CN110330550B

CN110330550B - Affinity peptide of PD-L1-IgV and application thereof

Info

Publication number: CN110330550B
Application number: CN201910711326.8A
Authority: CN
Inventors: 高艳锋; 李琬琼; 翟文杰; 周秀曼; 祁元明
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2019-08-02
Filing date: 2019-08-02
Publication date: 2021-04-13
Anticipated expiration: 2039-08-02
Also published as: WO2021023140A1; CN110330550A

Abstract

The invention relates to an affinity peptide of PD-L1-IgV and application thereof. The affinity peptide 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: a polypeptide shown in SEQ ID NO.4, or a mutant peptide of which the 4 th position, the 5 th position, the 10 th position and/or the 11 th position has point mutation; peptide c: the polypeptide shown in SEQ ID NO.8, an alanine scanning peptide thereof, or an N-terminal or C-terminal truncated peptide thereof, wherein 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 invention develops a new method, and the peptide obtained by repeated screening and optimization can better block the interaction between PD-1/PD-L1, thereby treating tumors or other types of diseases.

Description

Affinity peptide of PD-L1-IgV and application thereof

The technical field is as follows:

the invention belongs to the technical field of biological pharmacy, and particularly relates to a peptide with affinity to PD-L1-IgV, application of the peptide in the aspects of relevant diseases such as tumors and the like.

Background art:

tumors are serious diseases threatening human health, have high heterogeneity, diversity and variability, and understanding the mechanisms of tumorigenesis and development and finding ways to treat tumors are still huge challenges facing scientists. Surgery, radiotherapy and chemotherapy are common methods used for traditional tumor treatment, but the tumor cannot be completely eliminated after the visible tumor is removed by the surgery, and the radiotherapy and chemotherapy have the problem of non-specificity, namely the tumor cannot be targeted, so that serious side effects are easily generated. Compared with the three methods, the immunotherapy of the tumor can well activate the immune system of the body to specifically kill the tumor and enable immune cells of the body to generate immune memory, so the immunotherapy can effectively prevent the tumor from relapse and metastasis, and has small side effect. In 2013, the journal of science lists the immunotherapy of tumors as the first ten scientific breakthroughs, and the immunotherapy of tumors gradually becomes the fourth important therapy after three traditional treatment methods. Immunotherapy of tumors is a major hotspot in the scientific community, and the nobel physiology or medical prize in 2018 was also awarded to two immunologists in this field to highlight their "contribution to finding therapies for negative immune regulation to treat cancer".

T cells are the core of the anti-tumor immune response of the body's immune system, and their activation requires dual signals: the first signal is that the surface receptor TCR of the T lymphocyte recognizes a complex of an antigenic peptide and an MHC molecule; the second signal is the binding of the T cell surface receptor TCR to a costimulatory molecule on the antigen presenting cell. Immune checkpoints can control the intensity of T cell immune response by balancing co-stimulatory and co-inhibitory signals, inhibitory molecules expressed by immune cells in the tumor microenvironment, such as PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, and the like, interact with T cells or antigen presenting cell surface ligands, thereby depleting T cell function, inhibiting anti-tumor effects thereof, and causing immune escape. The blocking of inhibitory molecules such as PD-1, CTLA-4, TIGIT, LAG-3, TIM-3 and the like can reactivate tumor specific T lymphocytes in a tumor microenvironment, break tumor immune tolerance established by an organism and bring hope for tumor treatment.

PD-L1 is a main ligand of PD-1, tumor cells can cause lymphocyte function exhaustion and mediate immune escape of tumors by highly expressing PD-L1, but studies show that the tumor cells can secrete PD-L1 in an exocrine form and enter lymph nodes to inhibit T cell functions, and clinical data show that the expression level of PD-L1 is in a trend of negative correlation with prognosis of tumor patients. Research data also show that PD-L1 can be combined with B7-1 to inhibit the B7-1/CD28 pathway, so that T lymphocytes are trapped in an immunosuppressed state, the proliferation capacity and the capacity of secreting killer cytokines are weakened, and therefore, the inhibition signal mediated by PD-L1 is considered to be bidirectional. Under normal conditions, PD-1/PD-L1 can reduce the proliferation capacity of T lymphocytes and reduce the secretion of cytokines such as IL-2 and IFN-gamma in order to avoid over-activation of the immune system and the occurrence of autoimmune diseases. Research shows that the ratio of tumor infiltrating T lymphocytes can be increased by independently blocking a PD-1/PD-L1 channel, the secretion level of IFN-gamma is improved, meanwhile, the ratio of myeloid suppressor lymphocytes is also obviously reduced, and the functions of the T lymphocytes are recovered and improved.

