CN107226842B - PD-1 targeted polypeptide and application thereof - Google Patents

PD-1 targeted polypeptide and application thereof Download PDF

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CN107226842B
CN107226842B CN201610173921.7A CN201610173921A CN107226842B CN 107226842 B CN107226842 B CN 107226842B CN 201610173921 A CN201610173921 A CN 201610173921A CN 107226842 B CN107226842 B CN 107226842B
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cancer
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CN107226842A (en
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李洪林
刘晓峰
朱丽丽
李巧
全丽娜
吴方舒
赵振江
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East China University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A—HUMAN NECESSITIES
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Abstract

The invention discloses a PD-1 targeted polypeptide and application thereof. The invention also discloses a preparation method of the polypeptides and a pharmaceutical composition containing the polypeptides. The polypeptide can be combined with human PD-1 protein, so that the polypeptide can be used as a pilot peptide for developing human PD-1 receptor inhibitors, and provides a basis for the development of antitumor drugs. The polypeptide can be prepared by a chemical synthesis method, and has the advantages of high purity, small molecular weight, safety, reliability and the like.

Description

PD-1 targeted polypeptide and application thereof
Technical Field
The invention belongs to the field of biotechnology. In particular, the invention relates to a polypeptide targeting human PD-1 protein and application thereof.
Background
The existence of numerous costimulatory and costimulatory signals in the immune system precisely regulate the magnitude and quality of T cell responses, and these inhibitory signals are referred to as immune checkpoints. Tumor cells often overexpress these immune checkpoint proteins, thereby inhibiting activation of T cells and evading killing by the immune system. Enhancement of T cell activity by different strategies is of great interest for tumor immunotherapy, where blocking immune checkpoints is one of the effective strategies to enhance T cell activity. A series of immunosuppressive molecules, such as PD-1, CTLA-4, Tim-3, SLAM and the like, exist on the surface of the T cell, and can be combined with corresponding ligands to activate an immunosuppressive regulation pathway, so that the function of the T cell is exhausted, wherein the PD-1/PD-L1 pathway is the current research hotspot.
PD-1 is an important inhibitory receptor on the surface of T cells, and is known for its initial discovery in apoptotic T cell lymphomas and its ability to promote programmed cell death. PD-1 protein is inducibly expressed in activated T cells, B cells, macrophages, dendritic cells, monocytes, and the like. The two ligands are PD-L1(CD274, B7-H1) and PD-L2(CD273, B7-DC). PD-L1 is more widely distributed than PD-L2, PD-L1 can be expressed in cells such as B cells, DC, macrophages, bone marrow-derived mast cells, T cells, fibroblasts, epithelial cells and the like, while PD-L2 is expressed only in cells such as DC, macrophages, memory B cells and the like, and therefore, blocking the PD-1/PD-L1 pathway is more effective than blocking the PD-1/PD-L2 pathway. The mechanism of inhibition of the immune pathway by PD-1/PD-L1 is as follows: the tumor cells have a large number of gene mutations and abnormal expression of proteins, and can be used as tumor antigens to activate T cells. After the tumor-specific cytotoxic T cells reach the tumor site, the T cell antigen receptor (TCR) recognizes the tumor cells, releases interferon gamma (IFN-gamma) and T cell particles, and kills the tumor cells. But IFN-gamma can induce tumor cells to express PD-L1 besides the anti-tumor effect. These PD-L1 inhibit the anti-tumor effect of T cells after binding to PD-1 in activated T cells.
Although many drugs that block the PD-1/PD-L1 pathway are currently in clinical trials or individual drugs have been approved by the FDA in the united states, for example: nivolumab, pembrolizumab and pidilizumab targeting PD-1 protein, and MPDL3280A, MDX1105, MEDI4736 and the like targeting PD-L1, but most of the drugs are antibody drugs and have the defects of high production cost, poor tissue permeability, easy immunogenicity generation and the like.
