PBP peptides that specifically bind to PD-1, their screening methods and applications

Abstract

This invention discloses a PBP peptide that specifically binds to PD-1, its screening method, and its applications. The PBP peptide has the sequence shown in SEQ ID No. 1. This invention uses bacterial surface display technology to screen for PD-1-targeting PBP peptides. These peptides specifically bind to PD-1, exhibit cross-reactivity with human and mouse PD-1, and can competitively bind to PD-1 with PD-L1, effectively blocking the interaction between PD-1 and PD-L1, reversing the function of exhausted T cells, and exerting an anti-tumor effect. Compared to antibodies, the PBP peptide obtained in this invention has a small molecular weight, strong penetration ability at tumor sites, low production cost, simple preparation process, and does not cause serious immune-related side effects. While achieving corresponding therapeutic effects, it avoids the disadvantages of antibody drugs, providing a low-molecular-weight candidate drug for tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to a class of polypeptides, and more particularly to a PBP polypeptide that can specifically bind to PD-1, a screening method thereof, and its application in the preparation of drugs for anti-tumor therapy, belonging to the field of tumor immunotherapy technology. Background Technology

[0002] Immunotherapy has become an important means of cancer treatment. Immune checkpoint inhibitors, especially monoclonal antibodies targeting the PD-1 / PD-L1 signaling pathway, have achieved significant therapeutic effects in various cancers and show great promise in cancer treatment. Immune checkpoint inhibitors typically target immune cells in the body that can eliminate tumors, such as macrophages with phagocytic function and T cells with cytotoxic function. In recent years, tumor immunotherapy targeting T cells has been very popular and has achieved very important research progress, among which cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1) are the most well-known.

[0003] Studies have found that CTLA-4 plays a more fundamental role in regulating T cell responses to normal self-antigens. Targeting CTLA-4 can significantly induce T cell infiltration into normal organs, causing damage. Meanwhile, the interaction between PD-1 and PD-L1 primarily occurs in the tumor microenvironment (TME). Blocking the interaction between PD-1 and PD-L1 causes significantly less toxicity than blocking CTLA-4. Therefore, relieving T cell suppression by interfering with the interaction between PD-1 and PD-L1 has greater advantages in tumor treatment.

[0004] Currently, antibody drugs are the main way to block the PD-1 / PD-L1 pathway, but their application is limited by several drawbacks. For example, the large size of antibodies leads to poor penetration into tumor sites; antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) effects caused by antibody Fc fragments can damage immune cells and induce immune-related side effects, leading to treatment interruption and even threatening patient life; the relatively long half-life of antibodies also increases the difficulty of drug clearance when side effects occur; in addition, antibodies also suffer from high production costs and complex preparation processes. Therefore, the high immunogenicity and high cost significantly limit the clinical application of antibody drugs, making the search for and development of low-molecular-weight candidate non-antibody drugs to block the PD-1 / PD-L1 signaling axis urgent. Because the interaction plane between ligands and receptors is large and flat, without a defined binding pocket, developing small-molecule drugs to block protein-protein interactions remains a significant challenge. Peptides occupy the optimal position between proteins and small molecules, making it easier to mimic the interaction between PD-1 and PD-L1. Compared to antibodies, peptides have relatively low affinity, allowing them to quickly dissociate from the target and alleviate side effects when they occur. Furthermore, peptides offer advantages such as simple manufacturing processes and low production costs. Although significant progress has been made in peptide research targeting the PD-1 / PD-L1 signaling pathway, it remains in its early stages and lags far behind antibody research. Therefore, developing effective peptide inhibitors is of great significance. Summary of the Invention

[0005] The main objective of this invention is to provide a PBP polypeptide that specifically binds to PD-1, thereby overcoming the shortcomings of the prior art.

[0006] Another objective of this invention is to provide a method and application for screening PBP peptides that specifically bind to PD-1.

[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0008] This invention provides a PBP polypeptide that specifically binds to PD-1, having the sequence shown in SEQ ID No. 1.

[0009] Furthermore, the PBP peptide can specifically bind to both free PD-1 protein and PD-1 expressed on the cell surface.

