Cyclopeptide molecule specifically targeting EphA2 and application of cyclopeptide molecule in preparation of tumor diagnosis reagent or tumor treatment medicine

Abstract

The invention belongs to the technical field of biological medicines, and discloses a specific EphA2 targeting cyclic peptide molecule and application thereof in preparation of a tumor diagnosis reagent or a tumor treatment medicine. Eph receptor tyrosine kinase A2 (EphA2) is abnormally highly expressed in various malignant tumors, and is proved to be an important target for cancer diagnosis and treatment. According to the invention, a phage display technology is utilized to carry out multiple rounds of in-vitro screening on human EphA2 protein, and a monocyclic peptide molecule with patent medicine potential and high affinity with EphA2 is obtained through sequence analysis, chemical synthesis and combined characterization. In addition, the invention also comprises two cyclopeptide coupled radionuclide drugs, which show huge application potential in targeted imaging and treatment of tumors characterized by EphA2 overexpression, and provides a new choice for treatment of EphA2 related tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a cyclic peptide molecule specifically targeting EphA2 and an application thereof in preparing a tumor diagnostic reagent or a tumor therapeutic drug. Background Art

[0002] Eph receptor tyrosine kinase A2 (EphA2) belongs to the Eph receptor family and is a 130 kDa type I transmembrane glycoprotein. It consists of a ligand binding domain (LBD), a cysteine-rich Sushi domain, an epidermal growth factor (EGF)-like domain, and two fibronectin type III repeats. Various studies have shown that EphA2 is expressed at a low level in normal tissues, but is overexpressed and dysregulated in a variety of cancers (such as breast cancer, ovarian cancer, lung cancer, colorectal cancer, cervical cancer, prostate cancer, and skin cancer), and is associated with tumor occurrence, metastasis, invasion, and poor clinical prognosis. The wide expression pattern of EphA2 and its functional relevance in malignant tumors make it an attractive new anticancer therapeutic target.

[0003] At present, EphA2 targeted drugs mainly include antibody-drug conjugates (ADC), peptide-drug conjugates (PDC), tyrosine kinase inhibitors (TKI), chimeric antigen receptor T cells (CAR-T) cells, and nanocarriers using RNA interference technology, etc. Among them, antibody drugs, such as ADC drug MEDI-574 (doi: 10.1007 / s10637-012-9801-2), monoclonal antibody DS-8895a (doi: 10.1007 / s10637-022-01237-3), etc., and small molecule antagonists, such as dasatinib (doi: 10.1158 / 1078-0432.CCR-11-0288), etc., have not achieved satisfactory results in clinical trials. Compared with other targeted drugs such as monoclonal antibodies, linear peptides or small molecules, cyclic peptides have better tissue permeability, renal clearance, lower immunogenicity, and relatively higher safety. Cyclic peptides also have higher binding affinity and selectivity, are easier to chemically modify, and have a more stable rigid structure. These characteristics make cyclic peptide drugs a better choice for tumor diagnosis and treatment.

[0004] Recently, the EphA2-targeted PDC drug BT5528 developed by Bicycle therapeutics has shown preliminary anti-tumor activity in a phase I / II study of advanced solid tumors associated with EphA2 expression (doi:10.1158 / 1535-7163). In addition, similar bicyclic peptide ligands have also been applied to peptide-radionuclide conjugates (PRCs). Radionuclide 68 Ga / 177 The Lu-labeled PRC drug BCY18469 showed promising results in preclinical studies (doi:10.7150 / thno.96641).

