CN117024517B

CN117024517B - Alkaline phosphatase response and convertible supermolecule bispecific cell cement polypeptide and preparation method and application thereof

Info

Publication number: CN117024517B
Application number: CN202311038473.6A
Authority: CN
Inventors: 高洁; 杨志谋; 陈昱妙
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2023-08-17
Filing date: 2023-08-17
Publication date: 2024-06-25
Anticipated expiration: 2043-08-17
Also published as: CN117024517A

Abstract

The invention provides an alkaline phosphatase response and convertible supermolecule bispecific cell cement polypeptide, a preparation method and application thereof, and relates to the technical field of medicine and pharmacy. The polypeptide is obtained by co-assembling short peptide 1 and short peptide 2, and the like. The invention provides an innovative method, which utilizes the design of an ALP-responsive dual-specificity supermolecule cell engagement device (Supra-BiCE) to realize the targeted combination between cancer cells and immune cells so as to enhance the immune treatment effect. Through the co-assembly of blocking peptides, the Supra-BiCE can simultaneously recognize TIGIT on the surface of immune cells and PD-L1 on the surface of tumor cells, and through the conversion from ALP-guided nanofibers to long nanofibers, the binding affinity is improved, the recruitment of tumor-specific immune cells is ensured, and the retention in tumors is prolonged. This approach provides new strategies and tools for enhancing the effectiveness of immunotherapy.

Description

Alkaline phosphatase response and convertible supermolecule bispecific cell cement polypeptide and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicine and pharmacy, in particular to an alkaline phosphatase response and convertible supermolecule bispecific cell cement polypeptide, a preparation method and application thereof.

Background

Cancer immunotherapy has completely altered the field of clinical oncology by using checkpoint blocking antibodies and chimeric antigen receptor T cell therapies. However, current approaches have certain limitations, including low response rates of checkpoint inhibitors and limited efficacy of cell therapies on solid tumors. One of the key factors responsible for the poor efficacy of cancer immunotherapy is insufficient infiltration and impaired function of immune cells within solid tumors. Strategies that can enhance immune cell infiltration and activation are critical to improving the outcome of cancer immunotherapy.

Disclosure of Invention

In order to solve the above problems, the present invention provides an alkaline phosphatase-responsive and convertible supermolecule bispecific cell cement polypeptide, a method for preparing the same and application thereof, and the present invention provides a novel method for solving challenges related to infiltration and activation of immune cells in cancer immunotherapy, introducing an alkaline phosphatase (ALP) -responsive and convertible supermolecule bispecific cell cement (Supra-BiCE) which utilizes the potential of NK and T cells for effective cancer immunotherapy.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides an alkaline phosphatase response and convertible supermolecule bispecific cell cement polypeptide, which is prepared by co-assembling a short peptide 1 and a short peptide 2;

The short peptide 1 is as follows: 1-Ada-GFFpY-GGYFHWHRLNP;

The short peptide 2 is as follows: 1-Ada-GFFpY-NYSKPTDRQYHF;

the 1-Ada is 1-adamantaneacetic acid;

The configuration of the amino acid in the short peptide 1 and the short peptide 2 is D configuration.

Preferably, both the short peptide 1 and the short peptide 2 are synthesized by Fmoc-solid phase synthesis.

Preferably, the Fmoc-solid phase synthesis method comprises the following steps:

(1) The C-terminus of Fmoc-amino acid was bound to the resin;

(2) Removing Fmoc protecting groups and washing;

(3) The C-terminal of the next Fmoc-amino acid is coupled with the N-terminal of the amino acid or polypeptide on the resin, and the resin is washed;

(4) Repeating the steps (2) - (3), and finally adding 1-adamantane acetic acid for washing;

(5) Cutting the polypeptide derivative from the resin to obtain a crude product;

(6) Purifying the crude product by high performance liquid chromatography to obtain short peptide 1 or short peptide 2.