In addition, PD-1/PD-L1 is generally involved in the induction of T cell tolerance, and in addition to the above-mentioned broad-focused application in the tumor field, the research and therapeutic application of this pathway in the fields of autoimmune diseases, viral and bacterial infections is also progressing, so that the search for a reasonable and effective therapeutic strategy based on the PD-1/PD-L1 pathway is also a problem that scientists are facing and need to solve urgently.

At present, monoclonal antibodies aiming at a PD-1/PD-L1 pathway are already on the market and are applied to tumor immunotherapy, but antibody medicines have high production cost, poor tissue permeability and long half-life and can not stop adverse immune events quickly; the small molecular drugs such as polypeptide and the like are convenient to synthesize, have good tissue permeability and low immunogenicity, and have good development value and application prospect.

The invention content is as follows:

the invention develops a new method, obtains a peptide with affinity PD-L1-IgV by repeated screening and optimization, and proves that the affinity peptide has the activity of affinity PD-L1 and blocking the combination of PD-1/PD-L1, and can treat tumors or other types of diseases through experiments. In a first aspect, the present invention provides an affinity peptide for PD-L1-IgV, selected from the group consisting of the peptides defined by the following peptides a, b, c or combinations thereof:

peptide a, the amino acid sequence of which is selected from SEQ ID NOs:1, 2, 3 or 5(NOs represent a side-by-side listing of sequence numbers in the art, i.e., the amino acid sequences are SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.5, respectively);

peptide b, polypeptide shown in SEQ ID NO.4, or mutant peptide with point mutation of amino acid at position 4, position 5, position 10 and/or position 11;

peptide C, polypeptide shown in SEQ ID NO.8, alanine scanning peptide thereof, or N-terminal or C-terminal truncated peptide thereof, wherein the number of amino acids of the truncated peptide is 4, 5, 6, 7, 8, 9, 10 or 11, or alanine scanning peptide of the truncated peptide.

For example, peptide c can be classified as:

peptide c1, which is a polypeptide as shown in SEQ ID NO.8 or an alanine scanning peptide thereof;

peptide C2, which is a polypeptide shown in SEQ ID NO.8 or an N-terminal or C-terminal truncated peptide thereof, wherein the number of amino acids of the truncated peptide is 4, 5, 6, 7, 8, 9, 10 or 11;

peptide c3, which is the polypeptide shown in SEQ ID NO.32 or an alanine scanning peptide thereof.

Partial peptides of peptide c1 and peptide c2 can combine the peptide c 4: Arg-Val-Tyr-Ser-Phe and 12 peptides of which the N end is extended by 7 amino acids, wherein the 7 amino acids of the 12 peptides contain 1 alanine, and the sequence is Gly-Gln-Ser-Glu-His-His-Ala-Arg-Val-Tyr-Ser-Phe.

Alanine scanning peptide refers to a series of single mutation peptides obtained by replacing any amino acid of a protein parent with alanine in the field, such as a series of alanine scanning peptides of a polypeptide shown as a peptide c SEQ ID NO.8, with the sequences of SEQ ID NO.15 and SEQ ID NO.16 …

The truncated peptide refers to a series of shorter peptides obtained by cutting 1 to more amino acids from the N-terminal or C-terminal of a protein parent body in the field, such as a series of N-terminal truncated peptides of a polypeptide shown as a peptide C SEQ ID NO.8, and the sequences are SEQ ID NO.26 and SEQ ID NO.27 … …

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

1) glu is mutated into Gln, Asn and Asp;

2) his is mutated into Gln and Glu;

3) ala is mutated into Trp, Phe, Tyr, His, Ile, Gln and Glu;

4) ser is mutated to Arg.