[0010] Furthermore, the PBP peptide can enhance T cell proliferation and IFNγ secretion.

[0011] Furthermore, the PBP peptide can block the PD-1 / PD-L1 signaling pathway and reverse T cell function.

[0012] This invention also provides a method for screening PBP peptides that specifically bind to PD-1, comprising:

[0013] Peptides that can specifically bind to PD-1 were screened using a random peptide display library on the bacterial surface, and their conserved sequences were analyzed.

[0014] Using conserved sequences as the core, a bacterial surface bias library was constructed to screen for PBP peptides that specifically bind to PD-1.

[0015] This invention also provides the application of the aforementioned PBP peptide that specifically binds to PD-1 in the preparation of antitumor drugs.

[0016] Accordingly, embodiments of the present invention also provide an antitumor drug comprising the aforementioned PBP polypeptide that specifically binds to PD-1.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0018] This invention utilizes bacterial surface display technology to screen and obtain a PD-1-targeting PBP peptide. This peptide specifically binds to PD-1, exhibits cross-reactivity with both human and mouse PD-1, and competitively binds to PD-1 with PD-L1. It effectively blocks the PD-1 / PD-L1 interaction, reverses the function of exhausted T cells, and exerts an anti-tumor effect. Compared to antibodies, the PBP peptide obtained in this invention has a smaller molecular weight, stronger penetration at tumor sites, lower production costs, and a simpler preparation process. It does not cause severe immune-related side effects. While achieving corresponding therapeutic effects, it avoids the drawbacks of antibody drugs, providing a low-molecular-weight candidate drug for tumor immunotherapy. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the process of screening peptides using magnetic bead screening technology (MACS) and fluorescence activated cell sorting (FACS) in a typical embodiment of the present invention.

[0021] Figure 2 This is a diagram showing the results of conserved sequence analysis of a polypeptide in a typical embodiment of the present invention;

[0022] Figure 3This is a schematic diagram illustrating the construction principle of a PD-1 targeted peptide bias library in a typical embodiment of the present invention;

[0023] Figure 4 This is a schematic diagram illustrating the process of screening PD-1 targeting peptides from a biased library in a typical embodiment of the present invention;

[0024] Figure 5 This is a graph showing the experimental results of surface plasmon resonance (SPR) analysis of peptide kinetic constants in a typical embodiment of the present invention;

[0025] Figure 6 This is a graph showing the experimental results of detecting the specificity of free peptide binding to PD-1 using a fluorescence ELISA method in a typical embodiment of the present invention.

[0026] Figure 7 This is a graph showing the flow cytometry results of the ability of a peptide to competitively bind to PD-1 with PD-L1 in a typical embodiment of the present invention.

[0027] Figure 8A and Figure 8B These are, respectively, experimental results of measuring T cell proliferation using the CCK-8 cell proliferation kit in a typical embodiment of the present invention and experimental results of measuring IFNγ content using a human IFNγ detection kit.

[0028] Figure 9 This is a graph showing the experimental results of detecting the toxicity of PD-1 targeting peptides to Luci-CT26 cells in a typical embodiment of the present invention.

[0029] Figure 10 This is a graph showing the experimental results of determining the half-life of the PD-1 targeting peptide in a typical embodiment of the present invention;

[0030] Figures 11A-11E This is a graph showing the evaluation results of the anti-tumor effect of PD-1 targeting peptides in a typical embodiment of the present invention. Detailed Implementation

[0031] Peptides occupy an optimal position between small molecules and protein biopharmaceuticals, and have been widely used in medicine and biotechnology. Compared to antibodies, peptides can overcome the aforementioned drawbacks. In view of the shortcomings of existing technologies, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention. It mainly provides a peptide PBP that can specifically bind to PD-1 and its application in tumor immunotherapy. The invention attempts to use bacterial surface display technology to screen for peptides that target and bind to PD-1, hoping that these peptides can block the PD-1 / PD-L1 signaling pathway, reverse the exhaustion state of T cells, and exert an anti-tumor effect. After finally obtaining the PBP peptide, its physicochemical properties and physiological activities are tested.

