WO2021023140A1 - Peptide d'affinité de pd-l1-igv et son utilisation - Google Patents

Peptide d'affinité de pd-l1-igv et son utilisation Download PDF

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WO2021023140A1
WO2021023140A1 PCT/CN2020/106488 CN2020106488W WO2021023140A1 WO 2021023140 A1 WO2021023140 A1 WO 2021023140A1 CN 2020106488 W CN2020106488 W CN 2020106488W WO 2021023140 A1 WO2021023140 A1 WO 2021023140A1
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peptide
seq
affinity
affinity peptide
amino acid
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PCT/CN2020/106488
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Chinese (zh)
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高艳锋
李琬琼
翟文杰
周秀曼
祁元明
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郑州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the technical field of biopharmaceuticals.
  • the inhibitory molecule PD-1 expressed by immune cells in the tumor microenvironment interacts with T cells or antigen-presenting cell surface ligands to exhaust T cells, inhibit their anti-tumor effects, and cause immune escape. .
  • PD-L1 is the main ligand of PD-1.
  • PD-1/PD-L1 is usually involved in inducing T cell tolerance.
  • this pathway is used in the research and development of autoimmune diseases, viruses and bacterial infections.
  • Therapeutic applications are also constantly progressing. Therefore, based on the PD-1/PD-L1 pathway, finding a reasonable and effective treatment strategy is also a problem that scientists are facing and urgently need to solve.
  • Monoclonal antibodies against PD-1/PD-L1 pathways are currently on the market and are used in tumor immunotherapy, but the high production cost of antibody drugs, poor tissue permeability, and long half-life, can not quickly terminate immune adverse events; peptide synthesis is convenient , The tissue permeability is good, the immunogenicity is also low, and it has good development value and application prospects.
  • the present invention provides an affinity peptide of PD-L1-IgV, which is selected from the peptides defined by the following peptides a, b, c, or a combination thereof:
  • amino acid sequence is selected from SEQ ID NOs: 1, 2, 3, or 5 (NOs in the art indicate a parallel listing of sequence numbers, that is, the amino acid sequences are SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. .3 or SEQ ID NO.5);
  • Peptide b the polypeptide shown in SEQ ID NO. 4, or a mutant peptide with point mutations in the 4th, 5th, 10th, and/or 11th amino acids, where the point mutations include conservative substitutions of amino acids, Such as the exchange of Gln and Glu, the exchange of Asn and Asp;
  • Peptide c the polypeptide shown in SEQ ID NO. 8, a conservative mutant peptide obtained by conservative substitution of the above amino acids (such as the peptide shown in SEQ ID NO. 59), its alanine scanning peptide, or its N-terminal or C-terminal
  • the truncated peptide, the number of amino acids of the truncated peptide is 4-11, or the alanine scanning peptide of the truncated peptide.
  • peptide c can be divided into:
  • Peptide c1 which is the polypeptide shown in SEQ ID NO. 8 or its alanine scanning peptide
  • Peptide c2 which is the polypeptide shown in SEQ ID NO. 8 or its N-terminal or C-terminal truncated peptide, and the number of amino acids of the truncated peptide is 4-11;
  • Peptide c3 which is the polypeptide shown in SEQ ID NO. 32 or its alanine scanning peptide.
  • Partial peptides of peptide c1 and peptide c2 can be combined to form peptide c4: Arg-Val-Tyr-Ser-Phe and 12 peptides obtained by adding 7 amino acids to the N-terminus.
  • the 7 amino acids of the 12 peptides contain 1 alanine.
  • Such as the sequence is Gly-Gln-Ser-Glu-His-His-Ala-Arg-Val-Tyr-Ser-Phe.
  • the above-mentioned pentapeptide Arg-Val-Tyr-Ser-Phe derived peptide in addition to the 7 amino acid increase mentioned above, and the N-terminal increase of less than 7 amino acids, is also the affinity peptide of PD-L1-IgV, Also within the scope of protection of this patent, if the derived peptide sequence is selected from SEQ ID NOs: 39-57 (NOs: 39-57 is the abbreviated form of the NOs listed side by side, and the sequence numbers included are between 39-57 Any integer).
  • Alanine scanning peptide refers to a series of single mutant peptides obtained by replacing any one amino acid of the protein parent with alanine in the art, such as a series of alanine scanning peptides shown in peptide c SEQ ID NO.8, The sequence is SEQ ID NO.15, SEQ ID NO.16...
  • Truncated peptides in the art refer to a series of shorter peptides obtained by truncating 1 to more amino acids from the N-terminus or C-terminus of the protein parent, such as a series of N-terminus truncations of the peptide shown in the peptide c SEQ ID NO.8 Short peptide, the sequence is SEQ ID NO.26, SEQ ID NO.27...
