CN117447560A

CN117447560A - Bicyclic peptide ligands against EGFR, pharmaceutical formulations, screening methods and uses thereof

Info

Publication number: CN117447560A
Application number: CN202311401434.8A
Authority: CN
Inventors: 李景虹; 王凌霄; 李昊东
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2023-10-26
Filing date: 2023-10-26
Publication date: 2024-01-26

Abstract

The present invention relates to bicyclic peptide ligands against EGFR, pharmaceutical formulations, screening methods and uses thereof. In particular, the invention relates to a bicyclic peptide ligand of EGFR protein developed by phage display technology, which has a peptide sequence selected from the group consisting of SEQ ID NO:1-10 and mutants thereof, including single amino acid mutants, double amino acid mutants and optimized sequences. It has excellent binding specificity and affinity. The dicyclic peptide ligand can be used as a candidate targeting molecule of an anti-tumor drug for treating cancers caused by the overexpression of an Epidermal Growth Factor Receptor (EGFR). The invention also relates to a method for efficiently screening EGFR bicyclic peptide ligands with high affinity.

Description

Bicyclic peptide ligands against EGFR, pharmaceutical formulations, screening methods and uses thereof

Technical field:

the invention relates to the field of bioengineering, in particular to a bicyclic peptide ligand of an Epidermal Growth Factor Receptor (EGFR) protein developed by phage display technology, a screening method and application thereof and a pharmaceutical preparation containing the same.

The background technology is as follows:

EGFR (Epidermal Growth Factor Receptor ) protein is an important transmembrane receptor tyrosine kinase, and is involved in various biological processes such as cell proliferation, differentiation, survival and the like, and is expressed in various cells including epidermal cells, alveolar cells, hepatocytes, ovarian cells, breast cells and the like. EGFR proteins play important biological roles in normal cellular physiology, such as maintenance of epidermis and keratinocytes, peripulmonary cell proliferation, hepatocyte proliferation, and the like. However, in some diseases, such as tumors, EGFR proteins are activated abnormally, and cause excessive activation of EGFR signaling pathways, thereby promoting biological activities such as growth, division, invasion and metastasis of tumor cells, and thus becoming an important promoting factor for proliferation and progression of tumor cells, and the cancers involved include lung cancer, breast cancer, colon cancer, glioblastoma, head and neck tumor, and the like. Abnormal activation of EGFR in tumor cells can also promote control of angiogenesis and immune escape by tumor cells, thereby further promoting tumor growth and metastasis.

Based on the important role of EGFR in tumors, which has become one of the important targets for the treatment of cancer, the research and development of EGFR inhibitors or targeted drugs has become a hotspot in current cancer treatment research. EGFR inhibitors can inhibit the growth and progression of tumor cells by inhibiting activation of EGFR signaling pathways, thereby achieving the goal of treating cancer. Currently, the main EGFR inhibitors are divided into two types, namely a small molecule inhibitor and a monoclonal antibody, wherein the small molecule inhibitor mainly acts on EGFR kinase structural domains of tumor cells to cause inhibition of receptors, thereby inhibiting growth and division of cancer cells; whereas monoclonal antibodies effectively inhibit the activity of EGFR by binding to the external domain of EGFR, thereby preventing proliferation of cancer cells.

Although EGFR protein inhibitors have achieved some research results and clinical applications, there are some shortcomings. For example, antibodies inhibitors are relatively bulky, resulting in difficult internalization, inability to access all areas of tumor tissue, and difficult localization of cancer cells distal to functional vessels; slow dynamics; the development and production costs are high and the finished drug is more expensive for the patient. Whereas for small molecule inhibitors, their residence time in the body is short and they are easily cleared by the kidneys, which results in generally larger doses of the drug required; their poor specificity and this lack of selectivity may lead to unwanted side effects such as diarrhea, rash, nausea etc, thereby limiting the use of small molecule inhibitors.

