Universal polypeptide-PROTAC targeting multiple immune checkpoints and application thereof

Abstract

The invention relates to a universal polypeptide-PROTAC targeting multiple immune checkpoints and an application of the universal polypeptide-PROTAC. The existing immune checkpoint inhibitor has the risk that the compensatory expression of immune checkpoint molecules is up-regulated after administration so as to generate drug resistance. The general polypeptide-PROTAC has a structure of B-Z-L-V, B is a cell-penetrating peptide, Z is an SH2 structural domain of SHP2 or SHP1 or an active fragment of the SH2 structural domain, L is null or a connecting element, and V is VHL or a ligand of other E3 molecules. According to the present invention, the C-SH2 structural domain can simultaneously specifically bind to a variety of immune checkpoint molecules, and the E3 ligase ligand structural domain can perform ubiquitination degradation on the immune checkpoint, such that the compensatory expression up-regulation of the immune checkpoint molecules can be fundamentally avoided so as to produce the drug resistance;

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and in particular to a universal polypeptide targeting multiple immune checkpoints - PROTAC and applications thereof. Background Art

[0002] Immune checkpoint blockade is an important method of tumor immunotherapy, which uses the body's own immune system to resist and fight malignant tumors and substantially improves the patient's overall survival. However, the patient response rate is not high when the blocker targeting a single immune checkpoint is used alone. The reason is that due to the mutual influence between tumor cells and immune cells, cytokines, etc., there are intricate immune signaling pathways in the tumor microenvironment, resulting in limited immunotherapy effects targeting only one immunosuppressive molecule. Therefore, the combination of drugs targeting different immune checkpoints has become a new strategy to improve patient response rates, such as universal immune checkpoint inhibitors (UICB) targeting common targets of different immune checkpoints.

[0003] Patent ZL2020100339548 discloses an anti-tumor fusion protein and its preparation method and application, and prepares a mimetic polypeptide of the SHP2 C-SH2 domain that can bind to ITIM, containing TAT protein, optional connecting elements and the ITIM motif binding region of SH2 of SHP2 or SHP1 or its active fragment. The polypeptide has the versatility to bind to a variety of immune checkpoint molecules, can inhibit the ITIM motifs downstream of a variety of immunosuppressive molecules, has a broad spectrum, can enhance the function of T cells, NK cells, and macrophages, and has anti-tumor activity. However, the polypeptide only works by competitively binding to the ITIM motif, and there is a risk of causing compensatory upregulation of immune checkpoint molecules after administration, thereby producing drug resistance.

[0004] Therefore, it is necessary to design new universal immune checkpoint inhibitors to overcome the above-mentioned defects. Summary of the invention

[0005] The purpose of the present invention is to provide a universal polypeptide-PROTAC targeting multiple immune checkpoints and its application to solve the problems of existing immune checkpoint inhibitors in terms of drug resistance.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] Provided is a universal peptide-PROTAC targeting multiple immune checkpoints, wherein the structure of the universal peptide-PROTAC is BZLV;

[0008] in:

[0009] B is a cell-penetrating peptide;

[0010] Z is the SH2 domain of SHP2 or SHP1 or an active fragment thereof;

[0011] L is no or connected element;

[0012] V is the ligand of VHL or other E3 molecules.

[0013] Furthermore, the amino acid sequence of the B part is YGRKKRRQRRRPP.

[0014] Furthermore, the amino acid sequence of the Z portion is VRESQSHPGDFVL.

[0015] Furthermore, when the L portion is a connecting element, the amino acid sequence is GSGSGS.

[0016] Furthermore, the amino acid sequence of the V portion is ALAP(OH)YIP.

[0017] On the other hand, a pharmaceutical composition is provided, comprising the universal polypeptide-PROTAC and a pharmaceutically acceptable carrier thereof.

[0018] On the other hand, the invention provides a use of a universal polypeptide-PROTAC targeting multiple immune checkpoints as described above, wherein the universal polypeptide-PROTAC is used to prepare a drug for treating or preventing tumors.

[0019] On the other hand, the use of a universal polypeptide-PROTAC targeting multiple immune checkpoints as described is provided,

[0020] The universal polypeptide-PROTAC is used to prepare drugs that enhance the killing effect of T cells on tumor cells, promote the activation of T cells, promote the proliferation of T cells, and inhibit the apoptosis of T cells.

[0021] On the other hand, the application of a universal polypeptide-PROTAC targeting multiple immune checkpoints as described is provided, wherein the universal polypeptide-PROTAC is used to prepare a drug that enhances the killing effect of NK cells on tumor cells, increases the expression level of NK cell CD107a, perforin, granzyme secretion, and increases the activation of NK cells.

[0022] On the other hand, the invention provides an application of a universal polypeptide-PROTAC targeting multiple immune checkpoints as described above, wherein the universal polypeptide-PROTAC is used to prepare a drug that enhances the phagocytic killing effect of M cells on tumor cells, enhances the activation of M cells, and increases the M1 polarization of M cells.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a universal polypeptide-PROTAC targeting multiple immune checkpoints and its application, which can specifically bind to multiple immune checkpoint molecules at the same time through its C-SH2 domain, and ubiquitinate and degrade the immune checkpoints through its E3 ligase ligand domain, fundamentally avoiding the compensatory upregulation of immune checkpoint molecules to produce drug resistance. In addition, the universal polypeptide-PROTAC of the present invention is the first universal immune checkpoint degrader, which is functionally superior to the C-SH2 domain, can significantly enhance the anti-tumor activity of immune cells such as T lymphocytes, NK cells, and macrophages, has a broad spectrum of anti-tumor effects, and avoids the toxic side effects and increased treatment costs caused by various combined medications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, drawings of other embodiments can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a graph showing the purity of synthetic peptide-PROTAC detected by HPLC. In the graph, A is Y35, B is Y37, and C is Y39.