  • the point mutations are independently selected from the following mutations:
  • Ala mutations are Trp, Phe, Tyr, His, Ile, Gln, Glu;
  • the affinity peptide is a single point mutant peptide of the polypeptide shown in SEQ ID NO. 4, that is, an amino acid mutation occurs at the aforementioned 1 position (such as position 4) of the parent peptide.
  • the truncated peptide is the N-terminal truncated peptide of the polypeptide shown in SEQ ID NO. 8, and the number of amino acids is 5-11.
  • sequence of the affinity peptide is selected from SEQ ID NOs: 1-60.
  • the configuration of the amino acids of the affinity peptide is independently selected from D-type or L-type, and can be both D-type or L-type, for example, all amino acids are in D-configuration.
  • glycine is not divided into D and L types, in order to make the description of the configuration of each amino acid concise, glycine is also arbitrarily defined as: D type or L type).
  • the PD-L1 of the present invention refers to the ligand of mammalian PD-1 protein, such as human PD-L1 (hPD-L1) or mouse PD-L1 (mPD-L1), and PD-L1-IgV is PD-L1 The IgV-like domain.
  • PD-1 and PD-L1 can be wild-type or mutant proteins that still retain their activity, such as wild-type or mutant PD-1 in the applicant’s prior patent CN108794619.
  • affinity peptide of this patent can be changed in response to the need for a drug. It can exist in a free form or in the form of a pharmaceutically acceptable salt.
  • the aforementioned affinity peptide can also be chemically modified to extend Half-life, such as cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, albumin affinity peptide coupling, tumor homing peptide coupling, penetrating peptide coupling, nanocarrier coupling; Simple modifications based on the idea of this patent constitute equivalent infringement of this patent.
  • the present invention provides a drug, a pharmaceutical composition or a kit containing the affinity peptide described in the first aspect
  • the pharmaceutical composition may include a pharmaceutically acceptable excipient, and the drug or pharmaceutical composition may be used in:
  • Anti-tumor such as colon cancer or melanoma
  • the PD-L1-IgV affinity peptide obtained in the present invention can all block the binding of PD-1/PD-L1, and the peptide with high blocking rate can significantly inhibit the growth of colon cancer or melanoma without obvious toxic and side effects.
  • Figure 1 is a graph showing the experimental results of peptides H5S, H7, H9, H12, H14 blocking PD-1/PD-L1 protein binding;
  • Figure 2 is a diagram showing the experimental results of peptide H12 and H12 mutant peptides blocking PD-1/PD-L1 protein binding;
  • Figure 3 shows the results of a peptide cell-level affinity experiment
  • Figure 4 is a graph showing the effect of peptides on body weight changes of BABL/c mice vaccinated with CT26;
  • Figure 5 shows the effect of peptides on the volume changes of transplanted tumors in BABL/c mice inoculated with CT26;
  • Figure 6 is a graph showing the effect of peptide on the tumor volume of C57BL/6 mice inoculated with B16-OVA;
  • Figure 7 is a diagram showing the experimental results of blocking the binding of PD-1/PD-L1 protein by P8 alanine scanning peptide
  • Figure 8 is a diagram showing the experimental results of truncated peptides blocking PD-1/PD-L1 protein binding
  • Figure 9 is a diagram showing the experimental results of blocking the binding of PD-1/PD-L1 protein by the alanine scanning peptide of peptide P32;
  • Figure 10 is a graph showing the experimental results of blocking the binding of PD-1/PD-L1 protein by the conservative mutant peptide of P8;
  • Figure 11 shows the effect of truncated peptide on the volume change of transplanted tumor in BABL/c mice inoculated with CT26;
  • Figure 12 shows the blocking effect of the derived peptides on PD-1/PD-L1 protein binding
  • the antibody detects whether hPD-1 is stably expressed on the cell membrane, and the isotype antibody is used as a control (Mouse IgG1 ⁇ isocontrol PE) to ensure that the cells can be used for subsequent blocking experiments.
  • Wash antibody Wash once with 1mL ice-cold PBS7.2, centrifuge at 1800rpm for 5min, add 200 ⁇ L PBS7.2 to resuspend, transfer to a flow tube for flow detection of CHO-K1-hPD-1 cells and hPD-L1-Fc protein Combine the situation.
  • the blocking results show that the polypeptides (H5S, H7, H9, H12, H14) of the present invention can block the binding of hPD-L1 protein to CHO-K1-hPD-1 cells, and the blocking rate is shown in FIG. 1.
  • PEPstrMOD predicts the 3D structure of H12 peptide online: Open the PEPstrMOD webpage, enter the peptide sequence to be predicted, and click Submit and Go to Next Step. Since the peptide is in D configuration, change Steriochemistry to Dextro(D), enter the email below and click submit. After the system predicts, you can download the peptide structure in the email.