In recent years, polypeptide inhibitors have become a hotspot for drug development. The polypeptide medicine has the advantages of both small molecule inhibitor and antibody inhibitor. Firstly, the molecules are smaller and easy to cross the human body barrier, the quality control level of the molecular sieve is close to that of the traditional small molecular chemical drugs, but the binding force and the selectivity are far superior to those of the small molecules; secondly, the polypeptide drugs have the activity close to that of the antibody drugs, and simultaneously have the volume which is smaller by nearly 100 times, penetrate through tumor nodules more deeply, and have better distribution; in addition, polypeptide drugs are safe, their mechanism of action is usually reversible, which is advantageous in avoiding unnecessary side effects and toxic reactions, and the structure of polypeptide drugs is similar to that of natural biological molecules, so that their metabolism and excretion in vivo are usually easier.

However, the existing polypeptide drugs mainly comprise linear polypeptides and have a plurality of defects. For example, although binding forces are better than those of small molecules, they are weaker than antibodies, which can easily lead to off-target effects and side effects; polypeptide drugs are easily metabolized and degraded in vivo, meaning that their half-lives are short, requiring frequent dosing, and increasing the difficulty of treatment.

Recently, bicyclic peptides have been attracting attention in drug development as a new polypeptide form. A bicyclic peptide is a peptide compound having two rings that can be looped through disulfide bonds or bridging molecules. Compared to antibodies or linear polypeptides, bicyclic peptides have the following advantages: 1. structural stability. Linear polypeptides are susceptible to enzymatic degradation in the organism, thereby losing biological activity. The cyclic rigid structure of the bicyclic peptide, in turn, helps to resist degradation by enzymes, allowing it to remain stable in adverse circumstances. In addition, bicyclic peptides are also better able to accommodate changes in the in vivo environment due to their smaller molecular weight than antibodies. 2. High selectivity and affinity. Bicyclic peptides can form highly specific structures through interaction of two cyclic structures, resulting in highly selective interactions with target molecules. This benefits from the more favourable entropy effect of bicyclic peptides when bound, and the excellent properties of being able to mimic complex protein structures with minimal structural units. 3. Synthesizable properties. Bicyclic peptides can be prepared by solid phase peptide synthesis, which allows for a controlled production process and higher yields. In contrast, antibody production is often dependent on expression systems in organisms, and the production process is complex and difficult to control. 4. Low immunogenicity. Bicyclic peptides have a smaller molecular weight and therefore are less immunogenic in organisms. This means that the bicyclic peptide is not likely to trigger side effects of the immune system when used as a medicament, thereby improving the safety of the medicament. In contrast, antibodies are susceptible to eliciting immunogenic reactions in organisms due to their large molecular weight and complex structure. In summary, bicyclic peptides may be currently the best EGFR protein ligand candidates. However, the complex three-dimensional structure of bicyclic peptides increases the difficulty of rational design.

Phage display and panning techniques are an effective method for high throughput screening of polypeptide ligands for targeted proteins, which utilize phage surface displayed proteins or peptides as recognition molecules to achieve highly selective binding to specific targets. The technology is widely applied to the basic research fields of biology, pharmacology and the like, and has wide application prospects in the fields of development of targeted drugs, development of diagnostic reagents, preparation of biosensors and the like. Therefore, the rapid and efficient screening of the bicyclic peptide by utilizing phage display and panning technology is a powerful means for developing the ligand of the bicyclic peptide.

The invention comprises the following steps:

the invention aims to provide a double-ring peptide ligand of EGFR protein developed by phage display technology and a screening method thereof, so as to overcome the defects of the prior art.

To achieve the above object, embodiments of the present invention propose a bicyclic peptide obtained by panning against EGFR protein through a phage-displayed library of bicyclic peptides. The dicyclic peptide provided by the embodiment of the invention can realize specific high-affinity binding with EGFR protein, and provides precursors for the development of inhibitors and medicaments targeting EGFR protein.

In order to achieve the above task, the present invention adopts the following technical scheme:

in one aspect, the invention provides a bicyclic peptide ligand for EGFR, wherein the bicyclic peptide ligand has a peptide sequence selected from the group consisting of SEQ ID NOS: 1-10 and mutants thereof, including single amino acid mutants, double amino acid mutants, and optimized sequences.