Optionally, the affinity peptide is a single-site mutation peptide of the polypeptide shown in SEQ ID No.4, namely, the 1 site (such as the 4 th site) of the parent peptide is subjected to amino acid mutation.

Optionally, the truncated peptide is an N-terminal truncated peptide of the polypeptide shown in SEQ ID No.8, and the number of amino acids of the N-terminal truncated peptide is 5, 6, 7, 8, 9, 10 or 11.

Optionally, the sequence of the affinity peptide is selected from SEQ ID NOs: 4. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, 38.

Optionally, the configuration of the amino acids of the affinity peptide is independently selected from D or L, and may be both D or L, e.g. the amino acids are all in D configuration.

PD-L1 of the present invention refers to a ligand of a mammalian PD-1 protein, such as human PD-L1(hPD-L1) or mouse, and PD-L1-IgV is the IgV-like domain of PD-L1. PD-1 and PD-L1 may be wild type or mutant proteins which still retain their activity, such as wild type or mutant PD-1 in the applicant's prior patent CN 108794619.

It should be noted that the affinity peptides of this patent, either in free form or in the form of their pharmaceutically acceptable salts, constitute equivalent infringements of this patent based on a simple modification of the idea of this patent.

In a second aspect, the present invention provides a pharmaceutical composition or kit comprising an affinity peptide according to the first aspect as described above.

In a third aspect, the present invention provides the use of an affinity peptide according to the first aspect as hereinbefore described for the preparation of a pharmaceutical composition or kit.

The pharmaceutical composition of the second or third aspect may comprise a pharmaceutically acceptable excipient, and may be used for at least one of the following:

1) against tumors, e.g. colon cancer or melanoma

2) Treating infections caused by bacteria, viruses or fungi

3) Treatment of autoimmune diseases

4) Block the binding of PD-1 protein and PD-L1 ligand; the PD-1 and PD-L1 can be wild type of human or mouse or mutant protein still retaining the activity.

The polypeptides of the invention may be prepared by solid phase synthesis, e.g. using the Fmoc protocol.

The invention has the beneficial effects that:

the invention develops a new method, obtains PD-L1-IgV affinity peptide by repeated screening and optimization, and proves that the peptides can block the combination of PD-1/PD-L1 through an affinity blocking activity experiment. Further in vitro affinity experiments and in vivo anti-tumor experiments of mice prove that the high blocking rate peptide can obviously inhibit the growth of CT26 colon cancer and B16-OVA melanoma of the mice, and has no obvious toxic or side effect. Therefore, the peptide has good application prospect in the aspects of tumor treatment, autoimmune diseases and the like.

Description of the drawings:

FIG. 1 is a graph showing the results of experiments in which peptides H5S, H7, H9, H12, and H14 blocked the binding of PD-1/PD-L1 protein;

FIG. 2 is a graph showing the results of experiments in which mutant peptides of parent peptides H12 and H12 blocked the binding of PD-1/PD-L1 protein;

FIG. 3 shows the results of cell-level affinity assays for polypeptides;

FIG. 4 is a graph showing the effect of the peptide of the present invention on the body weight change of BABL/c mice inoculated with CT 26;

FIG. 5 is a graph showing the effect of the peptides of the invention on the change in the volume of transplanted tumors in BABL/c mice inoculated with CT 26;

FIG. 6 is a graph of the effect of the peptides of the invention on tumor volume in C57BL/6 mice vaccinated with B16-OVA;

FIG. 7 is a graph of the results of experiments in which the alanine scanning peptide of P8 blocked PD-1/PD-L1 protein binding;

FIG. 8 is a graph showing the results of experiments in which truncated peptides block the binding of PD-1/PD-L1 protein;

FIG. 9 is a graph of the results of an experiment in which the alanine scanning peptide of peptide P32 blocked PD-1/PD-L1 protein binding;

the significance analysis markers referred to in each figure indicate P <0.05, indicating P < 0.01.