The invention also provides a preparation method of the supermolecule bispecific cell cement polypeptide, which comprises the following steps:

1) Mixing the short peptide 1, the short peptide 2 and the phosphate buffer solution to obtain a short peptide solution, and regulating the pH value of the short peptide solution to 7.4 to obtain a solution to be assembled;

2) And (3) carrying out heating treatment and natural cooling on the solution to be assembled obtained in the step (1) to obtain the supermolecule bispecific cell cement polypeptide.

Preferably, the phosphate buffer in the step 1) is 1/2 x phosphate buffer, and the 1/2 x phosphate buffer contains 1% dimethyl sulfoxide by volume.

Preferably, the pH value of the short peptide solution is regulated by using a sodium carbonate solution, and the concentration of the sodium carbonate solution is 1mol/L.

Preferably, the ratio of the mass of the short peptide 1 and the mass of the short peptide 2 to the volume of the phosphate buffer solution in the step 1) is 1.157mg,1.192mg and 500 mu L.

Preferably, the heating treatment conditions in the step 2) include: the temperature was 90℃and the time was 10 minutes.

The invention also provides application of the supermolecule bispecific cell cement polypeptide in preparation of drugs for promoting NK and T cell immune infiltration of tumor cells.

Preferably, the tumor cells comprise the mouse breast cancer tumor cell line 4T1.

Supra-BiCE was constructed using a simple co-assembly strategy, combining two immune checkpoint blocking peptides: SA-P (short peptide 1), targeting and blocking programmed cell death ligand 1 (PD-L1) cells and SA-P (short peptide 2) on tumors, targets and blocks T cell immunoglobulins and ITIM domains (TIGIT) expressed by T cells and Natural Killer (NK) cells. This unique combination enables Supra-BiCE to selectively bind NK and T cells, promoting their activation and recruitment to enhance cancer immunotherapy.

Following administration via the tail vein, supra-BiCE self-assembles into nanoribbons and interacts with NK and T cells by binding to TIGIT. In tumor regions that overexpress ALP, the nanoribbon will be converted to long nanofibers in situ, resulting in an increased binding affinity of Supra-BiCE to PD-L1 and TIGIT. The transformation process is beneficial to the targeted enrichment and retention of NK cells and CD8+ T cells in a tumor area, and overcomes the limitation of poor infiltration of immune cells.

Advantageous properties and advantages: the Supra-BiCE design provides several significant characteristics and advantages in the field of cancer immunotherapy. First, it enables NK and T cells to bind locally to tumor cells, activating immune cells through dual immune checkpoint blockade. The method can remarkably improve the therapeutic effect of immunotherapy by overcoming limitations of immunocyte infiltration and immunosuppression tumor microenvironment.

Second, supra-BiCE demonstrates in situ enzyme-directed transformation behavior. The nanoribbons are converted into long nanofibers in the tumor area over-expressed by ALP, thereby increasing binding affinity to immune cells and tumor cells. This transformation process facilitates selective targeting, enrichment and retention of immune cells within the tumor, further enhancing the effectiveness of cancer immunotherapy.

Conclusion: in summary, alkaline phosphatase reactions and convertible Supra-BiCE represent a significant advance in the field of cancer immunotherapy. By addressing the limitations of immune cell infiltration and activation, this new approach makes it possible to significantly improve the therapeutic efficacy of solid tumor immunotherapy. The unique characteristics and advantages of Supra-BiCE, including its local binding to immune cells and tumor cells, in situ transformation, make it a promising tool for enhancing the effectiveness of cancer immunotherapy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a structural formula and a high performance liquid chromatography mass spectrum of the polypeptide of example 2;

FIG. 2 is a structural formula and a high performance liquid chromatography mass spectrum of the polypeptide of example 3;

FIG. 3 is a structural formula and a high performance liquid chromatography mass spectrum of the polypeptide of comparative example 2;

