WO2021023140A1

WO2021023140A1 - Affinity peptide of pd-l1-igv and application thereof

Info

Publication number: WO2021023140A1
Application number: PCT/CN2020/106488
Authority: WO
Inventors: 高艳锋; 李琬琼; 翟文杰; 周秀曼; 祁元明
Original assignee: 郑州大学
Priority date: 2019-08-02
Filing date: 2020-08-01
Publication date: 2021-02-11
Also published as: CN110330550A; CN110330550B

Abstract

The present invention provides an affinity peptide o f a PD-L1-IgV and an application thereof. The affinity peptide is selected from the polypeptide as represented by SEQ ID NO: 1, the same series of polypeptides and variants, which comprise: mutant peptides which are subjected to point mutation at position 4, position 5, position 10, and/or position 11 of the polypeptide as represented by SEQ ID NO: 4; and an N-terminal or C-terminal truncated peptide of the polypeptide as represented by SEQ ID NO: 8, or an alanine scanning peptide of the truncated peptide. The peptide obtained by screening and optimizing can block the interaction between PD-1 and PD-L1 well so as to treat tumor or other types of diseases.

Description

PD-L1 IgV affinity peptide and its application

Technical field:

The invention belongs to the technical field of biopharmaceuticals.

Background technique:

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. In addition to the application in the field of tumors, 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.

Summary of the invention:

In the first aspect, 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:

Peptide a, the 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.

Such as 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...

Optionally, the point mutations are independently selected from the following mutations:

1) Glu mutations into Gln, Asn, Asp;

2) His mutation is Gln, Glu;

3) Ala mutations are Trp, Phe, Tyr, His, Ile, Gln, Glu;

4) Ser is mutated to Arg.

Optionally, 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.

Optionally, 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.

Optionally, the sequence of the affinity peptide is selected from SEQ ID NOs: 1-60.

Optionally, 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. (Although 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.

In addition, it should be noted that the 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.

In the second aspect, 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:

1) Anti-tumor, such as colon cancer or melanoma

2) Treat infections caused by bacteria, viruses or fungi

3) Treatment of autoimmune diseases

4) Block the binding of PD-1 protein to PD-L1 ligand; the PD-1 and PD-L1 can be wild-type proteins of human or mouse origin or mutant proteins that still retain their activity.

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.

Description of the drawings:

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 sign of significance analysis involved in each figure * means P<0.05, and ** means P<0.01.

detailed description:

Unless otherwise specified, the reagents, biological materials, culture media and solutions used below are all commonly used in the field, available to the public or commercially available.

For the screening of PD-L1-IgV affinity peptides, each experiment is explained as follows:

1. Liquid phase screening of phage mirror display peptide library to obtain parent peptide H12, etc. The general screening process is as follows:

a) Fully chemically synthesize D-hPD-L1-IgV-biotin, and use SA magnetic beads to capture the D configuration protein, and use the liquid phase screening method to screen the phage display dodecapeptide library;

b) After multiple rounds of screening, phage monoclonal with affinity to the target protein D-hPD-L1-IgV are enriched round by round;

c) Select positive clones for sequencing, and obtain multiple inserted dodecapeptide sequences (mutate one of the peptides), and obtain linear parent peptides H5S, H7, H9, H12, H14 (sequences are shown in SEQ ID NO.1- As shown in 5, each amino acid configuration is synthesized as D type).

2. Blocking experiment:

a) Cultivate CHO-K1-hPD-1 cells in RMPI 1640 medium containing 10% FBS. After the cells grow to log phase, trypsinize and collect the cells in 1.5 mL EP tubes, each with 2 cells ×10 ⁵ , after washing twice with 1mL PBS7.2, place on ice for later use.

b) First, the antibody (Antihuman PD-1 PE) detects whether hPD-1 is stably expressed on the cell membrane, and the isotype antibody is used as a control (Mouse IgG1 κ isocontrol PE) to ensure that the cells can be used for subsequent blocking experiments.

c) Co-incubation of polypeptide and hPD-L1-Fc: weigh a certain amount of polypeptide and dissolve it to 100 μM in PBS7.2. Take 200μL of low-adsorption EP tube, mix 50ng hPD-L1-Fc with 50μL 100μM peptide by vortex and incubate on ice for 30min.

d) Add the above mixture to CHO-K1-hPD-1 cells, vortex to mix, and incubate on ice for 30 minutes.

e) Add 0.3 μL of antibody (Human Fcγ specific PE) to the mixture of the above three substances, vortex to mix, and incubate for 30 min on ice in the dark.

f) Wash antibody: Wash once with 1mL ice-cold PBS7.2, centrifuge at 1800rpm for 5min, add 200μL 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.