Compared with antibody drugs, polypeptide molecules are the most attractive antibody substitutes due to the advantages of high specificity and tissue affinity, low production cost, low possibility of generating immunogenicity, high tissue permeability and the like. Therefore, research and development of some low molecular weight organic small molecule or polypeptide drugs become important research directions for blocking the PD-1/PD-L1 pathway.
In view of the above, there is a great need in the art for low molecular weight small organic molecules or polypeptides as candidate drugs for blocking the PD-1/PD-L1 pathway.
Disclosure of Invention
The invention aims to provide low-molecular-weight organic small molecules or polypeptides capable of blocking a PD-1/PD-L1 pathway.
In a first aspect, the present invention provides a polypeptide represented by formula I:
[Xaa0]-[Xaa1]-Asp-Tyr-[Xaa2]-Arg-[Xaa3]-Tyr-[Xaa4](I)
wherein the content of the first and second substances,
[ Xaa0] is a peptide fragment consisting of Gly Asn, Thr Glu or Phe Asn, or none;
[ Xaa1] is Trp, Lys or Met;
[ Xaa2] is a peptide segment composed of Asn Ser Gln, Arg His Gly Asn Ile, Ser Leu Glu Glu Leu, Ser Trp Lys Ser Glu, Gln Asn, Cys Pro Cys, Cys His Gly Pro Cys, Cys Ala Glu Pro Leu and Ser Trp Cys Pro Cys;
[ Xaa3] is a peptide consisting of Ala Gln Leu, Met Lys Leu Ala, Glu Lys Ala Lys, Leu Lys Glu Ala, or Glu Lys Cys Lys;
[ Xaa4] is Asn Gln, Asp Leu, Lys, Asp Leu, or Arg;
and the polypeptide has PD-1 binding activity, and the length of the polypeptide is 15-18 amino acids.
In specific embodiments, the polypeptide is selected from the group consisting of:
(a) a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO 1-9;
(b) a polypeptide derived from (a) and having PD-1 binding activity, which is formed by substituting, deleting or adding 1 to 6 (preferably 1 to 4) amino acid residues to the polypeptide of (a).
In a preferred embodiment, the binding K of the polypeptide to PD-1DA value of 1-5. mu.M, preferably 1-4. mu.M, more preferably 1-3. mu.M, most preferably the binding K to native PD-L1 and PD-1DThe values are approximate.
In a specific embodiment, the polypeptide is selected from the group consisting of polypeptides having an amino acid sequence as set forth in any one of SEQ ID NOs 1-9.
In a specific embodiment, the polypeptide is a polypeptide having an amino acid sequence as set forth in SEQ ID NO 1 or 6.
In a second aspect, the present invention provides an isolated nucleic acid molecule encoding a polypeptide according to the first aspect of the invention.
In a third aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the second aspect of the invention.
In preferred embodiments, the host cells include, but are not limited to: bacteria, yeast, plant cells, insect cells, or mammalian cells.
In preferred embodiments, the bacteria include, but are not limited to: escherichia coli and Bacillus subtilis; such yeasts include, but are not limited to: pichia pastoris and Saccharomyces cerevisiae.
In a fourth aspect, the present invention provides a pharmaceutical composition comprising a polypeptide according to the first aspect of the invention and a pharmaceutically acceptable excipient.
In a preferred embodiment, the pharmaceutical composition further comprises: (c) other pharmaceutically acceptable drugs that block the PD-1/PD-L1 pathway.
In another preferred embodiment, the other agents that block the PD-1/PD-L1 pathway include, but are not limited to: nivolumab, pembrolizumab, pidilizumab targeting PD-1 protein, and MPDL3280A, MDX1105, MEDI4736, etc. targeting PD-L1.
In a preferred embodiment, the pharmaceutical composition may further comprise other anti-neoplastic agents including, but not limited to: iplilimumab, ramucirumab, trametinib, ceritini, dabrafenib, and the like.
In another preferred embodiment, the dosage form of the pharmaceutical composition is suitable for the following modes of administration, including but not limited to: injection, infusion, intraperitoneal, intratumoral, intramuscular administration.
