SR-B1 targeted polypeptide compound and application thereof in preparation of antitumor drugs and immunotherapy sensitizer

Abstract

The invention relates to an SR-B1 targeted polypeptide compound and application thereof in preparation of antitumor drugs and immunotherapy sensitizers, and belongs to the technical field of biological drug manufacturing. In order to solve the problems that an existing PD-L1 inhibitor is single in action mechanism and immune system dysfunction is easily caused, the invention provides an SR-B1 targeted polypeptide compound which is composed of SR-B1 targeted polypeptide and Resiquimod. The SR-B1 targeting polypeptide comprises an SR-B1 targeting peptide fragment, a self-assembly peptide fragment and a hydrophobic molecule which are connected in sequence. The SR-B1 targeting polypeptide can recognize overexpressed SR-B1 in tumor cells, block cholesterol uptake, inhibit tumor progression and inhibit expression of PD-L1 at the same time. The polypeptide compound accurately delivers Resiquimod to a tumor site, anti-tumor immune response is activated, and sensitivity of tumor immunotherapy is greatly enhanced through combination of cholesterol metabolism regulation and immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical manufacturing, and in particular relates to an SR-B1 targeting polypeptide complex and its application in the preparation of anti-tumor drugs and immunotherapy sensitizers. Background Art

[0002] Tumor cells often increase their extracellular cholesterol uptake by upregulating the expression of cholesterol-uptake-related proteins, such as scavenger receptor type 1 (SR-B1). This additional cholesterol supports rapid tumor cell proliferation, correlates with tumor malignancy and progression, and plays a multifaceted, critical role in the formation and maintenance of the tumor's immunosuppressive microenvironment.

[0003] Cholesterol metabolism does not exist in isolation within cells; rather, it is closely interconnected and regulated by a complex network of intracellular signaling pathways, some of which can directly or indirectly regulate the expression of programmed death receptor ligand 1 (PD-L1). PD-L1, a key protein located on the surface of tumor cells, plays a central role in tumor immune escape. Tumor cells express PD-L1 on their surfaces, which specifically binds to programmed death receptor 1 (PD-1) on immune cells, thereby activating a series of intracellular signaling events and transmitting immunosuppressive signals, significantly suppressing immune cell activity and enabling tumor cells to successfully evade immune surveillance and attack. Modulating cholesterol metabolism and its interactions with signaling pathways can influence PD-L1 expression. This regulatory effect is expected to enhance the immune system's ability to kill tumor cells and enhance immune surveillance, providing new strategies and approaches for cancer treatment.

[0004] Existing PD-L1 inhibitors, such as α-PD-L1, have a single mechanism of action and an average efficacy rate of only 10-20%. Most patients may not benefit significantly from treatment. Due to their lack of tumor tissue targeting, PD-L1 inhibitors cannot specifically accumulate at the tumor site, potentially disrupting the immune system and triggering a range of adverse reactions, such as immune pneumonia, immune hepatitis, immune enteritis, and endocrine disorders. In severe cases, these can affect patients' quality of life and even be life-threatening. Summary of the Invention

[0005] To address the problem that existing PD-L1 inhibitors have a single mechanism of action and are prone to causing dysfunction of the body's immune system, the present invention provides an SR-B1 targeting polypeptide complex and its use in the preparation of anti-tumor drugs and immunotherapy sensitizers. The SR-B1 targeting polypeptide complex can specifically target tumors with high SR-B1 expression, inhibit SR-B1-mediated cholesterol uptake, reduce the cholesterol content in tumor cells, and thus inhibit PD-L1 expression, which has the effects of inhibiting tumor progression and sensitizing tumor immunotherapy.

[0006] The technical solution of the present invention:

[0007] A SR-B1 targeting polypeptide complex, characterized in that it is composed of an SR-B1 targeting polypeptide and a Toll-like receptor 7 / 8 agonist Resiquimod; the SR-B1 targeting polypeptide comprises an SR-B1 targeting peptide segment, a self-assembling peptide segment, and a hydrophobic molecule connected in sequence; the amino acid sequence of the SR-B1 targeting peptide segment is shown in SEQ ID No. 1, and the amino acid sequence of the self-assembling peptide segment is shown in SEQ ID No. 2; and the hydrophobic molecule is a hydrophobic hexaalkyl compound.