[0027] Figure 2 This is a molecular weight diagram of synthetic peptide-PROTAC detected by mass spectrometry. In the figure, A is Y35, B is Y37, and C is Y39.

[0028] Figure 3 This is a laser confocal detection of synthetic peptide-PROTAC entering CD8+T cells.

[0029] Figure 4 This is the affinity diagram of peptide-PROTAC and PD-1 intracellular ITIM sequence detected by SPR. In the figure, A is Y35 and ITIM, B is Y37 and ITIM, and C is Y39 and ITIM.

[0030] Figure 5 This is a WB test of Y35, Y37, and Y39 degrading immunosuppressive receptors PD-1, NKG2A, and SIRPα in immune cells. In the figure, A is the degradation of PD-1, B is the degradation of NKG2A, C is the degradation of SIRPα, and D is the maximum degradation efficiency table.

[0031] Figure 6This is a diagram of Co-IP verification of the formation of the ternary complex of PD-1-Y39-VHL and BTLA-Y39-VHL. In the figure, A is the detection of the ternary complex of PD-1-Y39-VHL, and B is the detection of the ternary complex of BTLA-Y39-VHL.

[0032] Figure 7 This is a laser confocal microscope image of Y39 degrading the inhibitory receptor PD-1 in T cells.

[0033] Figure 8 This is a WB test of Y39 degrading inhibitory receptors PD-1 and BTLA in T cells. In the figure, A shows Y39 degrading PD-1 at different concentrations, B shows Y39 degrading BTLA at different concentrations, C shows Y39 degrading PD-1 at different times, D shows Y39 degrading BTLA at different times, and E shows the maximum degradation efficiency and half-degradation concentration table.

[0034] Fig. 9 This is a diagram of Co-IP verification of the formation of the NKG2A-Y39-VHL and PD-1-Y39-VHL ternary complex.

[0035] In the figure, A is the detection of the NKG2A-Y39-VHL ternary complex, and B is the detection of the PD-1-Y39-VHL ternary complex.

[0036] Fig.10 This is a laser confocal microscope image of Y39 degrading NKG2A.

[0037] Fig.11 This is a WB test of Y39 degrading inhibitory receptors NKG2A and PD-1 in NK cells. In the figure, A shows Y39 degrading NKG2A at different concentrations, B shows Y39 degrading PD-1 at different concentrations, and C shows the maximum degradation efficiency and half-degradation concentration.

[0038] Fig.12 This is a diagram of the formation of the SIRPα-Y39-VHL and PD-1-Y39-VHL ternary complex detected by Co-IP.

[0039] In the figure, A is the detection of SIRPα-Y39-VHL ternary complex, and B is the detection of PD-1-Y39-VHL ternary complex.

[0040] Fig.13 This is a confocal laser scan to detect SIRPα degradation by Y39.

[0041] Fig.14 This is a WB detection of Y39 degradation of SIRPα and PD-1 in macrophages. In the figure, A shows Y39 degradation of SIRPα at different concentrations, B shows Y39 degradation of PD-1 at different concentrations, and C shows the maximum degradation efficiency and half-degradation concentration table.

[0042] Fig.15 This is a diagram showing that proteasome inhibitors can reduce the degradation of immune checkpoints by Y39. In the figure, A is the reduction of Y39's degradation of PD-1 and BTLA, B is the reduction of Y39's degradation of NKG2A, and C is the reduction of Y39's degradation of SIRPα.

[0043] Fig.16 This is a diagram showing that Y39 cannot degrade inhibitory receptors in cells with VHL knockout. In the figure, A is a Jurkat cell line, B is a NK92 cell line, and C is a THP-1 cell line.

[0044] Fig.17 This is a diagram of the effect of peptide-PROTAC on CD8+T cells. In the figure, A and B are WB detections of Y39 upregulating activation signal molecule pathways, C is flow cytometry detection of Y39 promoting Ki67 expression on CD8+T cells, D is flow cytometry detection of Y39 promoting T cell proliferation, E is flow cytometry detection of Y39 promoting CD69 expression on CD8+T cells, F is RTCA detection of Y39 enhancing the killing ability of T cells, G is flow cytometry detection of Y39 downregulating the expression of Cleaved caspase-3, and H is flow cytometry detection of Y39 inhibiting CD8+T cell apoptosis.

[0045] Fig.18 This is a diagram showing the effect of peptide-PROTAC on NK cells. In the figure, A is WB detection of Y39 upregulating NK cell activation-related signaling pathways, B is flow cytometry detection of Y39 increasing NK cell activation and killing-related molecule expression, and C is RTCA detection of Y39 promoting NK cell killing function.

[0046] Fig.19 This is a diagram showing the effect of peptide-PROTAC on macrophages. In the figure, A is WB detection of Y39 upregulating macrophage activation-related signaling molecule pathways, B is flow cytometry detection of Y39 increasing the expression of M1 phenotype molecules and reducing the expression of M2 phenotype molecules, C is flow cytometry detection of Y39 promoting macrophage phagocytosis, and D is laser confocal detection of Y39 promoting macrophage phagocytosis.

