PD-1 affinity covalent amino acid modified peptide and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a PD-1 affinity covalent amino acid modified peptide and application thereof. According to the invention, PD-1 protein is taken as a target molecule, a PD-1 affinity peptide is obtained by screening through a phage display dodecapeptide library high-throughput screening technology, mutation modification and anti-enzymolysis modification are carried out on the affinity peptide, and covalent amino acid modification is further carried out, so that the retention time of the affinity peptide at an effect part is remarkably prolonged; the obtained covalent amino acid modified peptide can affine PD-1 and block interaction between PD-1 and PD-L1, can be used as a pilot peptide for developing a PD-1 / PD-L1 covalent inhibitor, and has a good application prospect and a relatively high economic value. The PD-1 affinity covalent amino acid modified peptide provided by the invention can be used for preparing antitumor drugs, molecular probes and the like, and provides a new choice for tumor diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a covalent amino acid modified peptide with affinity to PD-1 and an application thereof. Background Art

[0002] In recent years, tumor immunotherapy has attracted much attention. Science magazine selected it as the top ten scientific breakthroughs of 2013. Tumor immunotherapy mainly stimulates the body's own immune system to enhance anti-tumor ability. It can not only kill tumors, but also prevent tumor recurrence and metastasis. The effect of tumor immunotherapy is inseparable from the activation state of T cells, and the activation of T cells requires a classic dual-signal system. Among them, the first signal is generated by the binding of antigens to receptors, and the second signal is generated by the binding of antigen-presenting cells to regulatory receptors on the surface of T cells. The second signal is also called a co-stimulatory signal. According to 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). Negative co-stimulatory molecules are also called immune checkpoints. Immune checkpoint blockade therapy mainly inhibits the binding of programmed death receptors and their ligands to increase the host immune system's aggressiveness against tumor cells.

[0003] Programmed death-1 (PD-1) is an important molecule that transmits negative regulatory signals to T cells. Blocking the PD-1 / PD-L1 signaling pathway can block negative regulatory signals, relieve immunosuppression, and restore the function of T cells to kill tumor cells to a certain extent, thereby achieving the purpose of anti-tumor. At present, monoclonal antibodies targeting PD-1 have achieved good therapeutic effects in clinical applications. However, antibody drugs generally have disadvantages such as strong immunogenicity, poor tumor infiltration, and inability to withdraw drugs in time when adverse reactions occur. Peptide drugs have a relatively short half-life, good specificity and tumor permeability, especially covalent peptides can be enriched at the drug effect 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 that blocks PD-1 / PD-L1 binding, and its amino acid sequence is: HDWFKAFYDKINETYNK-OH. The polypeptide has a high binding affinity with PD-L1, can effectively inhibit the interaction between PD-1 / PD-L1, restore T cell activity, prevent tumor cell immune escape and thus produce an anti-tumor effect. Chinese patent CN111153961B (Zhengzhou University) discloses a group of polypeptides with affinity for PD-1, and its amino acid sequence is as follows: DDFRVWWPNFPA, DDFYVWWPNFPR, DDFRYWWPNFPR, 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 can also significantly inhibit mouse CT26 colon cancer, and has potential application prospects in tumor treatment, autoimmune diseases, inflammatory diseases, etc. However, the affinity of the above-mentioned polypeptides for PD-L1 or PD-1 protein needs to be improved. Therefore, it is necessary to develop a new peptide to strongly block the interaction between PD-1 / PD-L1 and improve the disease treatment effect of the peptide. Summary of the invention

[0005] The main technical problem solved by the present invention is to provide a covalent amino acid modified peptide with affinity to PD-1, which significantly increases its retention time at the effector site by covalent amino acid modification of the polypeptide. The polypeptide can be affinity to PD-1 and block the interaction between PD-1 / PD-L1, and can be used as a lead peptide for the development of PD-1 / PD-L1 covalent inhibitors, with good application prospects and high economic value.

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

[0007] Again, the present invention provides an application of a covalent amino acid modified peptide or polypeptide probe that is affinity to PD-1.

[0008] Therefore, the present invention provides a reagent for tumor diagnosis or tracing imaging.

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

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] A covalent amino acid modified peptide with affinity for PD-1, comprising one or more covalent amino acid (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 invention, 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 fluorosulfate tyrosine (Tyr(O-SO2F)), arylfluorosulfate tyrosine, and the like.