FIG. 4 is a structural formula and a high performance liquid chromatography mass spectrum of the polypeptide of comparative example 3;

FIG. 5 is an optical view of solutions configured in examples and comparative examples;

FIG. 6 is a graph of the microscopic morphology of PBS solutions for example and control polypeptides;

FIG. 7 is a two-stage structure of the polypeptides of examples 1, 2, 3 and example 1 before and after ALP catalysis;

FIG. 8 is a copolymerization Jiao Tu of Cy 5-labeled example 1 polypeptide to promote NK cell and tumor cell adhesion;

FIG. 9 is a photograph showing in vivo imaging of the aggregation ability of the Cy5.5-labeled polypeptide at a tumor site in Experimental example 4;

FIG. 10 is a statistical graph of tumor volumes in experimental example 5;

FIG. 11 is a statistical graph of tumor suppression rate in experimental example 5;

FIG. 12 is a statistical graph of body weight monitoring during treatment of mice in Experimental example 5.

Detailed Description

The invention provides an alkaline phosphatase response and convertible supermolecule bispecific cell cement polypeptide, which is prepared by co-assembling a short peptide 1 and a short peptide 2; the short peptide 1 is as follows: 1-Ada-GFFpY-GGYFHWHRLNP (SEQ ID No. 1); the short peptide 2 is as follows: 1-Ada-GFFpY-NYSKPTDRQYHF (SEQ ID No. 2); the 1-Ada is 1-adamantaneacetic acid; the configuration of the amino acid in the short peptide 1 and the short peptide 2 is D configuration.

In the present invention, the short peptide 1 and the short peptide 2 are preferably synthesized by adopting an Fmoc-solid phase synthesis method, the present invention is not particularly limited, and a person skilled in the art may adopt a conventional preparation method, and in a specific embodiment of the present invention, the Fmoc-solid phase synthesis method preferably includes the following steps:

(1) The C-terminus of Fmoc-amino acid was bound to the resin;

(2) Removing Fmoc protecting groups and washing;

The preparation method comprises the steps of mixing the short peptide 1, the short peptide 2 and the phosphate buffer solution to obtain a short peptide solution, and adjusting the pH value of the short peptide solution to 7.4 to obtain a solution to be assembled. In the present invention, the phosphate buffer is preferably a 1/2 Xphosphate buffer, and the 1/2 Xphosphate buffer contains 1% by volume of dimethyl sulfoxide. The invention preferably uses sodium carbonate solution to adjust the pH value of the short peptide solution, and the concentration of the sodium carbonate solution is preferably 1mol/L. In the present invention, the ratio of the mass of the oligopeptide 1, the mass of the oligopeptide 2 and the volume of the phosphate buffer is preferably 1.157mg,1.192mg and 500. Mu.L.

The invention carries out heating treatment and natural cooling on the obtained solution to be assembled to obtain the supermolecule bispecific cell cement polypeptide. In the present invention, the conditions of the heat treatment preferably include: the temperature was 90℃and the time was 10 minutes.

The invention also provides application of the supermolecule bispecific cell cement polypeptide in preparation of drugs for promoting NK and T cell immune infiltration of tumor cells. In the present invention, the tumor cells preferably comprise the mouse breast cancer cell line 4T1.

In the examples of the present invention, the sources of the formulations involved are as follows:

2-Cl-Trt resin: available from Jier Biochemical (Shanghai) Inc. with a substitution of 1.158mmol/g.

Amino acid: purchased from gill biochemistry (Shanghai) limited with a purity of 98%.

N, N-Diisopropylethylamine (DIEPA): purchased from Shanghai Ala Biochemical technologies Co., ltd, purity was 99%.

2- (7-Azabenzotriazol) -N, N' -tetramethyluronium Hexafluorophosphate (HATU): the purity was 98% obtained from Tianjin Sheen Sichuang Biochemical technology Co.