In a fifth aspect, the invention provides the use of a polypeptide according to the first aspect of the invention for the manufacture of a medicament for inhibiting the binding of PD-1 to PD-L1 or for inhibiting a tumor, or for treating an infection caused by a bacterium, virus or fungus, or for treating an inflammatory disease.
In particular embodiments, the tumor includes, but is not limited to: melanoma, lung cancer (preferably non-small cell lung cancer), kidney cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvis cancer, Central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, Pituitary adenomas, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and the like;
such viruses include, but are not limited to: hepatitis viruses (type a, type b and type c), herpesvirus, influenza virus, adenovirus, coronavirus, measles virus, dengue virus, poliovirus, rabies virus, etc.;
such bacteria include, but are not limited to: chlamydia, rickettsia, mycobacteria, staphylococci, pneumococci, cholera, tetanus, etc.;
such fungi include, but are not limited to: candida, aspergillus, dermatitides, etc.; or
Such inflammatory diseases include, but are not limited to: ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, pernicious anemia, polymyositis, etc.
In a sixth aspect, the present invention provides a method of inhibiting the binding of PD-1 to PD-L1, comprising the steps of: the polypeptide of the first aspect of the invention or the pharmaceutical composition of the fourth aspect of the invention is used to inhibit binding of PD-1 to PD-L1.
In a preferred embodiment, the method of inhibiting the binding of PD-1 to PD-L1 is non-therapeutic in vitro.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the results of PCR identification of human PD-1 recombinant plasmid constructed in example 3, in which marker represents DNA molecular weight marker; the band appearing in the lane is the band of interest (357 bp).
FIG. 2 shows the result of SDS-PAGE electrophoresis of the purified human PD-1 protein of example 4, in which M represents the protein molecular weight marker; lane 1 represents the human PD-1 protein, the band size of which is 13kDa, corresponding to the size of the theoretical value.
FIG. 3a shows the amino group of human PD-1 proteinSPR picture of polypeptide combination with SEQ ID NO. 1, and K of the polypeptide and PD-1 proteinDThe value was 1.38. mu.M.
FIG. 3b shows SPR graphs showing the binding of human PD-1 protein to the polypeptide having the amino acid sequence of SEQ ID NO. 2, the K of the polypeptide and PD-1 protein determinedDThe value was 3.13. mu.M.
FIG. 3c shows SPR graphs showing the binding of human PD-1 protein to the polypeptide having the amino acid sequence of SEQ ID NO. 3, the K of the polypeptide and PD-1 protein determinedDThe value was 3.14. mu.M.
FIG. 3d shows SPR graphs showing the binding of human PD-1 protein to the polypeptide having the amino acid sequence of SEQ ID NO. 4, the K of the polypeptide and PD-1 protein determinedDThe value was 3.39. mu.M.
FIG. 3e shows SPR graphs showing the binding of human PD-1 protein to the polypeptide having the amino acid sequence of SEQ ID NO. 5, the K measured for the polypeptide and PD-1 proteinDThe value was 3.32. mu.M.
FIG. 3f shows SPR plots showing binding of human PD-1 protein to human PD-L1 protein, and K was measured for human PD-L1 protein and PD-1 proteinDThe value was 1.15. mu.M.
FIG. 4 shows an SPR plot showing that human PD-L1 protein competes with the polypeptide having the amino acid sequence of SEQ ID NO. 1 for binding to human PD-1 protein.
FIG. 5a shows SPR graphs showing the binding of human PD-1 protein to a polypeptide having the amino acid sequence of SEQ ID NO. 6, the K measured for the polypeptide and PD-1 proteinDThe value was 2.08. mu.M.
FIG. 5b shows SPR graphs showing the binding of human PD-1 protein to the polypeptide having the amino acid sequence of SEQ ID NO. 7, the K of the polypeptide and PD-1 protein determinedDThe value was 3.96. mu.M.
FIG. 5c shows SPR graphs showing the binding of human PD-1 protein to the polypeptide having the amino acid sequence of SEQ ID NO. 8, the K of the polypeptide and PD-1 protein determinedDThe value was 3.11. mu.M.