[0005] In view of the above impressive research results, cyclic peptide drugs may become an ideal candidate for the diagnosis and treatment of tumors with high EphA2 expression. So far, there are still not many cyclic peptide drugs targeting EphA2 on the market. There is a lot of room and value for in-depth research on cyclic peptide molecules with high affinity, specific targeting of EphA2 and more diverse properties. Summary of the invention

[0006] The purpose of the present invention is to provide a cyclic peptide molecule that specifically targets EphA2 and its use in the preparation of tumor diagnostic reagents or tumor therapeutic drugs, so as to supplement new cyclic peptide molecules with high affinity, specific targeting of EphA2 and more diverse properties, and provide more possibilities for the targeted treatment of EphA2.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The present invention first provides a linear peptide or cyclic peptide molecule that specifically targets EphA2, which is selected from one of the molecules E001, E002, E101, E102, E201, E202, E301, E302, E303, E304, E401, E402, E501, E502, E301-DOTA or E303-DOTA, and the amino acid sequence of E001, E002, E101, E102, E201, E202, E301, E302, E303, E304, E401, E402, E501, E502, E301-DOTA or E303-DOTA is as follows:

[0009] (1) E001: Phe-Arg-Phe-Glu-Ala-Tyr-Pro-Asp-Ala-Gln-Arg-Leu; the sequence is shown in SEQ ID NO.1;

[0010] (2) E002: Ser-Val-Met-Tyr-Arg-Ser-Leu-Pro-Leu-Glu-Phe-Phe-Phe-Thr; the sequence is shown in SEQ ID NO. 2;

[0011] (3) E101: Phe-Cys-Ala-Tyr-Pro-Cys-Asn-His; the sequence is shown in SEQ ID NO. 3;

[0012] (4) E102: Lys-Arg-Trp-Cys-Ala-Phe-Pro-Cys-Leu; the sequence is shown in SEQ ID NO. 4;

[0013] (5) E201: Cys-Pro-Trp-Asp-Phe-Cys-Trp; the sequence is shown in SEQ ID NO. 5;

[0014] (6) E202: Ile-Thr-Leu-Thr-Cys-Pro-Cys-Thr-Leu-Gly-Ser-Trp; the sequence is shown in SEQ ID NO. 6;

[0015] (7) E301 / E501: Phe-Leu-Ala-Cys-Pro-Glu-Thr-His-Cys-Thr; the sequence is shown in SEQ ID NO.7;

[0016] (8) E302: Phe-Gln-Ala-Cys-Pro-Met-Asn-Cys-Arg-Gly-Ser-Trp; the sequence is shown in SEQ ID NO. 8;

[0017] (9) E303 / E502: Phe-Arg-Ala-Cys-Pro-Phe-Asp-Cys-Thr-Gly-Ser-Trp; the sequence is shown in SEQ ID NO. 9;

[0018] (10) E304: Gln-Phe-Leu-Ala-Cys-Pro-Met-Thr-Ser-Cys-Gly-Ser-Trp; the sequence is shown in SEQ ID NO. 10;

[0019] (11) E401: Leu-Arg-Phe-Cys-Asp-Tyr-Ile-Gly-Cys-Thr; the sequence is shown in SEQ ID NO.11;

[0020] (12) E402: Ser-Phe-Cys-Ala-Trp-Ile-Gly-Cys-Thr; the sequence is shown in SEQ ID NO.12;

[0021] (13)E301-DOTA:

[0022] Phe-Leu-Ala-Cys-Pro-Glu-Thr-His-Cys-Thr-Gly-Ser-Trp-Lys (DOTA); the sequence is shown in SEQ ID NO. 13;

[0023] (14)E303-DOTA:

[0024] Phe-Arg-Ala-Cys-Pro-Phe-Asp-Cys-Thr-Gly-Ser-Tyr-His-Lys (DOTA); the sequence is shown in SEQ ID NO.14.