[0032] The following is an explanation of the terminology used in this invention:

[0033] Programmed cell death protein 1 (PD-1), also known as CD279 (differentiation cluster 279), is an important immunosuppressive molecule. It modulates the immune system and promotes self-tolerance by downregulating the immune system's response to human cells and by suppressing T-cell inflammatory activity. This can prevent autoimmune diseases, but it can also prevent the immune system from killing cancer cells.

[0034] Programmed cell death ligand 1 (PD-L1), also known as CD274 (differentiation cluster 274), is a co-inhibitory factor of the immune response. It can bind to PD-1 to reduce the proliferation of PD-1-positive cells, inhibit their cytokine secretion, and induce apoptosis. PD-L1 also plays an important role in various malignant tumors, as it can attenuate the host's immune response to tumor cells.

[0035] Surface plasmon resonance (SPR) is an optical phenomenon that can be used to track interactions between biomolecules in their natural state in real time. This method causes no damage to biomolecules and requires no labeling.

[0036] The following will provide a further explanation of the technical solution, its implementation process, and its principles.

[0037] One aspect of this invention provides a PBP polypeptide that specifically binds to PD-1, having the sequence shown in SEQ ID No. 1, specifically: QRTSADCWEDHGWGS.

[0038] Furthermore, the PBP peptide can specifically bind to both free PD-1 protein and PD-1 expressed on the cell surface.

[0039] Furthermore, the PBP peptide can enhance T cell proliferation and IFNγ secretion.

[0040] Furthermore, the PBP peptide can block the PD-1 / PD-L1 signaling pathway and reverse T cell function.

[0041] Furthermore, the molecular weight of the PBP peptide is 1.7KD-1.8KD. Compared to antibodies, the PBP peptide obtained in this invention has a smaller molecular weight and stronger penetration ability at tumor sites.

[0042] Another aspect of the present invention provides a method for screening PBP peptides that specifically bind to PD-1, comprising:

[0043] Peptides that can specifically bind to PD-1 were screened using a random peptide display library on the bacterial surface, and the conserved sequence "DC / GWE / DD" was obtained through analysis.

[0044] Using conserved sequences as the core, a bacterial surface bias library was constructed to screen and obtain a PBP peptide that specifically binds to PD-1, namely the PD-1 targeted binding peptide PBP (sequence: QRTSADCWEDHGWGS). This peptide can block the interaction between PD-1 and PD-L1, providing a low-molecular-weight candidate drug for tumor immunotherapy.

[0045] In some preferred embodiments, the screening method includes: screening for peptides that specifically bind to PD-1 using magnetic bead screening technology and fluorescence activated cell sorting.

[0046] In some more preferred embodiments, in addition to screening for PD-1-targeting peptides using bacterial surface display technology, phage surface display technology, yeast surface display technology, or biochip (microarray) technology can also be used to screen for PD-1-targeting peptides.

[0047] Another aspect of the present invention provides the application of the aforementioned PBP peptide that specifically binds to PD-1 in the preparation of antitumor drugs.

[0048] Furthermore, the antitumor drug has the function of enhancing T cell proliferation and IFNγ secretion.

[0049] Furthermore, the antitumor drug has the function of blocking the PD-1 / PD-L1 signaling pathway and reversing T cell function.

[0050] Another aspect of the present invention provides an antitumor drug comprising the aforementioned PBP peptide that specifically binds to PD-1.

[0051] The results of ELISA, SPR, and flow cytometry in this invention show that the PBP peptide has excellent binding affinity to both free PD-1 protein and cell surface-expressed PD-1, and exhibits cross-reactivity with mouse PD-1 (mPD-1). ELISA and cell surface competitive binding experiments demonstrate that the PBP peptide can competitively bind to PD-1 with PD-L1 in a dose-dependent manner.

[0052] Molecular docking results showed that the PBP peptide binds to PD-1 at a site close to the PD-L1-PD-1 interaction site. T cell activation experiments demonstrated that the PBP peptide enhances T cell proliferation and IFNγ secretion. Furthermore, in a CT26 mouse xenograft model, immunohistochemical results demonstrated that the PBP peptide exerts its anti-tumor effect by activating T cells and promoting IFNγ secretion. This invention provides a promising low-molecular-weight candidate drug for tumor immunotherapy. In summary, the PBP peptide discovered in this invention can exert its anti-tumor effect by blocking the PD-1 / PD-L1 signaling pathway and reversing T cell function, thus providing a low-molecular-weight candidate drug for tumor immunotherapy.