[0008] Furthermore, the mass ratio of the SR-B1 targeting polypeptide to Resiquimod is 1:1.

[0009] Furthermore, the molecular structure of the SR-B1 targeting polypeptide is shown in Formula I:

[0010]

[0011] Formula I.

[0012] Furthermore, the SR-B1 targeting peptide solution was mixed with the Resiquimod solution. The hydrophobic ends of the SR-B1 targeting peptide aggregated together through hydrophobic interaction to form nanospherical micelles, and Resiquimod was wrapped in its hydrophobic core, resulting in an SR-B1 targeting peptide complex with a particle size of 35.52 nm ± 5.56 nm.

[0013] Application of an SR-B1 targeting polypeptide complex in the preparation of anti-tumor drugs.

[0014] Furthermore, the tumor is a tumor with specific high expression of SR-B1.

[0015] Furthermore, the tumor with specific high expression of SR-B1 is renal clear cell carcinoma, renal papillary cell carcinoma, melanoma, colorectal cancer or esophageal squamous cell carcinoma.

[0016] Furthermore, the anti-tumor drug is a parenteral dosage form, specifically an injectable dosage form.

[0017] Application of an SR-B1 targeting polypeptide complex in the preparation of an immunotherapy sensitizer.

[0018] Furthermore, the immunotherapy sensitizer is a sensitizer used for anti-tumor immunotherapy of tumors with high specific expression of SR-B1.

[0019] Furthermore, the tumor with specific high expression of SR-B1 is renal clear cell carcinoma, renal papillary cell carcinoma, melanoma, colorectal cancer or esophageal squamous cell carcinoma.

[0020] Furthermore, the immunotherapy sensitizer is a parenteral dosage form, specifically an injectable dosage form.

[0021] Beneficial effects of the present invention:

[0022] The SR-B1 targeting peptide complex provided by the present invention encapsulates Resiquimod within its hydrophobic core by forming nanospherical micelles formed by aggregation of the hydrophobic portions of the SR-B1 targeting peptides. The SR-B1 targeting peptide can specifically and accurately recognize overexpressed SR-B1 in tumor cells. By effectively binding to SR-B1, it directly blocks the key pathway for cholesterol uptake by tumor cells, effectively reducing cholesterol levels within tumor cells. This can inhibit tumor progression and PD-L1 expression, thereby disrupting the immune suppression of tumor cells against the immune system.

[0023] The SR-B1-targeting peptide complex provided by the present invention, under the action of the SR-B1 targeting peptide, enables precise delivery of Resiquimod to tumor sites, causing its specific accumulation at the tumor site and activating the body's anti-tumor immune response. By combining cholesterol metabolism regulation with immunotherapy, the sensitivity of tumor immunotherapy is greatly enhanced, opening up a highly promising combined treatment approach for tumor immunotherapy, which is expected to significantly improve the therapeutic efficacy and prognosis of cancer patients.

[0024] The SR-B1 targeting polypeptide complex provided by the present invention undergoes conformational changes within the tumor microenvironment and self-assembles into nanofibers. Due to its unique nanofiber retention mechanism, the SR-B1 targeting polypeptide's retention time within tumor tissue is significantly prolonged, effectively preventing the problem of rapid polypeptide clearance. This allows the SR-B1 targeting polypeptide to continuously inhibit tumor cells' cholesterol uptake, thereby producing a long-term and effective inhibitory effect on tumor cell proliferation, migration, and invasion.

