Covalent amino acid modified peptides that bind pd-1 and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a covalent amino acid modified peptide with affinity to PD-1 and application thereof. The application takes PD-1 protein as a target molecule, and obtains a PD-1 affinity peptide through high-throughput screening technology of phage display dodecapeptide library. The affinity peptide is subjected to mutation modification and anti-enzymatic modification, and is further subjected to covalent amino acid modification, so that the residence time of the affinity peptide in an effect site is significantly increased. The obtained covalent amino acid modified peptide can have affinity to PD-1 and block the interaction between PD-1 and PD-L1, and can be used as a lead peptide for developing a covalent inhibitor of PD-1 / PD-L1, and has good application prospect and high economic value. The covalent amino acid modified peptide with affinity to PD-1 provided by the application can be used for preparing an antitumor drug and a molecular probe, and provides a new option for tumor diagnosis and treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a covalent amino acid modified peptide for affinity PD-1 and application thereof. BACKGROUND

[0002] In recent years, tumor immunotherapy has attracted much attention, and Science magazine selected it as the first of the top ten scientific breakthroughs in 2013. Tumor immunotherapy mainly enhances the anti-tumor ability of the body's own immune system by stimulating the immune system, which can not only kill tumors, but also prevent tumor recurrence and metastasis. The effect of tumor immunotherapy is closely related to the activation state of T cells, and the activation of T cells requires a classic two-signal system. Among them, the first signal is generated by the combination of antigen and receptor, and the second signal is generated by the combination of antigen presenting cells and regulatory receptors on the surface of T cells, which is also called co-stimulatory signal. According to the different immune effects, the co-stimulatory molecules on the surface of T cells can be divided into positive co-stimulatory molecules (such as CD28, OX40, CD27) and negative co-stimulatory molecules (such as PD-1, CTLA-4, LAG-3, TIGIT, TIM-3), and the negative co-stimulatory molecules are also known as immune checkpoints. Immune checkpoint blockade therapy mainly inhibits the combination of programmed death receptors and their ligands, and improves the aggressiveness of the host immune system to tumor cells.

[0003] Programmed death-1 (PD-1) is an important molecule that transmits negative regulatory signals to T cells, and blocking the PD-1 / PD-L1 signaling pathway can block negative regulatory signals, relieve immune suppression, and restore the function of T cells to kill tumor cells to some extent, thereby achieving the purpose of anti-tumor. At present, the monoclonal antibody targeting PD-1 has achieved good efficacy in clinical application. However, antibody drugs generally have the disadvantages of strong immunogenicity, poor tumor infiltration, and inability to timely withdraw the drug when adverse reactions occur. The half-life of polypeptide drugs is relatively short, and they have good specificity and tumor penetration, especially covalent polypeptides can be enriched in the drug efficacy site, and have high research and clinical application value in the fields of tumor immunotherapy and tumor molecular imaging.

[0004] Chinese patent CN114685612B (Jiangsu University) discloses a polypeptide inhibitor blocking PD-1 / PD-L1 binding, the amino acid sequence of which is H-D-W-F-K-A-F-Y-D-K-I-N-E-T-Y-N-K-OH. The polypeptide has high binding affinity with PD-L1, can effectively inhibit PD-1 / PD-L1 interaction, restore T cell activity, prevent tumor cell immune escape and thus produce tumor inhibition effect. Chinese patent CN111153961B (Zhengzhou University) discloses a group of PD-1 affinity polypeptides, the amino acid sequences of which are as follows: D-D-F-R-V-W-W-P-N-F-P-A, D-D-F-Y-V-W-W-P-N-F-P-R, D-D-F-R-Y-W-W-P-N-F-P-R, and the configuration of each amino acid is independently selected from D type or L type. The polypeptide can block PD-1 / PD-L1 binding, and the high blocking rate peptide therein can also significantly inhibit mouse CT26 colon cancer, and has potential application prospect in tumor treatment, autoimmune diseases and inflammatory diseases. However, the affinity of the above-mentioned polypeptide to PD-L1 or PD-1 protein needs to be improved. Therefore, it is necessary to develop a new polypeptide to strongly block the interaction between PD-1 and PD-L1 and improve the disease treatment effect of the polypeptide. SUMMARY

[0005] The technical problem solved by the present application is to provide a PD-1 affinity covalent amino acid modified peptide, which can significantly increase the residence time of the polypeptide in the effector site by covalent amino acid modification of the polypeptide. The polypeptide can bind to PD-1 and block the interaction between PD-1 and PD-L1, and can be used as a lead peptide for developing PD-1 / PD-L1 covalent inhibitors, and has good application prospect and high economic value.