According to a specific embodiment of the invention, wherein the bicyclic peptide ligand has the following peptide sequence: SEQ ID NO. 1 or SEQ ID NO. 9.

According to a specific embodiment of the present invention, wherein the structure of the bicyclic peptide ligand comprises two cyclic structures, the cyclic structures are formed in a manner comprising disulfide bonds, preferably by disulfide bonds between cysteines in the bicyclic peptide ligand to form (Cys 2-Cys16/Cys4-Cys 10) and/or (Cys 2-Cys10/Cys4-Cys 16) bicyclic peptides.

According to a specific embodiment of the present invention, wherein the affinity constant (KD) of the bicyclic peptide ligand is less than or equal to 2.49 μm.

According to a specific embodiment of the present invention, wherein the bicyclic peptide ligand is developed by phage display technology.

In another aspect, the invention also provides a method of screening for a bicyclic peptide ligand of an EGFR protein having high affinity, comprising the steps of:

a. constructing a phage display library comprising a plurality of bicyclic peptide sequences, the peptides in the library having GCXC (X) ₅ C(X) ₅ C (SEQ ID NO: 11), wherein X is any amino acid, C is a cysteine, and the sequence-CXC-motif directs the formation of (Cys 2-Cys16/Cys4-Cys 10) and/or (Cys 2-Cys10/Cys4-Cys 16) bicyclic peptides between cysteines in the sequence via disulfide bonds;

b. incubating the phage display library with an immobilized EGFR protein to bind phage-displayed bicyclic peptides to the EGFR protein;

c. removing unbound phage by multiple washes;

d. recovering phage combined with EGFR protein by enzyme digestion or acidolysis;

e. the amino acid sequence of the bicyclic peptide ligand with high affinity is determined by amplifying the recovered phage and sequencing the phage.

According to a specific embodiment of the present invention, the bicyclic peptide sequences in the phage display library may be optimized by random mutagenesis, recombination or directed evolution.

According to a specific embodiment of the invention, the specificity and sensitivity of the screening can be improved by adjusting parameters such as washing conditions, incubation time or incubation temperature during the screening process.

In another aspect, the present invention provides a pharmaceutical formulation comprising as an active ingredient the bicyclic peptide ligand described above and a pharmaceutical carrier, preferably the pharmaceutical carrier comprises a polymer, nanoparticle or liposome.

In another aspect, the present invention provides the use of a bicyclic peptide ligand or pharmaceutical formulation as described above in the manufacture of a medicament for the treatment or prophylaxis of a tumor.

In yet another aspect, the invention provides a method of treating or preventing a tumor, the method comprising administering to a subject in need thereof an appropriate amount of a bicyclic peptide ligand or pharmaceutical formulation of the invention.

The beneficial effects are that:

the invention provides a double-ring peptide ligand of EGFR protein developed by phage display technology, which has excellent binding specificity and affinity, and provides a new targeting strategy for tumor treatment. Compared with the existing EGFR inhibitor, the bicyclic peptide ligand has the following advantages:

1. high affinity: the polypeptide ligand provided by the invention has high affinity binding (KD=2.49 mu M) with EGFR protein, and can effectively identify and target EGFR protein.

2. High specificity: in the screening process, the experimental conditions can be controlled to ensure that the obtained polypeptide ligand has high specificity and reduce the influence on non-target proteins.

3. Safety: compared with the traditional antibody and small molecule medicine, the polypeptide ligand has lower immunogenicity and better biocompatibility, and reduces side effects.

4. Flexibility: the polypeptide ligand can be modified chemically or optimized structurally to improve stability, affinity and bioactivity and meet different application requirements.

5. The application is wide: the polypeptide ligand provided by the invention can be used for diagnosing and treating diseases related to EGFR abnormal activation, such as tumors, inflammations and the like.

In addition, the invention also provides a method for effectively screening the double-ring peptide ligand with high affinity, which is helpful for searching more EGFR inhibitors with therapeutic potential.