[0047] Fig. 20 This is a test diagram of the tumor inhibition effect of peptide-PROTAC on mouse colon cancer. In the figure, A is a photo of mouse tumor; B is a mouse tumor growth curve, C is the average tumor weight statistics of mice, D / E is the expression of GZMB in tumor-infiltrating NK / T cells detected by flow cytometry, and F / G is the expression of TNFα / CD206 related molecules in tumor-infiltrating macrophages detected by flow cytometry.

[0048] Fig.21This is a test diagram of the tumor inhibition effect of peptide-PROTAC on mouse liver cancer. In the figure, A is a photo of mouse tumor, B is a mouse tumor growth curve, C is the average tumor weight of mice, D is the number of tumor-infiltrating T cells detected by flow cytometry, E is the expression of Ki67 in tumor-infiltrating T cells detected by flow cytometry, F is the ratio of M2 / M1 in mouse tumor tissue detected by flow cytometry, and G is the expression of M2-related molecule IL-10.

[0049] Fig. 22 This is a graph showing the tumor-suppressing effect of peptide-PROTAC on liver cancer in mice reconstructed with human immune system.

[0050] In the figure, A is a photo of mouse tumor, B is the mouse tumor growth curve, C is the average tumor weight statistics of mice, D is the CD8+T cell infiltration ratio in tumor tissue detected by flow cytometry, E is the CD8+T cell infiltration ratio in tumor tissue detected by immunofluorescence, and F / G is the expression of CD8+T cell killing-related molecules Perforin / TNFα in tumor tissue.

[0051] Fig.23 This is a test diagram of the tumor inhibition effect of peptide-PROTAC on PD-1-resistant colon cancer. In the figure, A is a photo of mouse tumor, B is a mouse tumor growth curve, C is the average tumor weight statistics of mice, D is the CD8+T cell infiltration ratio in tumor tissue detected by flow cytometry, E is the proliferation ability of infiltrating CD8+T cells in tumor tissue detected by flow cytometry, F is the M2 / M1 ratio in mouse tumor tissue, and G is the activation state of infiltrating NK cells in tumor tissue detected by flow cytometry.

[0052] Fig.24 This is a schematic diagram of the mechanism of action of the universal peptide PROTAC. DETAILED DESCRIPTION

[0053] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0054] It should be noted that similar reference numerals and letters represent similar items, so once an item is defined in one embodiment, it does not need to be further defined and explained in subsequent embodiments. In some embodiments, the specific implementation process is described in steps, which is only for clear and accurate expression and should not be understood as a limitation of the sequence. In addition, in the embodiment description process, the chemical substances used in the steps are all existing substances or commercially available products.

[0055] Proteolysis-targeting chimeras (PROTAC) are a drug development technology that uses the ubiquitin-proteasome system to degrade target proteins. PROTAC structurally includes an E3 ubiquitin ligase ligand and a target protein ligand, and the two parts are connected by a suitable linker in the middle. Peptide-PROTAC binds to the E3 ligase through its E3 ubiquitin ligase ligand, and targets the target protein through the target protein ligand to form a ternary complex, hijacking the activity of the E3 ubiquitin ligase to ubiquitinate the target protein and subsequently mediate the proteasome degradation of the target protein. The advantages of peptide-PROTAC are: 1. It has extremely high targeting, because the PROTAC molecule needs to bind to the target protein (Protein of Interest, POI) and the E3 ligase at the same time to form a ternary complex and induce degradation, which increases the selectivity requirements and can avoid some unnecessary off-target effects and toxic side effects; 2. PROTAC does not need to occupy the active site or binding site of POI, as long as it can bind to POI and induce its ubiquitination, it can achieve degradation. Therefore, the target range can be expanded, including some proteins without active sites or binding pockets, which can also be degraded by PROTAC; 3. In the process of forming a ternary complex to degrade the target molecule, the PROTAC molecule is not consumed and can be recycled and reused, with a catalytic degradation effect. Therefore, only a low dose is required to have extremely high activity, reducing the dosage and cost of the drug.

[0056] The present invention designs a universal polypeptide - PROTAC targeting multiple immune checkpoints. Unlike existing immunotherapy drugs that mostly target only one immunosuppressive molecule, PROTAC can specifically bind to multiple immune checkpoint molecules at the same time, including a transmembrane peptide sequence, a peptide sequence targeting the ITIM domain recognition peptide sequence, a Linker sequence and an E3 ligase recruitment peptide sequence. It can effectively target and bind to multiple immune checkpoint molecules containing ITIM sequences in cells, causing the latter's protein degradation and restoring the anti-tumor activity of immune cells, providing an effective solution for tumor immunotherapy, and can ubiquitinate and degrade multiple immune checkpoint molecules, fundamentally eliminating the immunosuppressive signals caused by immune checkpoints.

[0057] The structure of the universal polypeptide-PROTAC is BZLV, wherein B is a cell-penetrating peptide, Z is the SH2 domain of SHP2 or SHP1 or an active fragment thereof, L is no or a connecting element, and V is a ligand of VHL or other E3 molecules.

[0058] The cell-penetrating peptide in part B is a transmembrane peptide, and its amino acid sequence is SEQ ID NO: 1, specifically YGRKKRRQRRRPP.

[0059] The SH2 domain of SHP2 or SHP1 in the Z portion or its active fragment is a targeting ITIM domain recognition peptide, and its amino acid sequence is SEQ ID NO: 2, specifically VRESQSHPGDFVL.

