CN113583091B

CN113583091B - Specific targeting polypeptide of immunosuppressive cell tight junction protein and application thereof

Info

Publication number: CN113583091B
Application number: CN202110845210.0A
Authority: CN
Inventors: 秦志海; 曹红; 倪琛
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2021-07-26
Filing date: 2021-07-26
Publication date: 2023-05-26
Anticipated expiration: 2041-07-26
Also published as: CN113583091A

Abstract

The application belongs to the technical field of tumor gene immunity, and particularly relates to a specific targeting polypeptide of immunosuppressive cell tight junction protein and application thereof. The amino acid sequence of the specific targeting polypeptide is shown as SEQ NO.1, specifically YNXXLNXKXXP, wherein X is any one of 20 natural amino acids or D-type amino acid thereof. The specific targeting polypeptide can block immune suppression cells (MDSCs) from penetrating vascular endothelium, and has the effects of promoting various treatment methods such as tumor immunotherapy, tumor chemotherapy and the like.

Description

Specific targeting polypeptide of immunosuppressive cell tight junction protein and application thereof

Technical Field

The application belongs to the technical field of tumor gene immunity, and particularly relates to a specific targeting polypeptide of immunosuppressive cell tight junction protein and application thereof.

Background

Treatment resistance is a major challenge in tumor treatment. Immunosuppression is one of the key causes of the development of tumor therapeutic resistance. Myeloid-derived immunosuppressive cells (MDSCs) are a class of immunosuppressive cells that are widely present in a variety of tumors. MDSCs mainly play a role in tumor local, inhibit the killing function of tumor specific T cells, promote tumor blood vessels and can enhance tumor stem property. Research has shown that MDSCs are important factors that lead to failure of various tumor treatment methods. The combined targeting MDSCs can promote the effects of tumor chemotherapy, targeted therapy, tumor vaccine and immunotherapy (anti-CTLA-4/PD 1). Therefore, there is a need to devise strategies for targeting MDSCs to enhance tumor efficacy and benefit tumor patients.

The current targeting strategies for MDSCs are directed primarily to MDSCs mechanisms of development, amplification, activation and chemotaxis. The molecular mechanism by which MDSCs penetrate the blood vessel is not yet known. Previous studies by the inventors have found that the Claudin-12 (CLDN 12), a tight junction protein, is expressed on the cell surface of MDSCs, as well as on vascular endothelial cells. The CLDN12 on the MDSCs and the CLDN12 on the endothelial cells mediate the MDSCs cells to penetrate through tumor vascular endothelial cells and enter tumor tissues through homologous interaction, so that an immunosuppressive effect is exerted, the interaction between the CLDN 12-mediated MDSCs and the endothelial cells is blocked, the MDSCs can be blocked from entering the tumor tissues, and further the treatment effect of tumors is promoted.

Disclosure of Invention

In order to block the interaction of CLDN12 mediated MDSCs and endothelial cells and promote the treatment effect of tumors, the invention provides a specific targeting polypeptide of immunosuppressive cell tight junction protein, the amino acid sequence of which is shown as SEQ NO.1, specifically YNXXLNXKXXP; wherein X is any one of 20 natural amino acids or D-form amino acids thereof.

Preferably, a specific targeting polypeptide of the immunosuppressive cell tight junction protein has an amino acid sequence shown in SEQ NO.2, and specifically YNSHLNRKFEP.

Preferably, the tight junction protein is Claudin-12.

The application of specific targeting polypeptide of immunosuppressive cell tight junction protein in preparing antitumor preparation is provided.

Preferably, the formulation is for use in tumor immunotherapy or tumor chemotherapy.

Preferably, the preparation is injection, tablet, powder or capsule.

Compared with the prior art, the beneficial effects of this application are:

1) Current approaches to targeting MDSCs are directed primarily to the development, amplification, activation and chemotaxis of MDSCs. The present invention is directed to the process of vascular endothelial penetration of MDSCs mediated by the clan 12, a tightly-linked protein, based on a completely different molecular mechanism.