[0025] The SR-B1-targeting polypeptide complex provided by the present invention exhibits high tissue specificity, enhancing localized drug concentration in tumors while reducing toxicity to normal tissues. Experiments in mouse models have confirmed that the SR-B1-targeting polypeptide exhibits no significant toxicity or adverse effects on major mouse organs, demonstrating its high biosafety and promising broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the molecular structure of the SR-B1 targeting polypeptide NP and the control polypeptide NP-C in Example 1;

[0027] Figure 2 1 is the ESI-MS mass spectra of the SR-B1 targeting polypeptide NP and the control polypeptide NP-C in Example 1;

[0028] Figure 3 1 is a high performance liquid chromatogram of the SR-B1 targeting polypeptide NP and the control polypeptide NP-C in Example 1;

[0029] Figure 4 Transmission electron microscopic images of the SR-B1 targeting polypeptide NP and the control polypeptide NP-C before and after co-incubation with the SR-B1 solution in Example 2, scale bar: 200 nm;

[0030] Figure 5 CD spectra of the SR-B1 targeting polypeptide NP and the control polypeptide NP-C before and after incubation with the SR-B1 solution in Example 2;

[0031] Figure 6 CLSM fluorescence images of SR-B1 targeting polypeptide NP and control polypeptide NP-C after co-incubation with 786-O cells in Example 3, scale: 20 μm;

[0032] Figure 7 This is a comparison of the fluorescence intensity of the SR-B1 targeting polypeptide NP and the control polypeptide NP-C after co-incubation with 786-O cells in Example 3, and the fluorescence intensity of the positive control SR-B1;

[0033] Figure 8 CLSM fluorescence images of 786-O cells treated with SR-B1 targeting peptide NP and control peptide NP-C in Example 4 for 24 hours, 48 ​​hours, and 72 hours, respectively. Scale bar: 20 μm.

[0034] Figure 9 Fluorescence images of the mouse model injected with NP and NP-C at different administration times in Example 5;

[0035] Figure 10This is a comparison of fluorescence signals at different administration times in the mouse model injected with NP and NP-C in Example 5;

[0036] Figure 11 Fluorescence images of 786-O cells in different treatment groups in Example 6;

[0037] Figure 12 This is a comparison of the fluorescence intensity of NBD cholesterol in 786-O cells in different treatment groups in Example 6;

[0038] Figure 13 This is a comparison of cholesterol levels in 786-O cells in different treatment groups in Example 6;

[0039] Figure 14 This is a comparison of PD-L1 levels in 786-O cells in different treatment groups in Example 7;

[0040] Figure 15 This is a comparison chart of the particle size changes of the SR-B1 targeting polypeptide complex NPR and the control polypeptide complex NPR-C before and after co-incubation with the SR-B1 solution in Example 8;

[0041] Figure 16 This is an in vitro accumulation and release curve of Resiquimod after the SR-B1 targeting polypeptide complex NPR and the control polypeptide complex NPR-C were co-incubated with the SR-B1 solution in Example 9;

[0042] Figure 17 This is a comparison of tumor volumes of mice in different treatment groups in Example 10;

[0043] Figure 18 The figures are actual photos of tumors and comparison of tumor weights of mice in different treatment groups in Example 10;

[0044] Figure 19 This is a comparison of the number of immune cells in the tumor lymph nodes and tumor tissues of each group of mice in Example 11;

[0045] Figure 20 This is a comparison of the body weights of mice in each group in Example 12;

[0046] Figure 21 1 is a comparison chart of the blood biochemical test results of the peripheral blood of each group of mice in Example 12. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0048] Example 1

[0049] This embodiment provides an SR-B1 targeting polypeptide complex NPR and a preparation method thereof.

[0050] In this embodiment, the SR-B1 targeting peptide complex NPR is composed of SR-B1 targeting peptide NP and Toll-like receptor 7 / 8 agonist Resiquimod. The SR-B1 targeting peptide NP is composed of SR-B1 targeting peptide, self-assembling peptide and hydrophobic molecules connected in sequence. The molecular structure diagram is shown in FIG. Figure 1 The amino acid sequence of the SR-B1 targeting peptide is shown in SEQ ID No. 1, specifically FAEKFKEAVKDYFAKFWD; the amino acid sequence of the self-assembling peptide is shown in SEQ ID No. 2, specifically KLVFF; the hydrophobic molecule is a hydrophobic hexaalkyl compound (C6), specifically a carboxylic acid, carboxylate, or ester compound carrying a hexaalkyl chain. The hydrophobic hexaalkyl compound used in this example is hexanoic acid. The Toll-like receptor 7 / 8 agonist Resiquimod in this example was purchased from Med Chem Express (Shanghai, China) under the product model HY-13740.

