A neuropilin NRP-1 affinity peptide and its use

Abstract

The present application discloses a neuropilin NRP-1 affinity peptide and its uses. In particular, the polypeptide having affinity for NRP-1 of the present invention is selected from the sequences shown in SEQ ID NO: 1-5. Additionally, the present application also provides a bioactive substance containing the polypeptide sequence. The polypeptide or bioactive substance provided by the present invention can be used as a single agent or combined with other tumor therapeutic drugs for tumor treatment or detection by targeting and binding to NRP-1. The affinity peptide of the present invention has good specificity for NRP-1, strong affinity, and low production, purification, and preservation costs, providing new ideas for tumor targeted therapy and anti-angiogenesis therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to a polypeptide having affinity for targets on the surface of tumors and its applications, specifically to a polypeptide having affinity for neuropilin-1 (NRP-1) and its uses. Background Art

[0002] Neuropilins are a family of transmembrane glycoprotein receptors with a size of approximately 130 - 140 kDa. They consist of an N-terminal extracellular domain, a transmembrane domain, and a cytoplasmic domain. Among them, the extracellular domain mediates interactions with various growth factors to promote the activation of their homologous receptor tyrosine kinases. It plays a crucial role in the formation of the neural and vascular systems during embryonic development.

[0003] During the growth of tumors, a large amount of nutrients and oxygen are required. This strong nutritional demand and extremely fast growth rate will cause the tumor site to continuously induce the formation of new blood vessel networks to provide the oxygen and nutrients needed for its growth. Existing studies have shown that the NRP-1 protein is highly expressed on the surface of various types of tumors and can mediate the promotion of tumor proliferation, growth, and migration by vascular growth factors such as VEGF.

[0004] Therefore, the NRP-1 protein is an excellent target for developing tumor-targeted and therapeutic drugs. Summary of the Invention

[0005] The purpose of the present invention is to provide a polypeptide having high affinity for the NRP-1 protein. In particular, the polypeptide of the present invention can specifically bind to the extracellular domain of neuropilin, and it has the advantages of being convenient, efficient, practical, and safe. In addition, the present invention also provides the application of the polypeptide in drugs such as tumor diagnosis, targeting, and / or treatment.

[0006] Specifically, the embodiments of the present invention can be described as follows:

[0007] In one aspect, the present invention provides a polypeptide targeting neuropilin NRP-1, and the polypeptide is selected from the sequences shown in SEQ ID NO: 1 - 5. Preferably, the polypeptide of the present invention is SEQ ID NO: 1 (HAFKHHGHRNPP). Preferably, the polypeptide of the present invention is SEQ ID NO: 2 (WHHHWPYLRTAN). Preferably, the polypeptide of the present invention is SEQ ID NO: 3 (HWPFRHHASHAD). Preferably, the polypeptide of the present invention is SEQ ID NO: 4 (TVDLSLSSTTSV). Preferably, the polypeptide of the present invention is SEQ ID NO: 5 (FGPWKHTHHLQR).

[0008] In yet another aspect, the present invention provides a polypeptide targeting neuropilin NRP-1, which polypeptide is selected from the sequence shown in SEQ ID NO: 1.

[0009] In yet another aspect, the present invention provides an isolated polynucleotide that encodes the polypeptide of the present invention.

[0010] In yet another aspect, the present invention provides a recombinant vector that contains the isolated polynucleotide of the present invention.

[0011] In yet another aspect, the present invention provides a host cell that contains the recombinant vector of the present invention, or the isolated polynucleotide of the present invention is integrated into its genome.

[0012] In yet another aspect, the present invention provides a bioactive substance that contains the polypeptide of the present invention and is selected from: engineered phages, fusion proteins, and conjugates.

[0013] In yet another aspect, the present invention provides the use of the polypeptide, bioactive substance, isolated polynucleotide, recombinant vector, and / or host cell of the present invention in the preparation or screening of biomedical materials and / or drugs for tumor treatment by targeting and binding to NRP-1.