[0006] Secondly, the present application provides a polypeptide probe designed based on the PD-1 affinity covalent amino acid modified peptide.

[0007] Thirdly, the present application provides an application of the PD-1 affinity covalent amino acid modified peptide or the polypeptide probe.

[0008] Fourthly, the present application provides a reagent for tumor diagnosis or tracer imaging.

[0009] Finally, the present application provides a drug for preventing and / or treating tumors or autoimmune diseases.

[0010] To solve the above technical problems, the present application adopts the following technical solutions:

[0011] A PD-1 affinity covalent amino acid modified peptide comprises one or more covalent amino acids (X) modifications at any position of the following polypeptide:

[0012] Asp-Asp-Phe-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro-Arg, or a modified form thereof.

[0013] As a preferred embodiment of the present application, the covalent amino acid (X) is selected from fluorosulfur exchange-based covalent amino acids, including but not limited to one or more of fluorosulfate tyrosine (Tyr(O-SO2F)), aryl fluorosulfate tyrosine, and the like.

[0014] As a preferred embodiment of the present application, the covalent amino acid-modified peptide is selected from the following polypeptides or combinations thereof:

[0015] (1) Asp-Asp-X-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro-Arg, or a modified form thereof;

[0016] (2) Asp-Asp-Phe-Tyr-X-Trp-Trp-Pro-Asn-Phe-Pro-Arg, or a modified form thereof;

[0017] (3) Asp-Asp-Phe-Tyr-Val-Trp-X-Pro-Asn-Phe-Pro-Arg, or a modified form thereof;

[0018] wherein X is a covalent amino acid selected from fluorosulfur exchange-based covalent amino acids, including but not limited to one or more of fluorosulfate tyrosine (Tyr(O-SO2F)), aryl fluorosulfate tyrosine, and the like.

[0019] As a preferred embodiment of the present application, the configuration of each amino acid in the polypeptide is independently selected from the D configuration or the L configuration. When a certain amino acid or certain amino acids are specified to be in the D configuration without specifying the configuration of other amino acids, the configuration of the other amino acids is by default the L configuration.

[0020] More preferably, 2-3 consecutive amino acids at both ends of the polypeptide are in the D configuration.

[0021] As a preferred embodiment of the present application, the covalent amino acid-modified peptide is selected from the following polypeptides or combinations thereof:

[0022] (1) Asp D -Asp D -X-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D ;

[0023] (2) Asp D -AspD -Phe D -Tyr-X-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D ;

[0024] (3)Asp D -Asp D -Phe D -Tyr-Val-Trp-X-Pro-Asn-Phe-Pro D -Arg D ;

[0025] wherein X is fluorosulfate tyrosine (Tyr(O-SO2F)).

[0026] Specifically, when the D configuration of the corresponding amino acid is represented by a lower-case single letter, the above-mentioned polypeptide (1) can be written as: d-d-X-Y-V-W-W-P-N-F-p-r. The covalent amino acid modified polypeptide is a D configuration amino acid protected peptide at both ends, has good anti-enzymatic ability, and has a relative molecular weight of 1739.78, and the chemical structure is shown in the following formula (I):

[0027]

[0028] Specifically, when the D configuration of the corresponding amino acid is represented by a lower-case single letter, the above-mentioned polypeptide (2) and polypeptide (3) can be written as: d-d-f-Y-X-W-W-P-N-F-p-r and d-d-f-Y-V-W-X-P-N-F-p-r.

[0029] As a preferred embodiment of the present application, the 3, 5, and 7 positions of the polypeptide (1), (2), and (3) can also be replaced by other covalent amino acids; or, new covalent amino acid replacements are added to the 3, 5, or 7 positions of the polypeptide (1), (2), and (3). For example, new covalent amino acid replacements are added to the 5 and / or 7 positions of the polypeptide (1); or, the fluorosulfate tyrosine (Tyr(O-SO2F)) at the 3 position of the polypeptide (1) is replaced by other covalent amino acids based on fluorine-sulfur exchange, while new covalent amino acid replacements are added to the 5 and / or 7 positions.

[0030] As a preferred embodiment of the present application, the modification sites include but are not limited to N-terminal modification, C-terminal modification, backbone modification, side chain modification, amino acid modification, etc. The purposes of modification include prolonging half-life, increasing water solubility, reducing or eliminating toxic side effects, etc., provided that the activity of the polypeptide is retained.

[0031] Specifically, the modified forms include but are not limited to one or more of the following:

[0032] (1) cyclic peptide synthesis: head-to-tail cyclization, side chain cyclization (lactone, lactam, ether bond, etc.), multiple disulfide bonds, single thioether cyclization, etc.

