Preparation method of PD-L1 remodeling probe

Abstract

The invention discloses a preparation method of a PD-L1 remodeling probe, and belongs to the technical field of probe preparation. Dissolving galactose oxidase containing free sulfydryl in a phosphate buffer solution; the preparation method comprises the following steps: adding NHS-polyethylene glycol 1000-maleimide into galactose oxidase, and mildly stirring at room temperature to obtain a mixed solution; transferring the mixed solution into a dialysis membrane, and dialyzing with a phosphate buffer solution for 12-16 hours; after dialysis is completed, adding a peptide fragment, reacting at room temperature to promote the reaction of NHS ester and amino of the peptide fragment, and finally generating a peptide-polyethylene glycol 1000-galactose oxidase covalent complex; the covalent complex is analyzed through lauryl sodium sulfate-polyacrylamide gel electrophoresis, if the molecular weight of a band is increased and migration occurs, the complex is successfully coupled, and the PD-L1 remodeling probe is obtained. The PD-L1 remodeling probe prepared by the invention can be used for specifically and efficiently oxidizing galactose / N-acetylgalactosamine residues at the tail end of PD-L1 to generate aldehyde groups so as to be subjected to biological orthogonal reaction with hydrazide groups.

Description

A method for preparing a PD-L1 remodeling probe Technical Field

[0001] This invention belongs to the field of probe preparation technology, specifically relating to a method for preparing a PD-L1 remodeled probe. Background Technology

[0002] In the structural arrangement of topological biomolecules such as signaling complexes, DNA, and proteins, receptor aggregation plays a particularly crucial role—including T-cell and B-cell receptors. This aggregation can significantly enhance the activation efficiency of signaling pathways and cellular responses. Artificial topological nanostructures (ATNs), constructed from self-assembled nanomaterials through non-covalent ligand-receptor interactions, belong to a novel class of topological biomolecules.

[0003] However, non-covalent interactions are typically dynamic and prone to dissociation under complex physiological conditions such as shear stress, dilution effects, and protein competition, which limits their stability and practical applications. In contrast, covalent bonds exhibit superior stability and durability, maintaining stable interactions even in harsh environments, providing a more reliable technical path for the construction and functionalization of topological structures.

[0004] According to currently available research, there are no reports on a research direction that uses covalently driven self-assembly to form artificial topological nanostructures, thereby simultaneously alleviating immunosuppression and synergistically activating T cell function. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for preparing a PD-L1 remodeling probe. The method envisions remodeling PD-L1 on the surface of tumor cells through local oxidation to introduce covalent binding sites. Then, using bioorthogonal chemistry to drive the covalent assembly of functional artificial topological nanostructures, T cells can be recruited and activated. This "local oxidation-covalent assembly" strategy not only blocks the PD-1 / PD-L1 pathway but also promotes T cell recruitment and activation, thereby enhancing the anti-tumor immune response.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a PD-L1 remodeling probe disclosed in this invention includes the following steps: S1, dissolving galactose oxidase containing free thiol groups in phosphate buffer; S2, adding NHS-polyethylene glycol 1000-maleimide to the galactose oxidase and gently stirring at room temperature to obtain a mixture; S3, transferring the mixture to a dialysis membrane and dialyzing at 4°C with the phosphate buffer from step S1 for 12–16 hours; S4, after dialysis, adding an equimolar amount of peptide and reacting at room temperature to promote the reaction between the NHS ester and the amino group of the peptide, ultimately generating a peptide-polyethylene glycol 1000-galactose oxidase covalent complex; S5, analyzing the covalent complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. If an increase in the molecular weight of the band and migration are observed, it indicates that the complex has been successfully coupled, and the PD-L1 remodeling probe is obtained.

[0007] Furthermore, the pH of the phosphate buffer is 7.4, and the concentration of the phosphate buffer containing galactose oxidase is 100 μM.

[0008] Furthermore, the molar ratio of the galactose oxidase to NHS-polyethylene glycol 1000-maleimide is 1:1, and the stirring time is 30 minutes.

[0009] Furthermore, the dialysis membrane has a molecular weight cutoff of 10 kDa; the buffer solution is changed every 4–6 hours during dialysis, for a total of 2–3 changes.