[0053] Furthermore, in this invention, in addition to surface plasmon resonance (SPR), micro-thermophoresis (MST) can also be used to detect the peptide kinetic constant.

[0054] Furthermore, in this invention, when detecting the activating effect of peptides on T cells, in addition to detecting the T cell proliferation capacity and IFNγ secretion, the evaluation can also be carried out by detecting the killing effect of T cells on tumor cells.

[0055] Furthermore, in this invention, when detecting the inhibitory effect of peptides on mouse tumors, in addition to using the CT26 mouse model, other mouse tumor models, such as the MC38 and B16 mouse tumor models, can also be used for evaluation.

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be further described in detail below with reference to the accompanying drawings and several preferred embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Test methods in the following embodiments that do not specify specific conditions are all performed under conventional conditions. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0057] All reagents and raw materials used in the following examples are commercially available. Test methods without specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. Furthermore, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0058] Example 1

[0059] The specific screening method for PBP peptides that specifically bind to PD-1 in this embodiment is as follows:

[0060] I. Screening for PD-1-targeting peptides from bacterial random peptide libraries

[0061] Peptide screening was performed using a bacterial surface random display library containing the X15 peptide. Magnetic bead screening (MACS) and fluorescence activated cell sorting (FACS) were used sequentially for peptide screening.

[0062] 1. Magnetic cell sorting (MACS) technology

[0063] Take 5 OD (1 OD of bacterial culture contains 10) 9 One clone of bacteria was added to 150 mL of LB medium containing 34 μg / mL chloramphenicol (CM) and a final concentration of 0.2% (m / v) glucose, and incubated overnight at 37°C with shaking at 200 rpm. The next day, 5 OD of bacterial culture was taken from the overnight random peptide library and inoculated into 150 mL of LB medium containing 34 μg / mL CM. The culture was incubated at 37°C with shaking at 200 rpm for about 2 hours. When the OD value approached 0.6, arabinose was added to a final concentration, and the culture was induced at room temperature with shaking at 200 rpm for 1 hour. After induction, 10 times the volume of the bacterial culture was taken, washed with 10 mL of PBS, and the bacteria were resuspended in 0.9 mL of PBS and placed on ice for later use.

[0064] Oscillating and mixing SA-beads ( MyOne TM Streptavidin C1), according to a bacteria:beads ratio of 20:1, take 20 μL (approximately 1 × 10⁻⁶). 9SA-beads (10 mg / mL beads) were mixed with 1 mL of PBS by gentle inversion. The mixture was then magnetically separated for 2 min using a magnetic rack. The supernatant was removed from the pipette tip, and the washing process was repeated twice. The SA-beads were resuspended in 100 μL of PBS. The prepared bacterial culture was mixed with the SA-bead suspension, and the mixture was incubated at 4°C for 45 min to remove bacteria that could bind to the SA-beads, preventing non-specific adsorption during subsequent screening. The SA-beads were then magnetically separated, and the supernatant was collected. The bacteria were collected by centrifugation at 3000g and 4°C for 5 min. The precipitate was resuspended in 0.9 mL of PBS, and biotinylated PD-1 (PD-1) was added to a final concentration of 40 nM. The mixture was incubated at 4°C for 45 min. After incubation, the precipitate was collected by centrifugation for 5 min. The precipitate was resuspended in 1 mL of PBS, and the SA-beads washed as described above were added. Magnetic beads were added at a ratio of 20:1 (bacteria:magnetic beads). The mixture was incubated at 4°C for 45 min. Magnetic beads were magnetically separated, resuspended in 1 mL of PBS, and repeatedly inverted in the EP tube to ensure thorough mixing. The beads were then incubated by rotation for 2 min, and this washing step was repeated three times. The supernatant collected during washing was clearly named W1, W2, and W3. The beads, after three washes, were resuspended in LB medium and named MACS. 10 μL of each of W1, W2, W3, and MACS were added to 990 μL of LB medium, thoroughly mixed, and then 100 μL was spread onto LB plates and incubated overnight at 37°C. The next day, the number of single colonies on each plate was counted. Based on the spread volume and the dilution factor of the bacterial suspension, the library capacity of the bacterial polypeptide library after magnetic bead sorting was calculated. The remaining W1, W2, W3, and MACS were added to LB liquid medium, and glucose was added to a final concentration of 0.2%. The mixture was incubated overnight at 37°C and 200 rpm.