[0051] SR-B1 targeting peptide NP uses SR-B1 targeting peptide as the target head, which specifically binds to SR-B1 protein overexpressed in tumor cells to achieve SR-B1 targeted recognition; the self-assembling peptide is derived from β-amyloid protein, and due to the interaction of hydrogen bonds, it can self-assemble into water-insoluble nanofibers with β-sheet secondary structure, thereby achieving long-term retention of SR-B1 targeting peptide in tumor tissue; the hydrophobic hexaalkyl compound can maintain the hydrophilic and hydrophobic balance of SR-B1 targeting peptide.

[0052] The SR-B1 targeting polypeptide NP provided in this embodiment is synthesized by conventional solid-phase peptide synthesis method in the art, from C-terminus to

[0053] The N-terminus is connected to the hydrophobic hexaalkyl compound C6 and the required amino acids in the self-assembling peptide and SR-B1 targeting peptide sequence in sequence through an amide bond to obtain an artificially synthesized SR-B1 targeting polypeptide NP, the molecular structure of which is shown in Formula I:

[0054]

[0055] Formula I.

[0056] The preparation method of the SR-B1 targeting peptide complex NPR in this example is as follows: an NP solution with an NP concentration of 20 μmol / L is mixed with a Resiquimod solution, and the mass ratio of NP to Resiquimod is 1:1. The hydrophobic portion of the SR-B1 targeting peptide NP aggregates together through hydrophobic interactions to form nanospherical micelles, while simultaneously encapsulating Resiquimod in its hydrophobic core to obtain an SR-B1 targeting peptide complex NPR with a particle size of 35.52 nm ± 5.56 nm. After injection, the nanomicrosphere micelles are easily absorbed through the membrane, increasing the chance of binding between the peptide and the target, and enabling more efficient targeting.

[0057] This example also provides a non-conformable self-assembly control polypeptide NP-C, which is composed of a SR-B1 targeting peptide segment, a non-conformable peptide segment, and a hydrophobic molecule connected in sequence. The molecular structure diagram is shown in FIG. Figure 1 As shown; the amino acid sequence of the SR-B1 targeting peptide is shown as SEQ ID No. 1, specifically FAEKFKEAVKDYFAKFWD; the amino acid sequence of the invariant peptide is shown as SEQ ID No. 3, specifically KAAGG, and the hydrophobic molecule is a hydrophobic hexaalkyl compound (C6), specifically a carboxylic acid, carboxylate or ester compound containing a hexaalkyl chain.

[0058] The control polypeptide NP-C is synthesized by conventional solid-phase peptide synthesis methods in the art. The hydrophobic hexaalkyl compound C6 and the required amino acids in the constant conformation peptide segment and the SR-B1 targeting peptide segment are sequentially linked from the C-terminus to the N-terminus through amide bonds to obtain an artificially synthesized control polypeptide NP-C. Its molecular structure is shown in Formula II:

[0059]

[0060] Formula II.

[0061] In this example, the control peptide complex NPR-C was prepared by mixing a 20 μmol / L NP-C solution with a Resiquimod solution, with a mass ratio of NP-C to Resiquimod of 1:1. The hydrophobic portions of the control peptide NP-C aggregated together through hydrophobic interactions to form unique nanospherical micelles, encapsulating the Resiquimod within their hydrophobic cores. This yielded the control peptide complex NPR-C, which had a particle size similar to that of the complex NPR.