The specific implementation mode is as follows:

embodiments of the present invention will be described in detail below with reference to examples, but the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

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

The inventor screens the affinity peptide of PD-L1-IgV, and each experiment is described as follows:

1. the phage image display peptide library is subjected to liquid phase screening to obtain a parent peptide H12 and the like, and the general screening process is as follows:

a) D-hPD-L1-IgV-biotin is synthesized by complete chemistry, SA magnetic beads are adopted to capture the D-configuration protein, and a liquid phase screening method is adopted to carry out screening work of a phage display dodecapeptide library;

b) after multiple rounds of screening, the phage monoclonals with affinity to the target protein D-hPD-L1-IgV are enriched one by one;

c) selecting positive clones from the above-mentioned groups, sequencing them to obtain several inserted dodecapeptide sequences (1 of them is mutated), so as to obtain linear parent peptides H5S, H7, H9, H12 and H14 (the sequences are shown in SEQ ID No.1-5, and the amino acid configurations are all synthesized into D type).

2. Blocking experiments:

a) culturing CHO-K1-hPD-1 cells in RMPI 1640 medium containing 10% FBS until the cells grow to the logarithmic phase and collecting the cells in 1.5mL EP tubes in a cell amount of 2X 10 per tube⁵1mL PBS7.2 was washed twice and then placed on ice for use.

b) The antibody (Anti human PD-1 PE) was first tested hPD-1 for stable cell expression in the cell membrane and isotype antibody was used as a control (Mouse IgG1 kappa iso control PE) to ensure that the cells could be used for subsequent blocking experiments.

c) Co-incubation of the polypeptide with hPD-L1-Fc: a certain mass of polypeptide was weighed and dissolved in PBS7.2 to 100. mu.M. A200. mu.L low adsorption EP tube was used and 50ng hPD-L1-Fc was incubated with 50. mu.L 100. mu.M polypeptide by vortexing and incubating on ice for 30 min.

d) The mixture was added to CHO-K1-hPD-1 cells, vortexed, mixed and incubated on ice for 30 min.

e) Add 0.3. mu.L of antibody (Human Fc. gamma. specific PE) to the mixture of the three substances, vortex to mix well and incubate in the dark on ice for 30 min.

f) Antibody washing: 1mL of ice-cold PBS7.2 is washed once, centrifuged at 1800rpm for 5min, added with 200 μ L of PBS7.2 for resuspension, and transferred into a flow tube to detect the binding condition of CHO-K1-hPD-1 cells and hPD-L1-Fc protein in a flow mode.

The blocking results show that: the polypeptides (H5S, H7, H9, H12 and H14) of the invention can block the binding of hPD-L1 protein and CHO-K1-hPD-1 cells, and the blocking rate is shown in figure 1.

3. Taking H12 as a parent peptide, and carrying out subsequent optimization work, wherein the specific implementation method comprises the following steps:

a) PEPstrMOD online prediction of the 3D structure of H12 peptide: open PEPstMOD page, input the desired predicted polypeptide sequence, click on Submit and Go to Next Step. Because the polypeptide is in D configuration, Sterichemistry is changed into Dextro (D), and the polypeptide structure can be downloaded in a mailbox after the system forecast is finished by clicking submission after a mailbox is input from the lower part.

b) Carrying out Z-DOCK molecular docking: and (3) performing molecular docking on the predicted H12 peptide structure and hPD-L1(PDB ID:3BIK) respectively, performing Z-DOCK on-line docking on the molecular docking, analyzing the docking result by MOE, selecting a docking mode (comprehensively considering bond energy, distance and interaction sites), and operating 50ns molecular dynamics by using the MOE.

c) MOE single site mutation: selecting a docking mode after the molecular dynamics, carrying out high-low sequencing on the results after mutation by utilizing MOE (molecular dynamics) to operate single-site amino acid mutation, selecting the first nine mutant peptides with higher scores to carry out synthesis of subsequent mutant peptides, respectively naming the 9 mutant peptides as P6-P14 according to Fmoc (Fmoc solid phase synthesis) method, wherein the sequences are sequentially shown as SEQ ID NO.6-SEQ ID NO.14, each amino acid is D-type, detecting the blocking capacity of the mutant peptides according to the previous experiment 2-blocking experiment after mass spectrum identification, and showing that each peptide can block the combination of hPD-L1 protein and CHO-K1-hPD-1 cells, wherein the blocking rate is shown as figure 2. In addition, using peptide P8 as an example, the blocking of mPD-L1 in murine mPD-L1 (protein and cell responses were replaced with murine ones) was performed in reference to experiment 2, and the results showed that P8 also blocked mPD-1/mPD-L1 binding at a blocking rate of 30%.