Trifluoroacetic acid (TFA): commercially available from Shanghai Meilin Biochemical technologies Co., ltd.) at a purity of 99%.

Triisopropylsilane (TIS): purchased from Shanghai Ala Biochemical technologies Co., ltd, purity was 99%.

Anhydrous Dichloromethane (DCM): purchased from Tianjin chemical reagent company.

N, N-Dimethylformamide (DMF): purchased from Tianjin chemical reagent company.

Chromatographic pure methanol: commercially available from Tianjin Conscode technologies.

1-Adamantaneacetic acid: commercially available from Shanghai Ala Biochemical technologies Co.

Hoechst 33342 dye liquor: is commercially available from Shanghai Biyun biotechnology limited.

CFSE: is commercially available from Shanghai Biyun biotechnology limited.

Cy5: provided by the sameidie technology (Thermofisher Scientific).

Piperidine: purchased from Tianjin chemical reagent company.

Cell culture medium (DMEM): is provided by the Siemens technology (Thermofisher Scientific), and is sterile.

Fetal bovine serum: is provided by the Siemens technology (Thermofisher Scientific), and is sterile.

Balb/c mice: females at the age of 8 weeks were purchased from the company Vetolihua Biotechnology, inc.

The equipment involved in the embodiment of the invention is as follows:

High Performance Liquid Chromatograph (HPLC): manufactured by Lumtech in germany.

High performance liquid chromatography mass spectrometer: manufactured by shimadzu, model LC-MS2020.

An electronic balance: manufactured by Sarturious, germany, model BS124S.

Transmission Electron Microscope (TEM): tecnai G2F 20 System.

Freeze dryer: manufactured by Beijing Asia Taike Colon, model number is LGJ-1-50.

Laser confocal microscope: manufactured by Leica, germany, model number TCS SP8.

Round two chromatograph: manufactured by BioLogic, france, model number MOS-450.

The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.

Example 1

Synthesis of polypeptide 1-Ada-GFFpY-GGYFHWHRLNP (SA-T), polypeptide 1-Ada-GFFpY-NYSKPTDRQYHF (SA-P).

The polypeptide is SA-T or SA-P by adopting a classical Fmoc-short peptide solid phase synthesis method.

The method comprises the following specific steps:

In a solid phase synthesis tube, 0.5mmol of 2-Cl-Trt resin was taken, 15mL of DCM was added, and the mixture was placed on a shaker and shaken for 10 minutes to swell the 2-Cl-Trt resin.

DCM was extruded from a solid phase synthesizer loaded with 2-Cl-Trt resin using an ear-washing pellet.

0.75Mmol Fmoc protected amino acid was dissolved in 10mL DCM, 1.5mmol DIEPA was added, mixed well and added to a solid phase synthesizer and reacted at room temperature for 1 hour.

And (3) performing a sealing step: the reaction solution in the solid phase synthesis tube was removed, then washed with 10mL of DCM for 1 min each, 3 times, and then the prepared ratio of DCM was added: DIEPA: methanol was 17:1:2 in 16mL and reacted at room temperature for 15 minutes.

The reaction solution in the solid phase synthesis tube was removed, washed with DCM 10mL each for 1 min for 3 times, then DMF 10mL each for 1 min for 3 times. 15mL of DMF containing 20% piperidine was added and reacted for 30 minutes, followed by washing with DMF, 10mL each for 1 minute, and washing 5 times in total.

The second Fmoc protected amino acid 1mmol, HATU 1mmol, DIEPA2 mmol were weighed and dissolved in 10mL DMF, and the dissolved amino acid solution was added to the solid phase synthesis tube and reacted at room temperature for 1 hour.

The procedure of steps 5) and 6) was repeated with the amino acid and the blocking group 1-Ada being added sequentially. Then, the reaction mixture was washed with DMF for 5 times and dichloromethane for 5 times, and the reaction mixture was prepared for the next reaction.

