PROTAC, a universal peptide targeting multiple immune checkpoints, and its applications

Abstract

This invention relates to a universal peptide-PROTAC targeting multiple immune checkpoints and its applications. Existing immune checkpoint inhibitors pose a risk of compensatory upregulation of immune checkpoint molecule expression after administration, leading to drug resistance. The universal peptide-PROTAC of this invention has the structure B-Z-L-V, where B is a cell-penetrating peptide, Z is the SH2 domain of SHP2 or SHP1 or its active fragment, L is absent or a linker element, and V is a ligand for VHL or other E3 molecules. This invention, through its C-SH2 domain, can simultaneously and specifically bind to multiple immune checkpoint molecules, and through its E3 ligase ligand domain, it ubiquitinates and degrades immune checkpoints, fundamentally avoiding compensatory upregulation of immune checkpoint molecule expression and the resulting drug resistance.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, specifically to a universal polypeptide-PROTAC that targets multiple immune checkpoints and its applications. Background Technology

[0002] Immune checkpoint blockade is an important method in tumor immunotherapy, utilizing the body's own immune system to fight against malignant tumors and substantially improve patients' overall survival. However, the response rate of patients is low when using single-immune checkpoint blockers as monotherapy. This is because the tumor microenvironment contains intricate immune signaling pathways due to the interactions between tumor cells, immune cells, cytokines, etc., resulting in limited efficacy of immunotherapy targeting only one immunosuppressive molecule. Therefore, combination therapy targeting different immune checkpoints has become a new strategy to improve patient response rates, such as universal immune checkpoint blockades (UICBs) that target common sites of different immune checkpoints.

[0003] Patent ZL2020100339548 discloses an anti-tumor fusion protein, its preparation method, and its application. It describes the preparation of a mimic polypeptide with an SHP2 C-SH2 domain capable of binding to ITIM, containing a TAT protein, optional linker elements, and the ITIM motif binding region of SHP2 or SHP1's SH2, or its active fragment. This polypeptide exhibits versatility in binding to multiple immune checkpoint molecules, can inhibit the ITIM motif downstream of various immunosuppressive molecules, and possesses broad-spectrum activity, enhancing the function of T cells, NK cells, and macrophages, thus demonstrating anti-tumor activity. However, this polypeptide exerts its effect only through competitive binding to the ITIM motif, and there is a risk of compensatory upregulation of immune checkpoint molecule expression after administration, leading to drug resistance.

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

[0005] The purpose of this invention is to provide a universal polypeptide, PROTAC, that targets multiple immune checkpoints and its applications, in order to address the problem of drug resistance in existing immune checkpoint inhibitors.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A universal polypeptide-PROTAC targeting multiple immune checkpoints is provided, wherein the structure of the universal polypeptide-PROTAC is BZLV;

[0008] in:

[0009] B is a cell-penetrating peptide;

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

[0011] L represents no or no connecting element;

[0012] V is a ligand for VHL or other E3 molecules.

[0013] Furthermore, the amino acid sequence of part B 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, the pharmaceutical composition comprising the universal polypeptide-PROTAC and its pharmaceutically acceptable carrier.

[0018] On the other hand, the application of a universal peptide-PROTAC targeting multiple immune checkpoints as described above is provided, the universal peptide-PROTAC being used to prepare drugs for treating or preventing tumors.

[0019] On the other hand, it provides applications for PROTAC, a universal peptide targeting multiple immune checkpoints as described above.

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

[0021] On the other hand, the application of the universal polypeptide-PROTAC targeting multiple immune checkpoints as described above is provided. 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.

[0022] On the other hand, the application of the universal polypeptide-PROTAC targeting multiple immune checkpoints as described above is provided. 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.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention provides a universal peptide-PROTAC targeting multiple immune checkpoints and its applications. Through its C-SH2 domain, it can simultaneously and specifically bind to multiple immune checkpoint molecules, and through its E3 ligase ligand domain, it ubiquitinates and degrades immune checkpoints, fundamentally avoiding compensatory upregulation of immune checkpoint molecules and the resulting drug resistance. Furthermore, as the first universal immune checkpoint degrader, this universal peptide-PROTAC is functionally superior to the C-SH2 domain, significantly enhancing the antitumor activity of immune cells such as T lymphocytes, NK cells, and macrophages, exhibiting broad-spectrum antitumor activity while avoiding the toxic side effects and increased treatment costs associated with various combination therapies. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other embodiments can be obtained from these drawings without creative effort.

[0026] Figure 1 This is an HPLC chromatogram of the purity of the synthesized peptide-PROTAC. In the chromatogram, A represents Y35, B represents Y37, and C represents Y39.

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

[0028] Figure 3 This is a laser confocal microscopy detection image of the synthesized polypeptide PROTAC entering CD8+ T cells.