[0014] As a preferred embodiment of the present invention, the covalent amino acid modified peptide is selected from the following polypeptides or a combination 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, and the covalent amino acid is selected from covalent amino acids based on fluorine-sulfur exchange, including but not limited to one or more of fluorosulfate tyrosine (Tyr(O-SO2F)), arylfluorosulfate tyrosine, and the like.

[0019] As a preferred embodiment of the present invention, the configuration of each amino acid in the polypeptide is independently selected from D configuration or L configuration. When one or some amino acids are limited to D configuration without specifying the configuration of other amino acids, it is assumed that the other amino acids are L configuration.

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

[0021] As a preferred embodiment of the present invention, the covalent amino acid modified peptide is selected from the following polypeptides or a combination 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 a lowercase single letter is used to represent the D configuration of the corresponding amino acid, the above polypeptide (1) can be abbreviated as: ddXYVWWPNFpr. The covalent amino acid modified polypeptide is a D-configuration amino acid protective peptide at both ends, has good resistance to enzymatic hydrolysis, has a relative molecular weight of 1739.78, and has a chemical structure as shown in the following formula (I):

[0027]

[0028] Specifically, when a lowercase single letter is used to represent the D configuration of the corresponding amino acid, the above polypeptide (2) and polypeptide (3) can be abbreviated as: ddfYXWWPNFpr and ddfYVWXPNFpr.

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

[0030] As a preferred embodiment of the present invention, the modified sites include but are not limited to N-terminal modification, C-terminal modification, backbone modification, side chain modification, amino acid modification, etc. The purpose of modification includes extending 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, monosulfide cyclization, etc.;

[0033] (2) Isotope labeling: 13 C, 15 N, 18 O and other isotope labeling;

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

[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. Peptide-protein coupling: KLH coupling, albumin coupling (such as bovine serum albumin BSA, chicken ovalbumin OVA, mouse serum albumin MSA, human serum albumin HSA), albumin affinity peptide coupling, tumor homing peptide coupling, membrane-penetrating peptide coupling, antibody Fc fragment coupling, elastin-like coupling, etc.

[0038] b. Peptide-nanocarrier coupling;

[0039] (6) Modification of N-terminal or side chain amino acids: acetylation, formylation, biotin labeling, trifluoroacetylation, benzoylation, 2-aminobenzoylation, maleimidation, chloroacetylation, bromoacetylation, succinylation, palmitation, malication, fatty acidation, formalization, chelation reaction (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 formation, alcoholization, 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-phenylpropanylation, 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 radionuclides such as Zr;

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

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

[0045] A polypeptide probe comprises the sequence of the covalent amino acid modified peptide having affinity 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, and the like.

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

[0048] MLR;

[0049] Wherein, M is a photolabel or radionuclide complexing group;

[0050] L is a linking group;

[0051] R is the covalent amino acid modified peptide having affinity for PD-1 or its dimer or polymer form.

[0052] As a preferred embodiment of the present invention, the optical label 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] As a preferred embodiment of the present invention, the radionuclide probe is a radioactive I or F labeled amino acid on a covalent amino acid modified peptide that is affinity to PD-1.

[0054] As 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 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 chelating agent for radionuclide labeling includes but is not limited to NOTA, DOTA, DTPA, HYNIC, DFO, etc.

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

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

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

[0058] (1) Application in the preparation of reagents for tumor diagnosis or tracing imaging;

[0059] (2) Use in the preparation of drugs for preventing and / or treating tumors or autoimmune diseases;

[0060] (3) Use in the preparation of drugs for preventing and / or treating infections caused by bacteria, viruses or fungi;

[0061] (4) Use in the preparation of reagents for qualitatively and / or quantitatively detecting whether PD-1 protein is expressed, its expression location, or its expression amount;

[0062] (5) Use in the preparation of reagents for detecting affinity and / or blocking ability for PD-1 protein (e.g., blocking the binding between PD-1 / PD-L1).

[0063] As a preferred embodiment of the present invention, the reagents described in (1) include tumor diagnostic imaging agents, tumor boundary precise positioning imaging agents, intraoperative image navigation imaging agents, etc.

[0064] As a preferred embodiment of the present invention, the drugs include targeted gene therapy drugs, chemotherapy drugs, and the like.

[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 tracing imaging, comprising the covalent amino acid modified peptide or polypeptide probe having affinity to PD-1.