10ML of the cleavage solution was prepared, and the mixture was added to a solid phase synthesizer in a ratio of 95% TFA,2.5% TIS and 2.5% H ₂ O (volume ratio), and reacted at room temperature for 1 hour. The product was excised from the 2-Cl-Trt resin and the solvent removed by rotary evaporation to give the crude product. Subsequently, separation and purification are performed by HPLC to obtain SA-T or SA-P. As a result of LC-MS detection, as shown in FIGS. 1 and 2, the purity of SA-T and SA-P reaches 95% as can be seen from FIGS. 1 and 2.

Configuration of (SA-T & P):

1) Together, the lyophilized 1.157mg of SA-T and 1.192mg of SA-P were dissolved in 500. Mu.L of 1/2 XPBS buffer and 1% DMSO (volume ratio) was added to give a final concentration of SA-T & P solution of 500. Mu.M.

2) 1M Na ₂CO₃ was added to adjust the pH of the final solution to 7.4.

3) The solution was heated to 90 ℃ for 10 minutes and allowed to cool naturally to ensure adequate dissolution of the peptide.

4) In subsequent experiments, the prepared peptide-containing solution was diluted appropriately as needed to obtain the desired concentration, resulting in SA-T & P solution.

Example 2

Synthesis of polypeptide 1-Ada-GFFpY-GGYFHWHRLNP (SA-T).

The polypeptide is SA-T for short, which is synthesized by adopting a classical Fmoc-short peptide solid-phase synthesis method. The method comprises the following specific steps:

10ML of the cleavage solution was prepared, and the mixture was added to a solid phase synthesizer in a ratio of 95% TFA,2.5% TIS and 2.5% H ₂ O (volume ratio), and reacted at room temperature for 1 hour. The product was excised from the 2-Cl-Trt resin and the solvent removed by rotary evaporation to give the crude product. Subsequently, separation and purification were performed by HPLC to obtain SA-T.

Configuration of SA-T solution:

1) The lyophilized 1.157mg SA-T was dissolved in 500. Mu.L of 1/2 XPBS buffer and 1% DMSO was added (volume ratio) to give a final concentration of SA-T solution of 500. Mu.M.

2) 1M Na ₂CO₃ was added to adjust the pH of the final solution to 7.4.

4) In the subsequent experiments, the prepared peptide-containing solution was appropriately diluted as needed to obtain the desired concentration, resulting in SA-T solution.

Example 3

Synthesis of the polypeptide Ada-GFFpY-NYSKPTDRQYHF (SA-P).

The polypeptide is SA-P for short, which is synthesized by adopting a classical Fmoc-short peptide solid-phase synthesis method. The method comprises the following specific steps:

10ML of the cleavage solution was prepared, and the mixture was added to a solid phase synthesizer in a ratio of 95% TFA,2.5% TIS and 2.5% H ₂ O (volume ratio), and reacted at room temperature for 1 hour. The product was excised from the 2-Cl-Trt resin and the solvent removed by rotary evaporation to give the crude product. Subsequently, separation and purification were performed by HPLC to obtain SA-P.

Configuration of SA-P solution:

1) 1.192mg of lyophilized SA-P was dissolved in 500. Mu.L of 1/2 XPBS buffer and 1% DMSO (volume ratio) was added to give a final concentration of SA-P solution of 500. Mu.M.

2) 1M Na ₂CO₃ was added to adjust the pH of the final solution to 7.4.

4) In the subsequent experiments, the prepared peptide-containing solution was appropriately diluted as needed to obtain the desired concentration, resulting in a SA-P solution.

Comparative example 1

The Fmoc-short peptide solid phase synthesis method of example 1 was followed to prepare a polypeptide, abbreviated as T (GGYFHWHRLNP) or P (NYSKPTDRQYHF). In the synthesis of the control polypeptides, the gel forming factor consisting of 3 amino acids, 1-adamantane and alkaline phosphatase reaction sites were absent in the method of the comparative example. The polypeptide obtained in this comparative example is detected by high performance liquid chromatography mass spectrometry, the structural formula and the result of T are shown in figure 3, and the purity of T can reach 95% as can be seen from figure 3. The structural formula and the result of P are shown in FIG. 4, and the purity of P can be seen to reach 95% from FIG. 4.