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

[0030] Figure 5 This is a Western blotting graph showing the degradation of immunosuppressive receptors PD-1, NKG2A, and SIRPα in immune cells by Y35, Y37, and Y39 cells. In the graph, A represents PD-1 degradation, B represents NKG2A degradation, C represents SIRPα degradation, and D is a table showing the maximum degradation efficiency.

[0031] Figure 6This is a diagram illustrating the formation of the PD-1-Y39-VHL and BTLA-Y39-VHL ternary complexes as verified by Co-IP. In the diagram, A represents the detection of the PD-1-Y39-VHL ternary complex, and B represents the detection of the BTLA-Y39-VHL ternary complex.

[0032] Figure 7 This is a diagram of the inhibitory receptor PD-1 in Y39-degraded T cells detected by laser confocal microscopy.

[0033] Figure 8 This is a Western blotting graph showing the degradation of inhibitory receptors PD-1 and BTLA by Y39 in T cells. In the graph, A represents the degradation of PD-1 by Y39 at different concentrations, B represents the degradation of BTLA by Y39 at different concentrations, C represents the degradation of PD-1 by Y39 at different time points, D represents the degradation of BTLA by Y39 at different time points, and E is a table showing the maximum degradation efficiency and the half-maximal degradation concentration.

[0034] Figure 9 This is a diagram illustrating the formation of the NKG2A-Y39-VHL and PD-1-Y39-VHL ternary complex verified by Co-IP.

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

[0036] Figure 10 This is a laser confocal microscopy detection image of Y39 degradation NKG2A.

[0037] Figure 11 This is a Western blotting (WB) image showing the degradation of inhibitory receptors NKG2A and PD-1 in NK cells by Y39. In the image, A represents the degradation of NKG2A by Y39 at different concentrations, B represents the degradation of PD-1 by Y39 at different concentrations, and C is a table showing the maximum degradation efficiency and the half-maximum degradation concentration.

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

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

[0040] Figure 13 This is a SIRPα image of Y39 degradation detected by laser confocal microscopy.

[0041] Figure 14 This is a Western blot (WB) image showing the degradation of SIRPα and PD-1 in macrophages by Y39. In the image, A represents the degradation of SIRPα by Y39 at different concentrations, B represents the degradation of PD-1 by Y39 at different concentrations, and C is a table showing the maximum degradation efficiency and the half-maximum degradation concentration.

[0042] Figure 15 This diagram shows how proteasome inhibitors reduce the degradation of Y39 at immune checkpoints. In the diagram, A represents the reduction of Y39 degradation of PD-1 and BTLA, B represents the reduction of Y39 degradation of NKG2A, and C represents the reduction of Y39 degradation of SIRPα.

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

[0044] Figure 17 This is a graph showing the effect of peptide-PROTAC on CD8+ T cells. In the graph, A and B show the upregulation of activation signaling pathways by Y39 as detected by Western blotting; C shows the promotion of Ki67 expression on CD8+ T cells by Y39 as detected by flow cytometry; D shows the promotion of T cell proliferation by Y39 as detected by flow cytometry; E shows the promotion of CD69 expression on CD8+ T cells by Y39 as detected by flow cytometry; F shows the enhancement of T cell killing ability by Y39 as detected by RTCA; G shows the downregulation of Cleaved caspase-3 expression by Y39 as detected by flow cytometry; and H shows the inhibition of CD8+ T cell apoptosis by Y39 as detected by flow cytometry.

[0045] Figure 18 This is a diagram showing the effect of peptide-PROTAC on NK cells. In the diagram, A represents the upregulation of NK cell activation-related signaling pathways by Y39 as detected by Western blotting; B represents the increase in NK cell activation and killing-related molecule expression by Y39 as detected by flow cytometry; and C represents the promotion of NK cell killing function by Y39 as detected by RTCA.

[0046] Figure 19 This is a diagram showing the effect of peptide-PROTAC on macrophages. In the diagram, A represents the upregulation of macrophage activation-related signaling pathways by Y39 as detected by Western blotting; B represents the increase in M1 phenotype expression and decrease in M2 phenotype expression by Y39 as detected by flow cytometry; C represents the promotion of macrophage phagocytic function by Y39 as detected by flow cytometry; and D represents the promotion of macrophage phagocytic function by Y39 as detected by laser confocal microscopy.

[0047] Figure 20 This is a graph showing the tumor-suppressing effect of peptide-PROTAC on colon cancer in mice. In the graph, A is a photograph of mouse tumors; B is the tumor growth curve of mice; C is the average tumor weight of mice; D / E are the expression of GZMB in tumor-infiltrating NK / T cells detected by flow cytometry; and F / G are the expression of TNFα / CD206, related molecules in tumor-infiltrating macrophages, detected by flow cytometry.

