Active polypeptide with targeted degradation of cd47 function and application thereof

Abstract

The application discloses an active polypeptide with a function of targeted degradation of CD47 and application thereof, and belongs to the field of biological medicines. The amino acid sequence of the polypeptide is shown as SEQ ID NO:1. The polypeptide can efficiently penetrate a cell membrane, specifically combine with an intracellular domain of CD47, and guide CD47 to be degraded through a lysosome pathway, so that a CD47 / SIRP alpha 'don't eat me' signal path is blocked from the source. The polypeptide has the advantages of small molecular weight, low immunogenicity, simple synthesis, and an intracellular degradation mechanism capable of avoiding the blood toxicity risk of existing antibody therapy, and provides a powerful tool for developing a novel strategy for overcoming the defects of existing CD47 targeted therapy.

Description

Technical Field

[0001] This invention relates to an active polypeptide with targeted CD47 degradation function and its application, belonging to the field of biomedicine. Background Technology

[0002] Tumor immune checkpoint therapy, represented by PD-1 / PD-L1 inhibitors, has achieved significant breakthroughs, but challenges such as limited patient response rates and drug resistance remain. Therefore, developing therapies with novel targets and mechanisms of action is crucial. CD47 is a transmembrane protein overexpressed on the surface of various tumor cells. As a "don't eat me" signaling molecule, it directly inhibits the phagocytic function of macrophages by binding to the receptor SIRPα on the surface of myeloid cells such as macrophages, making it one of the key targets for tumor immune escape.

[0003] Currently, mainstream therapies targeting CD47 (such as monoclonal antibodies and SIRPα-Fc fusion proteins) focus on blocking the CD47 / SIRPα protein interaction in its extracellular region. However, such strategies have the following inherent limitations: (1) The interaction interface is relatively wide and shallow, making it difficult for antibodies to achieve efficient and complete blocking; (2) More importantly, CD47 is also widely expressed on the surface of normal blood cells such as erythrocytes and platelets, which often leads to dose-limiting hematologic toxicity side effects such as anemia and thrombocytopenia caused by antibody therapy, severely limiting its clinical safety window and therapeutic application scope.

[0004] Therefore, developing a novel therapy that can reduce CD47 protein abundance at its source (i.e., within tumor cells), thereby eliminating its immunosuppressive function, has significant clinical value. Compared to large molecule antibodies, peptide drugs, due to their small molecular weight, high structural designability, low immunogenicity, and ease of chemical synthesis, show unique potential in developing drugs targeting intracellular proteins. However, their successful application requires effectively overcoming challenges such as insufficient cell membrane penetration efficiency and poor in vivo stability. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide an active polypeptide with the function of targeting and degrading CD47 and its application.

[0006] Technical solution: The active polypeptide with targeted degradation function of CD47 described in this invention has the amino acid sequence shown in SEQ ID NO: 1.

[0007] Furthermore, the active polypeptide has a nucleotide sequence as shown in SEQ ID NO: 3.

[0008] Furthermore, the N-terminus, C-terminus, or side chain of the polypeptide is connected to a functional module selected from the group consisting of: membrane-penetrating peptides, cell-targeting peptides, fluorescent labeling groups, or modifying groups for increasing stability and / or prolonging half-life.

[0009] Furthermore, the N-terminus of the polypeptide is acetylated and the C-terminus is amidated.

[0010] The active polypeptide described in this invention is used in the preparation of drugs that degrade CD47 or as an adjuvant for antibodies that target the extracellular region of CD47.

[0011] The application of the active polypeptide described in this invention in the preparation of drugs for the prevention and / or treatment of tumors.

[0012] Furthermore, the tumor type is one in which CD47 is overexpressed on the tumor surface.

[0013] Furthermore, the tumor includes lung cancer, colon cancer, or breast cancer.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients and drug delivery carriers.

[0015] The pharmaceutical composition of the present invention comprises the above-mentioned active polypeptide as the active substance.

[0016] Furthermore, the pharmaceutical composition also includes an antibody that targets the extracellular region of CD47.