[0065] 2. Fluorescence-activated cell sorting (FACS)

[0066] Take 100 μL of overnight culture from MACS sorting and inoculate it into 5 mL of LB medium containing 34 μg / mL CM resistance. Incubate at 37°C with shaking at 200 rpm for 2–2.5 h. When the OD600 of the culture is between 0.5 and 0.6, place the tube in a 4°C freezer for 10 min. Then add arabinose to a final concentration of 0.02% (m / v) for induction and incubate at room temperature with shaking at 200 rpm for 1.2 h. After induction, measure the OD600 of each tube. The experiment includes a control group and an experimental group. For each group, add exactly 0.05 OD of culture, add 1 mL of pre-chilled PBS, and centrifuge at 3000g for 5 min. Wash once with PBS. Finally, concentrate the bacteria in approximately 100 μL of the system. For the first flow cytometry sorting, add PD-1 to a final concentration of 40 nM. Incubate at 4°C with rotation for 45 min. After incubation, add 1 mL of PBS solution, centrifuge at 3000 g for 5 min, and repeat the washing step once. Finally, add SAPE solution to a final concentration of 4 nM. Incubate at 4 °C with rotation for 30 min, wash twice with PBS, resuspend the bacterial culture in 0.5 mL of PBS, and store on ice for flow cytometry analysis.

[0067] Each round of sorting requires simultaneous comparison of fluorescence intensity and binding rate between the previous and next generations. As the number of generations increases, the binding efficiency of bacteria to PD-1 becomes increasingly higher. When the binding rate reaches over 40%, the increase in fluorescence intensity between generations becomes insignificant. At this point, the PD-1 concentration needs to be reduced by half in a gradient manner. To increase the specificity of the selected peptides, 1% human serum is added during incubation. When the bacterial library, under increased screening pressure and after multiple rounds of screening, no longer shows an increase in the binding rate of the bacterial library to PD-1 (as described in the screening process...), the process is complete. Figure 1 As shown in the image, the bacterial library enriched from the last generation was plated on LB agar plates containing 34 μg / mL CM and incubated overnight at 37°C. The plates were observed the following day, and single colonies were randomly picked and inoculated into 5 mL of LB agar containing CM. The culture was incubated at 37°C with shaking at 200 rpm for approximately 10 hours. Approximately 500 μL of the bacterial culture was then sent to Suzhou Genewiz Biotechnology Co., Ltd. for DNA sequencing. The sequencing plasmid was pBAD33, the antibiotic was chloramphenicol (CM), and the universal primer pBAD-Forward was used, requiring a sequencing fragment length of 1000 bp. Five polypeptide sequences were translated from the DNA sequence.

[0068] II. Analysis of Conserved Peptide Sequences

[0069] Five PD-1 targeting peptide amino acid sequences screened from a random peptide library were stored sequentially in FASTA format and imported into Weblogo 3.7.4 software for conserved sequence analysis. The results are as follows: Figure 2As shown.

[0070] III. Construction of PD-1 Targeting Peptide Bias Library

[0071] A biased library was constructed by inserting random amino acids (X5DC / GWE / DDX5) from both ends of a conserved sequence into the pBAD33-eCPX plasmid. The construction principle is as follows: Figure 3 As shown.