[0048] Figure 21This is a graph showing the tumor-suppressing effect of peptide-PROTAC on mouse liver cancer. In the graph, A is a photograph of a mouse tumor, B is the tumor growth curve of a mouse tumor, 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 M2 / M1 ratio in mouse tumor tissue detected by flow cytometry, and G is the expression of the M2-related molecule IL-10.

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

[0050] In the figure, A is a photograph of a mouse tumor, B is a growth curve of a mouse tumor, C is the average tumor weight of mice, D is the proportion of CD8+ T cells infiltrating the tumor tissue detected by flow cytometry, E is the proportion of CD8+ T cells infiltrating the tumor tissue detected by immunofluorescence, and F / G is the expression of Perforin / TNFα, a molecule related to CD8+ T cell killing, in the tumor tissue.

[0051] Figure 23 This is a graph showing the tumor-suppressing effect of peptide-PROTAC on PD-1 resistant colon cancer. In the graph, A is a mouse tumor photograph, B is a mouse tumor growth curve, C is the average tumor weight of mice, D is the proportion of CD8+ T cells infiltrating the tumor tissue as detected by flow cytometry, E is the proliferation capacity of infiltrating CD8+ T cells in the tumor tissue as detected by flow cytometry, F is the M2 / M1 ratio in the mouse tumor tissue, and G is the activation status of infiltrating NK cells in the tumor tissue as detected by flow cytometry.

[0052] Figure 24 This is a schematic diagram of the mechanism of action of the universal polypeptide PROTAC. Detailed Implementation

[0053] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0054] It should be noted that similar labels and letters indicate similar items; therefore, once an item is defined in one embodiment, it does not need to be further defined and explained in subsequent embodiments. The specific implementation process is described in steps in some embodiments only for clarity and accuracy and should not be construed as a limitation on the order. Furthermore, the chemical substances used in the steps described in the embodiments are all existing substances or commercially available products.

[0055] Proteolysis-targeting chimeras (PROTACs) are a drug development technology that utilizes the ubiquitin-proteasome system to degrade target proteins. Structurally, a PROTAC consists of an E3 ubiquitin ligase ligand and a target protein ligand, linked by a suitable linker. The peptide-PROTAC binds to the E3 ligase via its E3 ubiquitin ligase ligand and targets the target protein via its target protein ligand, forming a ternary complex. This complex hijacks the activity of the E3 ubiquitin ligase, ubiquitinates the target protein, and subsequently mediates its proteasome degradation. The advantages of peptide-PROTACs are: 1. Extremely high targeting specificity, as the PROTAC molecule needs to simultaneously bind to both the target protein (POI) and the E3 ligase to form the ternary complex and induce degradation. This increases selectivity and avoids unnecessary off-target effects and toxic side effects; 2. PROTACs do not need to occupy the active or binding sites of the POI; as long as they can bind to the POI and induce its ubiquitination, degradation can be achieved. Therefore, the target range can be expanded to include 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 target molecules, PROTAC molecules are not consumed and can be recycled and reused repeatedly. They have a catalytic degradation effect, so only a low dose is needed to achieve extremely high activity, reducing the dosage and cost of drugs.

[0056] This invention designs a universal polypeptide, PROTAC, that targets multiple immune checkpoints. Unlike most existing immunotherapeutic drugs that target only one immunosuppressive molecule, PROTAC can simultaneously and specifically bind to multiple immune checkpoint molecules, including transmembrane peptide sequences, ITIM domain-targeting peptide sequences, linker sequences, and E3 ligase recruitment peptide sequences. It can effectively target and bind to multiple intracellular immune checkpoint molecules containing ITIM sequences, leading to the degradation of these proteins and restoring the anti-tumor activity of immune cells. This provides an effective solution for tumor immunotherapy, and it can ubiquitinate and degrade multiple immune checkpoint molecules, fundamentally eliminating immunosuppressive signals caused by immune checkpoints.

[0057] The structure of the universal polypeptide-PROTAC is BZLV, where B is a cell-penetrating peptide, Z is the SH2 domain of SHP2 or SHP1 or its active fragment, L is absent or a linker 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 part, or its active fragment, is the peptide that targets the ITIM domain and recognizes it. Its amino acid sequence is SEQ ID NO: 2, specifically VRESQSHPGDFVL.

[0060] The linker element in the L part is a linker with an amino acid sequence of SEQ ID NO: 3, specifically GSGSGS.

[0061] The ligand of VHL or other E3 molecules in the V portion is the E3 ligase recruiting peptide, whose 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] The universal peptide-PROTAC also includes variant forms. These variant forms include (but are not limited to): deletions, insertions, and / or substitutions of 1-3 amino acids (typically 1-2, more preferably 1), and additions or deletions of one or more amino acids (typically up to 3, more preferably up to 2, and more preferably up to 1) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the function of the protein. Similarly, adding or deleting one or more amino acids at the C-terminus and / or N-terminus typically does not alter the structure and function of the protein. Furthermore, the universal peptide-PROTAC also includes monomeric and multimeric forms, as well as peptides including linear and non-linear forms (such as cyclic peptides).