[0017] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: Based on a functional fragment of the ING4 protein screened for the first time, this application designs an active polypeptide targeting the intracellular domain of CD47. By directly inducing its degradation via the lysosomal pathway, it eradicates the "don't eat me" signal at the source of protein expression, theoretically avoiding the problem of reduced efficacy due to incomplete competitive binding, and potentially avoiding hematologic toxicity caused by CD47 antibodies. The combination of this polypeptide and an antibody targeting the extracellular region of CD47 exhibits significant synergistic anti-tumor effects both in vivo and in vitro. Both exert a dual attack on CD47 from intracellular (degradation) and extracellular (blocking) perspectives, providing new and strong evidence for clinical combination therapy. Attached Figure Description

[0018] Figure 1 The GST pull-down assay results showed that the TY-4 peptide directly binds to the full-length CD47 (His-CD47), but does not bind to the CD47 mutant (His-CD47 / ΔC) which lacks the intracellular C-terminus, indicating that its binding site is located in the intracellular domain of CD47.

[0019] Figure 2Western blot results showed that CD47 protein levels in H520, HCT-116, and MCF-7 cells decreased in a concentration-dependent manner after treatment with different concentrations of TY-4 peptide.

[0020] Figure 3 Lysosomal component separation and Western blot analysis showed that TY-4 treatment significantly increased the CD47 content in lysosomes, suggesting that TY-4 promoted the transport of CD47 to lysosomes.

[0021] Figure 4 Flow cytometry analysis showed that TY-4 treatment significantly reduced the level of CD47 protein (mean fluorescence intensity) on the cell membrane surface of H520, HCT-116 and MCF-7 cells.

[0022] Figure 5 Flow cytometry analysis of in vitro phagocytosis experiments showed that after tumor cells were pretreated with TY-4, the percentage of phagocytosis of CFSE-labeled tumor cells by M1 macrophages was significantly increased.

[0023] Figure 6 The results of mouse xenograft model experiments showed that the treatment effect of TY-4 combined with CD47 antibody was the best, and the number of intratumoral macrophages (F4 / 80) was reduced. + ) phagocytosis of GFP + The proportion of tumor cells was the highest. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0025] Main experimental materials and reagents Cell lines: Human tumor cell lines (lung cancer H520, colon cancer HCT-116, breast cancer MCF-7), human embryonic kidney cells HEK-293T, and mouse tumor cell lines (lung cancer LLC, colon cancer CT-26, breast cancer 4T1) were all purchased from ATCC in the United States.

[0026] Main reagents: High-glucose DMEM medium, fetal bovine serum (FBS), 0.25% trypsin-EDTA (Gibco); mononuclear cell isolation kit (Dynabeads™ Untouched™, ThermoFisher); Turbofect transfection reagent (Thermo); pLenti-CMV-MCS-GFP-SV-puro vector, pGEX-6P-1 vector, pET28a vector (Addgene); CD47 primary antibody (Proteintech); fluorescently labeled secondary antibodies and flow cytometry antibodies: Alexa Fluor594-labeled goat anti-rat IgG, FITC-labeled anti-human CD47 antibody, PE-labeled anti-mouse F4 / 80 and CD11b antibodies (Elabscience / JacksonImmunoResearch); InVivoMab anti-mouse CD47 antibody αCD47 (BioX Cell); lysosomal inhibitor chloroquine (CQ), proteasome inhibitor MG132 (Sigma); recombinant human GM-CSF, IFN-γ, LPS (PeproTech). Puromycin.

[0027] Main instruments: CO2 incubator, high-speed centrifuge, clean bench, flow cytometer, protein electrophoresis and imaging system.

[0028] Example 1: Design and Synthesis of Active Peptides The core active polypeptide TY-4 (SEQ ID NO: 1) of this invention is based on the rational design and systematic optimization of a functional fragment (SEQ ID NO: 2: QKEKKAARARSKGKNSDEEAPKTA) of the ING4 protein with tumor-suppressing function obtained through screening.

[0029] First, to endow and enhance its cell membrane penetration ability, the N-terminal start sequence "QKE" was replaced with an arginine-rich "RRR" sequence. The positive charge of the arginine residues can effectively promote the peptide to enter the cell through multiple pathways (such as direct penetration and endocytosis), which is a prerequisite for its intracellular targeting function.