[0033] (2) isotope labeling: 13 C, 15 N, 18 O, etc. isotope labeling;

[0034] (3) polyethylene glycol (PEG) modification: PEG2, PEG4, PEG8, PEG12, PEG24, PEG36, PEG2000, PEG5000, PEG3400, PEG20K, PEG40K, etc. modification;

[0035] (4) phosphorylation modification: phosphorylation modification of L- or D-configuration amino acids (such as threonine T, serine S), phosphorylation modification of single or multiple amino acids;

[0036] (5) coupling protein or other carrier:

[0037] a. polypeptide-protein coupling: KLH coupling, albumin coupling (such as bovine serum albumin BSA, chicken egg white albumin OVA, mouse serum albumin MSA, human serum albumin HSA), albumin affinity peptide coupling, tumor homing peptide coupling, transmembrane peptide coupling, antibody Fc fragment coupling, elastin-like coupling, etc.

[0038] b. polypeptide-nanocarrier coupling;

[0039] (6) modification of N-terminal or side chain amino acids: acetylation, formylation, biotin labeling, trifluoroacetylation, benzoylation, 2-aminobenzoylation, maleimide, chloroacetylation, bromoacetylation, succinylation, palmitoylation, malate, fatty acid, formaldehyde, chelation (such as Hynic, DTPA, DOTA, NOTA modification), chloro, fluoro, bromo, nitro or methoxy substitution, fluorescent labeling (such as Cy series, Texas series, Alexa series, rhodamine, Bodipy, Rox, FAM, FITC, MCA, TAMRA, Dnp), PAS modification, etc.

[0040] (7) C-terminal modification: amidation, esterification, aldehyde group, alcohol group, succinylation, fluorescent labeling (such as Cy series, rhodamine, AMC, AFC, PNA, CMK, FMK), etc.

[0041] (8) alkylation modification: N-methylation, side chain methylation, N-ethylation, N-phenylpropylation, N-allylation, etc.

[0042] (9) radionuclide modification: 125 I, 131 I,18 F, 99m Tc, 68 Ga、 64 Cu、 67 Ga、 90 Y、 111 In、 177 Lu、 89 Modification with radioactive nuclides such as Zr;

[0043] (10) Other special modifications: glycopeptides, sulfonation, MAPS, etc.

[0044] In a preferred embodiment of the present invention, the polypeptide is prepared by chemical synthesis. Chemical synthesis methods include solid-phase synthesis (such as the Fmoc method), liquid-phase synthesis, and solid-liquid-phase synthesis. Synthetic strategies include C-terminal synthesis, N-terminal synthesis, and segmented synthesis. If conventional modification is performed at the C-terminus or N-terminus during synthesis, the polypeptide contains the conventionally modified group.

[0045] A polypeptide probe comprising the sequence of a covalently modified peptide that binds to PD-1.

[0046] As a preferred embodiment of the present invention, the polypeptide probe includes, but is not limited to, fluorescent molecular imaging probes, radionuclide probes, etc.

[0047] Specifically, the general structural formula of the polypeptide probe is as follows:

[0048] MLR;

[0049] In the formula, M is a photolabeled or radioactive nuclide complex group;

[0050] L is a linking group;

[0051] R is the covalently modified amino acid peptide of the affinity for PD-1 or its dimer or polymeric form.

[0052] As a preferred embodiment of the present invention, the optical labeling includes, but is not limited to, infrared fluorescent dyes, compounds containing organic chromophores, compounds containing organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, bioluminescent molecules, etc.

[0053] In a preferred embodiment of the present invention, the radionuclide probe is an amino acid on a covalently modified peptide that is radiolabeled with I or F and has affinity for PD-1.

[0054] In a preferred embodiment of the present invention, the radionuclide complexing group includes a radionuclide and a bifunctional chelating agent for radionuclide labeling. The radionuclide includes, but is not limited to, those mentioned above. 125 I, 131 I,18 F、 99m Tc、 68 Ga、 64 Cu、 67 Ga、 90 Y、 111 In、 177 Lu、 89 Zr, etc. The bifunctional chelator for radionuclide labeling includes but is not limited to NOTA, DOTA, DTPA, HYNIC, DFO, etc.

[0055] Specifically, the radionuclide complexing group further includes a radionuclide ligand selected from N-tris (hydroxymethyl) methylglycine (Tricine) and / or triphenylphosphine triisulfonic acid sodium salt (TPPTS).

[0056] As a preferred embodiment of the present application, the linking group L includes but is not limited to n is an integer from 0 to 10.