[0062] Electrospray ionization mass spectrometry (ESI-MS) analysis of SR-B1 targeting peptide NP and control peptide NP-C was performed. Figure 2 As shown, the molecular weight of the SR-B1 targeting polypeptide NP is 3390.01, and the molecular weight of the control polypeptide NP-C is 3011.45; the detected molecular weight corresponds to the theoretical molecular weight, proving that the mass spectrometry detection results of the samples are correct.

[0063] SR-B1 targeting peptide NP (20×10 -6 M) and control peptide NP-C (20×10 -6 M) was subjected to high performance liquid chromatography analysis, and the results were as follows Figure 3 As shown, there is only one main peak in the HPLC graph, the purity of SR-B1 targeting peptide NP is >95%, and the purity of the control peptide NP-C is >95%.

[0064] Example 2

[0065] This example demonstrates that the SR-B1 targeting polypeptide has the ability to undergo conformational change and self-assemble into water-insoluble nanofibers after co-incubation with SR-B1.

[0066] The SR-B1 targeting polypeptide NP and the control polypeptide NP-C obtained in Example 1 were incubated with the SR-B1 solution for 1 hour, and the NP and NP-C solution samples were observed using a transmission electron microscope before and after incubation. The results are as follows: Figure 4 As shown, after co-incubation with SR-B1 solution, NPs underwent allosteric behavior and formed hydrophobic nanofibers through self-assembly.

[0067] Circular dichroism (CD) analysis of NP and NP-C solution samples was performed before and after incubation. Figure 5 As shown, after NP was co-incubated with SR-B1 solution, the polypeptide formed a β-sheet nanofiber structure.

[0068] Example 3

[0069] This example demonstrates that the SR-B1 targeting polypeptide has a targeting effect on tumor cells with high expression of SR-B1.

[0070] The SR-B1-targeting peptide NP and the control peptide NP-C obtained in Example 1 were each labeled with cyanine 7 dye (Cy7). Renal clear cell carcinoma cells 786-O were incubated with Cy7-labeled NP and NP-C for 1 hour. Immunofluorescence detection of SR-B1 was then performed as a positive control, and CLSM fluorescence images were acquired to assess the colocalization of NP and NP-C with SR-B1.

[0071] like Figure 6 As shown in the CLSM fluorescence images, the positions of Cy7-labeled NP and NP-C are consistent with the positions of SR-B1 in tumor cells, indicating that both NP and NP-C can accurately target SR-B1 in tumor cells. Figure 7 The fluorescence intensity comparison shows that the fluorescence intensity of NP and NP-C is comparable to that of SR-B1, indicating that both NP and NP-C have good targeting properties and high binding efficiency with SR-B1, reducing the risk of off-target.

[0072] Based on this, this example confirms that the SR-B1 targeting peptide in NP and NP-C, as a target head, can specifically bind to the SR-B1 protein overexpressed in tumor cells, thereby achieving targeted recognition of SR-B1.

[0073] Example 4

[0074] This example demonstrates that the SR-B1 targeting polypeptide has the ability to form nanofiber structures on the cell membranes of tumor cells and retain them for a long time.

[0075] The SR-B1 targeting peptide NP and the control peptide NP-C obtained in Example 1 were labeled with cyanine 7 dye (Cy7). 786-O cells were seeded in a culture dish and incubated with Cy7-labeled NP and NP-C for 1 hour, respectively. The cells were then washed three times with phosphate buffered saline (PBS) and replaced with fresh culture medium for continued culture. Confocal laser scanning microscopy (CLSM) was used to image the cells at 24 hours, 48 ​​hours, and 72 hours to evaluate the intracellular retention of NP and NP-C. The results are shown in Figure 2. Figure 8 As shown in the results, NP can be retained in 786-O cells for a long time, and the retention effect is significantly better than that of NP-C.

[0076] This shows that SR-B1 targeting polypeptide NP, driven by self-assembling peptide segments, can form nanofiber structures on the cell membrane of tumor cells, thereby prolonging the retention time.

[0077] Example 5

[0078] This example demonstrates through animal model experiments that the SR-B1 targeting polypeptide has the ability to target ccRCC tumors in vivo and maintain long-term retention.