[0030] Secondly, to enhance its binding affinity and specificity to the intracellular domain of the target CD47, key amino acid substitutions were made in the core recognition region: alanine at position 20 (A) was replaced with tryptophan (W) with a large side chain and hydrophobicity; simultaneously, threonine at position 23 (T) was replaced with leucine (L), which is more hydrophobic. These substitutions aim to optimize the spatial conformation of the peptide and may stabilize its binding to the CD47 target by enhancing hydrophobic interactions.

[0031] Finally, to improve the peptide's protease resistance and in vivo stability, the optimized linear sequence (RRRKKAARARSKGKNSDEEWPKLA) was terminally modified: acetylation (Ac-) at the N-terminus and amidation (-NH2) at the C-terminus. This modification effectively shields the peptide chain ends, reducing the risk of degradation by intracellular and extracellular proteases.

[0032] The final sequence obtained through the above design is: Ac-RRRKKAARARSKGKNSDEEWPKLA-NH2. All peptides were synthesized using standard solid-phase synthesis methods and purified preparatively by high-performance liquid chromatography (HPLC). Their chemical structures were accurately identified by mass spectrometry (MS).

[0033] Example 2: Verification of the specific binding of TY-4 peptide to the intracellular C-terminal domain of CD47 To verify whether the TY-4 peptide directly binds to the intracellular region of CD47, a GST pull-down assay was used. The gene encoding the TY-4 peptide (SEQ ID NO: 3: CGTCGTCGTAAAAAAGCTGCTCGTGCTCGTTCTAAAGGTAAAAATTCTGATGAAGAAT GGCCTAAATTAGCT) was synthesized, inserted into the pGEX-6P-1 vector, and sequenced to confirm that it was GST-TY-4. Using the pET28a plasmid, following the method in the published literature (Q Gou, B Yan, Y Duan, Y Guo, J Qian, JShi, Y Hou. Ubiquitination of CD47 Regulates Innate Anti-Tumor Immune Response. Adv Sci (Weinh). 2025 Feb;12(5):e2412205), expression plasmids of His-labeled full-length CD47 (His-CD47) or CD47 without the intracellular C-terminus (His-CD47 / ΔC) were constructed. Recombinant plasmids GST-TY-4, His-CD47, and His-CD47 / ΔC were transformed into *E. coli* BL21(DE3) for protein induction, expression, and purification. The purified GST or GST-TY-4 protein was then incubated with GSH agarose beads at 4°C for 4 hours, followed by incubation with His-CD47 or His-CD47 / ΔC protein at 4°C for 1 hour. After thorough washing, the presence of CD47 protein in the pull-down complex was detected by Western blot using an anti-His antibody.

[0034] The results are as follows Figure 1As shown, a significant His-CD47 signal was detected only in samples co-incubated with GST-TY-4 and His-CD47; however, no corresponding signal was detected in the GST-TY-4 co-incubation group with His-CD47 / ΔC, or in the control GST protein group. This result indicates that the TY-4 peptide can specifically and directly bind to the CD47 protein, and this binding is strictly dependent on the intracellular C-terminal domain of CD47.

[0035] Example 3: Inhibitory effect of TY-4 peptide on total CD47 protein levels in tumor cells To investigate the effect of TY-4 on CD47 protein expression in tumor cells, H520, HCT-116, and MCF-7 cells were selected for treatment. TY-4 peptide was dissolved in sterile ultrapure water to prepare a 10 mM stock solution, which was then diluted to 0, 1, 5, and 10 μM with the corresponding complete culture medium before use. The 0 μM group was treated with an equal volume of sterile ultrapure water as a solvent control. H520, HCT-116, and MCF-7 cells were cultured in DMEM complete medium containing 10% FBS (fetal bovine serum) and treated with TY-4 at 37°C and 5% CO2 for 12 hours. After treatment, cells were collected, lysed, and total protein was extracted. CD47 protein expression was detected by Western blot, and standardized using Tubulin as an internal control.

[0036] The results are as follows Figure 2 As shown, TY-4 treatment reduced CD47 protein levels in a concentration-dependent manner across all three tumor cell lines, with the 10 μM TY-4 treatment group exhibiting the most significant downregulation. This directly demonstrates that TY-4 possesses highly efficient activity in inducing intracellular CD47 protein degradation.