[0057] The application of an affinity PD-1 covalent amino acid modified peptide or polypeptide probe includes but is not limited to one or more of the following aspects:

[0058] (1) for preparing a reagent for tumor diagnosis or tracer imaging;

[0059] (2) for preparing a drug for preventing and / or treating tumors or autoimmune diseases;

[0060] (3) for preparing a drug for preventing and / or treating bacterial, viral or fungal infections;

[0061] (4) for preparing a reagent for qualitatively and / or quantitatively detecting PD-1 protein expression, expression location or expression amount;

[0062] (5) for preparing a reagent for detecting the affinity and / or blocking ability to PD-1 protein (for example, blocking the binding between PD-1 / PD-L1).

[0063] As a preferred embodiment of the present application, the reagent in (1) includes tumor diagnosis imaging agents, tumor boundary precise positioning imaging agents, intraoperative image navigation imaging agents, etc.

[0064] As a preferred embodiment of the present application, the drug includes targeted gene therapy drugs, chemotherapy drugs, etc.

[0065] Specifically, the tumor includes, but is not limited to, colon cancer, breast cancer, liver cancer, lung cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, bone cancer, head and neck cancer, melanoma, lymphoma, leukemia, etc.

[0066] A reagent for tumor diagnosis or tracer imaging, comprising the covalently modified amino acid peptide or polypeptide probe with affinity to PD-1.

[0067] A medicine for preventing and / or treating tumor or autoimmune disease, comprising the covalently modified amino acid peptide or polypeptide probe with affinity to PD-1.

[0068] As a preferred embodiment of the present application, the medicine is a targeted gene therapy medicine or a chemotherapy medicine. The polypeptide probe is a radionuclide probe, which is used to prepare a tumor radionuclide targeted therapy medicine.

[0069] As a preferred embodiment of the present application, the covalently modified amino acid peptide or polypeptide probe with affinity to PD-1 in the medicine exists in a free form or in a form of a pharmaceutically acceptable salt thereof. The PD-1 protein can be a wild type or a mutant protein retaining its activity, which is of human or mouse origin.

[0070] As a preferred embodiment of the present application, the content of the covalently modified amino acid peptide or polypeptide probe with affinity to PD-1 in the medicine is a therapeutically effective amount, which can be in a range of 0.01wt%-99.99wt%.

[0071] As a preferred embodiment of the present application, in addition to the covalently modified amino acid peptide or polypeptide probe with affinity to PD-1, the medicine further comprises other therapeutically effective components capable of synergistically or complementarily preventing and / or treating tumor, for inhibiting tumor growth or eliminating tumor, so as to achieve combined prevention and / or treatment by combined administration.

[0072] As a preferred embodiment of the present application, the medicine further comprises a pharmaceutically acceptable excipient or carrier, including but not limited to excipients, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, etc. The type and amount of the excipient or carrier can be selected and adjusted according to different dosage forms of the medicine.

[0073] As a preferred embodiment of the present application, the dosage form of the medicine is a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection solution, tablet, pill, capsule, spray, dispersion, etc.

[0074] As a preferred embodiment of the present application, the dosage of the medicine is a pharmaceutically acceptable dosage.

[0075] The present application has the following beneficial effects:

[0076] The present application takes PD-1 protein as a target molecule, and a PD-1 affinity peptide is screened by using a phage display dodecapeptide library high-throughput screening technology. The affinity peptide is subjected to mutation modification and anti-enzymatic modification, and is further subjected to covalent amino acid modification, so that the residence time of the affinity peptide in an effect site is significantly increased. The obtained covalent amino acid modified peptide can bind to PD-1 and block the interaction between PD-1 and PD-L1, and can be used as a lead peptide for developing a PD-1 / PD-L1 covalent inhibitor, and has good application prospect and high economic value.

[0077] The covalent amino acid modified peptide provided by the present application has an antitumor effect and no obvious toxic side effect, and can be used for preparing an antitumor drug, and provides a new choice for tumor immunotherapy. In addition, the covalent amino acid modified peptide provided by the present application also has good application prospect in tumor diagnosis or tracer imaging, and can be used for preparing a reagent for tumor diagnosis or tracer imaging, and has potential clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0078] Figure 1 The covalent ability of the polypeptide and the PD-1 protein in the experimental example is detected by WB;

[0079] In the figure, CPBP-12 is the polypeptide (1) in Example 2, V5X is the polypeptide (2) in Example 2, W7X is the polypeptide (3) in Example 2, and X represents covalent amino acid fluorosulfate tyrosine (Tyr(O-SO2F)).