[0067] A drug for preventing and / or treating tumors or autoimmune diseases, comprising the covalent amino acid modified peptide or polypeptide probe having affinity to PD-1.

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

[0069] As a preferred embodiment of the present invention, the covalent amino acid modified peptide or polypeptide probe that is affinity to PD-1 in the drug exists in a free form or in the form of a pharmaceutically acceptable salt thereof. The PD-1 protein can be a wild type protein of human or mouse origin or a mutant protein that still retains its activity.

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

[0071] As a preferred embodiment of the present invention, the drug contains, in addition to a covalent amino acid modified peptide or polypeptide probe that is affinity for PD-1, other pharmacological ingredients that can synergize or assist in the prevention and / or treatment of tumors, and is used to inhibit tumor growth or eliminate tumors, thereby achieving combined prevention and / or treatment through combined medication.

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

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

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

[0075] Beneficial effects of the present invention:

[0076] The present invention uses PD-1 protein as the target molecule, screens out PD-1 affinity peptides through phage display twelve-peptide library high-throughput screening technology, performs mutation modification and anti-enzymatic modification on the affinity peptide, and further performs covalent amino acid modification to significantly increase its retention time at the effector site. The obtained covalent amino acid modified peptide can have affinity for PD-1 and block the interaction between PD-1 / PD-L1, can be used as a lead peptide for the development of PD-1 / PD-L1 covalent inhibitors, and has good application prospects and high economic value.

[0077] The covalent amino acid modified peptide with affinity to PD-1 provided by the present invention has an anti-tumor effect and no obvious toxic side effects, can be used to prepare anti-tumor drugs, and provides a new option for tumor immunotherapy. In addition, the covalent amino acid modified peptide with affinity to PD-1 provided by the present invention also has good application prospects in tumor diagnosis or tracing imaging, can be used to prepare reagents for tumor diagnosis or tracing imaging, and has potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is the WB detection of the covalent ability of the peptide and PD-1 protein in the experimental example;

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

[0080] Figure 2 This is the covalent ability of the fluorosulfate tyrosine (Tyr(O-SO2F)) in the covalent amino acid modified peptide CPBP-12 to bind to the PD-1 protein after replacing it with an aromatic fluorosulfate tyrosine in the experimental example.

[0081] Figure 3 This is the MST test for the affinity of the covalent amino acid modified peptide CPBP-12 to PD-1 in the experimental example.

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

[0083] Figure 5 This is the distribution of the covalent amino acid modified peptide CPBP-12 in various organs of mice in the experimental example.

[0084] Figure 6 This is the experimental result of the polypeptide inhibiting the growth of CT26 tumor in vivo in the experimental example.

[0085] In order to more clearly illustrate the technical solution to be protected by the present invention, the drawings in the embodiments or experimental examples are briefly introduced above. It should be understood that the above drawings should not be regarded as any limitation on the protection scope of the present invention. For ordinary technicians in this field, other related drawings can also be obtained based on these drawings without paying creative work. DETAILED DESCRIPTION

[0086] The technical solution of the present invention will be clearly and completely explained below in conjunction with specific embodiments and experimental examples.

[0087] Those skilled in the art should understand that the following embodiments and experimental examples are only used to illustrate the technical solutions and technical effects of the present invention and should not be regarded as any limitation on the protection scope of the present invention. Based on the following embodiments, other technical solutions obtained by those skilled in the art without creative work, such as technical solutions obtained after modification, deformation or simple replacement, all belong to the protection scope of the present invention.

[0088] Unless otherwise specified, the raw materials, reagents, and equipment used in the examples or experimental examples are all commercially available products.

[0089] Unless otherwise specified, the methods used in the embodiments or experimental examples are conventional methods.

[0090] The terms and abbreviations in the embodiments or experimental examples have the common meanings in the art.

[0091] Example 1

[0092] This embodiment provides a covalent amino acid modified peptide with affinity for PD-1, comprising 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 fluorosulfate tyrosine (Tyr(O-SO2F)), arylfluorosulfate tyrosine, and the like.

[0095] This embodiment also provides a covalent amino acid modified peptide with affinity to PD-1, 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 except for the marked amino acid which is in D configuration, the other amino acids are in L configuration.