[0037] Example 4: TY-4 peptide promotes CD47 degradation via lysosomal pathway To elucidate the specific cellular pathway of TY-4-induced CD47 degradation, H520 cells were treated with 10 μM TY-4 for 12 hours, followed by treatment with the lysosomal inhibitor CQ (30 μM), the proteasome inhibitor MG132 (20 μM), or the control solvent DMSO for another 4 hours. Cells were then collected and lysed, and CD47 protein expression levels were detected by Western blot. Furthermore, the accumulation of CD47 in lysosomes was analyzed. Using H520 cells as a model, cells were treated with 10 μM TY-4 or the control for 12 hours, followed by treatment with the lysosomal inhibitor chloroquine (CQ, 30 μM) for another 4 hours. This treatment inhibited lysosomal acidification and degradation, leading to the retention and accumulation of substrates to be degraded within the lysosomes. Subsequently, lysosomal components were isolated using a commercially available kit, and the CD47 content was analyzed by Western blot. The lysosomal marker protein LAMP1 was used as a component quality control, and the membrane protein E-Cadherin was used as a cross-contamination reference.

[0038] The results are as follows Figure 3 As shown, TY-4 treatment significantly reduced CD47 protein levels in cells, while the lysosomal inhibitor CQ reversed this effect, and the proteasome inhibitor MG132 had no significant effect. Figure 3 (A). Furthermore, compared to the control group, the TY-4 treatment group showed a significant increase in CD47 protein signaling in lysosomal fractions of cells, while the CD47 level in total cell lysate was significantly decreased (A). Figure 3 (B). These results indicate that TY-4 treatment promotes the transport and accumulation of CD47 protein into lysosomes, meaning that it is primarily degraded via the lysosomal pathway.

[0039] Example 5: TY-4 peptide reduces CD47 expression on tumor cell membrane surface CD47 exerts its immunosuppressive function primarily through its expression on the cell membrane surface. To verify the effect of TY-4 on functional CD47 levels, flow cytometry was used for detection. After treating tumor cells with 10 μM TY-4 or a control (sterile ultrapure water) for 12 hours, single-cell suspensions were prepared and incubated with FITC-labeled anti-human CD47 antibody. The mean fluorescence intensity (MFI) of CD47 on the cell membrane surface was then measured.

[0040] The results are as follows Figure 4 As shown, after TY-4 treatment, the fluorescence intensity of CD47 on the surface of the three types of tumor cells was significantly lower than that in the control group. This result is consistent with the decreasing trend of total protein levels. Figure 2 This study confirmed that TY-4 can effectively reduce the level of CD47 on the membrane surface of tumor cells, which is used for immune escape.

[0041] Example 6: TY-4 peptide enhances macrophage phagocytic function in vitro by degrading CD47. To verify the functional production of TY-4, an in vitro phagocytosis experiment was performed. CD14⁺ monocytes were isolated from peripheral blood of healthy individuals and induced to differentiate into macrophages using GM-CSF (50 ng / ml). These macrophages were then polarized into pro-inflammatory M1 macrophages using LPS (10 ng / ml) and IFN-γ (50 ng / ml). Simultaneously, tumor cells (H520, etc.) were labeled with CFSE fluorescent dyes. After pretreatment with 10 μM TY-4 or a control for 1 hour, the tumor cells were co-cultured with M1 macrophages at an effector cell:target cell ratio of 4:1 for 4 hours. Cells were collected, macrophages were labeled with anti-CD11b antibody, and CD11b was analyzed by flow cytometry. + CFSE + The proportion of double-positive cell populations, i.e., the percentage of macrophages that have been phagocytosed by tumor cells.

[0042] The results are as follows Figure 5 As shown, compared with the control group, the percentage of tumor cells pretreated with TY-4 that were phagocytosed by macrophages was significantly increased. This indicates that TY-4 effectively relieved the inhibition of macrophage phagocytic function by reducing the CD47 level of tumor cells, thus restoring the anti-tumor activity of macrophages.