[0014] In yet another aspect, the present invention provides the use of the polypeptide, bioactive substance, isolated polynucleotide, recombinant vector, and / or host cell of the present invention in the preparation or screening of kits or drugs for tumor detection by targeting and binding to NRP-1.

[0015] In yet another aspect, the present invention provides an anti-tumor drug complex: the drug complex contains the polypeptide and / or bioactive substance of the present invention, and a preparation or drug and drug carrier for tumor treatment.

[0016] The preparation or drug for tumor treatment includes biological agents, chemical drugs, radiotherapy drugs, photothermal therapy drugs, photodynamic therapy drugs, and cryotherapy drugs. In particular, proteins, peptides, nucleic acids, antibiotics, anti-inflammatory drugs, anti-tumor drugs, neuroprotective agents, chemotherapeutic agents, cytotoxins, radioisotopes, fluorescent markers, luminescent substances, chromogenic substances, or enzymes.

[0017] The drug carrier for tumor treatment includes nano-drug carriers and micro-drug carriers, such as any one of liposomes, inorganic particles, organic particles, polymeric micelles, polymeric vesicles, carbon materials, microorganisms, etc.

[0018] In yet another aspect, the present invention provides a detection / diagnostic reagent or a detection / diagnostic kit, characterized by comprising the polypeptide and / or bioactive substance described in the present invention.

[0019] In yet another aspect, the present invention provides a composite material for tumor detection and targeted therapy, characterized by comprising the polypeptide and / or bioactive substance described in the present invention, and additional biomedical materials for tumor detection and targeted therapy.

[0020] The polypeptide provided by the present invention and the bioactive substance containing the polypeptide sequence can be used as a single preparation for anti-angiogenic therapy of tumors, or form a composite preparation with other tumor therapeutic drugs. At the same time, the polypeptide can also be combined with other materials for the composite material of tumor targeted therapy and detection. The polypeptide of the present invention has strong affinity and good specificity for neuropilin-1 (NRP-1), providing a new technical solution for the research and development of tumor targeted therapy and anti-angiogenic therapy reagents.

[0021] In particular, compared with the prior art, the present invention has the following outstanding advantages:

[0022] (1) The polypeptide screened out by the present invention can specifically bind to neuropilin-1 (NRP-1) without binding to other proteins, showing specificity.

[0023] (2) The present invention uses phage biopanning technology to screen small molecule polypeptides that bind to neuropilin-1 (NRP-1), which is convenient, fast, simple and efficient, laying a foundation for the research and development and clinical application of tumor drugs.

[0024] (3) The neuropilin-1 affinity polypeptide screened out by the present invention can be synthesized by a standardized chemical synthesis process. Compared with other types of biological agents, its production, purification and preservation costs are greatly reduced, and the target drug can be obtained in large quantities in a short time; and

[0025] (4) The polypeptide provided by the present invention has a specific binding effect on neuropilin-1, and has both therapeutic and tumor targeting effects, providing new options for further research on tumor targets, new drug screening, vaccines and new tumor early diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shows the input / output ratio of phage in each of the 1st to 4th rounds of screening in Example 1.

[0027] Figure 2The statistical graph of the results of the phage enzyme-linked immunosorbent assay (ELISA) in Example 2 is shown, where WT: wild-type phage, HP: phage containing SEQ ID NO: 1, WN: phage containing SEQ ID NO: 2, HD: phage containing SEQ ID NO: 3, TV: phage containing SEQ ID NO: 4, FR: phage containing SEQ ID NO: 5. Detailed implementation mode

[0028] The present invention will be further described in detail below with reference to examples and drawings. The following examples are preferred examples of the present invention, and the present invention is not limited to the following examples. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0029] Examples of the present invention are as follows:

[0030] Example 1: Screening of an affinity peptide for NRP-1 protein by phage biopanning technology

[0031] 1) Coating of the extracellular domain of NRP-1 protein (NCBI accession number: NP_001019799.1)

[0032] a. Dilute the NRP-1 protein with 0.1 M NaHCO3 so that the final concentration of the protein is 100 μg / ml, and pipette 150 μl of the diluted NRP-1 protein and add it to a 24-well plate.