4. Micro calorimetric surge (MST) detection of the affinity of the polypeptide P8 with PD-L1 is carried out in the following steps:

a) NT647 dye tag hPD-L1-His protein (theory: 100. mu.L of protein at a concentration of 10. mu.M was added to 50. mu.L of dye at a concentration of 20. mu.M). The concentration of hPD-L1-His protein used in the experiment was 7.14. mu.M. First, NT647 dye with a concentration of 20 μ M and a volume of 50 μ L is mixed with protein well and reacted for 30min at room temperature in dark place. After the target protein hPD-L1-His is labeled by using the NT.115TM protein labeling kit RED, a dye protein complex can be effectively formed. After the labeling reaction is finished, the unreacted dye is removed by using a molecular sieve, so that the subsequent experiment can be smoothly carried out.

b) Column balancing: the column was equilibrated during the dye labeling process described above, with PBS7.2 being equilibrated for 3 column volumes, and care was taken to keep the column wet during column equilibration.

c) Separating protein by a chromatographic column: after the protein and the dye are sufficiently reacted for 30min in the dark, the mixture is added into a chromatographic column which is well balanced in advance, 400 mu L of PBS7.2 is added, when the two liquids are sufficiently permeated into the column, 600 mu L of PBS7.2 is added, the liquid dripped out of the column begins to be collected, each drop is collected into a new EP tube (if the molecular weight of the protein is small, the 3 drops dripped out at the beginning do not need to be collected), and the liquid is collected until the column does not drip out any more. After completion of collection, the capillary aspirates a small amount of liquid, MST detects the efficiency of the label and the column is rinsed clean and stored in 20% alcohol solution at 4 ℃. And (3) properly diluting the marked protein according to the fluorescence intensity and the protein concentration used in a specific experiment.

d) Preparing a sample to be tested: weighing and dissolving a proper amount of polypeptide by an electronic balance, diluting the polypeptide 2 times from 100 mu M, diluting the diluted polypeptide by 16 gradients, mixing the diluted polypeptide with the labeled fluorescent protein in an equal volume (10 mu L of target protein plus 10 mu L of polypeptide), sucking 10 mu L of the mixture by a capillary tube, and placing the mixture on a sample holder according to a corresponding position for further analysis.

e) And (3) detecting affinity: and clicking a start scan on an instrument operation interface to detect whether the fluorescence intensity of each tube is consistent, and if the consistency is better, clicking a start MST measurement to start measurement.

f) The experimental data were analyzed and collated.

The results of experiments with human PD-L1 show that the linear D peptide P8 has good affinity for hPD-L1 protein (Kd value of 0.9 μ M for binding to PD-L1) and an equivalent level of affinity for the positive ligand protein hPD-1(Kd value of 0.4 μ M). Affinity experiments for murine mPD-L1 were performed with reference to the above experiments (protein and cell correspondence was replaced with murine) with similar results: p8 also has a good affinity for mPD-L1(Kd 3.78. mu.M) and an equivalent level of affinity for mPD-1 (Kd 4.04. mu.M).

5. Cell level affinity assay for polypeptides

After coupling a fluorescent dye Cy5.5-NHS with an amino group of standard Fmoc solid-phase synthesized highly active peptide P8 (C-terminal is connected to Rink resin, and N-terminal is exposed), purifying and preparing, co-incubating fluorescent peptide P8 with maximum solubility of 10 μ M in PBS7.2 with CHO-K1, CHO-K1-hPD-L1 and CHO-K1-mPD-L1 respectively, detecting whether the mutant peptide has specific affinity hPD-L1 and mPD-L1 by a flow cytometer, wherein the statistical result of the affinity is shown in FIG. 3.