[0060] The connecting element of the L part is Linker, and its amino acid sequence is SEQ ID NO: 3, specifically GSGSGS.

[0061] The ligand of VHL or other E3 molecules in the V part is the E3 ligase recruitment peptide, and its amino acid sequence is SEQ ID NO: 4, specifically ALAP(OH)YIP.

[0062] After combination, the amino acid sequence of the universal polypeptide-PROTAC is SEQ ID NO: 5, specifically YGRKKRRQRRRPPVRESQSHPGDFVLGSGSGS ALAP(OH)YIP.

[0063] Universal polypeptide-PROTAC also includes variant forms. These variant forms include (but are not limited to): deletion, insertion and / or substitution of 1-3 (usually 1-2, preferably 1) amino acids, and addition or deletion of one or several (usually within 3, preferably within 2, and more preferably within 1) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus generally does not change the structure and function of the protein. In addition, universal polypeptide-PROTAC also includes monomeric and multimeric forms, as well as linear and non-linear polypeptides (such as cyclic peptides).

[0064] The present invention also includes active fragments, derivatives and analogs of the above-mentioned universal polypeptide-PROTAC. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the universal polypeptide-PROTAC of the present invention. The polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of an antigenic peptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6×His). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0065] The present invention also provides analogs of the universal polypeptide-PROTAC of the present invention. The difference between these analogs and SEQ ID NO: 5 may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0066] Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0067] The present invention provides a universal polypeptide-PROTAC, which may optionally contain a peptide linker. The size and complexity of the peptide linker may affect the activity of the protein. Generally, the peptide linker should have sufficient length and flexibility to ensure that the two connected proteins have sufficient spatial freedom to perform their functions. At the same time, the formation of α-helix or β-fold in the peptide linker should be avoided to affect the stability of the fusion protein.

[0068] The present invention also provides a pharmaceutical composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned universal polypeptide-PROTAC, and a pharmaceutically acceptable carrier, diluent, stabilizer and / or thickener, and can be prepared into a pharmaceutical type such as a lyophilized powder, tablet, capsule, syrup, solution or suspension.

[0069] "Pharmaceutically acceptable carrier or excipient" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.

[0070] The composition may be a liquid or solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.

[0071] Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers. Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0072] The composition may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0073] Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration. The pharmaceutical composition is used for (a) treating or preventing tumors; (b) enhancing the killing effect of T cells on tumor cells; (c) promoting the activation of T cells; (d) promoting the proliferation of T cells; (e) inhibiting the apoptosis of T cells. (a) enhancing the killing effect of NK cells on tumor cells; (b) increasing the expression level of CD107a, perforin, and granzyme secretion of NK cells; (c) increasing the activation of NK cells; (a) enhancing the phagocytic killing effect of M cells on tumor cells; (b) enhancing the activation of M cells; (c) increasing the M1 polarization of M cells.

[0074] Example 1: Three universal peptide-PROTAC molecules

[0075] 1. The structure of universal peptide-PROTAC is BZLV, in which B at the N-terminus is the cell-penetrating peptide TAT, and Z is the SH2 domain of SHP2 or its active fragment; the TAT cell-penetrating peptide and the ITIM motif binding region of the C-terminal SH2 domain of SHP2 are connected by two prolines, and the connection method is: TAT-Phe-Phe-C-SH2, and the amino acid sequence is YGRKKRRQRRR-PP-VRESQSHPGDFVL. The LV part is continued to be connected to its N-terminus, in which V is the peptide ligand ALAP(OH)YIP of the E3 ligase VHL; L is a connecting element, and the length of the peptide linker may affect the degradation activity of the PROTAC molecule, so three flexible GS peptide linkers of different lengths were designed. The three designed and synthesized universal peptide-PROTACs are named Y35, Y37, and Y39 respectively. Their amino acid sequences are as follows:

[0076] 1)Y35:YGRKKRRQRRRPPVRESQSHPGDFVL GS ALAP(OH)YIP

[0077] 2)Y37:YGRKKRRQRRRPPVRESQSHPGDFVL GSGS ALAP(OH)YIP

[0078] 3)Y39:YGRKKRRQRRRPPVRESQSHPGDFVL GSGSGS ALAP(OH)YIP

[0079] All were sent to the company for chemical synthesis.

[0080] 2. Identification of peptides

[0081] The purity of the synthesized peptides was analyzed by HPLC. 60 μL of sample was injected into the needle, the running time was 20 min, the flow rate was 1.0 mL / min, the detection wavelength was 214 nm, and the area of ​​the main peak was used to determine the purity of the sample. The results showed that the purity of the Y35 peptide was 97.700%, the purity of the Y37 peptide was 95.763%, and the purity of the Y39 peptide was 98.231% ( Figure 1 ).

[0082] The molecular weight of the synthesized peptides was detected by mass spectrometry. The results showed that the molecular weight of the Y35 peptide was 4076.10, the molecular weight of the Y37 peptide was 4220.10, and the molecular weight of the Y39 peptide was 4364.10, which were all consistent with the theoretical molecular weight ( Figure 2 ).

[0083] The results showed that the peptide was successfully synthesized and can be used for further in vitro and in vivo experimental studies.