[0100] This embodiment also provides the use of a covalent amino acid modified peptide with affinity for PD-1 in the preparation of a drug for preventing and / or treating tumors or autoimmune diseases.

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

[0102] In other embodiments of the present invention, X in the covalent amino acid modified peptide having affinity for PD-1 is arylfluorosulfate tyrosine.

[0103] Example 2

[0104] This example provides a covalent amino acid modified peptide with affinity for 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] This embodiment also provides the use of a covalent amino acid modified peptide with affinity for PD-1 in the preparation of a drug for preventing and / or treating tumors or autoimmune diseases.

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

[0110] In other embodiments of the present invention, a polypeptide modified with fluorosulfate tyrosine (Tyr(O-SO2F)) and a polypeptide modified with arylfluorosulfate tyrosine are mixed at a concentration ratio of 1:1 to prepare a drug for preventing and / or treating tumors or autoimmune diseases.

[0111] Example 3

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

[0113] MLR;

[0114] Wherein, M is infrared fluorescent dye;

[0115] L is n is 6;

[0116] R is the covalent amino acid modified peptide having affinity to PD-1 in Example 1 or Example 2.

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

[0118] This embodiment also provides a reagent for tumor diagnosis or tracing imaging, comprising an effective amount of fluorescent molecular imaging probes and an appropriate amount of pharmaceutically acceptable excipients.

[0119] Example 4

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

[0121] MLR;

[0122] Wherein, M is a radionuclide complexing group, and the radionuclide complexing group includes 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 having affinity to PD-1 in Example 1 or Example 2.

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

[0126] This embodiment also provides a tumor nuclide targeted therapeutic drug, which includes an effective amount of radionuclide probes and an appropriate amount of pharmaceutically acceptable excipients.

[0127] Experimental example

[0128] 1. Liquid phase screening of parent peptides using phage mirror display peptide library

[0129] The screening process is as follows: taking the eukaryotic protein rhPD-1 / Fc as the target, the phage displayed twelve-peptide library was screened by liquid phase differential screening method; after three rounds of screening, the phage monoclones with affinity to the target protein rhPD-1 / Fc were enriched round by round; the positive clones were selected from the third round for sequencing, and a total of multiple inserted twelve-peptide sequences were obtained, several of which had repeated clones, and the parent peptide R5 was one of the positive clones.

[0130] The amino acid sequence of the parent peptide R5 is shown below:

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

[0132] 2. Design and synthesis of enzyme-resistant peptide R4-Y ddf-pr

[0133] The preparation process is as follows: the parent peptide R5 is molecularly docked with the PD-1 protein, the best docking mode is selected and molecular dynamics simulation is performed, and after a single amino acid mutation, the mutant peptide is selected based on the Delta affinity value, mutation intersection, interaction and affinity blocking experiments to obtain the optimized peptide R4-Y, and the anti-enzymatic peptide R4-Y is obtained through the D-configuration amino acid protection strategy at both ends. ddf-pr .

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

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

[0136] Anti-enzyme peptide R4-Y ddf-pr The amino acid sequence is as follows:

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

[0138] 3. Design and synthesis of covalent amino acid modified peptides

[0139] R4-Y ddf-pr The covalent amino acid modified peptide was designed and synthesized as the parent peptide. The process is as follows: fluorosulfate tyrosine (Tyr(O-SO2F)) can generate a covalent bond with its adjacent lysine, tyrosine or histidine through a fluorine-sulfur exchange reaction; R4-Y ddf-pr Molecular docking and molecular dynamics simulation were performed between the peptide and PD-1 protein to observe the binding surface between the peptide and the protein, and the amino acids adjacent to lysine, tyrosine or histidine on the protein were selected for covalent amino acid Tyr (O-SO2F) replacement, and the peptides after amino acid replacement were synthesized; the three candidate covalent amino acid modified peptides labeled with biotin (SA) were co-incubated with the same amount of hPD-1 protein and then subjected to SDS-PAGE gel electrophoresis. After protein transfer, anti-Streptavidin-HRP was incubated, and chemiluminescence was used to detect protein blot (Western Blot) to detect whether the peptide and protein were successfully covalently bound. The results are as follows Figure 1 shown.

[0140] from Figure 1 It can be seen that the peptides CPBP-12 (f3X), V5X and W7X can all covalently bind to the PD-1 protein. At the same peptide concentration, the covalent ability of CPBP-12 (f3X) is stronger than that of other peptides, while R4Y ddf-pr It cannot covalently bind to the PD-1 protein.