The dicyclic peptide ligand can be used as a candidate targeting molecule of an anti-tumor drug for treating cancers caused by over-expression of an Epidermal Growth Factor Receptor (EGFR). The antitumor drug prepared by the bicyclic peptide ligand provided by the invention can slow down the growth of tumor cells, reduce toxicity and side effects, and provide better treatment options for cancer patients. The double-ring peptide can be used for developing a biosensor to realize the visualization of tumors.

The screening method can screen the double-ring peptide ligand of EGFR protein with high affinity, and can further optimize the double-ring peptide sequence in phage display library, thereby providing powerful support for developing novel antitumor drugs.

In summary, the development of the EGFR bicyclic peptide ligand has broad application prospects and important scientific value, and will help promote the development and progress of the biomedical field.

Description of the drawings:

in order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a graph of phage enrichment results for each of 5 rounds of panning on phage-displayed bicyclic peptide libraries according to an embodiment of the invention.

FIG. 2 is a graph of SPR data relating to interactions of bicyclic peptides with EGFR in an embodiment of the present invention.

FIG. 3 is a graph of HPLC chromatographic results for bicyclic peptides involved in the examples of the present invention.

FIG. 4 is a graph showing the mass spectrum of the bicyclic peptide according to the present invention.

The specific embodiment is as follows:

the invention adopts the following technical scheme:

1. construction of random bicyclic peptide libraries using phage display technology

In some embodiments, the bicyclic peptide is obtained by panning a phage displayed library of bicyclic peptides against EGFR protein, the peptides in the library of bicyclic peptides having GCXC (X) ₅ C(X) ₅ C (SEQ ID NO: 11), wherein X is any amino acid, C is a cysteine, and the sequence-CXC-motif directs the formation of (Cys 2-Cys16/Cys4-Cys 10) and/or (Cys 2-Cys10/Cys4-Cys 16) bicyclic peptides between cysteines in the sequence via disulfide bonds. The bicyclic peptide is linked to the gene 3 protein (pIII) in the M13 filamentous phage coat protein via a triple alanine (Ala-Ala) linker.

2. Screening of bicyclic peptide libraries using biotin-labeled EGFR proteins as targets

In some embodiments, the EGFR protein used for panning is the extracellular domain of the intact protein (Leu 25-Ser 645) and is linked to Biotin (Biotin) by adding an AVI tag (amino acid sequence: GLNDIFEAQKIEWHE) at the C-terminus of the protein and further immobilized on streptavidin or neutravidin coated magnetic beads, and interaction of the protein with phage library or washing, elution is achieved by a magnet during panning. The screening mainly comprises the following steps:

a. incubating the phage display library with an immobilized EGFR protein to bind phage-displayed bicyclic peptides to the EGFR protein;

b. removing unbound phage by multiple washes;

c. recovering phage combined with EGFR protein by enzyme digestion or acidolysis;

d. the recovered phage was amplified and screened as a new library for a new round until the appropriate enrichment (positive phage/negative phage) was reached.

3. Analysis of the bicyclic peptide sequences of the screened positive phage clones

In some embodiments, we extract the DNA of positive phage library collected in the last round of panning and achieve amplification of the gene of interest and construction of high throughput sequencing library by PCR. All sequence information of the positive phage library was obtained by high throughput sequencing. Sequencing data are analyzed by software, and then the target gene is translated into a polypeptide sequence, so that the optimal bicyclic peptide sequence is GCRCVTGPWCATWPGC (SEQ ID NO: 1) (the ratio of the sequence in the library is 98%). In addition, the first 10 polypeptide sequences are highly conserved among the sequence information collected, containing the consensus sequence GCRCVTGPXCATXXGC (SEQ ID NO: 12), where X is any amino acid.