[0084] Example 2: Comparison of three universal peptide-PROTAC PROTAC molecules

[0085] 1. Three universal peptides - PROTAC PROTAC molecules can enter T lymphocytes

[0086] The recombinant peptide can enter the T lymphocytes only if it binds to the intracellular segment of the inhibitory receptor ITIM on the cell membrane to exert its degradation effect. Using confocal microscopy, it was found that three universal peptides carrying fluorescent tags (FITC)-PROTAC PROTAC molecules can enter CD8 + In T cells, as shown in the figure ( Figure 3 ), the PROTAC molecules in the FITC-Control group without the transmembrane peptide TAT did not show fluorescence in the cells. The above results confirm that the TAT transmembrane sequence can guide the PROTAC molecules into the CD8 + Inside T cells.

[0087] 2. Surface plasmon resonance (SPR) detection of the affinity of three universal peptides-PROTACPROTAC to the ITIM sequence of the intracellular segment of PD-1

[0088] In order to verify that PROTAC molecules can interact with the inhibitory receptor PD-1 intracellular segment ITIM at the molecular level, the PD-1 ITIM sequence was synthesized. The synthesized sequence was subjected to SPR experiments with Y35, Y37, and Y39, respectively. The results showed that the KD (affinity constant) value of Y35 binding to PD-1-ITIM was 3.01X10 -5 M, the KD value of binding to Y37 is 5.36X10 -6 M, the KD value of binding to Y39 is 2.46X10 -6 M( Figure 4 ). This result shows that all candidate PROTAC molecules can bind to the ITIM of the intracellular segment of PD-1, and Y39 has the highest affinity.

[0089] 3. WB detection of three universal peptides - PROTACPROTAC's ability to degrade different immunosuppressive receptors

[0090] Jurkat, NK92 and THP-1 cell lines were selected, and the cells were stimulated with 100 nM / mL PROTAC drugs to extract proteins, and WB experiments were performed to detect the expression of immunosuppressive receptors ( Figure 5 ). It was found that Y39 had the highest degradation efficiency in PD-1, NKG2A, and SIRP-α (D max )Highest.

[0091] Example 3: Universal peptide-PROTACPROTAC degrades multiple checkpoint molecules through ubiquitin-dependent pathways

[0092] 1. Peptide PROTAC degrades CD8 + PD-1 and BTLA expressed on T cells

[0093] CD8 + T cells were stimulated with Y39 for 1 day, and the collected cells were incubated with VHL antibodies, and finally immunoprecipitated with PD-1 and BTLA. The results showed that when using VHL antibodies for immunoprecipitation, PD-1 and BTLA proteins were detected in the proteins pulled down by the Y39 group, while no protein precipitation was detected in the control group. The experimental results show that Y39 can form a ternary complex with PD-1 or BTLA, and can form a ternary complex with VHL ( Figure 6 ). PD-1 was labeled with CY3, and Hoechst was used to co-incubate with cells to label the cell nucleus. The cells were collected at different time points and fixed for immunofluorescence detection. The test results showed that red fluorescence could be detected in T cells incubated with PD-1 antibodies at 0 hours, indicating the presence of PD-1 expression on the cell membrane; however, the fluorescence intensity weakened over time until the fluorescence basically disappeared at 24 hours ( Figure 7 ), confirming that Y39 can degrade PD-1 over time. WB found that Y39 can degrade PD1 and BTLA in a dose-dependent manner (0nM, 5nM, 10nM, 50nM, 100nM) and time-dependent manner (0, 4, 12, 24, 48H). Figure 8 ).

[0094] 2. Peptide PROTAC degrades PD-1 and NKG2A expressed by NK cells

[0095] NK cells were stimulated with Y39 for 1 day, and the cells were collected and incubated with VHL antibodies, and finally immunoprecipitated with NKG2A and PD-1. The results showed that when immunoprecipitated with VHL antibodies, NKG2A and PD-1 proteins were detected in the proteins pulled down by the Y39 group, while no protein precipitation was detected in the control group. The experimental results show that Y39 can form a ternary complex with NKG2A or PD-1, and can form a ternary complex with VHL ( Fig. 9 ). NKG2A was labeled with CY3, and Hoechst was used to co-incubate with cells to label the cell nucleus. The cells were collected at different time points and fixed for immunofluorescence detection. The test results showed that red fluorescence could be detected at 0H in NK cells incubated with NKG2A antibody, indicating that NKG2A was expressed on the cell membrane; but the fluorescence intensity weakened over time until the fluorescence basically disappeared at 24H, confirming that Y39 can degrade NKG2A over time ( Fig.10WB experiments confirmed that with the increase of time and drug concentration, Y39 can degrade NKG2A and PD-1 in a dose-dependent manner (0nM, 5nM, 10nM, 50nM, 100nM) and time-dependent manner (0, 4, 12, 24, 48H), and the maximum degradation efficiency and half degradation concentration ( Fig.11 ).

[0096] 3. Peptide PROTAC degrades SIRPα and PD-1 expressed by macrophages

[0097] Macrophages were stimulated with Y39 for 1 day, and the cells were collected and incubated with VHL antibodies, and finally SIRPα and PD-1 were used for co-immunoprecipitation. The results showed that when VHL antibodies were used for co-immunoprecipitation, SIRPα and PD-1 proteins were detected in the proteins pulled down by the Y39 group, while no protein precipitation was detected in the control group. The experimental results showed that Y39 can form a ternary complex with SIRPα or PD-1, and can form a ternary complex with VHL ( Fig.12 ). SIRPα was labeled with CY3, and Hoechst was used to co-incubate with cells to label the cell nucleus. The cells were collected at different time points and fixed for immunofluorescence detection. The test results showed that red fluorescence could be detected in macrophages incubated with SIRPα antibody at 0H, indicating that SIRPα was expressed on the cell membrane; but the fluorescence intensity weakened over time until the fluorescence basically disappeared at 24H, confirming that Y39 can degrade SIRPα over time ( Fig.13 WB confirmed that with the increase of time and administration concentration, Y39 can degrade NKG2A and PD-1 in a dose-dependent manner (0nM, 5nM, 10nM, 50nM, 100nM) and time-dependent manner (0, 4, 12, 24, 48H). Fig.14 ).