[0141] Furthermore, another covalent amino acid aryl fluorosulfate tyrosine based on fluorine-sulfur exchange was used to replace the fluorosulfate tyrosine in CPBP-12, and WB was also used to monitor whether the peptide and protein were successfully covalently bound. Figure 2 shown.

[0142] from Figure 2It can be seen that after replacing the fluorosulfate tyrosine in the peptide CPBP-12 with aryl fluorosulfate tyrosine, the peptide can still covalently bind to the PD-1 protein.

[0143] 4. Microcalorimetry (MST) to detect the affinity of covalent amino acid modified peptides to hPD-1

[0144] The affinity of the covalent amino acid modified peptide to the hPD-1 protein was tested as follows:

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

[0146] (2) Washing the column: Place the protein separation column in a 15 mL centrifuge tube and wash it with MST Buffer (generally 10 column volumes, column volume is 500 μL);

[0147] (3) Isolation and purification of protein: The protein dye mixture (200 μL) in step (1) was added dropwise to the protein separation column, and then 300 μL of MST Buffer was added. When the column no longer dripped liquid, 500 μL MST Buffer was added. The samples were collected from the first drop of liquid. After the collection was completed, the fluorescence was detected on the machine, and the protein fluorescence value was diluted to about 600. The samples were stored at -80°C after aliquoting.

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

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

[0150] (6) On-machine testing: Turn on the computer and start the MO.Control software, and select the (Red) channel BindingAffinity mode for testing;

[0151] (7) Analysis results: The binding dissociation constant (K) was calculated using NanoTemper analysis software MO.AffinityAnalysis v2.2.4. D The result is Figure 3 shown.

[0152] from Figure 3 It can be seen that the covalent amino acid modified peptide CPBP-12 can bind to hPD-1 protein with affinity, K D The value is 50±10nM.

[0153] 5. In vitro blocking experiment

[0154] The ability of covalent amino acid modified peptides to block hPD-1 / hPD-L1 binding was tested as follows:

[0155] (1) Cell collection: CHO-K1-hPD-1 cells in good condition were collected, centrifuged and washed twice at 4000 rpm and 4°C, and placed on ice for later use;

[0156] (2) Incubation: The cells in step (1) were cultured at 5 × 10 5 Divide into different 1.5mL EP tubes, add 25μL of peptide blocker (set the concentration as needed) to the experimental group and incubate on ice for 30min; set up positive control tubes and negative control tubes, add 25μL PBS (pH=7.4) to both positive control tubes and negative control tubes;

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

[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 by centrifugation at 4000 rpm and 4°C with 1 mL of PBS (pH = 7.4);

[0160] (6) Flow cytometry: Add 200 μL FACS buffer to each tube to resuspend the cells, transfer to a flow cytometry tube away from light, and prepare for flow cytometry. Figure 4 shown.

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

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

[0163] The in vivo imaging process of mice is as follows: a single cell suspension of CT26 cells in good growth condition is collected, 2×10 5 cells / CT26 mouse with tumor on the right back, wait until the tumor grows to 100 mm 3Around 2 hours after the injection of peptide, FITC-labeled CPBP-12 peptide was intraperitoneally injected at a dose of 100 μg / mouse. Two hours after the injection of peptide, the organs of the mice were imaged using a small animal imaging device to observe the distribution of the peptide in the organs of the mice. Figure 5 shown.

[0164] from Figure 5 It can be seen that the peptide CPBP-12 has the highest fluorescence enrichment in the tumor site, indicating that CPBP-12 can be used for tumor imaging in subsequent applications to monitor tumor metastasis or PD-1 expression in tumor tissue.

[0165] 7. CT26 colon cancer transplant model to investigate the anti-tumor effect of covalent amino acid modified peptide CPBP-12

[0166] 1. Tumor

[0167] The right back of the mouse was shaved in advance to wait for tumor loading; the CT26 single cell suspension with good growth status was collected and the cell density was adjusted to 1×10 6 cells / mL and placed on ice; after disinfecting the tumor-bearing site, 100 μL of CT26 single cell suspension (1×105 cells) was drawn into the subcutaneous tumor using a 1 mL syringe.