[0072] The construction process of the biased library mainly includes: preparation of the insert DNA, preparation of the pBAD33-eCPX vector, ligation and desalting of the insert DNA and vector DNA, electroporation of the ligation product, and determination of the library volume and quality. After the biased library was constructed, an appropriate amount of bacterial culture was diluted and plated on CM-resistant LB plates. The next day, 10 single clones were picked for sequencing to determine the quality of the biased library. The specific results are shown in the sequence of SEQ ID No. 2 below:

[0073]

[0074] IV. Screening PD-1 targeting peptides from biased libraries

[0075] Based on the size of the biased library, FACS can be directly used for screening in the experiment. After thawing the frozen biased library from a -80℃ freezer, 20 times the library volume of bacterial culture was inoculated into 5 mL of LB medium containing 0.2% glucose CM resistance, and incubated overnight at 37℃ and 200 rpm for activation. The next day, 100 μL of the overnight culture was inoculated into 5 mL of LB medium containing 0.2% glucose CM resistance, and the subsequent culturing and arabinose induction methods were as described above. 0.05 OD of bacterial culture was used for flow cytometry sample preparation, and the subsequent washing and incubation methods were as described above. In the early screening stage, the biotinylated PD-1 concentration used was 40 nM. As the bacterial fluorescence intensity increased, the biotinylated PD-1 concentration was reduced to 20 nM to increase the screening pressure. The screening process was as follows. Figure 4 As shown in Table 1, 100 events were isolated from the last generation of bacterial strains and plated on CM-resistant LB plates, then incubated overnight at 37°C. The next day, 20 single clones were selected for sequencing. The polypeptide sequences were obtained, and the most frequently occurring polypeptide was named PBP.

[0076]

[0077] V. Surface Plasmon Resonance (SPR) Analysis of Peptide Kinetic Constants

[0078] The experiment was conducted using a CM5 sensor chip at 25°C. The specific procedure was as follows: First, ligand coupling was performed. PD-1 was diluted to 20 μg / mL using coupling buffer (10 mM acetate, pH 4.5) and injected at a rate of 10 μL / min for 420 s. Then, ethanolamine (1 M) was injected at a rate of 10 μL / min for 7 consecutive mins to block the remaining active sites in the chip. Next, the PD-1 targeting peptide was dissolved in PBS, and the peptide concentration was adjusted to 125, 250, 500, and 750 μg / mL using running buffer (PBS-P+, pH 7.4, 0.05% P20). The peptides were injected in ascending order of concentration at a rate of 30 μL / min for 120 s. After the highest concentration group was loaded, dissociation and regeneration were performed. Simultaneously, the channel coupled only to PD-1 served as a blank control group. The response value of the control group was subtracted to correct the SPR signal. The experimental results are as follows: Figure 5 As shown.

[0079] VI. Detection of the specificity of free peptide binding to PD-1 by fluorescence ELISA

[0080] Add 100 μL (1 μg / mL) of human PD-1 (hPD-1) or mouse PD-1 (mPD-1) in PBS (pH 7.4) to the corresponding wells of a 96-well high-absorption plate and incubate overnight at 4°C. The next day, discard the liquid in the plate and pat off any remaining liquid on a non-woven fabric. Wash each well twice with 200 μL of PBST for 1 min each time. Block the plate with PBST solution containing 2% BSA at room temperature for 1 h with shaking, then wash the plate with PBST solution for 1 min each time, for a total of 3 times. Then add 100 μL (15 μM) of FITC-labeled peptide to the corresponding wells, with 3 replicates per group, and incubate at 100 rpm in the dark for 1.5 h at room temperature. Wash the plate twice with PBST for 1 min each time. After the incubation, add 100 μL of PBS to each well, and measure the FITC fluorescence intensity in each well using Cytation3. The experimental results are shown below. Figure 6 As shown.

[0081] VII. Flow cytometry detection of the ability of peptides to competitively bind to PD-1 with PD-L1

[0082] After digestion of CHO-K1 / PD-1 cells, 1×10⁻⁶ cells were taken. 5After centrifuging at 1000 rpm for 5 min and discarding the supernatant, the cells were washed with PBS. Then, 100 μL of PBS solution containing 40, 10, or 0 μM PD-1 targeting peptide and 100 nM biotinylated PD-L1 was added to the CHO-K1 / PD-1 cells, and the cells were resuspended and incubated at 4°C for 1 h. After washing with PBS, 100 μL of SAPE PBS solution with a final concentration of 5 nM was added to resuspend the cells, and the cells were incubated at 4°C for 30 min. After washing with PBS, the cells were centrifuged and resuspended, and the positivity rate was analyzed by flow cytometry. The results are shown below. Figure 7 As shown.