[0079] In this example, Balb / c nude mice were used as experimental animals and renal cancer cell 786-O cells (5×10 6 cells) were inoculated into the right hip of the mouse. When the tumor volume reached 50 mm 3 When the 400×10⁻ 6 M) These mice were administered Cy7-labeled SR-B1 targeting peptide NP obtained in Example 1 and control peptide NP-C. Fluorescence imaging was performed using a small animal in vivo imaging system 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours after injection. The results are shown in Figure 2. Figure 9 and Figure 10 As shown in the figure, NP is mainly distributed in the tumor site, indicating that it has good SR-B1 targeting in the body, and NP can remain in the tumor site for a long time in the body, while although NP-C can target the tumor site, it cannot remain in the tumor site for a long time.

[0080] Example 6

[0081] This example demonstrates that the SR-B1 targeting polypeptide has the effect of inhibiting the uptake of cholesterol by tumor cells and reducing the intracellular cholesterol content.

[0082] In this example, NBD cholesterol (a fluorescent cholesterol derivative) was used to evaluate the cholesterol uptake ability of 786-O cells. 786-O cells were seeded in a culture dish and incubated with the SR-B1 targeting peptide NP obtained in Example 1 (20×10 -6 M) and control peptide NP-C (20×10 -6 M) were co-incubated for 1 hour, with the SR-B1 inhibitor BLT-1 as a positive control. Subsequently, the culture medium was replaced with fresh culture medium containing NBD cholesterol, and the cells were cultured for another 72 hours. After washing the cells with phosphate-buffered saline (PBS), the intracellular NBD fluorescence signal and fluorescence intensity were detected using a fluorescence microscope and a fluorescence microplate reader, respectively. The results are shown in Figure 2. Figure 11 As shown, in 786-O cells treated with NPs, only a weak green NBD fluorescence signal was observed, proving that NPs (20 × 10 -6 M) can significantly inhibit the uptake of cholesterol by cells. Figure 12 As shown, the cholesterol uptake in 786-O cells after NP treatment was the least and less than that in BLT-1, indicating that NP's ability to inhibit cholesterol uptake in tumor cells is better than that of the existing SR-B1 inhibitor BLT-1.

[0083] 786-O cells were incubated with the SR-B1 targeting polypeptide NPs (20×10 -6 M) and control peptide NP-C (20×10 -6M) were co-incubated for 1 hour. The SR-B1 inhibitor BLT-1 was used as a positive control. The cells were washed with phosphate-buffered saline (PBS) and the medium was replaced with fresh medium. The cells were then cultured for an additional 72 hours, and intracellular cholesterol was extracted to determine the effects of NP-C or NP on cholesterol metabolism in 786-O cells. The results are shown in Figure 2. Figure 13 As shown, NP (20×10 -6 The cholesterol content in 786-O cells treated with NP-C was significantly decreased, and the effect was better than that of NP-C and BLT-1.

[0084] Example 7

[0085] This example demonstrates that the SR-B1 targeting polypeptide has the effect of reducing the PD-L1 level in tumor cells.

[0086] 786-O cells were seeded in a culture dish, and the SR-B1 targeting polypeptide NP obtained in Example 1 (20×10 -6 M) and control peptide NP-C (20×10 -6 M) After 72 hours of co-incubation, the PD-L1 level in 786-O cells was detected by flow cytometry using the SR-B1 inhibitor BLT-1 as a positive control.

[0087] The results are as follows Figure 14 As shown, NP (20×10 -6 M) Significantly reduced PD-L1 expression in 786-O cells, suggesting that NP-mediated cholesterol inhibition can ameliorate the immunosuppressive phenotype of tumors.

[0088] Example 8

[0089] This example demonstrates that the SR-B1 targeting polypeptide complex NPR has the ability to undergo conformational change and self-assemble into water-insoluble nanofibers after co-incubation with SR-B1.