  • Z-DOCK molecular docking molecular docking of the predicted H12 peptide structure with hPD-L1 (PDB ID: 3BIK), molecular docking adopts Z-DOCK online docking, MOE analysis docking results, select docking mode (comprehensive Bond energy, distance, and interaction sites are considered), and use MOE to run 50ns molecular dynamics.
  • MOE unit point mutation select the docking mode after the above molecular dynamics, use MOE to run unit point amino acid mutations, sort the results of the mutations by affinity, and select the top nine with higher scores for subsequent mutation peptide synthesis
  • the 9 mutant peptides were named P6-P14, and the sequence correspondences are shown in SEQ ID NO.6-SEQ ID NO.14.
  • Each amino acid is of type D.
  • 2-blocking experiment the blocking ability of mutant peptides was tested, and the results showed that each peptide can block the binding of hPD-L1 protein to CHO-K1-hPD-1 cells.
  • the blocking rate is shown in Figure 2.
  • P8 refers to Experiment 2 to conduct an experiment to block murine mPD-L1 (replace the protein and cell correspondence with mouse-derived ones).
  • the results show that P8 can also block mPD-1/mPD-L1 binding.
  • the blocking rate is 30%.
  • the conservative mutant peptides P58, P59, and P60 of peptide P8 were further synthesized (the sequence also corresponds to SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60; each amino acid is also D type), and the resistance is also detected according to the above method. Break rate, the result is shown in Figure 10.
  • Micro thermal surge detects the affinity of peptide P8 and PD-L1. The detection process is as follows:
  • NT647 dye-labeled hPD-L1-His protein (theory: take 100 ⁇ L of 10 ⁇ M protein and add 50 ⁇ L of 20 ⁇ M dye). The hPD-L1-His protein concentration used in the experiment was 7.14 ⁇ M.
  • Preparation of the sample to be tested Weigh and dissolve an appropriate amount of peptides on an electronic balance, dilute the peptides from 100 ⁇ M down by 2 times, perform a total of 16 gradients of dilution, and mix them with the labeled fluorescent protein in equal volumes (10 ⁇ L target protein plus 10 ⁇ L polypeptide), use a capillary tube to suck 10 ⁇ L of the above mixture and place it on the sample holder according to the corresponding position for the next analysis.
  • Test affinity On the instrument operation interface, click start scan to check whether the fluorescence intensity of each tube is consistent. If the consistency is good, click start MST measurement to start the measurement.
  • the fluorescent peptide P8 is placed in PBS7.2 Co-incubate with CHO-K1, CHO-K1-hPD-L1, CHO-K1-mPD-L1 with its maximum solubility of 10 ⁇ M, and flow cytometry to detect whether the mutant peptide can specifically affinity hPD-L1 and mPD-L1 ,
  • affinity rate is shown in Figure 3.
  • Tumor-bearing CT26 colorectal cancer cells and B16-OVA melanoma cells in good growth condition were collected, and tumor-bearing BABL/c mice (2 ⁇ 10 5 cells/mouse) and C57BL/6 mice (5 ⁇ 10 5 cells/only).
  • the tumor volume curves shown in Figure 5 and Figure 6 show that P8 can well inhibit the growth of CT26 colorectal cancer xenografts and B16-OVA melanoma xenografts at a dose of 0.5 mg/kg, and the results are shown in Figure 4. It showed that the body weight of BABL/c mice did not decrease significantly at the 0.5 mg/kg dosage of P8. The mental state of the mice during the administration of the two transplanted tumor models was relatively good.
  • the peptide P8 was subjected to alanine scanning, and the blocking experiment was conducted to study the blocking ability of the alanine scanning peptide.
  • the specific implementation method is basically the same as that described in the above experiment 2.
  • P8's alanine scanning peptide P15-P25 is all at a concentration of 100 ⁇ M It can block PD-1/PD-L1 protein binding.
  • the peptide P8 was truncated, and the blocking experiment was conducted to study the blocking ability of the truncated peptide.
  • the specific implementation method was basically the same as that described in the above experiment 2. Taking the value of the blocking rate of P8 on hPD-L1 as 100%, calculate the relative blocking rate of other peptides relative to the peptide, as shown in Figure 8: P8 truncated peptide P26-P33 can block at a concentration of 100 ⁇ M Cut off the PD-1/PD-L1 protein binding.
  • the peptide P32 was subjected to alanine scanning, and the blocking experiment was conducted to study the blocking ability of the alanine scanning peptide.
  • the specific implementation method was basically the same as that described in the above experiment 2.