[0083] VIII. T-cell function detection experiment

[0084] 1. Isolation and expansion of peripheral blood mononuclear cells (PBMCs)

[0085] Peripheral blood cells (PBMCs) were isolated from human blood and induced to expand. 20 mL of peripheral blood from healthy volunteers was slowly added to an equal volume of Ficoll lymphocyte separation medium and centrifuged at room temperature, 500 g, for 30 min using a density gradient. The cells in the second white membrane layer (containing PBMCs) were carefully aspirated using a pipette and transferred to a new 50 mL centrifuge tube. PBS solution was added, and the cells were washed twice to obtain PBMCs. The PBMC density was adjusted to 1 × 10⁻⁶ cells using PBS. 7 Take 100 μL of the medium and seed it into a 6-well plate containing 2 mL of RPMI 1640 medium (with 10% FBS). Simultaneously, add 10 μg / mL anti-human CD3 antibody, 10 μg / mL anti-human CD28 antibody, and 20 ng / mL IL2 to the 6-well plate to continuously stimulate PBMCs for 5 days. During this period, add or replace the medium with fresh medium as needed. After 5 days, replace the medium with RPMI 1640 medium without CD3 antibody, CD28 antibody, and IL2, and allow the cells to rest for one day.

[0086] 2. T cell activation experiment

[0087] The experiment was divided into 6 groups: negative control group (cells and culture medium), CD3 antibody activation group, PD-L1 inhibition of CD3 antibody activation group, positive control group (Keytruda antibody blocks the PD-1 / PD-L1 pathway), PBP peptide group, and CP (control peptide) group. Except for the negative control group and CD3 antibody activation group, which did not receive PD-L1 to block the activation of T cells by CD3 antibodies, all other groups received PD-L1. 100 μL of anti-human CD3 antibody (10 μg / mL) was added to a clear 96-well plate and incubated overnight at 4°C. After discarding the antibody solution and washing with PBS, 100 μL of PD-L1 (40 μg / mL) diluted in PBS was added to the corresponding wells and incubated at 37°C for 4 h. Discard the liquid, wash the plate twice with PBS, and then add 150 μL of resting cells (1 × 10⁻⁶) containing or without PD-1 targeting peptide (50 μg / mL), control peptide (50 μg / mL), or Keytruda (25 μg / mL). 5 (Each well) was seeded and incubated at 37°C for 72 hours. Three replicates were performed per group. Finally, the proliferation of T cells was measured using a CCK-8 cell proliferation assay kit. The experimental results are shown below. Figure 8A As shown; cell culture supernatant was collected, and the IFNγ content was determined using a human IFNγ detection kit. The experimental results are as follows. Figure 8B As shown.

[0088] IX. Detection of PD-1 targeting peptide toxicity to Luci-CT26 cells

[0089] After digestion, the Luci-CT26 cells were adjusted to a concentration of 5 × 10⁻⁶. 4 / mL, 100μL was seeded into 96-well clear cell culture plates. After overnight culture, the culture medium was discarded, and DMEM complete medium containing PD-1 targeting peptides at final concentrations of 5μM, 10μM, and 20μM was added to each group, respectively. A negative control group (cells only) was also set up. After incubation for 24h, 48h, and 72h, the cell-killing effect of the peptides was detected using a CCK-8 assay kit. The experimental results are as follows: Figure 9 As shown.

[0090] 10. Determination of the half-life of PD-1 targeting peptides

[0091] 100 μL of FITC-labeled PD-1 targeting peptide (4 mg / kg) was injected into mice via the tail vein. Blood samples (20 μL) were collected from the tail at different time points (0, 0.5, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120 min) into 1.5 mL centrifuge tubes containing heparin. All samples were then centrifuged at 1000 g for 5 min to obtain plasma. Before detection, the plasma was diluted to 100 μL with PBS and added to a black 96-well plate. Fluorescence intensity was measured using Cytation3. Data were fitted using a two-compartment model with PK Solver software. Experimental results are shown below. Figure 10 As shown.

