X3 is a basic or acidic amino acid, and X4, X5, X6 and X7 are hydrophobic amino acids.

Further, X1 is one of threonine, serine and cysteine; x2 is one of serine, glutamic acid, histidine and aspartic acid; x3 is one of lysine, arginine, glutamic acid, histidine and aspartic acid; x4 is one of benzo amino acid, leucine and isoleucine; x5 is one of benzo amino acid, tyrosine and glycine; x6 is one of leucine, valine, tryptophan and benzo amino acid; x7 is one of leucine, valine, tryptophan and phenylalanine; x8 is one of serine, glycine and cysteine; x9 is one of asparagine and glutamine; x10 is one of lysine, glutamic acid, aspartic acid, histidine and arginine.

Further, the amino acid sequence of the polypeptide is: TSRFAAFSQK.

Further, the amino acid sequence of the polypeptide is: TSRFVFFSQK.

A polypeptide nanotube is formed by self-assembly of the polypeptide.

Furthermore, the thickness of the wall of the polypeptide nanotube is 2.4nm, and the polypeptide is assembled in an antiparallel manner by forming a beta-sheet secondary structure in the self-assembly process; active residues on both sides of the polypeptide chain are exposed on the surface of the nanotube.

A method for producing a polypeptide comprising the steps of:

coupling amino acids at each position to a carrier one by using resin as a solid-phase carrier through a mixed splitting method;

the polypeptide is cut off on the resin through the lysate, and the required polypeptide can be obtained after separation and purification.

Application of polypeptide nanotubes, and application of polypeptide nanotubes as tumor therapeutic drugs or imaging agent carriers.

Further, the tumor is a PD-L1 expression positive tumor.

Further, the tumor is one of melanoma, non-small cell lung tumor, prostate tumor, colorectal tumor, pancreatic tumor, liver tumor, stomach tumor, esophageal tumor, breast tumor and bile duct tumor.

The beneficial effects of the invention are as follows:

(1) The polypeptide obtained by the invention has high affinity to PD-L1;

(2) The polypeptide obtained by the invention is self-assembled to form a nanotube, and residues with recognition function can be displayed on the surface of the nanotube, so that the ability of specifically combining with PD-L1 and blocking PD-L1/PD-1 is shown;

(3) The polypeptide obtained by the invention has inclination of about 40 degrees when self-assembled into a nanotube;

(4) The polypeptide self-assembled nanotube obtained by the invention has excellent capability of loading anti-tumor drugs and imaging reagents, and can be used for immune combination treatment and imaging of tumors.

Drawings

FIG. 1 is a schematic diagram of a method for producing a polypeptide of the present invention.

FIG. 2 is a mass spectrum of a polypeptide LY obtained by the present invention and a purity profile of a high Performance liquid HPLC.

FIG. 3 is a schematic diagram showing affinity between polypeptide LY and nanotube obtained by the present invention and PD-L1.

FIG. 4 is a schematic representation of the binding of the polypeptide of the invention to PD-L1.

FIG. 5 is a schematic representation of the binding of the resulting polypeptide of the invention to MC38 and 293T cells.

FIG. 6 is a schematic representation of nanotubes assembled from the polypeptides of the present invention.

FIG. 7 is a schematic illustration of nanotubes obtained after 2 hours of sonication in an embodiment of the present invention.

FIG. 8 is a circular dichroism spectrum of a nanotube formed by self-assembly of the polypeptide obtained by the present invention.

FIG. 9 is an SEM image of the binding wrap of the polypeptide self-assembled nanotubes on the surface of PD-L1 positive and PD-L1 negative cells obtained according to the present invention.

FIG. 10 is a schematic representation of model mice treated with the polypeptide nanotube drug carriers of the present invention for various periods of time.

Detailed Description

The invention will be further described with reference to the drawings and specific examples.

A targeted PD-L1 polypeptide having the general formula:

X1X2X3X4X5X6X7X8X9X10

X1 is one of threonine, serine and cysteine; x2 is one of serine, glutamic acid, histidine and aspartic acid; x3 is one of lysine, arginine, glutamic acid, histidine and aspartic acid; x4 is one of benzo amino acid, leucine and isoleucine; x5 is one of benzo amino acid, tyrosine and glycine; x6 is one of leucine, valine, tryptophan and benzo amino acid; x7 is one of leucine, valine, tryptophan and phenylalanine; x8 is one of serine, glycine and cysteine; x9 is one of asparagine and glutamine; x10 is one of lysine, glutamic acid, aspartic acid, histidine and arginine.

The polypeptide is designed from the beginning based on the Bola type structure polypeptide, and the general formula of the polypeptide is shown as above. Wherein four residues of X4, X5, X6 and X7 are hydrophobic residues, and form a hydrophobic core of the polypeptide. X1, X2, X3, X8, X9 and X10 are polar residues, and the whole molecule is in a symmetrical state.