4. The binding activity to EGFR was tested by synthesizing, expressing and purifying bicyclic peptides

In some embodiments, we synthesized the primary sequence of the bicyclic peptides (SEQ ID NO:1, SEQ ID NO: 9), i.e., the linear peptides, by a microwave solid phase synthesizer, and further oxidized the linear peptides to bicyclic peptides by oxidative glutathione (GSSG), DMSO, and guanidine hydrochloride phosphate buffer, and purified by reverse-phase High Performance Liquid Chromatography (HPLC) and validated by Mass Spectrometry (MS). Further, we determined the affinity constants (KD) of the bicyclic peptides (SEQ ID NO:1, SEQ ID NO: 9) to EGFR proteins by surface plasmon resonance (Surface Plasmon Resonance, SPR) and determined to be 5.62. Mu.M, 2.49. Mu.M, respectively.

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Example 1 screening for bicyclic peptide ligands with high affinity

In this example, the original phage bicyclic peptide library formed by linear peptides under the guidance of CXC motif is used as experimental material, phage display technology is used to perform high-throughput panning on the bicyclic peptide which can be strongly bound to EGFR protein (in this example, extracellular domain of EGFR protein (Leu 25-Ser 645) is used, and the C-terminal thereof is connected to Biotin through AVI tag), and phage with strong binding capacity is enriched through five rounds of "adsorption-elution-amplification" steps.

1. Phage titer assay

(1) TG1 glycerol bacteria (commercially available from Biyun Tian Co.) stored at-80℃were removed, streaked on 2YT-Tet plates (wherein 2YT-agar powder is commercially available from Kulebur Co., ltd.), dissolved in ultrapure water and sterilized, and the plates were formed after pouring into a petri dish for cooling, and then cultured upside down at 37℃for 12 hours.

(2) TG1 single colonies were picked with a sterile gun head in 20ml 2yt-Tet broth, incubated in a shaker at 37 ℃, 220rpm to logarithmic growth phase (od600=0.5).

(3) The sterilized 2YT agar was added to ampicillin and poured into a 9mm dish, and the mixture was allowed to stand and cooled until it was completely coagulated to prepare a solid medium (2 YT agar-Amp).

(4) The initial phage bicyclic peptide pool was diluted with 2YT gradients, each gradient replacing the gun head. Briefly describing the construction of the initial phage bicyclic peptide library, we will have the random peptide-GCXC (X) ₅ C(X) ₅ C-was linked to the gene 3 protein (pIII) in phage coat protein by a triple alanine (Ala-Ala-Ala) linker, glycine residues were added to the N-terminus of the random peptide to ensure removal of the signal sequence. Then the phagemid vector was transformed into E.coli to produce a vector containing 4.5X10 ⁹ A library of independent transformants can produce approximately 1.8X10 s per ml of culture ¹⁰ And (3) infecting phage.

(5) The TG1 bacterial liquid in logarithmic growth phase is split into 180 mu L each tube. mu.L of phage bicyclic peptide samples of different dilutions were added to each tube, vortexed and incubated for 30min at 37 ℃.

(6) Phage-infected bacteria were plated onto 2YT agar-Amp solid medium at 10. Mu.L each according to dilution.

(7) After the bacterial liquid is completely absorbed by the solid culture medium, the bacterial liquid is inversely cultured in a 37 ℃ incubator for 12 hours.

(8) The plates were removed from the incubator and the initial phage bicyclic peptide library concentration was estimated based on each gradient phage titer.

2. Phage display panning

First round panning:

(1) 100 mu L of streptavidin-coated magnetic beads are taken into a centrifuge tube, the centrifuge tube is washed twice with 1mL of TBS buffer solution, the centrifuge tube is placed on a magnetic rack for about 1min, and the supernatant is sucked.

(2) The beads were resuspended in 100. Mu.L TBS buffer and aliquoted into two centrifuge tubes of 50. Mu.L each.

(3) To one of the tubes, 8. Mu.g of biotin-labeled target protein was added, which is the experimental group. The other tube was added with an equal amount of TBS buffer, which was the control group.

(4) The two centrifuge tubes were placed on a shaker and incubated at room temperature for 10min. The centrifuge tube was placed on a magnetic rack for 1min and the supernatant was discarded.

(5) The beads in both tubes were washed three times with 1mL of TBS buffer, respectively.