[0033] b. Place the 24-well plate in a wet box and coat it overnight in a shaker at 4 °C.

[0034] 2) Biopanning of the phage library for the NRP-1 protein affinity peptide

[0035] a. Blocking: Take out the 24-well plate coated overnight, aspirate the liquid, add 2 ml of 5% BSA blocking solution, block at room temperature for 2 h, and then add 2 ml of PBST washing solution and wash 5 times to remove the blocking solution.

[0036] b. Phage library binding: Pipette 10 μl of liquid from the Ph.D. TM -12 phage random peptide library (from New England Biolabs), add it to 90 μl of PBST, and then add the mixture to the blocked and washed well plate, place it on a shaker, and incubate at room temperature for 1 h for the binding of the phage library and the protein.

[0037] c. Washing: Wash the well plate 10 times with TBST solution for 5 min each time to remove the phages that have not bound to the protein.

[0038] d. Elution: Add the eluent (0.2 M glycine - HCl, pH 2.2) to the washed well plate, incubate on a shaker for 8 min to elute the phages with high affinity for NRP-1 protein.

[0039] e. Neutralization: Add 1 M Tris-HCl, pH 9.1 to the eluent to make the final pH of the mixture 7.4.

[0040] f. Phage library amplification: Add the eluted phage solution to 20 ml of LB medium containing exponentially growing ER2738 bacteria, let it stand for 15 min for infection, and culture with shaking at 37 °C for 4 h.

[0041] g. Phage purification: Centrifuge the amplified 20 ml of bacterial - phage mixture at 12,000 g to remove bacteria. Add 4 ml of PEG / NaCl solution (20% PEG, 2.5 M NaCl) to the supernatant containing phages, let it precipitate overnight on ice. Resuspend the obtained precipitate with PBS to obtain the purified phage solution.

[0042] h. Second to fourth round of screening: Amplify and purify the phages eluted in the previous round, and use them as the phage library input for the next round. Incubate them with NRP-1 protein. The protein treatment and phage addition steps are as described in a - g above. Perform the second to fourth round of screening steps in sequence. When performing the screening, ensure that the amount of phages input in each round is the same. Record the amount of phages output in each round, and calculate the phage input amount / output amount in each round.

[0043] The results of the phage input amount / output amount in each round are as Figure 1 shown. When the initial phage input amount in each round is the same, as the number of screening rounds increases, the output amount of phages increases round by round, indicating that during the screening process, phages with affinity for NRP-1 protein are effectively enriched.

[0044] i. Obtain the NRP-1 affinity polypeptide sequence by phage DNA sequencing: Gradient dilute the phages eluted in the third and fourth rounds of screening, add them to exponentially growing Escherichia coli, infect at room temperature for 5 min. Pipette 200 μl of the phage - bacteria mixture and drop it onto an IPTG / Xgal solid LB plate, and spread it evenly with a spreader. Invert and place it in an incubator for culture. After plaques appear on the plate, pick single - clone plaques, add them to 5 ml of LB medium for overnight amplification culture, and perform DNA sequencing of the bacterial solution. By phage DNA sequencing, the affinity polypeptide sequence displayed on the phage surface can be determined. The high - frequency polypeptide sequences obtained by the fourth - round phage DNA sequencing are shown in Table 1 below.

[0045] Table 1 Frequency statistics of affinity polypeptides obtained from the fourth-round phage library screening

[0046]

[0047] As can be seen from the above table, in the fourth-round screening, the frequencies of five affinity polypeptide sequences were greater than 3.

[0048] Example 2: Verification of the affinity of polypeptides for the target protein NRP-1 by phage enzyme-linked immunosorbent assay (ELISA)

[0049] 1) Coating of the target protein: Add 50 µL of 2 µg / mL protein diluted with 0.1 M NaHCO3 to a 96-well ELISA plate. Place the ELISA plate in a wet box and incubate overnight at 4 °C.