[0064] This invention also includes active fragments, derivatives, and analogs of the aforementioned universal peptide-PROTAC. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a peptide that substantially retains the function or activity of the universal peptide-PROTAC of this invention. The peptide fragments, derivatives, or analogs of this invention may be (i) peptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) peptides having substituent groups in one or more amino acid residues, or (iii) peptides formed by fusing an antigenic peptide with another compound (e.g., a compound that prolongs the half-life of the peptide, such as polyethylene glycol), or (iv) peptides formed by fusing an additional amino acid sequence to this peptide sequence (fusion proteins formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6×His). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0065] This invention also provides analogs of the general polypeptide-PROTAC of this invention. These analogs may differ from SEQ ID NO: 5 in that they may be differences in amino acid sequence, differences in modifications that do not affect the sequence, or both. Analogs also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the polypeptides of this invention are not limited to the representative polypeptides exemplified above.

[0066] Modifications (typically without altering the primary structure) include chemically derived forms of peptides, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the peptide to glycosylating enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include peptides modified to improve their resistance to proteolysis or optimize their solubility.

[0067] This invention provides a universal peptide-PROTAC, which optionally contains a peptide linker. The size and complexity of the peptide linker can affect the activity of the protein. Generally, the peptide linker should have sufficient length and flexibility to ensure that the two linked proteins have sufficient spatial freedom to perform their functions. At the same time, the formation of α-helices or β-sheets in the peptide linker should be avoided to prevent the formation of these structures that could negatively impact 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 containing the aforementioned universal polypeptide-PROTAC, as well as a pharmaceutically acceptable carrier, diluent, stabilizer, and / or thickener, and can be prepared into a dosage form such as a lyophilized powder, tablet, capsule, syrup, solution, or suspension.

[0069] "Pharmaceutical-acceptable carrier or excipient" refers to one or more compatible solid or liquid fillers or gel substances that 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 and with the active ingredient of the present invention without significantly reducing the efficacy of the active ingredient.

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

[0071] Pharmaceutically acceptable examples of carrier components 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, glycerin, mannitol, sorbitol, etc.), and emulsifiers. Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

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

[0073] These substances are typically formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH typically around 5-8, preferably around 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration. The pharmaceutical composition is used for (a) the treatment or prevention of tumors; (b) enhancing the killing effect of T cells on tumor cells; (c) promoting T cell activation; (d) promoting T cell proliferation; and (e) inhibiting T cell apoptosis. (a) Enhancing the killing effect of NK cells on tumor cells; (b) Increasing NK cell CD107a expression levels, perforin, and granzyme secretion; (c) Increasing NK cell activation; (a) Enhancing the phagocytic killing effect of M cells on tumor cells; (b) Enhancing M cell activation; and (c) Increasing M1 polarization of M cells.

[0074] Example 1: Three Universal Peptide-PROTAC Molecules

[0075] 1. The structure of the universal peptide-PROTAC is BZLV, where B at the N-terminus represents the cell-penetrating peptide TAT, and Z represents the SH2 domain of SHP2 or its active fragment. The TAT membrane-penetrating peptide and the ITIM motif binding region of the C-terminal SH2 domain of SHP2 are linked by two proline residues in the following manner: TAT-Phe-Phe-C-SH2, with the amino acid sequence YGRKKRRQRRR-PP-VRESQSHPGDFVL. The LV portion is then added to the N-terminus, where V represents the peptide ligand ALAP(OH)YIP of the E3 ligase VHL, and L represents the linker element. The length of the peptide linker may affect the degradation activity of the PROTAC molecule; therefore, three flexible GS peptide linkers of different lengths were designed. The three designed and synthesized universal peptide-PROTACs were named Y35, Y37, and Y39, respectively. Their amino acid sequences are shown below:

[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 of it was sent to the company for chemical synthesis.

[0080] 2. Identification of polypeptides

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

[0082] The molecular weights of the synthesized peptides were determined using mass spectrometry. The results showed that the molecular weights of peptide Y35 were 4076.10, Y37 were 4220.10, and Y39 were 4364.10, all consistent with the theoretical molecular weights. Figure 2 ).

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

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

[0085] 1. All three universal polypeptides—PROTAC—are capable of entering T lymphocytes.

[0086] The ability of recombinant peptides to enter T lymphocytes is a prerequisite for their binding to the intracellular segment of their inhibitory receptor ITIM on the cell membrane, thereby exerting their degradation effect. Confocal microscopy revealed that three universal peptides carrying fluorescent tags (FITC), namely PROTAC, can all enter CD8+. + In T cells, as shown in the figure ( Figure 3 In the FITC-Control group, PROTAC molecules without the transmembrane peptide TAT did not exhibit intracellular fluorescence. These results confirm that the TAT transmembrane sequence can guide PROTAC molecules into CD8. + Within T cells.