FIG. 5d shows the SPR graph of the binding of human PD-1 protein to the polypeptide having the amino acid sequence of SEQ ID NO. 9, the K of the polypeptide and PD-1 protein determinedDThe value was 4.81. mu.M.
Detailed Description
The inventor unexpectedly discovers a group of polypeptides targeting human PD-1 protein through extensive and intensive research, and the group of polypeptides can be competitively combined with the human PD-1 protein, thereby becoming a polypeptide lead medicament for researching a PD-1/PD-L1 immune checkpoint blocking agent and further providing a basis for the development of anti-tumor medicaments. The present invention has been completed based on this finding.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. For the purpose of understanding the present invention, the following definitions will be made for terms related to the present invention, but the scope of the present invention is not limited to these specific definitions.
PD-1 protein
The term "PD-1 protein" as used herein refers to an important inhibitory receptor on the surface of T cells in humans. It has two ligands, PD-L1 and PD-L2. PD-L1 acts as an antitumor agent by inhibiting T cells after binding to PD-1 in activated T cells. Therefore, the blocking of the PD-1/PD-L1 pathway has positive effect on inhibiting or killing tumors.
It is well known to those skilled in the art that proteins in the human body may undergo certain mutations while retaining their activity. Thus, the PD-1 protein of the present invention includes a PD-1 protein containing a certain mutation as long as the mutated PD-1 protein performs the same function as that of the wild-type PD-1 in a human body. For example, in a specific embodiment, the PD-1 protein of the invention has the amino acid sequence shown in SEQ ID NO. 14 (PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE), and its corresponding coding nucleotide sequence is shown in SEQ ID NO. 13.
The PD-1 protein has a ligand (PD-L1) that binds specifically thereto. For example, in a specific embodiment, PD-L1 of the invention has the amino acid sequence shown in SEQ ID NO. 12.
Polypeptides of the invention
The present invention provides polypeptides capable of competitively binding to human PD-1 protein, which polypeptides have structural similarity, i.e. have specific amino acid residues at specific positions.
In a specific embodiment, the invention provides a polypeptide represented by formula I:
[Xaa0]-[Xaa1]-Asp-Tyr-[Xaa2]-Arg-[Xaa3]-Tyr-[Xaa4](I)
wherein [ Xaa0] is a peptide fragment consisting of Gly Asn, Thr Glu or Phe Asn, or is absent; [ Xaa1] is Trp, Lys or Met; [ Xaa2] is a peptide segment composed of Asn Ser Gln, Arg His Gly Asn Ile, Ser Leu Glu Glu Leu, Ser Trp Lys Ser Glu, Gln Asn, Cys Pro Cys, Cys His Gly Pro Cys, Cys Ala Glu Pro Leu and Ser Trp Cys Pro Cys; [ Xaa3] is a peptide consisting of Ala Gln Leu, Met Lys Leu Ala, Glu Lys Ala Lys, Leu Lys Glu Ala, or Glu Lys Cys Lys; [ Xaa4] is Asn Gln, Asp Leu, Lys, Asp Leu, or Arg; these polypeptides are 15-18 amino acids in length.
These polypeptides have good binding affinity to the human PD-1 protein, wherein the polypeptide with the highest binding affinity is equivalent to the natural ligand PD-L1 of the human PD-1 protein. In a preferred embodiment, the polypeptide is a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs 1-9; or a derivative polypeptide which is formed by substituting, deleting or adding 1-6 (preferably 1-4) amino acid residues in the polypeptide with the amino acid sequence shown in any one of SEQ ID NO 1-9 and has PD-1 binding activity.
In a specific embodiment, the binding K of the polypeptide of the invention to PD-1DA value of 1-5. mu.M, preferably 1-4. mu.M, more preferably 1-3. mu.M, most preferably the binding K to native PD-L1 and PD-1DThe values are approximate.
In a preferred embodiment, the polypeptide of the invention is selected from the group consisting of polypeptides having an amino acid sequence as set forth in any one of SEQ ID NOs 1-9; more preferably a polypeptide having an amino acid sequence as shown in SEQ ID NO 1 or 6.