Preparation of T & P solution:

1) 7.43mg of T and 7.78mg of P were dissolved together in 500. Mu.L of 1/2 XPBS buffer, and 1% DMSO (volume ratio) was added to give a final concentration of T & P solution of 500. Mu.M.

2) 1M Na ₂CO₃ was added to adjust the pH of the final solution to 7.4.

3) In subsequent experiments, the prepared peptide-containing solution was diluted appropriately as needed to obtain the desired concentration.

Comparative example 2

The Fmoc-short peptide solid phase synthesis method of example 1 was followed to prepare a polypeptide, abbreviated T (GGYFHWHRLNP). In the synthesis of the control polypeptides, the gel forming factor consisting of 3 amino acids, 1-adamantane and alkaline phosphatase reaction sites were absent in the method of the comparative example. The polypeptide obtained in this comparative example is detected by high performance liquid chromatography mass spectrometry, the structural formula and the result of T are shown in figure 3, and the purity of T can reach 95% as can be seen from figure 3.

Preparation of T solution:

1) 7.43mg of T was dissolved in 500. Mu.L of 1/2 XPBS buffer and 1% DMSO was added (volume ratio) to give a final concentration of 500. Mu.M.

2) 1M Na ₂CO₃ was added to adjust the pH of the final solution to 7.4.

Comparative example 3

The Fmoc-short peptide solid phase synthesis method of example 1 was followed to prepare a polypeptide, abbreviated P (NYSKPTDRQYHF). In the synthesis of the control polypeptides, the gel forming factor consisting of 3 amino acids, 1-adamantane and alkaline phosphatase reaction sites were absent in the method of the comparative example. The polypeptide obtained in this comparative example was detected by high performance liquid chromatography mass spectrometry, the structural formula and the result of P are shown in FIG. 4, and it can be seen from FIG. 4 that the purity of P reaches 95%.

Preparation of P solution:

1) 7.78mg of P was dissolved in 500. Mu.L of 1/2 XPBS buffer and 1% DMSO was added (volume ratio) to give a final concentration of 500. Mu.M in the P solution.

2) 1MNA ₂CO₃ was added to adjust the pH of the final solution to 7.4.

Experimental example 1

Transmission electron microscope experiment

The solutions of examples 1,2 and 3 were prepared and electron microscopy samples were prepared. First, 10. Mu.L of a 10. Mu.M polypeptide self-assembly solution was dropped onto a copper mesh, left for one minute, and then the excess solution was removed from the edges using filter paper. Next, 10 μl of phosphotungstic acid was added dropwise, left on the copper mesh for one minute for negative staining, and the excess dye solution was removed from the edge again using filter paper. And placing the treated copper net in a drying oven, and shooting by using an electron transmission microscope after the copper net is completely dried. ALP was then added to examples 1,2 and 3 at a rate of 10U/mL, and the mixture was placed in an incubator at 37℃and after 24 hours of reaction, the photograph of the solution was shown in FIG. 5, and the microstructure of examples 1,2 and 3 and the microstructure of example 1 converted by ALP catalysis were observed by a transmission electron microscope, and the result was shown in FIG. 6.

FIG. 6 shows that example 2 (SA-T) and example 3 (SA-P) form nanoparticles and short nanofibers, respectively. However, unlike example 2 (SA-T) and example 3 (SA-P), the co-assembled SA-T & P formed short nanoribbon structures, which demonstrated the occurrence of self-assembly. After the addition of ALP, example 1 undergoes a morphology transformation under the action of ALP, which is manifested as long nanofibers, which are then entangled to form a dense nanofiber network. The above results demonstrate that the addition of ALP does trigger the morphology transition of example 1.