[0010] Furthermore, the entire preparation process is carried out under light-protected conditions.

[0011] Furthermore, the peptide is the corresponding PD-L1 targeting peptide.

[0012] The beneficial effects of the present invention are as follows: 1. The PD-L1 remodeling probe prepared by the present invention can specifically and efficiently oxidize the galactose / N-acetylgalactosamine residues at the end of PD-L1 to generate aldehyde groups so as to undergo a bioorthogonal reaction with acylhydrazine groups.

[0013] 2. In the preparation of this invention, galactose (Ga1) / N-acetylgalactosamine (Ga1NAc) is a common terminal monosaccharide residue in glycoproteins and glycolipid oligosaccharide chains. PD-L1, as a highly glycosylated transmembrane protein, contains four well-defined N-glycosylation sites, and its glycosyl terminus is usually predominantly galactose. Galactose oxidase, a copper-containing metalloenzyme, can selectively oxidize the C6 hydroxyl group of terminal galactose (Gal) / N-acetylgalactosamine to the corresponding aldehyde group. By introducing a PD-L1-specific targeting peptide to construct a PD-L1 remodeling probe, galactose oxidase can be precisely activated, thereby achieving selective editing of PD-L1 glycans while avoiding non-specific oxidation of other glycoproteins.

[0014] 3. The PD-L1 remodeling probe prepared in this invention can be used as an anchoring point to trigger self-assembly on the surface of tumor cells to form functional ATNs. The ATNs contain anti-CD3 units that mimic bispecific T cell adaptor proteins, which can effectively recruit and activate T cells.

[0015] Other advantages, objectives, and features of the invention will be set forth in the following description and will be apparent to those skilled in the art in some respects, or may be learned by practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0016] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 is a schematic diagram of the synthetic routes of the three probes of this invention and the corresponding PD-L1 targeting peptide structures; Figure 2 is a schematic diagram of the binding and dissociation constants of the three probes of this invention; Figure 3 is an SDS-PAGE characterization result of GAO, PEG1000-GAO (PG), and P1 of this invention; Figure 4a is a CLSM diagram of cells of this invention after incubation with GAO-Cy5 (I), PG-Cy5 (II), and P1-Cy5 (III); Figures 4b and 4c are flow cytometry analysis results of MDA-MB-231 of this invention; Figures 4d and 4e are flow cytometry analysis results of SKBR-3 of this invention. Detailed Implementation

[0017] As shown in Figures 1-4, this invention discloses a method for preparing a PD-L1 remodeling probe.

[0018] S1. Dissolve galactose oxidase (GAO) containing free thiol groups in phosphate-buffered saline (PBS) at H 7.4 to a concentration of 100 μM; S2. Mix galactose oxidase (GAO) and NHS-polyethylene glycol 1000-maleimide at a molar ratio of 1:1 and gently stir at room temperature for 30 minutes to obtain a mixture, allowing the maleimide groups to specifically react with the thiol groups of galactose oxidase (GAO) to form stable thioether bonds; S3. Transfer the mixture to a container with a molecular weight cutoff of 10... In a kDa dialysis membrane, dialyze with phosphate buffer at 4°C for 12–16 hours, changing the buffer every 4–6 hours for a total of 2–3 times to remove unreacted crosslinking agents and low molecular weight impurities; S4, after dialysis, add an equimolar amount of peptides (peptide 1, peptide 2, and peptide 3), and react at room temperature for 1 hour to allow the NHS ester to react with the amino groups of the peptides, ultimately generating a peptide-polyethylene glycol 1000-galactose oxidase covalent complex; S5, analyze the product by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. If an increase in the molecular weight of the bands and migration are observed, it indicates that the complex has been successfully coupled; S6, determine the protein concentration of the peptide-polyethylene glycol 1000-galactose oxidase complex at a wavelength of 562 nm using the BCA protein quantification method to provide accurate protein content data for subsequent cell surface functionalization experiments.

[0019] The entire preparation process must be carried out under sterile and light-protected conditions to minimize thiol oxidation and maintain the enzyme activity and coupling efficiency of galactose oxidase (GAO).