The invention provides, on the basis of the polypeptides of the invention, an isolated nucleic acid molecule encoding a polypeptide of the invention and a host cell comprising said isolated nucleic acid molecule.
The skilled artisan knows how to select host cells and express proteins therein, for example, including but not limited to: bacteria, yeast, plant cells, insect cells, or mammalian cells. In particular embodiments, the bacteria include, but are not limited to: escherichia coli and Bacillus subtilis; such yeasts include, but are not limited to: pichia pastoris and Saccharomyces cerevisiae.
Because the polypeptide of the invention has good binding affinity with human PD-1 protein, the polypeptide can be used as an inhibitor of PD-1/PD-L1 binding or a regulator for regulating the binding degree of PD-1/PD-L1. Accordingly, the present invention also provides a pharmaceutical composition comprising a polypeptide of the invention and a pharmaceutically acceptable excipient.
The polypeptide of the invention can be obviously combined with other mechanism drugs for blocking the PD-1/PD-L1 pathway and other anti-tumor drugs to enhance the effects of each other. Therefore, the pharmaceutical composition can also comprise other pharmaceutically acceptable medicaments for blocking the PD-1/PD-L1 pathway. For example, such other drugs that block the PD-1/PD-L1 pathway include, but are not limited to: nivolumab, pembrolizumab, pidilizumab targeting PD-1 protein, and MPDL3280A, MDX1105, MEDI4736, etc. targeting PD-L1. In a preferred embodiment, the pharmaceutical composition may further comprise other anti-neoplastic agents including, but not limited to: iplilimumab, ramucirumab, trametinib, ceritini, dabrafenib, and the like.
It is well known to those skilled in the art that the specific dosage form of the pharmaceutical composition of the present invention depends on the mode of administration to be employed. For example, the pharmaceutical compositions of the present invention may be administered by modes of administration including, but not limited to: injection, infusion, intraperitoneal, intratumoral, intramuscular, and the like. The content of the active ingredient in the pharmaceutical composition may be determined by those skilled in the medical field on their own accord with, for example, the sex, age, general health condition of the patient, and the like.
Based on the activity of the polypeptide of the present invention, those skilled in the art can easily conceive of preparing a drug which inhibits the binding of PD-1 to PD-L1 or a drug which inhibits tumor. In particular embodiments, the tumor includes, but is not limited to: melanoma, lung cancer (preferably non-small cell lung cancer), kidney cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvis cancer, Central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, Pituitary adenomas, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma.
In other embodiments, the medicament that inhibits the binding of PD-1 to PD-L1 may also be used to treat a bacterial, viral, or fungal infection. For example, the following viruses cause infections: hepatitis viruses (type a, type b and type c), herpesvirus, influenza virus, adenovirus, coronavirus, measles virus, dengue virus, poliovirus, rabies virus, etc.; infections caused by the following bacteria: chlamydia, rickettsia, mycobacteria, staphylococci, pneumococci, cholera, tetanus, etc.; or pathogenic infections caused by the following fungi: candida, aspergillus, dermatitides, etc.
In other embodiments, the medicament that inhibits the binding of PD-1 to PD-L1 may also be used to treat inflammatory diseases, including but not limited to: ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, pernicious anemia, polymyositis, etc.
The invention further provides a method for inhibiting the binding of PD-1 and PD-L1 on the basis of the polypeptide or the pharmaceutical composition of the invention, which comprises the step of inhibiting the binding of PD-1 and PD-L1 by using the polypeptide or the pharmaceutical composition of the invention. In a specific embodiment, the method of inhibiting the binding of PD-1 to PD-L1 is non-therapeutic in vitro.
The invention has the advantages that:
1. the present invention provides polypeptides capable of competitively binding to human PD-1 protein;
2. the polypeptide can be well combined with human PD-1 protein, wherein the combination condition of the polypeptide with the highest combination affinity and the human PD-1 protein is not different from that of natural PD-L1 protein;
3. the polypeptide of the invention has higher specificity and tissue affinity, is not easy to generate immunogenicity and has better tissue permeability; and
4. the polypeptide of the invention has low production cost.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Examples
Example 1 Synthesis of the polypeptide of the present invention
(1) Laboratory instruments and materials
Dimethylformamide (DMF), piperidine, resin, Dichloromethane (DCM), ninhydrin reagent, benzotriazole-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU), Triisopropylsilane (TIS), ethylene glycol dithiol (EDT), dehydrated ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), methanol, ethanol, 20 amino acids, polypeptide solid phase synthesis tube, mass spectrometer MicroTOOF-Q11 (Bruker Daltonics).