[0168] 2. Grouping and drug administration of tumor-bearing mice

[0169] When the tumor of the tumor-bearing mice grows to 50-100mm 3 The mice were grouped in an "S" shape (8 mice in each group). The specific grouping and dosing conditions are shown in Table 1 below. The mice were given drugs for 14 days, during which they were fed freely. The mice were weighed every other day, and the tumor volume of the mice was measured (V = 1 / 2 × a (length) × b (width) × c (height)). The tumor volume of the mice was recorded and plotted. Figure 6 shown.

[0170] Table 1 Drug administration in tumor-bearing mice

[0171]

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

[0173] Although the technical scheme and technical effects of the present invention have been described in detail above with general descriptions, specific embodiments and experimental examples, for those skilled in the art, any modification, replacement or improvement made without departing from the spirit of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A covalent amino acid modified peptide with affinity for PD-1, characterized in that: Contains one or more covalent amino acid modifications at any position in the following polypeptides: Asp-Asp-Phe-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro-Arg, or a modified form thereof.

2. The covalent amino acid modified peptide with affinity to PD-1 according to claim 1, characterized in that: The covalent amino acid is selected from covalent amino acids based on fluorine-sulfur exchange, including but not limited to fluorosulfate tyrosine and arylfluorosulfate tyrosine.

3. The covalent amino acid modified peptide with affinity to PD-1 according to claim 1, characterized in that: Selected from the following polypeptides or combinations thereof: (1) Asp-Asp-X-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro-Arg, or a modified form thereof; (2) Asp-Asp-Phe-Tyr-X-Trp-Trp-Pro-Asn-Phe-Pro-Arg, or a modified form thereof; (3) Asp-Asp-Phe-Tyr-Val-Trp-X-Pro-Asn-Phe-Pro-Arg, or a modified form thereof; Wherein, X is a covalent amino acid selected from covalent amino acids based on fluorine-sulfur exchange, including but not limited to fluorosulfate tyrosine and arylfluorosulfate tyrosine.

4. The covalent amino acid modified peptide with affinity to PD-1 according to claim 3, characterized in that: The configuration of each amino acid in the polypeptide is independently selected from the D configuration or the L configuration; And / or, 2-3 consecutive amino acids at both ends of the polypeptide are in D configuration.

5. The covalent amino acid modified peptide with affinity to PD-1 according to claim 4, characterized in that: Selected from the following polypeptides or combinations thereof: (1)Asp D -Asp D -X-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D ; (2)Asp D -Asp D -Phe D -Tyr-X-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D ; (3)Asp D -Asp D -Phe D -Tyr-Val-Trp-X-Pro-Asn-Phe-Pro D -Arg D ; Wherein, X is fluorosulfate tyrosine.

6. A polypeptide probe, characterized in that: A sequence comprising a covalent amino acid modified peptide having affinity for PD-1 as described in any one of claims 1 to 5.

7. The polypeptide probe according to claim 6, characterized in that: The general structural formula of the polypeptide probe is as follows: MLR; Wherein, M is a photolabel or radionuclide complexing group; L is a linking group; R is a covalent amino acid modified peptide having affinity for PD-1 according to any one of claims 1 to 4, or a dimer or polymer thereof.

8. A use of a covalent amino acid modified peptide having affinity for PD-1 as claimed in any one of claims 1 to 5, or a polypeptide probe as claimed in any one of claims 6 to 7, characterized in that: Including but not limited to one or more of the following aspects: (1) Application in the preparation of reagents for tumor diagnosis or tracing imaging; (2) Use in the preparation of drugs for preventing and / or treating tumors or autoimmune diseases; (3) Use in the preparation of drugs for preventing and / or treating infections caused by bacteria, viruses or fungi; (4) Use in the preparation of reagents for qualitatively and / or quantitatively detecting whether PD-1 protein is expressed, its expression location, or its expression amount; (5) Use in the preparation of reagents for detecting affinity and / or blocking ability for PD-1 protein.

9. A reagent for tumor diagnosis or tracing imaging, characterized in that: A covalent amino acid modified peptide having affinity for PD-1 as described in any one of claims 1 to 5, or a polypeptide probe as described in any one of claims 6 to 7.

10. A drug for preventing and / or treating tumors or autoimmune diseases, characterized in that: A covalent amino acid modified peptide having affinity for PD-1 as described in any one of claims 1 to 5, or a polypeptide probe as described in any one of claims 6 to 7.

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