Experimental example 2

Round two-chromatographic determination secondary structure

For CD signal detection, the sample (SA-T & P) was diluted to a concentration of 50. Mu.M according to the preparation method described in Experimental example 1. Then, 200. Mu.l of the diluted sample was put into a quartz cell having a diameter of 0.1 cm. CD spectra were recorded in the 190-260nm range using a circular dichroism spectrometer (MOS-450, bioLogic), as shown in FIG. 7.

SA-P shows two characteristic peaks, located at 210 and 221 nm, respectively, tending to form an alpha helix structure. SA-T shows a positive peak, employing a beta-sheet conformation at 223 nm. These results are consistent with FT IR. In addition, SA-T & P shows two positive peaks, located at 213 and 233 nm, respectively, indicating that the alpha helix structure is dominant. A strong positive value around 226 nm indicates that SA-T & P is dominant in the phosphatase-catalyzed beta sheet structure.

Experimental example 3

Laser confocal microscope study of attachment process of SA-BiCE and cells

Co-culture observations of NK and 4T1 cells: first, mouse NK cells were labeled with CFSE (Thermofisher C34554). Briefly, cells were co-incubated with CFSE for 10 minutes and then washed three times with PBS. Next, the experimental group was incubated with 10. Mu.M Cy 5-labeled example 1 for NK cells. Control groups were incubated with PBS. After 2 hours of incubation, the incubated NK cells were inoculated onto 4T1 cells (Hoechst-labeled) previously cultured to a density of about 80% together with Cy 5-labeled example 1, and the number ratio of NK cells to 4T1 cells was 4:1. then, an image was taken at the same voltage using a laser confocal microscope (LEICA TSC SP) as shown in fig. 8.

As can be seen from fig. 8, after the co-culture of cd8+ T and NK cells with example 1 for 12 hours, the non-adherent cells were washed out, and a large number of cd8+ T and NK cells were observed to be tightly bound to tumor cells and to be significantly adhered to the cell membranes of 4T1 cells. The control group had a lower number of CD8 ⁺ T and NK cells interacting with tumor cells than the control group.

Experimental example 4

Ability of SA-BiCE to aggregate at tumor sites

The ability of examples and controls to aggregate at tumor sites was evaluated using a small animal living imaging system. The polypeptide self-assembly solution of the example, as well as the solutions of the control and Cy5.5, were prepared according to the dosage calculation of the cyanine dye Cy5.5-NHS at a dosage of 2.09. Mu.M kg ^-1. The administration mode is tail vein injection, and the administration volume is 150 mu L. The mark was 0h (0 h) after the completion of the administration. Living imaging was performed at different time points (1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours) using an excitation wavelength of 645nm. The results are shown in FIG. 9.

All examples showed significantly enhanced fluorescence signal one hour after mouse tail intravenous injection compared to the control. This is probably because the assembled peptide has a greater resistance to biodegradation. Examples 2, 3 and 1 exhibited stronger tumor targeting and retention than comparative examples 2, 3 and 1. This is because the nanostructure formed by the assembly of the polypeptide can reach the tumor site through EPR effect in blood circulation and self-assemble according to ALP overexpressed around the tumor, thereby enhancing the retention ability at the tumor site. Interestingly, the retention capacity of example 1 in mice after 48 hours was significantly higher than for the groups of examples 2 and 3. This is believed to be due to the stronger binding capacity to PD-L1 of example 1, and the stronger aggregation capacity under ALP catalysis in the tumor microenvironment.