2) The CLDN12 targeting polypeptide has specificity, generates fewer side effects and has less influence on tumor specific immune response relative to the processes of targeting MDSCs development, chemotaxis and the like.

3) The CLDN12 targeting polypeptide blocks the MDSCs from penetrating vascular endothelium and has the effect of promoting various treatment methods such as tumor immunotherapy, tumor chemotherapy and the like.

Drawings

FIG. 1 shows a schematic diagram of the structure of CLDN12 protein and the sequence of CLDN2-pm, control Con-p polypeptide;

FIG. 2 human and murine CLDN12 protein sequences are compared, the marker sequence being ECL2;

FIG. 3 human and Xenopus CLDN12 sequences compared, the marker sequence being ECL2;

FIG. 4 CLDN12-pm polypeptide treatment of MDSCs with reduced CLDN12 distribution on its surface;

FIG. 5 CLDN12-pm polypeptide blocks adhesion of MDSCs to endothelial cells, MDSCs are labeled with CFSE, and the adhered MDSCs are detected by flow;

FIG. 6 is a graph of the statistical results of FIG. 5 showing that CLDN12-pm polypeptide blocks the adhesion of MDSCs to endothelial cells;

FIG. 7 CLDN12-pm polypeptide blocks penetration of MDSCs into endothelial cell monolayers, MDSCs are labeled with PKH26, and the penetrated MDSCs are photographed by confocal microscopy;

FIG. 8 is a graph of the statistical results of FIG. 7 showing that CLDN12-pm polypeptide blocks the penetration of MDSCs through endothelial cell monolayers;

FIG. 9 is a schematic diagram showing the experimental animal treatment process at the top and the tumor growth curve at the bottom, wherein the CLDN12-pm polypeptide inhibits tumors and is used in combination with anti-PD1 to promote the therapeutic effect.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

Examples

1. Test materials

1. Cell lines and laboratory animals

2. Main reagent

3. Commonly used reagents

1×PBS Buffer：137 mM NaCl，2.7 mM KCl，10 mM Na ₂ HPO ₄ ，2 mM KH ₂ PO ₄ ；

4% paraformaldehyde solution: weighing 40g of paraformaldehyde, dissolving in 1L of 1 XPBS, adding a proper amount of NaOH solution to promote dissolution, and finally adjusting the pH value to 7.2-7.4; preserving at normal temperature in a dark place;

cell culture fluid: 1) RPMI1640 cell culture medium (RPMI 1640 containing 10% fetal bovine serum) contains 2mM glutamine, 100U/mL penicillin, 100 μg/mL streptomycin; 2) DMEM cell culture medium (DMEM containing 10% fetal bovine serum) containing 2mM glutamine, 100U/mL penicillin, 100 μg/mL streptomycin;

immunofluorescence staining reagent: 1) A sealing liquid; 2% BSA,0.1% Triton X-100,1 XPBS dilution; 2) An anti-dilution liquid; 0.5% BSA,1 XPBS dilution;

flow buffer PBS+0.5% BSA+2mM EDTA.

2. Test method

1. Cell immunofluorescent staining

The cells were plated in a Confocal dish at 37℃with 5% CO ₂ Culturing; after the cell density reached 90%, the supernatant was discarded and the cells were washed three times with PBS; 4% PFA fixation for 10-15 min; washing with PBS three times for 5 minutes once; sealing with sealing liquid, and standing at room temperature for 1h or overnight at 4deg.C; discarding the sealing liquid, and incubating the primary antibody overnight; incubating the primary antibody cells, and restoring to room temperature; discarding the primary antibody, and washing with PBS for three times for at least 10 minutes each time; incubating the secondary antibody at 37 ℃ for 1h; discarding the secondary antibody, washing with PBS for three times for at least 10 minutes each time; DAPI (4', 6-diamidino-2-phenylindole) was incubated for 10 min at room temperature, washed three times with PBS for at least 10 min each time; and shooting by a laser confocal microscope.