[0087] 2. Surface plasmon resonance (SPR) assay to detect the affinity of three universal peptides—PROTAC and its affinity for the intracellular ITIM sequence of PD-1.

[0088] To verify that PROTAC molecules can interact with the intracellular ITIM segment of the inhibitory receptor PD-1 at the molecular level, the ITIM sequence of PD-1 was synthesized. The synthesized sequence was subjected to SPR experiments with Y35, Y37, and Y39, respectively. The results showed that the KD (affinity constant) for PD-1-ITIM binding at Y35 was 3.01 x 10⁻⁶. -5 M, with a KD value of 5.36 x 10⁻⁶ when combined with Y37. -6 M, with a KD value of 2.46 x 10⁻⁶ when combined with Y39. -6 M( Figure 4 This result indicates that all candidate PROTAC molecules can bind to ITIM in the intracellular domain of PD-1, with Y39 showing the highest affinity.

[0089] 3. Western blot analysis of the degradation ability of three universal peptides—PROTAC—on different immunosuppressive receptors.

[0090] Jurkat, NK92, and THP-1 cell lines were selected. After stimulation with 100 nM / mL PROTAC, proteins were extracted, and Western blotting was performed to detect the expression of immunosuppressive receptors. Figure 5 Y39 was found to have the highest degradation efficiency (D) in PD-1, NKG2A, and SIRP-α. max )Highest.

[0091] Example 3: Universal Peptide-PROTAC PROTAC degrades multiple checkpoint molecules via a ubiquitin-dependent pathway.

[0092] 1. Peptide PROTAC degrades CD8 + T cell expression of PD-1 and BTLA

[0093] CD8 + T cells were stimulated with Y39 for 1 day, and the cells were collected and incubated with VHL antibody. Finally, PD-1 and BTLA were used for immunoprecipitation. The results showed that PD-1 and BTLA proteins were detected in the proteins precipitated by the Y39 group during immunoprecipitation with VHL antibody, while no protein precipitation was detected in the control group. The experimental results indicate that Y39 can form a ternary complex with either PD-1 or BTLA and VHL. Figure 6 PD-1 was labeled with CY3, and cell nuclei were labeled by co-incubation with Hoechst. Cells were collected at different time points, fixed, and then subjected to immunofluorescence detection. The results showed that T cells incubated with PD-1 antibody showed red fluorescence at 0H, indicating the presence of PD-1 expression on the cell membrane; however, the fluorescence intensity decreased over time, and the fluorescence essentially disappeared after 24H. Figure 7 This study confirmed that Y39 can degrade PD-1 over time. Western blotting revealed that Y39 can degrade PD-1 and BTLA in a dose-dependent manner (0 nM, 5 nM, 10 nM, 50 nM, 100 nM) and time-dependent manner (0, 4, 12, 24, 48 H). Figure 8 ).

[0094] 2. The peptide PROTAC degrades PD-1 and NKG2A expressed on NK cells.

[0095] NK cells were stimulated with Y39 for 1 day, and the cells were collected and incubated with VHL antibody. Finally, NKG2A and PD-1 were co-precipitated using immunoprecipitation. The results showed that NKG2A and PD-1 proteins were detected in the proteins precipitated by the Y39 group during VHL antibody co-precipitation, while no protein precipitation was detected in the control group. The experimental results indicate that Y39 can form a ternary complex with either NKG2A or PD-1 and VHL. Figure 9 NKG2A was labeled with CY3, and cell nuclei were labeled by co-incubation with Hoechst. Cells were collected at different time points, fixed, and then subjected to immunofluorescence detection. The results showed that NK cells incubated with NKG2A antibody exhibited red fluorescence at 0H, indicating the presence of NKG2A expression on the cell membrane; however, the fluorescence intensity decreased over time, and the fluorescence essentially disappeared after 24H, confirming that Y39 can degrade NKG2A over time. Figure 10Western blot experiments confirmed that Y39 could degrade NKG2A and PD-1 in a dose-dependent manner (0 nM, 5 nM, 10 nM, 50 nM, 100 nM) and time-dependent manner (0, 4, 12, 24, 48 H) with increasing time and dosage. The maximum degradation efficiency and half-maximal concentration (MCC) were calculated. Figure 11 ).