[0080] Figure 2 The covalent ability of the polypeptide and the PD-1 protein in the experimental example is detected by WB;

[0081] Figure 3 The affinity of the covalent amino acid modified peptide CPBP-12 and PD-1 is detected by MST in the experimental example.

[0082] Figure 4 The blocking effect of the covalent amino acid modified peptide CPBP-12 on PD-1 / PD-L1 is detected by flow cytometry in the experimental example.

[0083] Figure 5 The distribution of the covalent amino acid modified peptide CPBP-12 in each organ of a mouse in the experimental example.

[0084] Figure 6 The experimental results of the polypeptide in inhibiting the growth of CT26 tumor in vivo in the experimental example.

[0085] In order to more clearly illustrate the technical solutions of the present application, the above drawings in the examples or experimental examples are briefly introduced. It should be understood that the above drawings should not be regarded as any limitation on the protection scope of the present application. For those skilled in the art, other related drawings can also be obtained from these drawings without creative labor. DETAILED DESCRIPTION

[0086] The technical solutions of the present application will be clearly and completely explained below in combination with specific examples and experimental examples.

[0087] Those skilled in the art should understand that the examples and experimental examples described below are only used to illustrate the technical solutions and technical effects of the present application, and should not be regarded as any limitation on the protection scope of the present application. Based on the examples described below, other technical solutions obtained by those skilled in the art without creative labor, such as modified, deformed or simply replaced technical solutions, all belong to the protection scope of the present application.

[0088] The raw materials, reagents, equipment, etc. used in the examples or experimental examples are all commercially available goods, unless otherwise specified.

[0089] The methods used in the examples or experimental examples are all conventional methods, unless otherwise specified.

[0090] The terms, abbreviations involved in the examples or experimental examples are all conventional meanings in the art.

[0091] Example 1

[0092] The present example provides a covalent amino acid modified peptide affinity to PD-1, which contains one or more covalent amino acid (X) modifications at any position of the following polypeptide:

[0093] Asp-Asp-Phe-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro-Arg, or a modified form thereof;

[0094] The covalent amino acid (X) is selected from covalent amino acids based on fluorine-sulfur exchange, including but not limited to one or more of fluorine-sulfur acid salt tyrosine (Tyr(O-SO2F)), aryl fluorine-sulfur acid salt tyrosine, etc.

[0095] The present example also provides a covalent amino acid modified peptide affinity to PD-1, which is selected from any one of the following polypeptides:

[0096] (1) Asp D -Asp D -X-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D ;

[0097] (2) Asp D -Asp D -Phe D -Tyr-X-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D ;

[0098] (3) Asp D -Asp D -Phe D -Tyr-Val-Trp-X-Pro-Asn-Phe-Pro D -Arg D ;

[0099] wherein X is fluorosulfate tyrosine (Tyr(O-SO2F)), and other amino acids are in L configuration except the indicated amino acids in D configuration.

[0100] The embodiment also provides application of the covalent amino acid modification peptide with affinity to PD-1 in preparation of a medicament for preventing and / or treating tumors or autoimmune diseases.

[0101] The embodiment also provides a medicament for preventing and / or treating tumors, comprising an effective amount of the covalent amino acid modification peptide with affinity to PD-1, and an appropriate amount of pharmaceutically acceptable adjuvant for preparing the medicament into an injection dosage form.

[0102] In other embodiments of the present application, the covalent amino acid modification peptide with affinity to PD-1 has X as aryl fluorosulfate tyrosine.

[0103] Embodiment 2

[0104] The embodiment provides a covalent amino acid modification peptide with affinity to PD-1, which is composed of the following polypeptides in a concentration ratio of 1:1:1:

[0105] (1) Asp D -Asp D -Tyr(O-SO2F)-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D ;

[0106] (2) Asp D -Asp D -Phe D -Tyr-Tyr(O-SO2F)-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D ;

[0107] (3) Asp D -Asp D -Phe D -Tyr-Val-Trp-Tyr(O-SO2F)-Pro-Asn-Phe-Pro D -Arg D .

[0108] The embodiment also provides application of the covalent amino acid modification peptide with affinity to PD-1 in preparation of a drug for preventing and / or treating a tumor or an autoimmune disease.

[0109] The embodiment also provides a drug for preventing and / or treating a tumor, comprising an effective amount of the covalent amino acid modification peptide with affinity to PD-1, and an appropriate amount of a pharmaceutically acceptable excipient for preparing the drug into a powder injection dosage form.

[0110] In other embodiments of the present application, the fluorosulfate tyrosine (Tyr(O-SO2F)) modified polypeptide is mixed with the aryl fluorosulfate tyrosine modified polypeptide at a concentration ratio of 1:1 for preparing a drug for preventing and / or treating a tumor or an autoimmune disease.