6. The anti-tumor effect of P8 is respectively researched in a CT26 colon cancer transplantation tumor model and a B16-OVA melanoma transplantation tumor model, and the specific implementation method is as follows:

a) carrying out tumor loading: collecting good-growth CT26 colorectal cancer cells and B16-OVA melanoma cells, and respectively carrying BABL/c mice (2X 10)⁵cells/mouse) and C57BL/6 mice (5X 10)⁵cells/only).

b) The tumor volume of the mouse is about 40-80mm after the tumor is loaded for about one week³The mice are weighed by an electronic balance every other day during continuous administration for 2 weeks, the tumors of the mice are measured by a digital vernier caliper, and the volume change of the tumors of the mice is calculated and recorded according to the formula V which is 1/2 × a (length) × b (width) × c (height);

the tumor volume curves shown in FIGS. 5 and 6 show that P8 can well inhibit the growth of CT26 colorectal cancer transplanted tumor and B16-OVA melanoma transplanted tumor under the administration dose of 0.5mg/kg, and the results shown in FIG. 4 show that the weight of BABL/c mice is not obviously reduced under the administration dose of 0.5mg/kg for P8. The mental state of the mice was good during the administration of both models of transplants.

7. Degradation stability test of affinity peptidase

a) Dissolving peptide P8 PBS7.2 to 100 MuM mother liquor, rapidly mixing 190 μ L of 10% human serum solution with 10 μ L of the A10Y mother liquor, taking out 20 μ L of the mixed liquor, timing to be 0h, placing the rest in a 37 ℃ incubator for enzymolysis reaction, and respectively taking out 20 μ L of reaction products in a 1.5mL EP tube for detection of enzymolysis conditions at each time point after 0.5h, 1h, 2h, 4h, 8h, 16h, 24h and 48 h;

b) carefully adding 90 mu L of acetonitrile into the samples taken out at different time points by using a pipette, immediately shaking and uniformly mixing, placing on ice for 10min, and adding 90 mu L of 0.5% glacial acetic acid solution into the pipette to stop the enzymolysis reaction;

c) pre-cooling at 4 deg.C, centrifuging at 10000g for 20min, collecting supernatant, and packaging in new EP tube for subsequent use

An enzyme degradation stability experiment of RP-HPLC analysis shows that the polypeptide P8 still exists stably for 48 hours and has remarkable enzymolysis resistance.

8. Peptide P8 was subjected to alanine scanning and blocking experiments to investigate its alanine scanning peptide blocking ability, in substantially the same manner as described in experiment 2 above.

The relative blocking rate of each of the other peptides with respect to this peptide was calculated using the value of the blocking rate of P8 with respect to hPD-L1 as 100%, as shown in FIG. 7: the alanine scanning peptide P15-P25 of P8 can block the binding of PD-1/PD-L1 protein at the concentration of 100 mu M.

9. Peptide P8 was truncated and blocking experiments were performed to study the ability of truncated peptides to block, in essentially the same manner as described in experiment 2 above.

The relative blocking rate of each of the other peptides with respect to this peptide was calculated using the value of the blocking rate of P8 with respect to hPD-L1 as 100%, as shown in FIG. 8: the truncated peptide P26-P33 of P8 can block the binding of PD-1/PD-L1 protein at a concentration of 100 mu M.

10. Peptide P32 was subjected to alanine scanning and blocking experiments to investigate its alanine scanning peptide blocking ability, in substantially the same manner as described in experiment 2 above.

The relative blocking rate of each of the other peptides with respect to this peptide was calculated using the value of the blocking rate of P32 with respect to hPD-L1 as 100%, as shown in FIG. 9: alanine scanning peptide P34-38 of P32(8-12) all blocked PD-1/PD-L1 protein binding at a concentration of 100. mu.M.