Experimental example 5

Animal tumor model

To evaluate the inhibitory effect of SA-BiCE on tumor growth, 4T1 cells were injected under mammary fat pad of 8-week-old BALB/c mice, each of which was injected with 2X 10 ⁵ cells, and a 4T1 xenograft tumor model was established. The calculation formula for tumor volume was v=lw ²/2, where L and W represent the length and width of the tumor, respectively, and tumor size was monitored using vernier calipers. When the tumor volume reached 30-50mm ³, the mice were weighed and randomly divided into different groups. The experiments were performed by tail vein injection, and the mice were injected with the polypeptide derivative (6.5. Mu.M kg-1) and PBS every other day from day 6 for a total of 5 doses, and the tumor growth trend and the weight change of the mice in each group were monitored, as shown in FIG. 10. The experiment has obtained SYXK (jin) 2019-0003 certification No. issued by the ministry of the Tianjin city.

The results in FIG. 10 show that example 1 (SA-T & P) exhibited the best ability to resist tumor growth, reaching the highest level among all groups. The results according to fig. 11 show that the tumor suppression rate of example 1 was about 64.47% higher by 1.43-fold, 4.61-fold, 6.14-fold, 9.66-fold and 5.13-fold than that of example 2, example 3, comparative example 1, comparative example 2 and comparative example 3, respectively.

Fig. 12 is a statistical result of the monitoring of the body weight of mice during the treatment period, and the change of the body weight of the mice is found to be negligible, which indicates that none of the drugs shows obvious systemic toxicity, and the polypeptides provided by the invention have good biocompatibility.

The invention provides an innovative method, which utilizes the design of an ALP-responsive dual-specificity supermolecule cell engagement device (Supra-BiCE) to realize the targeted combination between cancer cells and immune cells so as to enhance the immune treatment effect. Through the co-assembly of blocking peptides, the Supra-BiCE can simultaneously recognize TIGIT on the surface of immune cells and PD-L1 on the surface of tumor cells, and through the conversion from ALP-guided nanofibers to long nanofibers, the binding affinity is improved, the recruitment of tumor-specific immune cells is ensured, and the retention in tumors is prolonged. This approach provides new strategies and tools for enhancing the effectiveness of immunotherapy.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. An alkaline phosphatase responsive and transformable supramolecular bispecific cell cement polypeptide, characterized in that it is co-assembled from short peptide 1 and short peptide 2;

The short peptide 1 is as follows: 1-Ada-GFFpY-GGYFHWHRLNP;

The short peptide 2 is as follows: 1-Ada-GFFpY-NYSKPTDRQYHF;

the 1-Ada is 1-adamantaneacetic acid;

2. The supramolecular bispecific cell cement polypeptide of claim 1, wherein both short peptide 1 and short peptide 2 are synthesized using Fmoc-solid phase synthesis.

3. The supramolecular bispecific cell cement polypeptide of claim 2, wherein the Fmoc-solid phase synthesis method comprises the steps of:

(1) The C-terminus of Fmoc-amino acid was bound to the resin;

(2) Removing Fmoc protecting groups and washing;

4. A method for preparing the supramolecular bispecific cell cement polypeptide of any one of claims 1-3, comprising the steps of:

5. The method according to claim 4, wherein the phosphate buffer in step 1) is 1/2×phosphate buffer, and the 1/2×phosphate buffer contains 1% dimethyl sulfoxide by volume.

6. The method according to claim 4, wherein the pH of the short peptide solution is adjusted using a sodium carbonate solution having a concentration of 1mol/L.

7. The method according to claim 4, wherein the ratio of the mass of the short peptide 1 to the mass of the short peptide 2 to the volume of the phosphate buffer solution in the step 1) is 1.157 mg/1.192 mg/500. Mu.L.

8. The method according to claim 4, wherein the conditions of the heat treatment in step 2) include: the temperature was 90℃and the time was 10 minutes.

9. The use of the supramolecular bispecific cell cement polypeptide of any one of claims 1-3 in the preparation of a medicament for promoting NK and T cell immune infiltration of tumor cells.

10. The use according to claim 9, wherein the tumour cell is a mouse breast cancer cell line 4T1.