[0090] The SR-B1 targeting polypeptide complex NPR and the control polypeptide complex NPR-C prepared in Example 1 were incubated with the SR-B1 solution for 1 hour, respectively. Dynamic light scattering (DLS) analysis was performed on the NPR and NPR-C solution samples before and after incubation. The results were as follows: Figure 15 As shown in the figure, the particle size of NPR increased significantly after co-incubation with SR-B1, indicating that NPR can undergo conformational change and self-assemble to form hydrophobic nanofibers after binding to SR-B1 protein. However, the particle size did not change significantly after co-incubation with NPR-C, indicating that NPR-C did not undergo structural changes.

[0091] Example 9

[0092] This example investigates the in vitro release of Resiquimod from the SR-B1 targeting polypeptide complex.

[0093] The in vitro release of resiquimod from the SR-B1-targeted peptide complex was evaluated using dialysis. Specifically, the SR-B1-targeted peptide complex NPR prepared in Example 1, either co-incubated with or without SR-B1, and the control peptide complex NPR-C were placed in dialysis tubing at different pH conditions. Released fluids were collected at predetermined time points for determination of resiquimod content. The amount of R848 released was quantified using UV-visible spectroscopy (wavelength 300 nm).

[0094] The results are as follows Figure 16 As shown, after incubation with SR-B1 solution, NPR showed rapid release of Resiquimod, releasing 51.56±1.43% within 3 hours and more than 80% within 48 hours. This proves that the SR-B1 targeting peptide NP in the SR-B1 targeting peptide complex undergoes conformational change after binding to SR-B1, self-assembling to form hydrophobic nanofibers, releasing Resiquimod originally encapsulated in the hydrophobic core of the nanospherical micelles.

[0095] The NPR-C group had the lowest resiquimod release because NPR-C lacked a self-assembling peptide segment and could not undergo conformational changes, thus failing to release resiquimod from the hydrophobic core.

[0096] Example 10

[0097] This example demonstrates that the SR-B1 targeting polypeptide complex has the effect of inhibiting tumor proliferation.

[0098] In this example, Renca cells overexpressing SR-B1 were used to establish a tumor xenograft mouse model. 3 The mice were then treated with different treatments: phosphate buffered saline (PBS, 200 μL, injected through the tail vein every other day, for a total of 5 injections), anti-programmed death ligand 1 antibody (α-PD-L1, 3 mg / kg, injected intraperitoneally once every 3 days, for a total of 5 injections), NPs prepared in Example 1 (200 μL, 400×10 −6 M, injected via tail vein every 3 days, for a total of 5 injections), NPR-C prepared in Example 1 (200 μL, 400×10 −6 M, injected via tail vein every 3 days, for a total of 5 injections) and NPR (200 μL, 400 × 10 −6 M, injected via the tail vein every 3 days for a total of 5 injections). Tumor volume and mouse body weight were recorded regularly throughout the experiment. On day 21 after the first treatment, mice were sacrificed, and tissue samples were collected for subsequent analysis.

[0099] The results are as follows Figure 17 and Figure 18 As shown, the tumor volume of mice in the NPR-treated group grew slowly, and after 21 days, the tumor weight was significantly lower than that in the PBS control group and the NPR-C group. Therefore, the SR-B1 targeting peptide complex NPR can significantly inhibit tumor proliferation. At the same time, the tumor growth rate and weight in the NPR group were lower than those in the NP group, indicating that the combined action of Resiquimod carried in NPR and NP peptide on tumor tissue can further enhance the inhibitory effect on tumor proliferation on the basis of Np.

[0100] Example 11

[0101] This example demonstrates that the SR-B1 targeting polypeptide complex has the effect of improving the tumor immune microenvironment.

[0102] In this example, tumors and draining lymph node tissues of mice in each group in Example 10 were collected and digested, and flow cytometry was used to detect immune cell maturation and tumor infiltration.