3. Use H12 as the parent peptide for subsequent optimization work. The specific implementation methods are as follows:

a) 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.

b) 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.

c) 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 According to the Fmoc solid-phase synthesis method, 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. After the mass spectrometry identification is correct, According to the previous experiment 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. In addition, taking peptide P8 as an example, refer 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.

4. Micro thermal surge (MST) detects the affinity of peptide P8 and PD-L1. The detection process is as follows:

a) NT647 dye-labeled hPD-L1-His protein (theory: take 100 μL of 10 μM protein and add 50 μL of 20 μM dye). The hPD-L1-His protein concentration used in the experiment was 7.14μM. First, mix NT647 dye with a concentration of 20 μM and a volume of 50 μL with the protein thoroughly, and then react for 30 minutes at room temperature in the dark. After using NT.115TM protein labeling kit RED to label the target protein hPD-L1-His, the dye-protein complex can be effectively formed. After the labeling reaction is completed, molecular sieves are used to remove unreacted dyes to facilitate subsequent experiments.

b) Column balance: balance the chromatography column during the above dye labeling process. PBS7.2 is enough to balance 3 column volumes. Pay attention to keeping the column moist during the column balance.

c) Chromatographic column separation of protein: After the protein and dye are fully reacted for 30 minutes in the dark, add the above mixture to the pre-equilibrated chromatographic column, and then add 400 μL of PBS7.2, and wait for the above two liquids to fully penetrate the column When the time, add 600μL PBS7.2 and start to collect the liquid dripped from the column, and collect each drop into a new EP tube (if the protein molecular weight is small, you do not need to collect the first 3 drops), and collect it to the column. Then drip the liquid. After the collection is completed, the capillary sucks a small amount of liquid, MST detects the labeling efficiency and rinses the column, and stores it in a 20% alcohol solution at 4°C. According to the fluorescence intensity and protein concentration used in the specific experiment, the labeled protein can be diluted appropriately.

d) Preparation of the sample to be tested: Weigh and dissolve an appropriate amount of peptides on an electronic balance, dilute the peptides from 100μM down by 2 times, perform a total of 16 gradients of dilution, and mix them with the labeled fluorescent protein in equal volumes (10μL target protein plus 10μL polypeptide), use a capillary tube to suck 10μL of the above mixture and place it on the sample holder according to the corresponding position for the next analysis.

e) 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.

f) Analyze and organize experimental data.

The experimental results on human PD-L1 show that the linear D peptide P8 can have a good affinity for hPD-L1 protein (the affinity Kd value for binding to PD-L1 = 0.9μM), and it has a good affinity with the positive ligand protein hPD-1 (Kd Value = 0.4μM) has the same level of affinity. Refer to the above experiment to carry out the affinity experiment for mouse mPD-L1 (replace the protein and cell corresponding to the mouse source), and get similar results: P8 can also have a good affinity for mPD-L1 (Kd=3.78μM), and is compatible with mPD -1 has the same level of affinity (Kd=4.04μM).

5. Peptide cell level affinity experiment

After the fluorescent dye Cy5.5-NHS and the high activity peptide P8 synthesized by standard Fmoc solid phase synthesis (the C terminal is connected to Rink resin, the N terminal is exposed) amino group, after purification and preparation, 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 10μM, and flow cytometry to detect whether the mutant peptide can specifically affinity hPD-L1 and mPD-L1 , The statistical results of affinity rate are shown in Figure 3.

6. Explore the anti-tumor effects of P8 in CT26 colon cancer transplantation tumor model and B16-OVA melanoma transplantation tumor model respectively. The specific implementation methods are as follows:

a) 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).

b) After tumor-bearing for about one week, when the tumor volume of the mice grows to about 40-80mm ³ , start S-shaped grouping and intraperitoneal administration every day for 2 consecutive weeks. Use an electronic balance to weigh the mice at intervals between the administration periods. Measure mouse tumors with digital vernier calipers, calculate and record the volume changes of mouse tumors according to the formula V=1/2×a(length)×b(width)×c(height);

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.