The experimental apparatus and materials used for the synthesis of polypeptides and testing are as follows:

fluorescein Isothiocyanate (FITC), thioflavin T,1 xps, 10 xps, skim milk, tween-20, N-methylmorpholine, piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethyluronium Hexafluorophosphate (HBTU), piperidine, triisopropylsilane (TIS), ethanedithiol (EDT), N Dimethylformamide (DMF), dehydrated diethyl ether, tentagel resin, king resin, cyanogen bromide, 20 Fmoc-amino acids, methanol, polypeptide synthesis tubes, shaker, cell culture box, surface Plasmon Resonance Imager (SPRi), vacuum water pump, rotary evaporator, laser confocal microscope (ZEISS LSM 710), scanning electron microscope, transmission electron microscope, atomic force microscope, all of the above reagents and materials were obtained commercially.

Preparing a solvent:

deprotection reagent-20% piperidine; reaction liquid-N-methyl morpholine, N dimethylformamide=1:24. Lysates-trifluoroacetic acid (92.5%), triisopropylsilane (2.5%), ethanedithiol (2.5%), ultrapure water (2.5%);

trione test solution->

Trione: vitamin C: phenol=1:1:1.

The synthesis of peptide library is carried out by Fmoc solid phase synthesis method, tenagel resin is used as solid phase carrier, amino acid at each position is coupled to carrier one by adopting mixed cleavage method, finally peptide chain is cut off from resin by strong acid and protecting group is removed, and the synthesis process is shown in figure 1.

Weighing 0.2g of tentagel resin, circulating according to the solid-phase polypeptide synthesis program, and according to the amino acid: resin=5:1 mass ratio, and HBTU equivalent to amino acid was added for coupling reaction, and ninhydrin chromogenic reagent was used for detection before and after the reaction. Finally, through methanol replacement and shrinkage steps, vacuum pumping is carried out, and the dry resin is obtained for standby. The peptide was cleaved from the resin with a solution containing 95% TFA, precipitated with glacial ethyl ether, and the resulting preliminary peptide was isolated and purified by HPLC and mass-detected to determine if synthesis was accurate.

Screening for a polypeptide having PD-L1 targeting

After peptide pool synthesis, the resin was collected, washed twice with 1×pbs, blocked with 5% skim milk for 2 hours, and washed three times with PBS. Biotin-labeled PD-L1 protein was added, incubated at 37℃for 2 hours, and then positive resins were selected using a magnetic sorting method. The polypeptide on the positive resin is cleaved by cyanogen bromide, and the positive sequence is identified by secondary mass spectrometry through Maldi-TOF-MS. The polypeptide separated and purified by HPLC is identified by mass spectrum, the obtained polypeptide sequence is TSRFAAFSQK (shown as SEQ ID NO: 1), the polypeptide sequence is named LY, the sequence is synthesized by Fmoc solid phase, and the identification purity result is shown as figure 2. Identification of LY Polypeptides for affinity with PD-L1

1mg/mL of the polypeptide solution was printed on the chip, incubated overnight at 4℃and then washed 3 times with 10 XPBS, 3 times with 1 XPBS, and 2 times with ultrapure water. With 5%Skim milk was blocked overnight, washed 3 times with 10×,1×pbs, and finally blow-dried with nitrogen and mounted on a chip machine for testing. The mobile phase was PD-L1 solution prepared from PBST at a concentration of 192nM,96nM,48nM,24nM,12nM. As shown in FIG. 3, the final polypeptide has an affinity for PD-protein of 2.6X10 ^- ⁸ M, indicating very high affinity.

Binding site of polypeptide LY on PD-L1 protein

The structure of the polypeptide LY was generated using PEP-FOLD, the crystal structure of PD-L1 was obtained from the PDB database, and molecular docking and analysis was performed using software ZDOCK 3.0.2, the results of which are shown in FIG. 4. From the figure, it can be seen that the polypeptide can be stably bound on the planar structure of PD-L1 in contact with PD-1, and the force of binding to PD-L1 is mainly hydrogen bond, electrostatic effect and pi-pi accumulation.

Polypeptide LY selectivity for PD-L1 positive cells at the cellular level

PD-L1 highly expressed colon cancer cell line MC38 was grown at a density of 1X 10 using 10% 1640 medium ⁵ The density per mL was seeded in confocal dishes. Normal cells 293T were cultured in 1X 10 medium with 10% DMEM ⁵ The density per mL was seeded in confocal dishes.

The cells were incubated at 37℃for 24 hours under 5% carbon dioxide. The medium was discarded, 100. Mu.L of FITC-LY solution in serum-free medium was added and incubated for 1 hour in the dark. Then, the cells were washed three times with 1 XPBS, incubated for 15 minutes with 1. Mu.M Hoechst 3342 staining reagent, washed three times with 1 XPBS, and the distribution of the polypeptide on both cells was observed by confocal laser.

As a result, as shown in FIG. 5, there was bright fluorescence on the cell membrane of MC38, a PD-L1 positive cell, and no fluorescence was observed on 293T cells, a PD-L1 negative cell. The screened polypeptide LY has good targeting and selectivity on PD-L1 positive primary amine.

The polypeptide LY is self-assembled to form nanotubes by the following method

The synthetically purified LY polypeptide was dissolved in hexafluoroisopropanol, incubated for 6 hours at room temperature, and then slowly volatilized clean with nitrogen. Fully dissolved in water and the pH was adjusted to 7.0 with NaOH. Standing in a refrigerator at 4 ℃ for a week to enable the polypeptide to be completely self-assembled.