(6) To each of the two tubes, 300. Mu.L of TBS buffer and 150. Mu.L of TBS buffer containing BSA protein were added to block the beads, and the two tubes were placed on a shaker and incubated at room temperature for 120min. Will be greater than 10 ¹² The phage library of pfu was added to the mixture as well and incubated on a shaker at room temperature for 120min.

(7) The blocked phage library was divided into two equal parts, each 2.25mL, into two tubes, which were added to the magnetic beads of the experimental and control groups, respectively, and the two tubes were placed on a shaker and incubated for 30min at room temperature.

(8) After incubation, two centrifuge tubes containing phage library and beads were placed on a magnetic rack for 1min, the supernatant was aspirated, the beads in both tubes were washed 9 times with 1mL of TBST buffer (containing 0.5% Tween-20), and finally washed 2 times with 1mL of TBS buffer, and during the washing, the centrifuge tube was replaced at least three times to reduce the amount of phage non-specifically adsorbed to the tube wall.

(9) After 200. Mu.L of glycine-HCl buffer (pH 2.2) was added to each of the two tubes to resuspend the beads, and incubated at room temperature for 5min, both centrifuge tubes were placed on a magnetic rack for 1min, and the supernatants were rapidly transferred to each of the two centrifuge tubes containing 45. Mu.L of Tris-HCl buffer (pH 8.0).

(10) The phage solution after elution was left in an appropriate amount for titer measurement (the procedure was the same as step 1 "phage titer measurement"), and the remaining phage solution was added to 20mL of TG1 bacterial solution in the logarithmic growth phase, and incubated at 37℃for 90 minutes under resting conditions in an incubator.

(11) 4500g of the bacterial liquid was centrifuged at 4℃for 15 minutes, and the supernatant was discarded.

(12) Bacterial pellet was resuspended in 500. Mu.L of 2YT liquid medium. Uniformly spreading on a 2YT agar-Amp plate culture medium with diameter of 15cm, and culturing in a 37 deg.C incubator overnight

(13) All colonies on the plates were scraped off and added to the centrifuge tube, and glycerol was added to a final volume of 30% and frozen in a-80 ℃ freezer as phage glycerol library for the next round of screening.

(14) A suitable amount of the stored phage glycerol stock was taken into 100mL of 2YT liquid medium, and 100. Mu.g/mL of ampicillin was added. Placed in a shaking table at 37℃at 220rpm and incubated until OD600 was 0.5.

(15) An appropriate amount of helper phage M13KO7 (NEB Co.) was added to the culture, and the culture was placed in a shaking table at 37℃and incubated at 220rpm for 30 minutes.

(16) The bacterial suspension was centrifuged at 4000rpm at 4℃for 10 minutes, and the supernatant was carefully discarded.

(17) The pellet was resuspended in 100mL of 2YT (ampicillin and kanamycin sulfate) liquid medium and incubated overnight at 220rpm in a shaker at 30 ℃.

(18) The bacterial solution was centrifuged at 6000rpm at 4℃for 10 minutes and the supernatant carefully poured into a sterile beaker.

(19) PEG/NaCl solution with a volume of 1/5 of the culture volume was added to the beaker, and the mixture was stirred and ice-washed for 6 hours.

(20) Centrifugation at 10000rpm for 25 minutes to pellet phage, re-suspension of phage pellet with 1mL 10mM PBS, and split charging for use.

(21) The first round of phage product titers after amplification were determined in the manner of step 1 "phage titer determination".

Second to fifth panning rounds:

(1) According to the calculation, the titer of the amplified product of the phage in the previous round is calculated to be greater than 10 ¹² The amount of solution required for phage library of pfu.

(2) The procedure was repeated for the first round, but with the following changes:

the addition of EGFR protein was decreased with each round of screening, e.g., 8. Mu.g, 6. Mu.g, 4. Mu.g, 2. Mu.g, respectively, were added for the second round through the fifth round.

b. The magnetic beads used in the second round and the fourth round are changed into magnetic beads coated with neutravidin so as to reduce nonspecific adsorption. The magnetic beads are prepared by reacting neutral avidin with activated methylsulfonyl coated magnetic beads, and are operated in a dark place during screening. FIG. 1 is a graph of phage enrichment results for each of 5 rounds of panning.