7. Affinity peptidase degradation stability test

a) Dissolve peptide P8 PBS7.2 into 100μM mother liquor, mix 190μL of 10% human serum solution with 10μL of the above A10Y mother liquor quickly, take out 20μL of the mixture and count it for 0h, and place the rest in a 37℃ incubator for enzymolysis Reaction, and take out 20μL of reaction product in 1.5mL EP tube at 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, 48h, and use it for the detection of enzymolysis at each time point;

b) Add 90 μL of acetonitrile to the samples taken at the above different time points carefully with a pipette, shake and mix them immediately, place them on ice for 10 minutes, and add 90 μL 0.5% glacial acetic acid solution to the pipette to terminate the enzymatic hydrolysis reaction ；

c) Pre-cooled centrifuge at 4°C, centrifuged at 10000g for 20 minutes and collected the supernatant in a new EP tube for subsequent RP-HPLC analysis of enzyme degradation stability experiments showed that peptide P8 still exists stably at 48h, and is resistant to enzymatic degradation Significantly.

8. 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.

Taking the value of the blocking rate of P8 on hPD-L1 as 100%, calculate the relative blocking rate of other peptides relative to this peptide, as shown in Figure 7: P8's alanine scanning peptide P15-P25 is all at a concentration of 100μM It can block PD-1/PD-L1 protein binding.

9. 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 100μM Cut off the PD-1/PD-L1 protein binding.

With reference to the aforementioned experiment 6, the anti-cancer effect of P32 in mice was further tested, and the results found that: at a dose of 0.5 mg/kg, the mice were in good mental state and had no significant weight loss during the administration period, which can inhibit colon cancer. The effect of tumor growth on the volume change of transplanted tumor in BABL/c mice inoculated with CT26. The results are shown in Figure 11.

10. 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.

Taking the value of the blocking rate of P32 on hPD-L1 as 100%, calculate the relative blocking rate of other peptides relative to this peptide, as shown in Figure 9: P32(8-12) alanine scanning peptide P34-38 It can block PD-1/PD-L1 protein binding at a concentration of 100μM.

11. Add a D-type amino acid to the N-terminus of peptide P32 to obtain 19 derived peptides, the peptide number is P39-57 (the numbers in the sequence list also correspond to SEQ ID NOs: 39-57), also refer to the above experiment 2 for detection The blocking ability and the effect of each peptide on blocking PD-1/PD-L1 protein binding are shown in Figure 12.

Claims

The affinity peptide of PD-L1-IgV, which is selected from the peptides defined by the following peptides a, b or c or a combination thereof:

Peptide a: the amino acid sequence is selected from SEQ ID NOs: 1, 2, 3 or 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 acid;

Peptide c: The polypeptide shown in SEQ ID NO. 8, its alanine scanning peptide, or its N-terminal or C-terminal truncated peptide, the number of amino acids of the truncated peptide is 4, 5, 6, 7, 8, 9 , 10 or 11, or the alanine scanning peptide of the truncated peptide.
The affinity peptide of claim 1, wherein the point mutation is independently selected from the following mutations:

1) Glu mutation is Gln, Asn or Asp;

2) His mutation is Gln or Glu;

3) Ala mutation is Trp, Phe, Tyr, His, Ile, Glu or Gln;

4) Ser is mutated to Arg.
The affinity peptide according to any one of the preceding claims, wherein the affinity peptide is a single point mutant peptide of the polypeptide shown in SEQ ID NO. 4.
The affinity peptide of claim 1, wherein 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, 6, 7, 8, 9. 10 or 11.
The affinity peptide of claim 1, wherein the sequence of the affinity peptide is selected from SEQ ID NOs: 1-38.
The affinity peptide of PD-L1-IgV is selected from the peptide P32 shown in SEQ ID NO. 32, its alanine scanning peptide or its N-terminally increased 1-7 amino acid derivative peptide.
The affinity peptide of claim 6, wherein the derivative peptide is a polypeptide obtained by adding 1 amino acid to the N-terminus of the polypeptide P32, for example, its sequence is selected from SEQ ID NOs: 31, 39-57.
The affinity peptide of claim 6, wherein the sequence of the affinity peptide is selected from SEQ ID NOs: 31, 32, 34-38, 39-57, 58-60.
The affinity peptide of any of the preceding claims, wherein the configuration of each amino acid of the affinity peptide is independently selected from D-type or L-type, and each amino acid can be D-type or L-type, For example, all amino acids are in D configuration.
A pharmaceutical composition or kit containing the affinity peptide of any of the preceding claims.
Use of the affinity peptide of any of the preceding claims in the preparation of medicines or kits.
The application according to claim 9, wherein the medicine or pharmaceutical composition is used for at least one of the following purposes:

1) Anti-tumor, tumors can include colon cancer or melanoma

2) Treat infections caused by bacteria, viruses or fungi

3) Treatment of autoimmune diseases

4) Block the binding of PD-1 protein to PD-L1 ligand; the PD-1 and PD-L1 can be wild-type proteins of human or mouse origin or mutant proteins that still retain their activity.