(2) Experimental procedure
Weighing the resin, putting the resin into a polypeptide solid phase synthesis tube, and adding a proper amount of DMF (dimethyl formamide) to swell for more than half an hour. The DMF was taken off, and Fmoc deprotection was performed using deprotection solution (piperidine: DMF. RTM. 1:4) and placed on a shaker for 10 min. The deprotection solution is pumped out, washing is carried out for three times by DMF and DCM, a small amount of resin is taken out from the solid phase synthesis tube and is washed for 2 times by ethanol, the color is detected and recorded by the indetrione method, and feeding is prepared for carrying out amino acid condensation reaction. Taking corresponding amino acids and HBTU according to the amino acid sequences shown in SEQ ID NO. 1-9, dissolving with a small amount of DMF, putting into a solid phase synthesis tube, adding 10 times of NMM, and stirring for reaction. After 1-2 hours, a small amount of resin is taken out of the solid phase synthesis tube and washed with ethanol 2 times, and detected by the indetrione method. The liquid in the solid phase synthesis tube was removed and washed with DMF, methanol, DCM, 2 times each to give the first amino acid condensed peptide resin. Repeating the Fmoc deprotection and amino acid condensation reaction steps on the obtained peptide resin until the last amino acid reaction is finished to obtain the peptide with the amino acid sequence shown as SEQ ID NO. 1-9. After the reaction, DMF, methanol and DCM are washed for 2 times respectively, and the air is continuously pumped for 10-15 minutes. The synthesized partial peptide resin was removed from the solid phase synthesis tube and cleaved in a cleavage solution (TFA 94.5%, water 2.5%, EDT 2.5%, TIS 1%) for two hours at room temperature. After filtering the resin, evaporating to dryness in a rotary evaporator, washing with anhydrous ether for 6 times, and then volatilizing at normal temperature. The crude peptide was purified using analytical grade HPLC with > 90% purity using HPLC. The resulting pure peptides were identified using mass spectrometry. And finally, freeze-drying the purified solution to obtain a pure product. Table 1 shows the theoretical values of the molecular weight of the polypeptide of the invention and the actual values determined by mass spectrometry.
Example 3 construction of human PD-1 recombinant plasmid
The gene of interest is a nucleic acid encoding amino acids 34-150 of the PD-1 protein. The target fragment was cloned into pET-28a vector (Novagen) using specific primers designed using the NcoI and NdeI sites. The forward primer is: 5'-TTTTCCATGGGTCCCCCCACCTTCTCCCCAG-3' (SEQ ID NO: 10); the reverse primer is: 5'-GCCGCCGCATATGTTATTCTGCCCTTCTCTCTG-3' (SEQ ID NO: 11). Human PD-1 gene is used as a template to carry out PCR amplification. The PCR system is as follows: mu.L PD-1 plasmid, 2. mu.L forward primer, 2. mu.L reverse primer, 5. mu.L 10 Xbuffer solution, 4. mu.L dNTP, 1. mu.L pfu polymerase, 34. mu.L ddH2And O. PCR amplification conditions: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 48s for 30 cycles; 10min at 72 ℃. And after the PCR amplification is finished, performing nucleic acid electrophoresis, cutting off a target band, and recovering a PCR product by using a PCR product recovery kit. The resulting PCR product and plasmid pEThe T-28a vector was double-digested with restriction endonucleases NcoI and NdeI, respectively, and then ligated by T4 ligase to transform into E.coli DH5 alpha competent cells. After overnight culture, single clones were picked for PCR identification of the bacterial fluid (see figure 1), and then positive recombinants were sequenced, leaving the strain and glycerol. Human PD-1 constructed by the recombinant plasmid has a nucleotide sequence shown as SEQ ID NO. 13.