[0111] Embodiment 3

[0112] The embodiment provides a polypeptide probe, specifically a fluorescent molecular imaging probe, with the following structural formula:

[0113] M-L-R;

[0114] In the formula, M is an infrared fluorescent dye;

[0115] L is n is 6;

[0116] R is the covalent amino acid modification peptide with affinity to PD-1 in Embodiment 1 or Embodiment 2.

[0117] The embodiment also provides application of the fluorescent molecular imaging probe in preparation of a reagent for tumor diagnosis or tracer imaging.

[0118] The embodiment also provides a reagent for tumor diagnosis or tracer imaging, comprising an effective amount of the fluorescent molecular imaging probe, and an appropriate amount of a pharmaceutically acceptable excipient.

[0119] Embodiment 4

[0120] The embodiment provides a polypeptide probe, specifically a radionuclide probe, with the following structural formula:

[0121] M-L-R;

[0122] wherein M is a radionuclide complexing group comprising a radionuclide 18 F, bifunctional chelator NOTA for radionuclide labeling;

[0123] L is n is 2;

[0124] R is the covalent amino acid modified peptide of affinity to PD-1 in Example 1 or Example 2.

[0125] The embodiment also provides an application of the radionuclide probe in preparation of a drug for preventing and / or treating a tumor or an autoimmune disease.

[0126] The embodiment also provides a tumor nuclide targeted therapy drug, comprising an effective amount of the radionuclide probe and an appropriate amount of pharmaceutically acceptable excipients.

[0127] Experimental Example

[0128] I. Liquid phase screening of phage mirror image display peptide library for parent peptide

[0129] The screening process is as follows: the liquid phase difference phase screening method is used to screen the phage display dodecapeptide library with the eukaryotic protein rhPD-1 / Fc as the target; after 3 rounds of screening, the phage monoclonal with affinity to the target protein rhPD-1 / Fc is enriched round by round; the positive clones are selected from the 3rd round for sequencing, and a plurality of inserted dodecapeptide sequences are obtained, in which a plurality of have repeated clones, and the parent peptide R5 is one of the positive clones.

[0130] The amino acid sequence of the parent peptide R5 is as follows:

[0131] Asp-Asp-Phe-Arg-Val-Trp-Trp-Pro-Asn-Phe-Pro-Arg.

[0132] II. Design and synthesis of anti-enzymatic polypeptide R4-Y ddf-pr

[0133] The preparation process is as follows: the parent peptide R5 is subjected to molecular docking with the PD-1 protein, the best docking mode is selected and subjected to molecular dynamics simulation, after single amino acid mutation, the mutant peptides are selected according to the Delta affinity value, mutation intersection, interaction and affinity blocking experiment, the optimized peptide R4-Y is obtained, and the anti-enzymatic polypeptide R4-Y is obtained through the protection strategy of D configuration amino acids at both ends ddf-pr .

[0134] The amino acid sequence of the optimized peptide R4-Y is as follows:

[0135] Asp-Asp-Phe-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro-Arg.

[0136] Anti-enzymatic polypeptide R4-Y ddf-pr The amino acid sequence of R4-Y is as follows:

[0137] Asp D -Asp D -Phe D -Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D .

[0138] III. Design and synthesis of covalently modified peptides

[0139] Taking R4-Y ddf-pr as the parent peptide, covalently modified peptides were designed and synthesized, and the process was as follows: fluorosulfate tyrosine (Tyr(O-SO2F)) can produce a covalent bond with its adjacent lysine, tyrosine or histidine through fluorosulfur exchange reaction; the polypeptide R4-Y ddf-pr was replaced with Tyr(O-SO2F) to produce a covalent bond with its adjacent lysine, tyrosine or histidine. The polypeptide with the replaced amino acid was synthesized; after the biotin (SA)-labeled three candidate covalently modified peptides were co-incubated with the same amount of hPD-1 protein, SDS-PAGE gel electrophoresis was performed, and after the protein was transferred to the membrane, anti-Streptavidin-HRP was incubated, and Western Blot was detected by chemiluminescence method to detect whether the polypeptide and the protein successfully produced a covalent bond. The results are shown in Figure 1 .

[0140] As can be seen from Figure 1 , the polypeptides CPBP-12(f3X), V5X and W7X can all produce covalent bonds with PD-1 protein, and under the same polypeptide concentration, the covalent ability of CPBP-12(f3X) is stronger than that of other polypeptides, while R4Y ddf-pr cannot produce a covalent bond with PD-1 protein.