[0103] The results are as follows Figure 19 As shown, the proportion of mature dendritic cells (CD80⁺CD86⁺) in NPR-treated tumor lymph nodes was as high as 77.26 ± 2.08%, the proportion of CD3⁺CD4⁺ T cells in tumor tissue increased by 2.15 ± 0.03 times, the proportion of CD3⁺CD8⁺ T cells increased by 5.02 ± 0.26 times, and the proportion of CD25⁺FOXP3⁺ regulatory T cells decreased to 4.09 ± 0.45%. These results indicate that NPR effectively improves the immunosuppressive tumor microenvironment and enhances the infiltration of immune cells in the tumor site.

[0104] Example 12

[0105] This example demonstrates the biosafety of the SR-B1 targeting polypeptide complex through animal experiments.

[0106] In this example, the body weights of the mice in each group in Example 10 were compared and analyzed, and peripheral blood was collected from the mice for blood biochemical tests.

[0107] The results are as follows Figure 20 As shown in Figure 2, there was no significant difference in body weight among the mice groups. Figure 21As shown, the blood biochemical test of peripheral blood showed that there were no significant differences in liver function indicators shown by ALT, AST and ALP, protein indicators shown by TP, ALB and GLOB, renal function indicators shown by BUN and Cre; white blood cell count shown by WBC; red blood cell count shown by RBC, hemoglobin count shown by HGB and platelet count shown by PLT, which proved that NPR-C and NPR are biosafe.

Claims

1. An SR-B1 targeting polypeptide complex, characterized in that: The invention is composed of an SR-B1 targeting polypeptide and a Toll-like receptor 7 / 8 agonist, Resiquimod; the SR-B1 targeting polypeptide comprises an SR-B1 targeting peptide segment, a self-assembling peptide segment, and a hydrophobic molecule connected in sequence; the amino acid sequence of the SR-B1 targeting peptide segment is shown in SEQ ID No. 1, and the amino acid sequence of the self-assembling peptide segment is shown in SEQ ID No. 2; and the hydrophobic molecule is a hydrophobic hexaalkyl compound.

2. The SR-B1 targeting polypeptide complex according to claim 1, characterized in that: The mass ratio of the SR-B1 targeting polypeptide to Resiquimod is 1:

1.

3. The SR-B1 targeting polypeptide complex according to claim 1 or 2, characterized in that: The molecular structure of the SR-B1 targeting polypeptide is shown in Formula I: ； Formula I.

4. The SR-B1 targeting polypeptide complex according to claim 3, characterized in that: When the SR-B1 targeting peptide solution is mixed with the Resiquimod solution, the hydrophobic ends of the SR-B1 targeting peptide will aggregate together through hydrophobic interactions to form nanospherical micelles, and wrap the Resiquimod in its hydrophobic core, resulting in an SR-B1 targeting peptide complex with a particle size of 35.52 nm ± 5.56 nm.

5. Use of the SR-B1 targeting polypeptide complex according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.

6. Use of the SR-B1 targeting polypeptide complex according to claim 5 in the preparation of anti-tumor drugs, characterized in that: The tumor is a tumor that specifically and highly expresses SR-B1.

7. Use of the SR-B1 targeting polypeptide complex according to claim 6 in the preparation of anti-tumor drugs, characterized in that: The tumor with specific high expression of SR-B1 is renal clear cell carcinoma, renal papillary cell carcinoma, melanoma, colorectal cancer or esophageal squamous cell carcinoma.

8. Use of the SR-B1 targeting polypeptide complex according to any one of claims 1 to 4 in the preparation of an immunotherapy sensitizer.

9. Use of the SR-B1 targeting polypeptide complex according to claim 8 in the preparation of an immunotherapy sensitizer, characterized in that: The immunotherapy sensitizer is a sensitizer used for anti-tumor immunotherapy of tumors with high specific expression of SR-B1.

10. Use of the SR-B1 targeting polypeptide complex according to claim 9 in the preparation of an immunotherapy sensitizer, characterized in that: The tumor with specific high expression of SR-B1 is renal clear cell carcinoma, renal papillary cell carcinoma, melanoma, colorectal cancer or esophageal squamous cell carcinoma.

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