Example 4 expression and purification of human PD-1 protein
The human PD-1 protein used in the invention is obtained after prokaryotic expression and purification. The specific experimental steps are as follows: and transforming the recombinant plasmid with correct sequencing into E.coli BL21 competent cells for induced expression. Individual colonies were picked and inoculated into TB medium containing kanamycin, and cultured overnight with shaking at 37 ℃. The following day, the small culture product was expanded into 1L TB medium and cultured to OD6000.5-0.6, adding inducer IPTG to a final concentration of 0.5mM, and inducing at 37 deg.C for 5-7 h. The bacteria were harvested at 4,000rpm, lysed with lysis buffer (50mM Tris-HCl, pH 8.0, 50mM NaCl, 1mM DTT, 0.5mM EDTA, 5% glycerol), then disrupted at high pressure, and the pellet was centrifuged at 12,000 rpm. The pellet was washed twice with a wash buffer (20mM Tris-HCl, pH 8.0, 2M urea, 2.5% Triton X-100). Then, the protein was dissolved in a lysis buffer (20mM Tris-HCl, pH 8.0, 8M urea), and the supernatant was centrifuged. The protein was concentrated to about 5mL in a 3kDa protein ultrafiltration concentration tube, added to 1L of a renaturation buffer (50mM Tris-HCl, pH 8.0, 50mM L-Arg, 24mM NaCl, 1mM KCl, 1mM EDTA) and renatured by dilution at 4 ℃ for 24 hours. The protein was concentrated to about 20mL using a 3kDa concentration tube, filled into dialysis bags, dialyzed overnight in dialysis buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1mM DTT), and concentrated. Purifying the obtained protein by cation exchange column and molecular sieve. And (3) carrying out 14% SDS-PAGE gel electrophoresis on the protein after the molecular sieve treatment, and identifying the purity of the purified protein. The obtained electrophoretogram is shown in FIG. 2, the protein size is about 13KDa, and the size is consistent with the theoretical value of human PD-1 protein, which indicates that the human PD-1 protein is successfully obtained.
Example 5 SPR binding assay of human PD-1 protein to Polypeptides of the invention
The binding of the polypeptide of the present invention to human PD-1 protein was determined by Biacore T200 Surface Plasmon Resonance (SPR) experiments. The specific experimental steps are as follows: the purified PD-1 protein was diluted to 50. mu.g/ml with 10mM sodium acetate solution, and the PD-1 protein was coupled to a CM5 chip using an amino coupling kit at a flow rate of 10. mu.l/min for a binding time of 420s, and the final coupling amount was approximately 5000 RU. The binding assay was performed in PBS containing 0.05% surfactant P20 (137mM NaCl, 2.7mM KCl, 8mM Na)2HPO4，2mM KH2PO4pH 7.4). The experimental temperature was set at 25 ℃, the flow rate was 30 μ l/min, the binding time was 90s, and the dissociation time was 120 s. After solubilization of the polypeptide with the running solution, it was diluted in multiples to different concentrations and bound to PD-1 with the running solution. The binding data obtained were analyzed using BIAevaluationionon 2.0 software, and affinity K was obtained using steady state fittingDThe value is obtained. The results of SPR binding experiments of the polypeptide of the present invention and human PD-1 protein are shown in FIGS. 3a-3e and FIGS. 5a-5 d. Table 1 shows the sequences of the polypeptides of the invention and the K determined by SPR experimentsDThe value is obtained.
The inventor simultaneously detects the binding affinity K of the human PD-1 protein and the human PD-L1 proteinDValue, which is 1.15 μ M (as shown in FIG. 3 f).