And sucking 10 mu L of the assembled polypeptide solution, dripping the solution onto a clean mica sheet, absorbing for 20 minutes, sucking off excessive liquid, and drying by using nitrogen. And observing the morphology of the assembly under an atomic force microscope, wherein the scanning parameters are as follows: 512×512, scan speed: 1.0HZ, scan angle: 0 °, mode: and (5) taping. Testing to obtain FIG. 6, it can be seen from the graph that nanotubes with uniform diameters are formed, and the thickness of the tube wall is 2.4nm; the mechanism proves that the polypeptide undergoes a molecular inclined assembly process, and residues with affinity to PD-L1 can be exposed on the surface of the nanotube.

After the polypeptide is assembled, the length of the nanotube can be controlled by ultrasonic method after ultrasonic treatment for 2 hours under different power, so that the nanotube can meet the requirement of the required length. As shown in fig. 7, after 2 hours of ultrasound, the length of the nanotubes was changed, and the length of the nanotubes could be controlled by this method.

Upon assembly, the polypeptide LY is tilted by about 40℃and exposes an active site with affinity for PD-L1 to the surface of the assembly.

Based on the polypeptide LY, the residue V, F with more hydrophobicity is selected to replace the central two A residues of the polypeptide LY. The hydrophobicity of the polypeptide is increased, so that the solvation effect is utilized to regulate and control the inclination angle of the beta-sheet. The polypeptide sequences formed were: TSRFVFFSQK (SEQ ID NO 2).

Characterization of the secondary Structure of polypeptide LY nanotubes

The method for detecting the secondary structure of the assembly by adopting the circular dichroism spectrometer comprises the following specific processes: a MOS450 circular dichroism spectrometer is selected, a CD mode is selected, the scanning range is 190-260 nm, the interval time is set to be 0.5s, and the cuvette optical path used in the experiment is 0.1mm. The results are shown in FIG. 8.

Targeting and selectivity of polypeptide LY assemblies on cell surfaces

The method comprises the following steps:

MC38 and 293T cells were grown on glass slides for 24 hours, then incubated with 100. Mu.L of the assembled peptide solution for 1 hour at 37℃and washed three times with PBS.

Cells were fixed overnight with 4% glutaraldehyde.

Critical point drying (replacing absolute ethyl alcohol with carbon dioxide)

Metal spraying for 30s

Scanning electron microscope imaging. The scan results are shown in fig. 9.

As can be seen from FIG. 9, PD-L1 negative cells 293T were smooth in surface and no polypeptide assemblies were present. A large number of assemblies were attached to the surface of PD-L1 positive MC38 cells. The polypeptide assembly has good targeting ability and selectivity.

Results of in situ treatment of polypeptide LY assemblies

MC38 cells were subcutaneously injected into C57BL/6N mice at 7 weeks of age until tumors grew to 100mm ³ As model mice. Mice were divided into experimental and control groups.

Experimental group: mice were injected intravenously with 100 μl of drug (wherein LY assembly (nanotubes obtained as shown in FIG. 6): 4mg/kg, ce6 (chlorin): 3 mg/kg) every day.

Control group: mice were injected intravenously with 100 μl of PBS every day, and after 6 hours of injection, were irradiated with 808nm laser for 10 minutes.

The results are shown in FIG. 10, from which it can be seen that the tumors of the mice in the experimental group significantly decreased, while the tumor sizes of the control group gradually increased. The nano tube obtained by the invention has good treatment effect when carrying anticancer drugs.

The polypeptide LY obtained by using the method of 'de novo design' and combinatorial chemistry peptide library screening can be self-assembled into the nanotube with the function of targeting and recognizing PD-L1 by manipulating the inclination of polypeptide molecules. The excellent capability of loading anti-tumor drugs and imaging agents can be used for immune combination treatment and imaging of tumors.

Claims

1. A targeted PD-L1 polypeptide, wherein the polypeptide has the sequence TSRFAAFSQK or TSRFVFFSQK.

2. A polypeptide nanotube, wherein the polypeptide nanotube is self-assembled from the polypeptide of any one of claims 1.

3. The polypeptide nanotube of claim 2, wherein the polypeptide nanotube has a wall thickness of 2.4nm, and wherein the polypeptide is assembled in an antiparallel manner by forming a β -sheet secondary structure during self-assembly; active residues on both sides of the polypeptide chain are exposed on the surface of the nanotube.

4. The method of producing a polypeptide according to claim 1, comprising the steps of:

5. The use of polypeptide nanotubes as claimed in claim 2 as a carrier, wherein the carrier is a tumor therapeutic drug carrier or an imaging agent carrier.

6. The use according to claim 5, wherein the tumor is a PD-L1 expression positive tumor.

7. The use according to claim 5, wherein the tumor is one of melanoma, non-small cell lung tumor, prostate tumor, colorectal tumor, pancreatic tumor, liver tumor, gastric tumor, esophageal tumor, breast tumor and bile duct tumor.