[0043] Example 7: TY-4 peptide synergistically inhibits tumor growth with anti-CD47 antibody in an animal model. To evaluate the in vivo antitumor activity of TY-4 and its potential for combination therapy, this study constructed several mouse syngeneic xenograft models. HEK293T cells were transfected with the pLenti-CMV-MCS-GFP-SV-puro plasmid using Turbofect transfection reagent, and lentiviral particles were packaged and harvested. Subsequently, CT-26, LLC, and 4T1 cells were infected with the obtained lentiviruses, respectively. After selection with puromycin (1 µg / mL), stable GFP-expressing LLC, CT-26, and 4T1 cell lines were successfully obtained, resulting in LLC-GFP, CT-26-GFP, and 4T1-GFP cells. Subsequently, LLC-GFP cells in logarithmic growth phase (5 × 10⁻⁶ cells) were... 5 CT-26-GFP cells (5 × 10⁻⁶) were subcutaneously inoculated into the right axilla of 5-week-old female C57BL / 6 mice; CT-26-GFP cells in logarithmic growth phase (5 × 10⁻⁶ cells) were then injected into the right axilla. 5 ) and 4T1-GFP (5×10 5 Cells were subcutaneously inoculated into the right axilla of 5-week-old female BALB / c mice. All experimental animals were purchased from the Institute of Comparative Medicine, Yangzhou University. Seven days after inoculation, tumor-bearing models (tumor volume approximately 50 mm²) were... 3Mice were randomly divided into 4 groups (n=5): PBS control group, TY-4 monotherapy group (2 mg / kg, intratumoral injection, every other day, for a total of 7 doses, 14 days in total), anti-mouse CD47 antibody (αCD47) monotherapy group (100 μg / mouse, intratumoral injection, every other day, for a total of 7 doses, 14 days in total), and TY-4 and αCD47 combination therapy group (TY-4 2 mg / kg + αCD47 100 μg / mouse, same dosing regimen as the monotherapy group). Tumor volume was measured on day 14 after the last administration. Mice were then sacrificed, and tumor tissue was collected to prepare a single-cell suspension. Tumor-infiltrating macrophages (F4 / 80) were analyzed by flow cytometry. + GFP in ) + The proportion of cells was used to assess phagocytic activity in vivo.

[0044] The results are as follows Figure 6 As shown: Tumor growth ( Figure 6 (A) TY-4 monotherapy showed some tumor growth inhibition effect. The anti-CD47 antibody monotherapy group had relatively limited tumor inhibition effect. The combination therapy group showed the most significant inhibition of tumor growth, with tumor volume significantly smaller than either monotherapy group (P<0.01), demonstrating a clear synergistic anti-tumor effect. (Phlophagy in vivo) Figure 6 (B) Flow cytometry analysis showed that macrophages in the tumor tissue of the combined treatment group engulfed GFP. + The proportion of tumor cells was significantly higher than in other groups. This is consistent with the results of in vitro experiments, confirming that the combination therapy can maximize the activation of macrophage phagocytic function in the tumor microenvironment, thereby producing a stronger therapeutic effect.

[0045] In summary, this invention provides a novel peptide, TY-4, that targets and induces the degradation of the CD47 intracellular domain. This peptide, through a novel mechanism of action, effectively reduces CD47 protein levels in tumor cells, reverses immunosuppression, enhances macrophage-mediated anti-tumor immunity, and exhibits a synergistic effect with existing CD47 antibody therapies. This invention provides new candidate molecules and strategies for developing highly effective and low-toxicity CD47-targeted anti-tumor drugs.

Claims

1. An active polypeptide with targeted CD47 degradation function, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The active polypeptide according to claim 1, characterized in that, The peptide has a functional module selected from the group consisting of a transmembrane peptide, a cell-targeting peptide, a fluorescent labeling group, or a modifying group for increasing stability and / or prolonging half-life at its N-terminus, C-terminus, or side chain.

3. The active polypeptide according to claim 1, characterized in that, The peptide is acetylated at its N-terminus and amidated at its C-terminus.

4. The use of the active polypeptide according to any one of claims 1 to 3 in the preparation of a drug that degrades CD47 or as an adjuvant for an antibody targeting the extracellular region of CD47.

5. The use of the active polypeptide according to any one of claims 1 to 3 in the preparation of a medicament for the prevention and / or treatment of tumors.

6. The application according to claim 5, characterized in that, The tumor type is a tumor that overexpresses CD47 on its surface.

7. The application according to claim 5, characterized in that, The tumors include lung cancer, colon cancer, or breast cancer.

8. The application according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients and drug delivery carriers.

9. A pharmaceutical composition, characterized in that, The active substance is the active polypeptide as described in any one of claims 1 to 3.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition also includes an antibody that targets the extracellular region of CD47.

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