TABLE 1 sequence Listing of amino acids (amino acid configuration is D-form) of each peptide of the present invention

Sequence listing

<110> Zhengzhou university

<120> PD-L1-IgV affinity peptide and application thereof

<160> 38

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

His Pro Trp Ser Ser Gly Leu Arg Leu Asp Leu Arg

1 5 10

<210> 2

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ser Ser His Leu Thr Asn Trp Trp Arg Asn Gly Ile

1 5 10

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Val Val Ser Pro Asp Met Asn Leu Leu Leu Thr Asn

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gly Gln Ser Glu His His Met Arg Val Ala Ser Phe

1 5 10

<210> 5

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ala Ser Ser Ala Tyr Leu Lys Ser Met Asp Pro Ala

1 5 10

<210> 6

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gly Gln Ser Glu His His Met Arg Val Trp Ser Phe

1 5 10

<210> 7

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Gln Ser Glu His His Met Arg Val Phe Ser Phe

1 5 10

<210> 8

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gly Gln Ser Glu His His Met Arg Val Tyr Ser Phe

1 5 10

<210> 9

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gly Gln Ser Glu His His Met Arg Val His Ser Phe

1 5 10

<210> 10

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Gln Ser Glu Glu His Met Arg Val Ala Ser Phe

1 5 10

<210> 11

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Gln Ser Glu His His Met Arg Val Ala Arg Phe

1 5 10

<210> 12

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gly Gln Ser Glu His His Met Arg Val Ile Ser Phe

1 5 10

<210> 13

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gly Gln Ser Glu His His Met Arg Val Glu Ser Phe

1 5 10

<210> 14

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gly Gln Ser Asn His His Met Arg Val Ala Ser Phe

1 5 10

<210> 15

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Gln Ser Glu His His Met Arg Val Tyr Ser Phe

1 5 10

<210> 16

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gly Ala Ser Glu His His Met Arg Val Tyr Ser Phe

1 5 10

<210> 17

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Gly Gln Ala Glu His His Met Arg Val Tyr Ser Phe

1 5 10

<210> 18

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gly Gln Ser Ala His His Met Arg Val Tyr Ser Phe

1 5 10

<210> 19

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gly Gln Ser Glu Ala His Met Arg Val Tyr Ser Phe

1 5 10

<210> 20

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Gly Gln Ser Glu His Ala Met Arg Val Tyr Ser Phe

1 5 10

<210> 21

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Gly Gln Ser Glu His His Ala Arg Val Tyr Ser Phe

1 5 10

<210> 22

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Gly Gln Ser Glu His His Met Ala Val Tyr Ser Phe

1 5 10

<210> 23

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Gly Gln Ser Glu His His Met Arg Ala Tyr Ser Phe

1 5 10

<210> 24

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Gly Gln Ser Glu His His Met Arg Val Tyr Ala Phe

1 5 10

<210> 25

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gly Gln Ser Glu His His Met Arg Val Tyr Ser Ala

1 5 10

<210> 26

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Gln Ser Glu His His Met Arg Val Tyr Ser Phe

1 5 10

<210> 27

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Ser Glu His His Met Arg Val Tyr Ser Phe

1 5 10

<210> 28

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Glu His His Met Arg Val Tyr Ser Phe

1 5

<210> 29

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

His His Met Arg Val Tyr Ser Phe

1 5

<210> 30

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

His Met Arg Val Tyr Ser Phe

1 5

<210> 31

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Met Arg Val Tyr Ser Phe

1 5

<210> 32

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Arg Val Tyr Ser Phe

1 5

<210> 33

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Val Tyr Ser Phe

1

<210> 34

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Ala Val Tyr Ser Phe

1 5

<210> 35

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Arg Ala Tyr Ser Phe

1 5

<210> 36

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Arg Val Ala Ser Phe

1 5

<210> 37

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Arg Val Tyr Ala Phe

1 5

<210> 38

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Arg Val Tyr Ser Ala

1 5

Claims

An affinity peptide of PD-L1-IgV, the sequence of said affinity peptide being selected from the group consisting of SEQ ID NOs: 6. 7, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, each amino acid of the affinity peptide being in the D configuration.
2. A pharmaceutical composition or kit comprising an affinity peptide according to claim 1.
3. Use of an affinity peptide for the manufacture of a medicament for use against colon cancer or melanoma, the affinity peptide having a sequence selected from the group consisting of SEQ ID NOs: 4. 6-25, wherein each amino acid of the affinity peptide is in D configuration.