2. Oriental induction of myeloid cells into MDSCs

Dilution of WT with DMEM complete MediumClaudin-12Bone marrow cells of genetically deficient mice; taking 100 mu m bone marrow cell dilution, and centrifuging at 1500rpm for 3 minutes; adding 1mL of erythrocyte lysate, splitting red for 1 minute, neutralizing and filtering by using a DMEM complete medium, and centrifuging at 1500rpm for 3 minutes; cells were resuspended in DMEM complete medium containing 40ng/mL GM-CSF and counted; cell of 10 ⁶ The holes are arranged in a 24-hole plate, and liquid is exchanged every other day; adherent cells can be collected for subsequent experiments after 5 days of continuous induction.

3. MDSCs Trans-endothesium experiment

The upper layer of a 5 μm transwell cell was followed by a 2X 10 ⁴ Hole injection of different vascular endothelial cells; after endothelial cells grow and fuse into a monolayer, the membrane is washed by changing liquid, and MDSCs after fluorescent marking of yellow-orange fluorescent dye PKH26 is 10 percent ⁵ A concentration of 200 uL/well was added to the chamber; serum gradient chemotactic cells, wherein the upper chamber adopts DMEM medium of 2% FBS, and the lower chamber adopts DMEM medium of 20% FBS; 37 ℃ 5% CO ₂ After the culture condition is 6-8hs, the upper cell layer of the cell is gently wiped off; shooting tumor cells penetrating to the lower layer of the cell by using a forward fluorescence microscope; image J counts the number of cells in each field.

4. PKH26/CSFE fluorescent labeled cells

Binding PKH26 with a longer lipid tail to the cell membrane lipid region using a fluorescent cell attachment kit; collecting cells, and fully dispersing the cells into single suspension cells; washing once with serum-free medium, and centrifuging 400g for 5 min; discarding the supernatant, adding diluted solution C (G8278, or serum-free medium) with half of the staining volume, and gently blowing and mixing to obtain 2×cell suspension; diluting PKH26 dye (P9691) with half of the volume of the dilution C to obtain 2 XPKH 26 dye solution, mixing with 2 Xcell suspension at equal volume, and final concentration of stained cells of 10 ⁷ /mL; staining at 4 ℃ for 1-5 minutes, adding 10 times of volume of DMEM complete medium, and incubating for 1 minute; after centrifugation for 10 minutes, 10mL of DMEM complete medium was washed twice for use.

5. Cell adhesion in solid phase

Spread in 12-well plate 1.5X10 ⁵ sEND.1 endothelial cells; washing and changing liquid after the cells are fused into a single-layer membrane; WT is combined withClaudin-12The MDSCs induced by the bone marrow orientation of the gene-deficient mice are respectively marked by fluorescent dyes with different colors and resuspended in a serum-free culture medium; at 1.5X10 ⁵ Cells/well were spread on the cell layer, 5% CO at 37 ℃C ₂ Culturing under the culture condition for 30 minutes; the PBS was washed three times to remove non-adherent cells, and then flow-through or fluorescent photographing was performed to count the proportion or number of adherent cells.

6. Subcutaneous tumor-bearing experiments in mice

Pancreatin digests tumor cells in log phase, PBS washes three times; count after sterile PBS was resuspended and cell concentration was adjusted to 1X 10 ⁶ 0.1 mL PBS; the left abdomen was injected with 0.1 mL cell suspension/mouse (C57 bl/6, 6-8 weeks); after tumor inoculation, from the firstMeasuring the length, width and height of the tumor once every other day after six days; according to the formula: v=abc (a is length, b is width, c is height) and tumor volume is calculated.

7. Polypeptide synthesis

The specific targeting polypeptides CLDN12-pm (YNSHLNRKFEP) and Con-p (LYQY) used in this study were synthesized by Shanghai sho biotechnology limited (purity > 95%), and stock was dissolved in 30% dmso; before use, dilute to use concentration with PBS; for the in vitro cell stimulation experiments, the final polypeptide concentration was 400. Mu.M, for 24 hours.