[0098] 4. Peptide PROTAC degrades multiple immune checkpoint molecules through ubiquitin-dependent pathways

[0099] CD8 + T cells were co-incubated with Y39 and protease inhibitor MG132. After the cells were lysed, the lysate was incubated with PD-1 antibody overnight to detect PD-1 levels. Incubation of immune cells with Y39 alone can reduce the expression of inhibitory receptors, but co-incubation of cells with Y39 and MG132 will inhibit the degradation of inhibitory receptors, further confirming that Y39 degrades immunosuppressive receptors through the ubiquitination pathway ( Fig.15). VHL was knocked out in different cell lines (Jurkat, NK92, THP-1) using plasmids. Subsequently, the VHL knockout cell lines were co-incubated with Y39, and the degradation of immunosuppressive receptors was detected. It was found that in the VHL knockout cell lines, Y39 could not degrade inhibitory receptors, confirming that Y39 ubiquitin-linked immunosuppressive receptors on immune cells through VHL ubiquitin ligase, and then degraded inhibitory receptors through proteasomes ( Fig.16 ).

[0100] Example 4: Universal Peptide-PROTAC Effects of PROTAC on Immune Cell Function

[0101] 1. Universal peptide-PROTACPROTAC for CD8 + T cell effects

[0102] T cells were collected 24 hours after Y39 stimulation to detect the phosphorylation levels of AKT and ERK, the downstream signaling molecules of inhibitory receptors. The experimental results showed that after Y39 stimulation, the total protein expression levels of SHP2, AKT and ERK did not change significantly, but the expression levels of phosphorylated proteins p-SHP2, p-AKT and p-ERK increased ( Fig.17 A, B). Y39 was co-incubated with T cells for 2 days and then flow cytometry was performed. The results showed that the Y39 treatment group could significantly increase the expression of Ki67 on T cells ( Fig.17 C), which indicates that Y39 can enhance the proliferation of T cells; CFSE results also show that compared with the control group, Y39 significantly enhances the proliferation of T cells ( Fig.17 D). Flow cytometry results showed that the Y39 treatment group could significantly increase the expression of CD69 on T cells ( Fig.17 E), indicating that Y39 can activate T cells. RTCA detection was used to detect the ability of Y39 to kill human colon cancer cells HCT116 with a T cell-to-target ratio of 10:1, and the killing rate was calculated ( Fig.17 F). The experimental results showed that the T cell cytotoxicity of the Y39 group was significantly increased compared with the control group (P<0.001). Flow cytometry results showed that the Y39 treatment group could significantly reduce the expression of Cleaved caspase-3 on T cells ( Fig.17 G), confirming that Y39 can inhibit T cell apoptosis. Flow cytometry staining of Annexin V and 7AAD was used to detect T cell apoptosis in different treatment groups. Compared with the control group, T cell apoptosis in the Y39 treatment group was significantly reduced ( Fig.17 H).

[0103] 2. Universal peptide-PROTAC The effect of PROTAC on NK cells

[0104] NK cells were collected 24 hours after Y39 stimulation to detect the phosphorylation of SHP2 bound to inhibitory receptors. The experimental results showed that the expression levels of phosphorylated proteins p-SHP2 (Y580) and p-SHP2 (Y542) were reduced after Y39 stimulation. T cells were collected 24 hours after Y39 stimulation to detect the phosphorylation levels of inhibitory receptor downstream signaling molecules SYK and ZAP70. The experimental results showed that the total protein expression levels of SYK and ZAP70 did not change significantly after Y39 stimulation, but the expression levels of phosphorylated proteins p-SYK and p-ZAP70 increased ( Fig.18 A). NK cells were collected after Y39 stimulation for 3 days, and the levels of granzyme B, perforin, and CD107a were detected by flow cytometry. Compared with the control group, Y39 significantly promoted the expression of granzyme B, perforin, and CD107a in NK cells ( Fig.18 B). The ability of Y39 to kill human colon cancer cells HCT116 with NK cells was tested by RTCA under the condition of effector cell: target cell ratio of 2.5:1, and the killing rate was calculated ( Fig.18 C). The experimental results showed that the NK cell cytotoxicity in the Y39 group was significantly increased compared with the control group (P<0.001).

[0105] 3. Universal peptide-PROTACPROTAC's effect on macrophages

[0106] Macrophages were collected after Y39 stimulation for 1 day to detect the phosphorylation of SHP2 bound to inhibitory receptors. The experimental results showed that the total protein expression level of SHP2 did not change significantly after Y39 stimulation, but the expression levels of phosphorylated proteins p-SHP2 (Y580) and p-SHP2 (Y542) decreased. The total protein expression levels of SYK and AKT in macrophages after Y39 stimulation for 24 hours did not change significantly, but the expression levels of phosphorylated proteins p-SYK and p-AKT increased ( Fig.19 A). Macrophages were collected after Y39 stimulation for 3 days, and the expression levels of tumor-promoting macrophage markers CD206 and IL-10, as well as tumor-suppressing macrophage markers CD86 and iNOS were detected by flow cytometry. Compared with the control group, Y39 promoted the transformation of macrophages into tumor-suppressing macrophages ( Fig.19 B). Flow cytometry was used to detect the phagocytic effect of macrophages on human colon cancer cells HCT116 at an effector-target ratio (effector cells: target cells) of 1:1. Fig.19 C. The results showed that compared with the control group, the phagocytic efficiency of the Y39 stimulation group was significantly improved (P<0.001). The experimental results showed that Y39 can enhance the phagocytic ability of macrophages on human colon cancer cells HCT116 ( Fig.19C).