3. High throughput sequencing of phage libraries and determination of bicyclic peptide sequences

(1) The phage display peptide library after 1-5 rounds of panning and the original phage peptide library are used to extract the corresponding phage library DNA by using the kit.

(2) The target gene was amplified by PCR, and the reaction system and reaction conditions are shown in tables 1 and 2.

TABLE 1

TABLE 2

Wherein SEQ ID NO. 13 is:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTC CGATC*T

SEQ ID NO. 14 is:

CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTG TGCTCTTCCGATC*T

* Representing phosphorothioate modifications

And (3) purifying a PCR product: the PCR product was purified and recovered by using DNA-sorting beads (the next holy organism). The gel was verified by 2% agarose gel electrophoresis.

(3) Next generation sequencing: the target gene fragment obtained by PCR was sequenced by Jin Weizhi Biotechnology Co., ltd. Based on the Illumina platform.

(4) Sequencing result analysis

After completion of sequencing, the sequencing data was analyzed by software (chem. Sci.,2022,13,7780-7789) to translate the target gene into the primary structural sequence of the bicyclic peptide, and the obtained partial sequence information is shown in table 3.

TABLE 3 Table 3

Sequence numbering	Sequence(s)	Relative abundance of
			SEQ ID NO:1	GCRCVTGPWCATWPGC	98.18％
SEQ ID NO:2	GCRCVTGPGCATWPGC	0.34％
			SEQ ID NO:3	GCRCVTGPWCATGPGC	0.33％
SEQ ID NO:4	GCRCVTGPWCATWPGG	0.19％
			SEQ ID NO:5	GCRCVTGPWCATWRGC	0.05％
SEQ ID NO:6	GCRCVTGPWCATRPGC	0.04％
			SEQ ID NO:7	GCRCVTGPRCATWPGC	0.03％
SEQ ID NO:8	GCRCGTGPWCATWPGC	0.03％
			SEQ ID NO:9	GCRCWRPELCLTTKAC	0.03％
SEQ ID NO:10	GCRCVAGPWCATWPGC	0.02％

As shown in Table 3, the highest abundance of SEQ ID NO. 1 is approximately 98.18% and far better than the rest of the sequences, the rest of the polypeptides (SEQ ID NO. 2-8, 10) differ from SEQ ID NO. 1 by only a single amino acid, SEQ ID NO. 9 and SEQ ID NO. 1 have different consensus sequences, possibly binding to another active site of EGFR.

Example 2 protein-polypeptide binding force assay

In this example, EGFR protein and bicyclic peptides SEQ ID NO. 1 and SEQ ID NO. 9 were used as experimental materials, and the binding affinity of the bicyclic peptides SEQ ID NO. 1 and SEQ ID NO. 9 to EGFR protein (in this example, the extracellular domain of EGFR protein (Leu 25-Ser 645) was used) was determined using Surface Plasmon Resonance (SPR). The bicyclic peptide SEQ ID NO. 1 is the most abundant sequence in the screening pool, while the bicyclic peptide SEQ ID NO. 9 is an example of another active site ligand for EGFR.

1. SEQ ID NO. 1 and SEQ ID NO. 9, determined in example 1, were synthesized using conventional solid phase peptide synthesis.

The linear polypeptides were synthesized by solid phase synthesis (SPPS) from Nanjing Jinsri Biotech and Hefei Jing Tai Biotech Inc. Fmoc protected amino acids were sequentially attached to the resin to form peptide chains. After the synthesis is completed, the N-terminal Fmoc group is deprotected, then each amino acid side chain protecting group is deprotected, and the peptide is separated from the resin. The polypeptide was purified by reverse phase chromatography and the purity of the resulting polypeptide was delivered at >95% purity.