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Abstract

La présente invention concerne un peptide d'affinité de PD-L1-IgV et son utilisation. Le peptide d'affinité est choisi parmi un polypeptide tel que représenté par SEQ ID NO : 1, la même série de polypeptides et de variants, qui comprennent : des peptides mutants qui sont soumis à une mutation ponctuelle en position 4, position 5, position 10 et/ou position 11 du polypeptide tel que représenté par SEQ ID NO : 4 ; et un peptide tronqué à extrémité N-terminale ou C-terminale du polypeptide tel que représenté par SEQ ID NO : 8, ou un peptide de balayage d'alanine du peptide tronqué. Le peptide obtenu par criblage et optimisation peut bloquer l'interaction entre PD-1 et PD-L1 de manière à traiter une tumeur ou d'autres types de maladies.
PCT/CN2020/106488 2019-08-02 2020-08-01 Peptide d'affinité de pd-l1-igv et son utilisation WO2021023140A1 (fr)

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CN201910711326.8A CN110330550B (zh) 2019-08-02 2019-08-02 PD-L1-IgV的亲和肽及其应用

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Publication number Priority date Publication date Assignee Title
CN110330550B (zh) * 2019-08-02 2021-04-13 郑州大学 PD-L1-IgV的亲和肽及其应用
WO2021116079A1 (fr) 2019-12-10 2021-06-17 Université de Mons Peptides se liant au récepteur des ldl en tant que véhicules pour traverser la barrière hémato-encéphalique
CN111153961B (zh) * 2020-01-08 2022-02-18 郑州大学 一种亲和pd-1的肽及其应用
CN112409450B (zh) * 2020-03-29 2023-01-24 郑州大学 TIGIT-IgV的亲和剂及其应用
CN112724199B (zh) * 2020-12-30 2023-01-24 郑州大学 亲和Clec9a的多肽及其应用
CN112812174A (zh) * 2021-01-15 2021-05-18 新乡学院 猪pd-l14qn-af表位多肽及其应用
CN114044808B (zh) * 2021-11-19 2024-01-30 郑州大学 白蛋白亲和剂及其应用

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CN103936835A (zh) * 2014-04-29 2014-07-23 郑州大学 具有抗肿瘤活性的靶向PD-L1IgV亲和肽D1
CN108350082A (zh) * 2016-06-13 2018-07-31 爱迈博 Pd-l1抗体及其用途
CN110330550A (zh) * 2019-08-02 2019-10-15 郑州大学 PD-L1-IgV的亲和肽及其应用

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CN104086627B (zh) * 2014-05-29 2016-06-08 郑州大学 具有抗肿瘤活性的PD-L1 IgV亲和肽S10
CN104098651B (zh) * 2014-06-30 2016-06-29 郑州大学 具有抗肿瘤活性的PD-L1 IgV亲和肽及其应用
WO2018133873A1 (fr) * 2017-01-23 2018-07-26 苏州康宁杰瑞生物科技有限公司 Polypeptide ou composite liant pd-l1

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103936835A (zh) * 2014-04-29 2014-07-23 郑州大学 具有抗肿瘤活性的靶向PD-L1IgV亲和肽D1
CN108350082A (zh) * 2016-06-13 2018-07-31 爱迈博 Pd-l1抗体及其用途
CN110330550A (zh) * 2019-08-02 2019-10-15 郑州大学 PD-L1-IgV的亲和肽及其应用

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