[0141] Further, another kind of covalent amino acid aryl fluorosulfate tyrosine was used to replace the fluorosulfate tyrosine in CPBP-12, and WB was also used to monitor whether the polypeptide and the protein successfully produced a covalent bond. The results are shown in Figure 2 .

[0142] As can be seen from Figure 2It can be seen that after the aryl fluorosulfate tyrosine is substituted for the fluorosulfate tyrosine in the polypeptide CPBP-12, the polypeptide can still covalently bind to the PD-1 protein.

[0143] IV. Microscale Thermophoresis (MST) detection of the affinity of covalent amino acid modified peptides to hPD-1

[0144] The affinity of covalent amino acid modified peptides to hPD-1 protein is detected by the following steps:

[0145] (1) Labeling hPD-1 protein: dilute the dye to 3 times the concentration of the labeled protein with PBS (pH = 7.4) before use, mix the dye and the protein at a volume ratio of 1:1 (100 μL of protein at a concentration of 10 μM, 100 μL of dye at a concentration of 30 μM), incubate at room temperature for 30 min in the dark;

[0146] (2) Wash the column: place the protein separation column in a 15 mL centrifuge tube and wash with MST Buffer (usually wash for 10 column volumes, column volume is 500 μL);

[0147] (3) Separate and purify the protein: add the protein-dye mixture (200 μL) in step (1) to the protein separation column by air drop, then add 300 μL of MST Buffer, and when the column no longer drips liquid, add 500 μL of MST Buffer. Start collecting from the first drop of liquid, and after collection is complete, detect the fluorescence on the machine, dilute the protein fluorescence value to about 600, and store at -80°C after aliquoting;

[0148] (4) Wash the column: wash with MST Buffer for at least 10 column volumes, and finally store the column in 20% alcohol;

[0149] (5) Sample preparation: dissolve the polypeptide to an appropriate concentration, perform 15-fold dilution, and obtain 16 concentration gradient samples, with a system of 10 μL; add 10 μL of labeled protein sample to each tube, mix well, and centrifuge to remove air bubbles; incubate on ice for 5 min, use a MST special capillary to suck the incubated liquid, and place it in the instrument holder;

[0150] (6) Machine detection: open the computer and start the MO. Control software, select the (Red) channel Binding Affinity mode for detection;

[0151] (7) Analyze the results: use NanoTemper analysis software MO. AffinityAnalysis v2.2.4 to calculate the binding dissociation constant (K D value). The results are shown in Figure 3 .

[0152] FromFigure 3 It can be seen that the covalently modified amino acid peptide CPBP-12 has an affinity for hPD-1 protein, K D The value is 50±10nM.

[0153] V. In vitro blocking experiment

[0154] The following steps were taken to detect the ability of covalently amino acid-modified peptides to block hPD-1 / hPD-L1 binding:

[0155] (1) Cell collection: Collect CHO-K1-hPD-1 cells in good condition, centrifuge twice at 4000 rpm and 4℃, and place on ice for later use;

[0156] (2) Incubation: The cells from step (1) are incubated at a rate of 5 × 10⁻⁶. 5 The samples were divided into different 1.5 mL EP tubes. 25 μL of peptide blocking agent (concentration set as needed) was added to the experimental group and incubated on ice for 30 min. Positive control tubes and negative control tubes were also set up, with 25 μL of PBS (pH = 7.4) added to both.

[0157] (3) Incubation of protein: Add 25 μL (50 ng) of hPD-L1-Fc protein and incubate on ice for 30 min. Add 25 μL of PBS (pH=7.4) to the negative control tube.

[0158] (4) Incubation with secondary antibody: Add 10 μL of Anti-Human IgG Fc PE fluorescent secondary antibody and incubate on ice in the dark for 30 min;

[0159] (5) Washing: Wash with 1 mL PBS (pH = 7.4) at 4000 rpm and 4 °C by centrifugation;

[0160] (6) Flow cytometry: Resuspend cells in 200 μL LFACS buffer in each tube, transfer to flow cytometry tubing in the dark, and prepare for flow cytometry analysis. Results are as follows: Figure 4 As shown.

[0161] from Figure 4 It can be seen that the covalently modified amino acid peptide CPBP-12 can block the binding between hPD-1 and hPD-L1, IC50 50 It is 0.84 μM.

[0162] VI. In vivo imaging of covalently modified amino acid peptide CPBP-12 in small animals

[0163] The in vivo imaging process in mice is as follows: A suspension of CT26 single cells in good growth condition was collected, 2 × 10⁻⁶ cells. 5 cells / only CT26 mice bearing tumors on the right back, until the tumor volume reaches 100 mm. 3When the left and right times, intraperitoneal injection of FITC-labeled CPBP-12 polypeptide, the injection dose is 100 μg / one; 2h after injection of polypeptide, using small animal imaging instrument for imaging of each organ of mice, to observe the distribution of polypeptide in each organ of mice. The results are shown in Figure 5 .