[0107] Example 5: Study on the anti-tumor effect of PROTAC in vivo

[0108] 1. Universal peptide-PROTAC Detection of the tumor inhibition effect of PROTAC on mouse colon cancer

[0109] A subcutaneous transplant tumor model of MC38 colon cancer was established in mice. The tumor growth curve of C57BL / 6 mice showed that compared with the control group, the tumor growth of mice in the Y39 group and the C-SH2 group was significantly inhibited (P<0.05). After 7 doses of administration, the mice were killed, and the tumors were dissected out and weighed. Compared with the control group, the average tumor weight of the Y39 group was significantly reduced (P<0.05), and the tumor inhibition rate of the 5μg group was the highest. The experimental results showed that Y39 significantly inhibited the growth of MC38 colon cancer tissue, and was superior to the therapeutic effect of C-SH2 ( Fig. 20 AC). The mouse MC38 transplanted tumor tissue was digested and prepared into a cell suspension, and the levels of the effector molecule GZMB of T cells and NK cells were detected by flow cytometry. Fig. 20 As shown in D and E, compared with the control group, the expression of GZMB in T and NK cells in the Y39 group was significantly increased. The results showed that compared with the control group and the C-SH2 group, Y39 enhanced the expression of CD8 + Expression of T and NK cell killing-related molecules. Flow cytometry detected that the tumor-suppressing molecule TNF-α on macrophages was significantly reduced, and the tumor-promoting molecule CD206 was significantly increased. The results showed that compared with the control group and C-SH2 group, Y39 increased the expression of tumor-suppressing molecules in mouse tumor tissues and reduced the expression of tumor-promoting molecules ( Fig. 20 F, G).

[0110] 2. Universal peptide-PROTAC Detection of the tumor inhibition effect of PROTAC on mouse liver cancer

[0111] A Hepa1-6 liver cancer subcutaneous transplant tumor model was established in mice. The tumor growth curve of C57BL / 6 mice showed that compared with the control group, the tumor growth of mice in the Y39 group, C-SH2 group and PD-1 group was significantly inhibited (P<0.05). After 7 doses of administration, the mice were killed, and the tumors were dissected out and weighed. Compared with the control group, the average tumor weight of the Y39 group, C-SH2 group and PD-1 group was significantly reduced (P<0.05). The results showed that the tumor weight of the Y39 (5μg) group was the lightest. The experimental results showed that Y39 significantly inhibited the growth of Hepa-1-6 tumor tissue, and was superior to the therapeutic effect of C-SH2 and PD-1 antibodies ( Fig.21 AC). The mouse transplanted tumor tissue was digested and prepared into a cell suspension, and the number of T cells and the expression level of the proliferation marker Ki67 were detected by flow cytometry. Fig.21As shown in D and E, compared with the control group, the number of T cells in the Y39 group increased significantly, and the expression of Ki67 also increased significantly. The results showed that compared with the control group, C-SH2 group and PD-1 group, Y39 could increase the CD8 + T infiltration, and has stronger proliferation ability than C-SH2 group. Flow cytometry detection showed that the tumor suppression-related molecule CD86 on macrophages was significantly reduced, and the tumor promotion-related molecules CD206 and IL-10 were significantly increased. The results showed that compared with the control group and C-SH2 group, Y39 increased the expression of tumor suppression-related molecules in mouse tumor tissues and reduced the expression of tumor promotion molecules ( Fig.21 F, G).

[0112] 3. Universal peptide-PROTAC Detection of the tumor inhibition effect of PROTAC on liver cancer in mice reconstructed with human immune system

[0113] PBMCs were injected into severely immunodeficient NCG mice through the tail vein, and human liver cancer cells (HepG2) were inoculated subcutaneously. After two weeks, blood was collected to detect the number of hCD45. If it reached more than 10-15% of the mouse PBMC, it was considered that the model was successfully constructed. We selected mice with successfully constructed models for drug administration and recorded tumor growth. Subsequently, after a strong graft-versus-host reaction occurred in some mice, the mice were killed and the tumor tissue was peeled off for detection. The mouse tumor growth curve showed that compared with the control group, the tumor growth of mice in the Y39 group, C-SH2 group and PD-1 group was significantly inhibited (P<0.05). After 6 doses of administration, the mice were killed, the tumors were dissected out and weighed. Compared with the control group, the average tumor weight of the Y39 group, C-SH2 group and PD-1 group was significantly reduced (P<0.05). The results showed that the tumor weight of the Y39 group was the lightest. The experimental results showed that Y39 significantly inhibited the growth of human tumor tissue, and was superior to the therapeutic effect of C-SH2 and PD-1 antibodies ( Fig. 22 AC). The tumor tissue was digested and prepared into a cell suspension, and the number of T cells was detected by flow cytometry and immunofluorescence. Fig.23 As shown in D and E, compared with the control group, the number of T cells in the Y39 group increased significantly. The results showed that Y39 could increase the CD8 + T infiltration. The expression levels of Perforin and TNF-α on T cells were detected by flow cytometry. Fig. 22 As shown in F and G, compared with the control group, the expression of killing-related molecules on T cells in the Y39 group was significantly increased. The results showed that compared with the control group, C-SH2 group and PD-1 group, Y39 had a stronger killing ability than the C-SH2 group ( Fig. 22 F, G).