[0050] 2) Blocking of the coated wells: Aspirate the protein solution in the wells and add 200 μL of 5% BSA solution to the wells. Incubate at 4 °C for 1 h.

[0051] 3) Washing: Discard the blocking solution. Invert the ELISA plate on filter paper and gently tap to drain all the liquid. Then add 200 µL of 0.5% PBST washing solution and incubate for 6 min. Discard the liquid and gently tap on the filter paper again to drain all the liquid. Repeat the washing 6 times.

[0052] 4) Phage incubation: Add 50 µL of phage at 2×109 pfu / mL and incubate at room temperature for 1 h.

[0053] 5) Washing: Aspirate the phage solution and repeat the washing 6 times with 0.5% PBST washing solution according to step (3).

[0054] 6) Addition of primary antibody: Add 100 µL of phage pVIII coat protein antibody diluted with PBST to the wells and incubate at 37 °C for 1 h. Then aspirate the antibody and add 200 µL of 0.5% PBST and wash 3 times according to step (3);

[0055] 7) Addition of secondary antibody: Add 100 µL of horseradish peroxidase (HRP)-conjugated secondary antibody diluted with PBST and incubate at 37 °C for 1 h. Then aspirate the antibody and add 200 µL of 0.5% PBST and wash 3 times according to step (3);

[0056] 8) Color development: Add 100 µL of TMB substrate solution and incubate for 10 - 20 min.

[0057] 9) Termination reaction: Add 100 µL of termination solution 2M H2SO4 (diluted with water) to terminate the reaction, and measure the absorption peak at 450 nm using an ELISA reader.

[0058] The ELISA results are as Figure 2 shown. Among the screened high-frequency polypeptides, the phage displaying the polypeptide HAFKHHGHRNPP (HP, SEQ ID NO: 1) has the strongest affinity for the NRP-1 protein, higher than the phages displaying other high-frequency polypeptide sequences and also higher than the wild-type phage. Additionally, compared with the wild-type phage, the phages displaying the polypeptides WHHHWPYLRTAN (WN, SEQ ID NO: 2), HWPFRHHASHAD (HD, SEQ ID NO: 3), TVDLSLSSTTSV (SEQ ID NO: 4), and FGPWKHTHHLQR (SEQ ID NO: 5) also respectively show higher affinities for the NRP-1 protein, where * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

[0059] The gene and protein sequences involved in the present invention are as follows:

[0060] SEQ ID NO: 1;

[0061] Name: Amino acid sequence of NRP-1 affinity peptide

[0062] Source: Artificial Sequence

[0063] HAFKHHGHRNPP

[0064] SEQ ID NO: 2;

[0065] Name: Amino acid sequence of NRP-1 affinity peptide

[0066] Source: Artificial Sequence

[0067] WHHHWPYLRTAN

[0068] SEQ ID NO: 3;

[0069] Name: Amino acid sequence of NRP-1 affinity peptide

[0070] Source: Artificial Sequence

[0071] HWPFRHHASHAD

[0072] SEQ ID NO: 4;

[0073] Name: Amino acid sequence of NRP-1 affinity peptide

[0074] Source: Artificial Sequence

[0075] TVDLSLSSTTSV

[0076] SEQ ID NO: 5;

[0077] Name: Amino acid sequence of NRP-1 affinity peptide

[0078] Source: Artificial Sequence

[0079] FGPWKHTHHLQR

Claims

1. A polypeptide having an affinity for neuropilin NRP-1, characterized in that, The amino acid sequence of the polypeptide is SEQ ID NO:

1.

2. An isolated polynucleotide, characterized in that, Encoding the polypeptide according to claim 1.

3. A recombinant vector, characterized in that, Comprising the isolated polynucleotide according to claim 2.

4. A cell, characterized in that, Comprising the recombinant vector according to claim 3, or having the isolated polynucleotide according to claim 2 integrated into its genome.

Images