2. Oxidation by oxidized glutathione to form bicyclic peptides and separation by HPLC

The linear peptide of SEQ ID No. 1, SEQ ID No. 9 (50. Mu.M) was dissolved in 100mM phosphate buffer (500. Mu.L, pH 7.4) containing 6M guanidine hydrochloride (Gu. HCl), 0.5mM oxidized glutathione (GSSG) and 10% DMSO (v/v). The reaction mixture was stirred at 37 ℃ for 12 hours. After the reaction was completed, the oxidized peptide was purified in HPLC using a C-18 column using a gradient of 10-50% acetonitrile (0.1% TFA) and water (0.1% TFA) over 30 minutes. After analysis by MALDI-TOF MS, the oxidized peptide was purified and lyophilized to give a white powder. The HPLC results are shown in FIG. 3.

3. Structure validation

The polypeptides were verified by mass spectrometry prior to use. High resolution ESI mass spectra were measured on an Agilent 6210 time-of-flight mass spectrometer. Normal ESI mass spectra were measured on Shimadzu LC/MS-2020 system. The mass spectrum results are shown in FIG. 4.

4. Determination of the binding affinity of bicyclic peptides to proteins using Surface Plasmon Resonance (SPR)

In the present invention, an interface plasmon resonance experiment was performed using a GE Healthcare Biacore K plus instrument. Biotinylated EGFR was immobilized on SA chips by using PBST buffer to a fixed amount of about 3000 RU. The bicyclic peptides SEQ ID NO. 1, SEQ ID NO. 9 were then diluted to concentrations of 0.0625, 0.125, 0.25, 0.5, 1, 2, 4 and 8. Mu. Mol/L, respectively, with PBST as binding buffer. Dissociation tests were performed using 4mM NaOH as the regeneration solution, setting a binding time of 120 seconds, a dissociation time of 360 seconds and a regeneration time of 30 seconds. All experimental data were collected and analyzed by Biacore 8K plus software.

Experimental results show that the binding constants KD of the bicyclic peptides SEQ ID NO. 1, SEQ ID NO. 9 and EGFR protein (Leu 25-Ser 645) are 5.62X10 respectively ^-6 M、2.49×10 ^-6 M. The results are shown in FIG. 2.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A bicyclic peptide ligand for EGFR, wherein the bicyclic peptide ligand has a peptide sequence selected from the group consisting of SEQ ID NOs 1-10, and mutants thereof, including single amino acid mutants, double amino acid mutants, and optimized sequences.

2. The bicyclic peptide ligand of claim 1, wherein the bicyclic peptide ligand has the peptide sequence: SEQ ID NO. 1 or SEQ ID NO. 9.

3. The bicyclic peptide ligand according to claim 1 or 2, wherein the structure of the bicyclic peptide ligand comprises two cyclic structures formed in a manner comprising disulfide linkages, preferably between cysteines in the bicyclic peptide ligand by disulfide linkages to form (Cys 2-Cys16/Cys4-Cys 10) and/or (Cys 2-Cys10/Cys4-Cys 16) bicyclic peptides.

4. The bicyclic peptide ligand of claim 1 or 2, wherein the affinity constant (KD) of the bicyclic peptide ligand is less than or equal to 2.49 μΜ.

5. The bicyclic peptide ligand of claim 1 or 2, wherein the bicyclic peptide ligand is developed by phage display technology.

6. A method of screening for bicyclic peptide ligands of EGFR protein having high affinity comprising the steps of:

c. removing unbound phage by multiple washes;

7. The method of claim 6, wherein the bicyclic peptide sequences in the phage display library are optimized by random mutagenesis, recombination or directed evolution.

8. The method of claim 6, wherein the specificity and sensitivity of the screening is increased during the screening by adjusting parameters such as washing conditions, incubation time or incubation temperature.

9. A pharmaceutical formulation, characterized in that it comprises as active ingredient a bicyclic peptide ligand according to any one of claims 1 to 5 and a pharmaceutical carrier, preferably the pharmaceutical carrier comprises a polymer, nanoparticle or liposome.

10. Use of a bicyclic peptide ligand of any one of claims 1 to 5 or a pharmaceutical formulation of claim 9 in the manufacture of a medicament for the treatment or prophylaxis of a tumour.