TABLE 1 polypeptide sequences, molecular weights and their Ks binding to human PD-1 protein of the inventionDValue of
Figure BDA0000949524020000121
Example 6 SPR experiment of human PD-L1 protein competitive binding to human PD-1 protein with the polypeptide of SEQ ID NO. 1
According to the SPR experiment result in example 5, the polypeptide shown in SEQ ID NO. 1 has the highest binding affinity with human PD-1 protein. Thus, the inventors of the present invention have studied the ability of the peptide to block the binding of human PD-1 protein to human PD-L1 protein, taking the polypeptide shown in SEQ ID NO. 1 as an example. Human PD-L1 protein was purchased from Beijing Yi Qiao Shen Biotechnology, Inc.
The specific experimental steps are as follows: firstly, coupling 50 mu g/ml of human PD-L1 protein to the surface of a CM5 chip by an amino coupling method; then, polypeptides (125nM, 62.5nM, 31.25nM, 15.60nM, 0nM) represented by SEQ ID NO:1 at different concentrations were incubated with 100nM human PD-1 protein, respectively, on ice for 15min, and passed over the chip surface with running buffer (10mM phosphate buffer, 2.7mM KCl, 137mM NaCl and 0.05% surfactant P20, pH 7.4) at a flow rate of 30. mu.l/min, a binding time of 90s, and a dissociation time of 120 s; finally, the experimental results were analyzed by BIAevaluationionon 2.0 software. As shown in FIG. 4, the concentration of unbound PD-1 protein in the solution gradually decreases with the increase of the concentration of the polypeptide, so that the binding of PD-1 to PD-L1 is weakened, and the response value is reduced. The result of the SPR experiment is shown in FIG. 4, and the result shows that the polypeptide shown in SEQ ID NO. 1 can block the combination of human PD-1 and PD-L1.
Discussion: the present invention provides polypeptides that target human PD-1 protein, i.e., that are capable of competitively binding human PD-1 protein, each polypeptide having a specific amino acid residue at a specific position and thus being capable of having a similar structure. The polypeptides can be well combined with human PD-1 protein, wherein the polypeptide shown in SEQ ID NO. 1 has the highest combination affinity with the human PD-1 protein, and the K of the polypeptideDValue of 1.38. mu.M binding K to PD-L1 and PD-1DThe values are comparable. Therefore, the polypeptides can be used as an inhibitor of PD-1/PD-L1 binding or a regulator for regulating the binding degree of PD-1/PD-L1, and provide a polypeptide lead medicament for researching PD-1/PD-L1 immune checkpoint blockers and designing and developing blockade PD-1/PD-L1 pathways.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Figure IDA0000949524100000011
Figure IDA0000949524100000021
Figure IDA0000949524100000031
Figure IDA0000949524100000041

Claims (8)

1. A polypeptide selected from the group consisting of polypeptides having an amino acid sequence as set forth in any one of SEQ ID NOs 1-9.
2. The polypeptide of claim 1, wherein the polypeptide is a polypeptide having an amino acid sequence as set forth in SEQ ID No. 1 or 6.
3. An isolated nucleic acid molecule encoding the polypeptide of claim 1 or 2.
4. A host cell comprising the isolated nucleic acid molecule of claim 3.
5. A pharmaceutical composition comprising the polypeptide of claim 1 or 2 and a pharmaceutically acceptable excipient.
6. The application of the polypeptide with the amino acid sequence shown as SEQ ID NO. 1 in preparing the medicines for inhibiting the combination of PD-1 and PD-L1 or inhibiting tumors, treating infections caused by bacteria, viruses or fungi or treating inflammatory diseases.
7. The use of claim 6, wherein the tumor includes but is not limited to: melanoma, lung cancer, kidney cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphomas, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, cervical cancer, cervical, Epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma;
such viruses include, but are not limited to: hepatitis virus, herpesvirus, influenza virus, adenovirus, coronavirus, measles virus, dengue virus, poliovirus, rabies virus;
such bacteria include, but are not limited to: chlamydia, rickettsia, mycobacteria, staphylococci, pneumococci, cholera, tetanus;
such fungi include, but are not limited to: candida, aspergillus, dermatitides; or
Such inflammatory diseases include, but are not limited to: ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, pernicious anemia, polymyositis.
8. The use of claim 7, wherein the lung cancer is non-small cell lung cancer and the hepatitis virus is hepatitis A, B or C virus.
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