[0164] From Figure 5 It can be seen that the fluorescence of polypeptide CPBP-12 is most enriched at the tumor site, which shows that CPBP-12 can be used for tumor imaging in subsequent applications to monitor tumor metastasis or tumor tissue PD-1 expression.

[0165] Seven, CT26 colon cancer transplanted tumor model to explore the anti-tumor effect of covalent amino acid modified peptide CPBP-12

[0166] 1, tumor-bearing

[0167] The right side of the mouse was shaved in advance and waited for tumor-bearing; collect CT26 single cell suspension with good growth state, adjust the cell density to 1×10 6 cells / mL, and place on ice; after disinfecting the tumor-bearing site, use a 1 mL syringe to suck 100 μL of CT26 single cell suspension (1×105 cells) for subcutaneous tumor-bearing.

[0168] 2, tumor-bearing mouse grouping and administration

[0169] When the tumor volume of tumor-bearing mice grows to 50-100 mm 3 , group according to "S" type (8 mice per group), and the specific grouping and administration are shown in Table 1; administer for 14 days, during which the mice are free to eat, and the mouse body weight is weighed every other day, the tumor volume of mice (V = 1 / 2 × a (length) × b (width) × c (height)) is measured, and the mouse tumor volume graph is recorded and drawn. The results are shown in Figure 6 .

[0170] Table 1 Administration of tumor-bearing mice

[0171]

[0172] From Figure 6 It can be seen that, compared with the R4-Y ddf-pr group, covalent amino acid modified peptide CPBP-12 can significantly inhibit the growth of CT26 tumor in vivo, and has no toxic side effects on mice.

[0173] Although the technical solutions and technical effects of the present application have been described in detail above with general descriptions, specific embodiments and experimental examples, modifications, substitutions or improvements made by those skilled in the art without departing from the spirit and scope of the present application are also within the protection scope of the present application.

Claims

1. An affinity PD-1 covalent amino acid modified peptide, characterized in that: The amino acid sequence of the modified peptide is: Asp-Asp-X-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro-Arg; wherein X is a covalent amino acid selected from a fluorosulfate tyrosine or an aryl fluorosulfate tyrosine.

2. The affinity PD-1 covalent amino acid modified peptide according to claim 1, characterized in that: The configuration of each amino acid in the polypeptide is independently selected from a D configuration or an L configuration.

3. The covalent amino acid modified peptide of affinity PD-1 according to claim 2, characterized in that: The 2-3 consecutive amino acids at both ends of the polypeptide are in a D configuration.

4. The covalent amino acid modified peptide of affinity PD-1 according to claim 3, characterized in that: The amino acid sequence of the modified peptide is: Asp D -Asp D -X-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D ; wherein X is a fluorosulfate tyrosine.

5. A polypeptide probe, characterized by: The sequence of the covalent amino acid modified peptide with affinity to PD-1 as claimed in any one of claims 1-4.

6. The polypeptide probe of claim 5, wherein: The general structure of the polypeptide probe is as follows: M-L-R; wherein M is a photo label or a radionuclide complexing group; L is a linking group; R is the covalent amino acid modified peptide with affinity to PD-1 as claimed in any one of claims 1-4 or a dimeric, multimeric form thereof.

7. Use of an affinity PD-1 covalent amino acid modified peptide according to any one of claims 1-4, or a polypeptide probe according to any one of claims 5-6, characterized in that: One or more of the following aspects are included: (1) use in the preparation of a reagent for tumor diagnosis or tracer imaging; (2) use in the preparation of a medicament for preventing and / or treating colon cancer; (3) use in the preparation of a reagent for qualitatively and / or quantitatively detecting whether PD-1 protein is expressed, where it is expressed, or how much it is expressed; (4) use in the preparation of a reagent for detecting the affinity and / or blocking ability to PD-1 protein.

8. An agent for use in tumor diagnosis, or tracer imaging, characterized in that: The covalent amino acid modified peptide with affinity to PD-1 as claimed in any one of claims 1-4 or the polypeptide probe as claimed in any one of claims 5-6.

9. A medicament for preventing and / or treating colon cancer, characterized by comprising the compound of claim 1. The covalent amino acid modified peptide with affinity to PD-1 as claimed in any one of claims 1-4 or the polypeptide probe as claimed in any one of claims 5-6.

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