[0114] 4. Universal peptide-PROTACPROTAC tumor inhibition effect detection on PD-1 resistant colon cancer

[0115] A PD-1-resistant colon cancer mouse tumor model was constructed to detect whether Y39 could enhance the tumor inhibition effect of PD-1-resistant tumors in mice. The mouse tumor growth curve showed that compared with the control group, the tumor growth of mice in the Y39 group, C-SH2 group and PD-1 group was significantly inhibited (P<0.05). After 7 doses of administration, the mice were killed, and the tumors were dissected out and weighed. Compared with the control group, the average tumor weight of the Y39 group, C-SH2 group and PD-1 group was significantly reduced (P<0.05), and the results showed that the tumor weight of the Y39 group was the lightest. The experimental results showed that Y39 significantly inhibited the growth of human tumor tissue, and was superior to the therapeutic effect of C-SH2 and PD-1 antibodies. The experimental results confirmed that PD-1 antibodies did not fully exert their anti-tumor effects in PD1-resistant tumors, while Y39 still had anti-tumor ability against PD1-resistant tumors, confirming that Y39 can activate immune cells through multiple immune checkpoint pathways ( Fig.23 AC). Further analysis of the cause showed that the tumor tissue was digested into a cell suspension, and the number of T cells was detected by flow cytometry and immunofluorescence. Fig.23 As shown in D and E, compared with the control group, the number of T cells in the Y39 group increased significantly. The results showed that Y39 could increase the CD8 + The results showed that Y39 could increase CD8 T infiltration in mouse tumor tissues compared with the control group, C-SH2 group and PD-1 group. + T infiltration, and has stronger proliferation ability than C-SH2 group. Flow cytometry detected that the tumor suppression-related molecule CD86 on macrophages increased significantly, and the tumor promotion-related molecule CD206 decreased significantly. The results showed that compared with the control group and C-SH2 group, Y39 increased the expression of tumor suppression-related molecules in mouse tumor tissues and reduced the expression of tumor promotion molecules ( Fig.23 F). Activation-related molecule CD69 on NK cells was detected by flow cytometry. The results showed that compared with the control group and C-SH2 group, the expression of activation-related molecule CD69 on NK cells in the Y39 group was significantly increased ( Fig.23 G). The above results indicate that Y39 can exert anti-tumor effects on PD1-resistant tumors by affecting the functions of multiple immune cells.

[0116] The universal polypeptide PROTAC constructed by the present invention can specifically bind to multiple immune checkpoint molecules at the same time through its C-SH2 domain, and ubiquitinate and degrade the immune checkpoints through its E3 ligase ligand domain, fundamentally avoiding the compensatory upregulation of immune checkpoint molecules and the generation of drug resistance.

[0117] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. Universal peptide targeting multiple immune checkpoints - PROTAC, characterized by: The structure of the universal peptide-PROTAC is BZLV; in: B is a cell-penetrating peptide; Z is the SH2 domain of SHP2 or SHP1 or an active fragment thereof; L is no or connected element; V is the ligand of VHL or other E3 molecules.

2. The universal polypeptide-PROTAC targeting multiple immune checkpoints according to claim 1, characterized in that: The amino acid sequence of part B is YGRKKRRQRRRPP.

3. The universal polypeptide-PROTAC targeting multiple immune checkpoints according to claim 1, characterized in that: The amino acid sequence of the Z portion is VRESQSHPGDFVL.

4. The universal polypeptide-PROTAC targeting multiple immune checkpoints according to claim 1, characterized in that: When the L portion is a connecting element, the amino acid sequence is GSGSGS.

5. The universal polypeptide-PROTAC targeting multiple immune checkpoints according to claim 1, characterized in that: The amino acid sequence of the V portion is ALAP(OH)YIP.

6. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains the universal polypeptide-PROTAC according to claim 1 and a pharmaceutically acceptable carrier thereof.

7. The use of a universal polypeptide-PROTAC targeting multiple immune checkpoints as claimed in claim 1, characterized in that: The universal polypeptide-PROTAC is used to prepare a drug for treating or preventing tumors.

8. The use of a universal polypeptide-PROTAC targeting multiple immune checkpoints as claimed in claim 1, characterized in that: The universal polypeptide-PROTAC is used to prepare drugs that enhance the killing effect of T cells on tumor cells, promote the activation of T cells, promote the proliferation of T cells, and inhibit the apoptosis of T cells.

9. The use of a universal polypeptide-PROTAC targeting multiple immune checkpoints as claimed in claim 1, characterized in that: The universal polypeptide-PROTAC is used to prepare drugs that enhance the killing effect of NK cells on tumor cells, increase the expression level of NK cell CD107a, perforin, granzyme secretion, and increase the activation of NK cells.

10. The use of a universal polypeptide-PROTAC targeting multiple immune checkpoints as claimed in claim 1, characterized in that: The universal polypeptide-PROTAC is used to prepare drugs that enhance the phagocytic killing effect of M cells on tumor cells, enhance the activation of M cells, and increase the M1 polarization of M cells.

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