[0096] 3. The 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 antibody. Finally, SIRPα and PD-1 were co-precipitated using immunoprecipitation. The results showed that SIRPα and PD-1 proteins were detected in the proteins precipitated by the Y39 group during VHL antibody co-precipitation, while no protein precipitation was detected in the control group. The experimental results indicate that Y39 can form a ternary complex with either SIRPα or PD-1 and VHL. Figure 12 SIRPα was labeled with CY3, and cell nuclei were labeled using Hoechst. Cells were collected at different time points, fixed, and then subjected to immunofluorescence detection. The results showed that macrophages incubated with SIRPα antibody exhibited red fluorescence at 0H, indicating SIRPα expression on the cell membrane; however, the fluorescence intensity decreased over time, and the fluorescence essentially disappeared after 24H, confirming that Y39 can degrade SIRPα over time. Figure 13 Western blot analysis confirmed that Y39 could degrade NKG2A and PD-1 in a dose-dependent manner (0 nM, 5 nM, 10 nM, 50 nM, 100 nM) and time-dependent manner (0, 4, 12, 24, 48 H) with increasing time and dosage. Figure 14 ).

[0098] 4. The peptide PROTAC degrades various immune checkpoint molecules via a ubiquitin-dependent pathway.

[0099] CD8 + T cells were co-incubated with Y39 and the protease inhibitor MG132. After cell lysis, the lysate was incubated overnight with PD-1 antibody, and PD-1 levels were measured. Incubation of immune cells with Y39 alone reduced the expression of inhibitory receptors, but co-incubation with Y39 and MG132 inhibited the degradation of inhibitory receptors, further confirming that Y39 degrades immunosuppressive receptors via the ubiquitination pathway. Figure 15VHL 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 Y39 could not degrade the inhibitory receptors in the VHL knockout cell lines, confirming that Y39 ubiquitinates the immunosuppressive receptors on immune cells via VHL ubiquitin ligase, and subsequently degrades the inhibitory receptors via the proteasome. Figure 16 ).

[0100] Example 4: Effects of the universal polypeptide PROTAC on immune cell function

[0101] 1. Universal peptide-PROTACPROTAC against CD8 + The effect of T cells

[0102] T cells were collected 24 hours after Y39 stimulation to detect the phosphorylation levels of AKT and ERK, downstream signaling molecules of their inhibitory receptors. The results showed that the total protein expression levels of SHP2, AKT, and ERK did not change significantly after Y39 stimulation, but the expression levels of phosphorylated proteins p-SHP2, p-AKT, and p-ERK increased. Figure 17 A, B). Flow cytometry analysis after co-incubating Y39 with T cells for 2 days showed that the Y39 treatment group significantly increased Ki67 expression on T cells. Figure 17 C), this result indicates that Y39 can enhance the proliferation capacity of T cells; CFSE results also show that, compared with the control group, Y39 significantly increased T cell proliferation (C). Figure 17 D). Flow cytometry results showed that the Y39 treatment group significantly increased the expression of CD69 on T cells. Figure 17 E), indicating that Y39 can induce T cells into an activated state. RTCA assay was performed to detect the ability of Y39 to kill human colon cancer cells HCT116 under an effector-to-target ratio (effector cells:target cells) of 10:1, and the killing rate was calculated. Figure 17 F). Experimental results showed that, compared with the control group, the T cell cytotoxicity of the Y39 group was significantly increased (P<0.001). Flow cytometry results showed that the Y39 treatment group could significantly reduce the expression of Cleaved caspase-3 on T cells (F). Figure 17 G), confirming that Y39 can inhibit T cell apoptosis. Flow cytometry staining with 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. Figure 17 H).

[0103] 2. Effects of the universal peptide PROTAC on NK cells

[0104] NK cells were collected 24 hours after Y39 stimulation to detect the phosphorylation of SHP2, which binds to the inhibitory receptor. Results showed that the expression levels of phosphorylated proteins p-SHP2 (Y580) and p-SHP2 (Y542) decreased after Y39 stimulation. T cells were also collected 24 hours after Y39 stimulation to detect the phosphorylation levels of downstream signaling molecules SYK and ZAP70, which are associated with the inhibitory receptor. 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. Figure 18 A). NK cells were collected 3 days after Y39 stimulation, 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. Figure 18 B). The ability of Y39 to kill human colon cancer cells HCT116 by NK cells was detected by RTCA at an effector-to-target ratio (effector cells:target cells) of 2.5:1, and the killing rate was calculated. Figure 18 C). The experimental results showed that the NK cell cytotoxicity of the Y39 group was significantly increased compared with that of the control group (P<0.001).