8. Tumor immunotherapy with polypeptide in combination with PD1 antibodies

About one week after tumor-bearing, the tumor size of the mice was about 100 mm ³ Mice were given CLDN12-pm or control polypeptide Con-p via the tail vein (500 μg of polypeptide per mouse, 200 μl of PBS in which the polypeptide was dissolved), once every 2 days, for a total of 6 administrations. Simultaneously with the second polypeptide treatment, PD1 antibody (200 μg per mouse, antibody in 200 μl PBS) was given intraperitoneally, once every 2 days, 4 total times. The size and body weight of the tumor of the mice were recorded.

3. Results and analysis

CLDN12 is a four-time transmembrane structural protein, the structure of which comprises two U-shaped loop structures ECL1 and ECL2 from the N-terminal to the C-terminal, and the specific structural schematic diagram is shown in fig. 1. Recognition and binding between MDSCs cells and endothelial cells is primarily dependent on CLDN12-CLDN12 homology interactions, which we speculate to be primarily dependent on ECL2 based on Claudins family protein structure.

CLDN12 proteins were highly conserved, with human and murine homologies greater than 90% (fig. 2), and human and xenopus' CLDN12 sequence controls are shown in fig. 3. Based on the conservation of CLDN12 gene among species, we predict that motif polypeptide ynxxlnxkkxp is an active polypeptide, where X is any one of 20 natural amino acids or D-type amino acids thereof, and adding 1-3 amino acids at both ends of motif may still be active.

In this example, a homologous polypeptide of the CLDN12 targeting polypeptide, designated CLDN12-pm, was verified based on the mouse ECL2 sequence, and the specific amino acid sequence was YNSHLNRKFEP.

Bone marrow cells were taken and induced to MDSCs. In vitro induced MDSCs cells were plated in Jiao Xiao dishes for incubation, and clDN12-pm or control polypeptide Con-p (400. Mu.M) was added and stimulated for 24 hours. Cells were stained for Gr1 and CLDN 12. The results indicate that CLDN12-pm can bind to CLDN12 on MDSCs, resulting in a reduced distribution on the membrane (fig. 4).

In vitro induced MDSCs or snend.1 were labeled with CFSE or PKH26, respectively. Then stimulated with CLDN12-pm or control polypeptide Con-p (400 uM) for 24 hours. The stimulated MSC2 was added to the endothelial cell monolayer to detect adherent cells, or the Tranwell culture system was used to detect penetration of MDSCs through the endothelial cell monolayer. Adherent cells were detected by flow cytometry, and confocal microscopy was performed across cells. As a result, MDSCs or endothelial cells after CLDN12-pm treatment were found to reduce the adhesion of MDSCs to endothelial cells (fig. 5, fig. 6), and to reduce the penetration of MDSCs through endothelial cell monolayers (fig. 7, fig. 8).

The CLDN12-pm polypeptide is used in combination with PD1 antibody for treating mice bearing LLC tumors, and the results are shown in fig. 9, where CLDN12-pm alone is found to produce an anti-tumor effect, which can be effectively enhanced when used in combination with PD1 antibody.

SEQUENCE LISTING

<110> Zhengzhou university first affiliated hospital

<120> specific targeting polypeptide of immunosuppressive cell tight junction protein and application thereof

<130> NONE

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 11

<212> PRT

<213> Synthesis

<400> 1

Tyr Asn Xaa Xaa Leu Asn Xaa Lys Xaa Xaa Pro

1 5 10

<210> 2

<211> 11

<212> PRT

<213> Synthesis

<400> 2

Tyr Asn Ser His Leu Asn Arg Lys Phe Glu Pro

1 5 10

Claims

1. A specific targeting polypeptide for an immunosuppressive cell-tight junction protein, characterized in that: the amino acid sequence is specifically YNSHLNRKFEP.

2. Use of a specific targeting polypeptide according to claim 1 for the preparation of an anti-LLC tumor formulation.

3. The use according to claim 2, wherein: the antitumor preparation is injection, tablet, powder or capsule.