[0020] As shown in Figure 1, the PD-L1 targeting peptide was coupled with N-hydroxysuccinimide-polyethylene glycol 1000-maleimide, and then reacted with the thiol group (-SH) of galactose oxidase (GA0). When peptide 1 was added, peptide 1-polyethylene glycol 1000-galactose oxidase (P1) was synthesized; when peptide 2 was added, peptide 2-polyethylene glycol 1000-galactose oxidase (P2) was synthesized; and when peptide 3 was added, peptide 3-polyethylene glycol 1000-galactose oxidase (P3) was synthesized.

[0021] The binding affinity and dissociation constant (Kd) of the three probes to the PD-L1 protein were then determined using surface plasmon resonance (SPR) technology. As shown in Figure 2, the binding and dissociation constants (Kd) of P1, P2, and P3 were 4.50 × 10⁻⁵ M, 7.29 × 10⁻⁵ M, and 2.02 × 10⁻⁵ M, respectively, with P1 exhibiting the strongest binding affinity.

[0022] P1 exhibits a significant binding advantage, and its higher affinity promises to provide a more reliable experimental basis for functional analysis, thus it will be used for subsequent experiments.

[0023] As shown in Figure 3, polyethylene glycol 1000-galactose oxidase (PG), which lacks PD-L1 targeting ability, was also synthesized as a control group. To further verify the structural integrity and purity of P1 and PG, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for analysis. The results showed that both exhibited clear bands at the expected molecular weight positions, confirming that the probes were successfully synthesized and their structures were correct.

[0024] To evaluate the cell-specific binding ability of the probe, we labeled galactose oxidase (GAO), control group (PG), and target probe (P1) with Cy5, and observed them using laser confocal scanning microscopy (CLSM).

[0025] As shown in Figure 4a, in SKBR-3 cells with low PD-L1 expression, GAO-Cy5, PG-Cy5, and P1-Cy5 all showed extremely weak fluorescence signals; while in MDA-MB-231 cells with high PD-L1 expression, significant fluorescence signals appeared on the cell surface after treatment with P1-Cy5, indicating that the probe can selectively bind to cells with positive PD-L1 expression.

[0026] As shown in Figures 4b and 4e, flow cytometry further quantified this interaction, revealing a significant increase in the fluorescence intensity of P1-Cy5 in MDA-MB-231 cells; similarly, Figures 4d and 4e show that flow cytometry further quantified this interaction, revealing a significant increase in the fluorescence intensity of P1-Cy5 in SKBR3-3 cells. These results are consistent with observations obtained using laser confocal scanning microscopy (CLSM).

[0027] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a PD-L1 remodeling probe, characterized in that: Includes the following steps, S1. Dissolve galactose oxidase containing free sulfhydryl groups in phosphate buffer; S2. Add NHS-PEG 1000-maleimide to galactose oxidase and gently stir at room temperature to obtain a mixture; S3. Transfer the mixture to a dialysis membrane and dialyze at 4°C with the phosphate buffer from step S1 for 12–16 hours; S4. After dialysis, add an equimolar amount of peptide and react at room temperature to promote the reaction between the NHS ester and the amino group of the peptide, ultimately generating a peptide-PEG 1000-galactose oxidase covalent complex; S5. Analyze the covalent complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. If an increase in the molecular weight of the band and migration are observed, it indicates that the complex has been successfully coupled, and the PD-L1 remodeling probe has been obtained.

2. The method for preparing a PD-L1 remodeling probe according to claim 1, characterized in that: The phosphate buffer has a pH of 7.4 and contains 100 μM galactose oxidase.

3. The method for preparing a PD-L1 remodeling probe according to claim 2, characterized in that: The molar ratio of galactose oxidase to NHS-polyethylene glycol 1000-maleimide is 1:1, and the stirring time is 30 minutes.

4. The method for preparing a PD-L1 remodeling probe according to claim 3, characterized in that: The dialysis membrane has a molecular weight cutoff of 10 kDa; the buffer solution is changed every 4–6 hours during dialysis, for a total of 2–3 changes.

5. The method for preparing a PD-L1 remodeling probe according to claim 4, characterized in that: The entire preparation process is carried out under light-protected conditions.

6. The method for preparing a PD-L1 remodeling probe according to claim 1, characterized in that: The peptide segment can be peptide segment 1, peptide segment 2, or peptide segment 3.

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