[0105] 3. Effects of the universal peptide PROTAC on macrophages

[0106] Macrophages stimulated with Y39 for 1 day were collected to examine the phosphorylation of SHP2 bound to the inhibitory receptor. Results showed no significant change in total SHP2 protein expression after Y39 stimulation, but the expression levels of phosphorylated proteins p-SHP2 (Y580) and p-SHP2 (Y542) decreased. Macrophages stimulated with Y39 for 24 hours showed no significant change in total SYK and AKT protein expression, but the expression levels of phosphorylated proteins p-SYK and p-AKT increased. Figure 19 A). Macrophages were collected 3 days after Y39 stimulation. Flow cytometry was used to detect the expression levels of tumor-promoting macrophage markers CD206 and IL-10, and tumor-suppressing macrophage markers CD86 and iNOS. Compared with the control group, Y39 promoted the transformation of macrophages into tumor-suppressing macrophages. Figure 19 B). Flow cytometry was used to detect the phagocytic activity of macrophages on human colon cancer cells HCT116 at an effector-to-target ratio (effector cells:target cells) of 1:1. Figure 19 As shown in Figure C. The results showed that, compared with the control group, the phagocytic efficiency of the Y39-stimulated group was significantly improved (P<0.001). The experimental results indicate that Y39 can enhance the phagocytic ability of macrophages against human colon cancer cells HCT116. Figure 19C).

[0107] Example 5: In vivo antitumor activity study of the universal polypeptide PROTAC

[0108] 1. Detection of the antitumor effect of the universal peptide PROTAC on colon cancer in mice.

[0109] A subcutaneous xenograft model of MC38 colon cancer was established in mice. Tumor growth curves in C57BL / 6 mice showed that tumor growth in both the Y39 and C-SH2 groups was significantly inhibited compared to the control group (P<0.05). After seven administrations, mice were sacrificed, and tumors were dissected and weighed. Compared to the control group, the average tumor weight in the Y39 group was significantly reduced (P<0.05), with the 5 μg group showing the highest tumor inhibition rate. The experimental results indicate that Y39 significantly inhibited the growth of MC38 colon cancer tissue and was superior to the therapeutic effect of C-SH2. Figure 20 AC). Mouse MC38 xenograft tissue was digested and prepared into a cell suspension. The levels of the effector molecule GZMB in T cells and NK cells were detected by flow cytometry. Figure 20 As shown in Figures D and E, compared with the control group, the expression of GZMB in T and NK cells was significantly increased in the Y39 group. The results showed that, compared with the control group and the C-SH2 group, Y39 enhanced CD8 expression in mouse tumor tissue. + The expression of T and NK cell killing-related molecules was investigated. Flow cytometry analysis revealed a significant decrease in the tumor-suppressing molecule TNF-α and a significant increase in the tumor-promoting molecule CD206 on macrophages. The results showed that, compared with the control group and the C-SH2 group, Y39 increased the expression of tumor-suppressing molecules and decreased the expression of tumor-promoting molecules in mouse tumor tissues. Figure 20 F, G).

[0110] 2. Detection of the antitumor effect of the universal peptide PROTAC on mouse liver cancer.

[0111] A subcutaneous xenograft model of Hepa-1-6 hepatocellular carcinoma was established in mice. Tumor growth curves in C57BL / 6 mice showed that tumor growth was significantly inhibited in the Y39, C-SH2, and PD-1 groups compared to the control group (P<0.05). Mice were sacrificed after seven administrations, and tumors were dissected and weighed. Compared to the control group, the average tumor weight was significantly reduced in the Y39, C-SH2, and PD-1 groups (P<0.05), with the Y39 (5 ​​μg) group showing the lightest tumor weight. The experimental results indicate that Y39 significantly inhibited the growth of Hepa-1-6 tumor tissue and was superior to the therapeutic effects of C-SH2 and PD-1 antibodies. Figure 21 AC). Mouse xenograft tissue was digested and prepared into a cell suspension. The number of T cells and the expression level of the proliferation marker Ki67 were detected by flow cytometry. Figure 21As shown in Figures D and E, compared with the control group, the Y39 group showed a significant increase in the number of T cells and a significant increase in Ki67 expression. The results indicate that compared with the control group, C-SH2 group, and PD-1 group, Y39 can increase CD8+ expression in mouse tumor tissue. + T cells infiltrated and exhibited stronger proliferative capacity compared to the C-SH2 group. Flow cytometry analysis revealed a significant decrease in the tumor suppressor molecule CD86 and a significant increase in the tumor-promoting molecules CD206 and IL-10 on macrophages. The results showed that, compared to the control group and the C-SH2 group, Y39 increased the expression of tumor suppressor-related molecules and decreased the expression of tumor-promoting molecules in mouse tumor tissues. Figure 21 F, G).

[0112] 3. Detection of the antitumor effect of the universal peptide PROTAC on liver cancer in mice with reconstructed human immune system.