[0092] XI. Evaluation of the antitumor effects of PD-1 targeted peptides

[0093] A mouse subcutaneous tumor model was constructed using Luci-CT26 cells in the experiment. 3×10 5 One Luci-CT26 cell was injected subcutaneously into the right thigh of a mouse. When the average tumor size reached 50-100 mm... 3 Mice were randomly divided into four groups (n=4 per group) and administered the drugs as follows: Blank control group: (100 μL PBS, intratumoral injection, daily); Positive control group: (10 mg / kg anti-mouse PD-1 antibody, intraperitoneal injection, every other day, for a total of 5 times); Experimental group: (4 mg / kg PD-1 targeting peptide, intratumoral injection, daily for two weeks); Negative control group: (4 mg / kg control peptide, intratumoral injection, daily for two weeks). Tumor size was measured every other day using calipers and calculated according to the formula (minor diameter). 2 The tumor size was calculated as (long diameter × 0.5). When the mouse tumor volume reached 2000 mm², the tumor size was... 3Mice were considered dead at the designated time and were euthanized. Tumor tissue was then fixed in 4% fixative. Furthermore, tumor evaluation was performed using the IVIS Lumina II system starting on day 8 post-inoculation, once a week for a total of four weeks. Mice were anesthetized with an intraperitoneal injection of 4% chloral hydrate, followed by an intraperitoneal injection of D-fluorescein potassium solution (15 mg / mL) at a dose of 10 μL / g. Approximately 8 minutes later, the anesthetized mice were placed face down on a black cardboard plate, their limbs secured with tape, and then placed on the sample stage for photographing in Luminenscence mode. All remaining mice were sacrificed on day 30, and tumors were collected and fixed. Blood was collected, centrifuged to obtain serum, and stored at -80°C. Simultaneously, other internal organs (heart, liver, spleen, lung, and kidney) were also fixed in fixative for later use. To investigate the antitumor mechanism of PD-1 targeting peptides, immunohistochemistry (IHC) was used to detect the expression of IFNγ in tumor tissues from each group. Finally, after taking digital photographs using an upright microscope, the positive rate was analyzed using ImageJ software. The experimental results are as follows: Figures 11A-11E As shown.

[0094] In summary, this invention utilizes bacterial surface display technology to screen and obtain the PD-1-targeting peptide PBP. This peptide specifically binds to PD-1, exhibits cross-reactivity with both human and mouse PD-1, and competitively binds to PD-1 with PD-L1. It effectively blocks the PD-1 / PD-L1 interaction, reverses the function of exhausted T cells, and exerts an anti-tumor effect. Compared to antibodies, the PBP peptide obtained in this invention has a smaller molecular weight, stronger penetration at tumor sites, lower production costs, and a simpler preparation process. It does not cause severe immune-related side effects. While achieving corresponding therapeutic effects, it avoids the drawbacks of antibody drugs, providing a low-molecular-weight candidate drug for tumor immunotherapy.

[0095] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0096] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A PBP polypeptide that specifically binds to PD-1, characterized in that, The amino acid sequence of the PBP polypeptide is shown in SEQ ID No.

1.

2. The PBP polypeptide according to claim 1, characterized in that: The PBP peptide can specifically bind to both free PD-1 protein and PD-1 expressed on the cell surface.

3. The PBP polypeptide according to claim 1, characterized in that: The PBP polypeptide can enhance T cell proliferation and IFNγ secretion.

4. The PBP polypeptide according to claim 1, characterized in that: The PBP peptide can block the PD-1 / PD-L1 signaling pathway.

5. The use of the PBP peptide that specifically binds to PD-1 according to any one of claims 1-4 in the preparation of an anti-colon cancer drug.

6. The application according to claim 5, characterized in that: The anti-colon cancer drug has the function of enhancing T cell proliferation and IFNγ secretion.

7. The application according to claim 5, characterized in that: The anti-colon cancer drug has the function of blocking the PD-1 / PD-L1 signaling pathway.

8. An anti-colon cancer drug, characterized in that... It comprises the PBP polypeptide that specifically binds to PD-1 as described in any one of claims 1-4.

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