[0113] Human hepatocellular carcinoma cells (HepG2) were subcutaneously inoculated into severely immunodeficient NCG mice via tail vein injection. Two weeks later, blood samples were collected to measure hCD45 levels. A successful model was considered to have reached 10-15% or more of the PBMC levels. We selected mice with successfully established models for drug administration and recorded tumor growth. Subsequently, mice exhibiting strong graft-versus-host disease were sacrificed, and tumor tissue was dissected for analysis. Tumor growth curves showed that tumor growth was significantly inhibited in the Y39, C-SH2, and PD-1 groups compared to the control group (P<0.05). After six administrations, mice were sacrificed, and tumors were dissected and weighed. Compared to the control group, the average tumor weight was significantly reduced in the Y39, C-SH2, and PD-1 groups (P<0.05), with the Y39 group showing the lightest tumor weight. The experimental results indicate that Y39 significantly inhibited the growth of human tumor tissue and was superior to the therapeutic effects of C-SH2 and PD-1 antibodies. Figure 22 (AC). Tumor tissue was digested and prepared into a cell suspension. The number of T cells was detected by flow cytometry and immunofluorescence. Figure 23 As shown in Figures D and E, the number of T cells in the Y39 group was significantly increased compared to the control group. The results indicate that Y39, compared to the control group, C-SH2 group, and PD-1 group, can increase CD8+ in mouse tumor tissue. + T cell infiltration. The expression levels of Perforin and TNF-α on T cells were detected by flow cytometry. Figure 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 Y39 had a stronger killing ability than the C-SH2 group compared with the control group, C-SH2 group, and PD-1 group. Figure 22 F, G).

[0114] 4. Detection of the antitumor effect of the universal peptide PROTAC on PD-1 resistant colon cancer.

[0115] A mouse model of PD-1-resistant colon cancer was established to investigate whether Y39 could enhance the tumor-suppressing effect on PD-1-resistant tumors in mice. Tumor growth curves showed that, compared with the control group, tumor growth in the Y39, C-SH2, and PD-1 groups was significantly inhibited (P<0.05). After seven administrations, mice were sacrificed, tumors were dissected and weighed. Compared with the control group, the average tumor weight in the Y39, C-SH2, and PD-1 groups was significantly reduced (P<0.05), with the Y39 group showing the lightest tumor weight. The experimental results indicate that Y39 significantly inhibited the growth of human tumor tissue and was superior to the therapeutic effects of C-SH2 and PD-1 antibodies. These results confirm that PD-1 antibodies did not fully exert their anti-tumor effect in PD-1-resistant tumors, while Y39 still possessed anti-tumor capabilities against PD-1-resistant tumors, demonstrating that Y39 can activate immune cells through multiple immune checkpoint pathways. Figure 23 Further analysis revealed that the tumor tissue was digested into a cell suspension, and the number of T cells was detected by flow cytometry and immunofluorescence. Figure 23 As shown in Figures D and E, the number of T cells in the Y39 group was significantly increased compared to the control group. The results indicate that Y39, compared to the control group, C-SH2 group, and PD-1 group, can increase CD8+ in mouse tumor tissue. + T infiltration was observed, and Ki67 expression was also significantly increased. Results showed that compared to the control group, C-SH2 group, and PD-1 group, Y39 could increase CD8+ expression in mouse tumor tissue. + T cells infiltrated and exhibited stronger proliferative capacity compared to the C-SH2 group. Flow cytometry analysis revealed a significant increase in the tumor suppressor molecule CD86 and a significant decrease in the tumor-promoting molecule CD206 on macrophages. The results showed that, compared to the control group and the C-SH2 group, Y39 increased the expression of tumor suppressor-related molecules and decreased the expression of tumor-promoting molecules in mouse tumor tissues. Figure 23 F). Flow cytometry analysis of CD69, an activation-related molecule on NK cells, showed that compared with the control group and the C-SH2 group, the expression of CD69 on NK cells in the Y39 group was significantly increased. Figure 23 G). The above results indicate that Y39 can exert an anti-tumor effect on PD1-resistant tumors by affecting the function of various immune cells.

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

[0117] The above examples illustrate the present invention only to aid in understanding it and are not intended to limit the scope of the invention. Those skilled in the art can make various simple deductions, modifications, or substitutions based on the principles of this invention.

Claims

1. PROTAC, a universal polypeptide targeting multiple immune checkpoints, characterized by: The structure of the universal polypeptide-PROTAC is BZLV; in: B is a cell-penetrating peptide; Z is the SH2 domain of SHP2 or SHP1 or its active fragment; L represents no or no connecting element; V is a ligand for VHL or other E3 molecules; The amino acid sequence of part B is YGRKKRRQRRRPP; The amino acid sequence of the Z portion is VRESQSHPGDFVL; When the L portion is a connecting element, the amino acid sequence is GSGSGS; The amino acid sequence of the V portion is ALAP(OH)YIP.

2. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains the universal polypeptide-PROTAC of claim 1 and its pharmaceutically acceptable carrier.

3. The application of PROTAC, a universal polypeptide targeting multiple immune checkpoints as described in claim 1, is characterized in that: The general-purpose polypeptide-PROTAC is used to prepare drugs for the treatment or prevention of colon cancer and liver cancer.

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