[0025] In one embodiment of the present invention, the molecule E001, E002, E101, E102, E201, E202, E301, E302, E303, E304, E401, E402, E501, E502, E301-DOTA or E303-DOTA is selected from one of the following structures:

[0026]

[0027]

[0028]

[0029]

[0030] The cyclic peptide molecules specifically targeting EphA2 provided by the present invention all contain two cysteines. The side chain thiol of cysteine ​​undergoes affinity substitution reaction with the active halogen Br on a specific chemical cross-linking agent, thereby being modified into a cyclic peptide. The amino acid sequences and binding affinities of E001, E002, E101, E102, E201, E202, E301, E302, E303, E304, E401, E501, E502, and E301-DOTA of the present invention are specifically shown in Table 1.

[0031] Table 1 Amino acid sequences and binding affinities of cyclic peptide molecules specifically targeting EphA2

[0032]

[0033]

[0034] Note: K D Values ​​are expressed as mean or mean ± standard deviation.

[0035] The present invention further provides a method for obtaining a cyclic peptide molecule specifically targeting EphA2: the cyclic peptide molecule can be obtained based on phage display technology.

[0036] Specifically, multiple rounds of in vitro screening were carried out using a phage cyclic peptide library targeting the extracellular region of the human EphA2 protein. The phage titer after each round of screening was measured mainly by infection counting to track the screening process. The specifically bound phages enriched in each round of screening were sequenced using the Illumina second-generation sequencing platform, and the second-generation sequencing data were processed using Matlab scripts, including sample sorting, sequence translation, cluster analysis, etc. The sequence and abundance of the target EphA2 specific binding peptide were separated based on the data processing results.

[0037] The present invention further provides a method for modifying and purifying the cyclic peptide molecule that specifically targets EphA2.

[0038] The operation is simple, the cost is low, and the practicability is strong, and it mainly relies on a high performance liquid chromatograph and a liquid chromatography-mass spectrometer. In one embodiment of the invention, the modification and purification method of the cyclic peptide molecule that specifically targets EphA2 is as follows:

[0039] The specific modification reaction system is 50% ACN / NH4HCO3 (100mM, pH 8.0), 0.5mM polypeptide and 1mM corresponding chemical cross-linking agent for 1h at 30°C. The pure cyclic peptide molecules were purified by preparative liquid phase in a water (containing 0.1% TFA) and ACN (containing 0.1% TFA) mobile phase system, and the molecular weight was verified by mass spectrometry and then freeze-dried.

[0040] The present invention uses surface plasmon resonance technology to characterize the binding affinity of 14 linear peptides or cyclic peptide molecules to the target EphA2. According to the measurement, the affinity is between nanomolar and micromolar, which provides a broad prospect for the research and development of inhibitors targeting EphA2.

[0041] The present invention further provides a radioactive metal nuclide 68 Ga, 177 Lu-labeled EphA2-targeting cyclic peptide-radionuclide conjugate 68 Ga / 177 Lu-DOTA-Cyclic E301 and 68 Ga-DOTA-Cyclic E303 lays a foundation for the preparation of EphA2-targeted tumor screening, diagnosis, tracing or prognosis assessment reagents or therapeutic drugs for diseases or conditions related to EphA2 expression.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] The present invention provides a cyclic peptide molecule that specifically targets EphA2 and its use in the preparation of tumor diagnostic reagents or tumor therapeutic drugs. Eph receptor tyrosine kinase A2 (EphA2) has been proven to be a potential tumor therapeutic target. The present invention uses phage display technology to carry out multiple rounds of in vitro screening for human EphA2 protein, and obtains a single cyclic peptide molecule with drug-making potential and high affinity to EphA2 through sequence analysis, chemical synthesis and combined characterization. In addition, the present invention also includes two cyclic peptide-coupled radionuclide drugs, which show great application potential in targeted imaging and treatment of tumors characterized by EphA2 overexpression, providing a new option for the treatment of EphA2-related tumors.

[0044] (1) Compared with traditional antibodies or small molecule inhibitors, the cyclic peptide molecules of the present invention have the advantages of high affinity, strong targeting, small size, easy synthesis and modification, low cost, simple operation, and flexible administration route.

[0045] (2) The cyclic peptide molecule of the present invention is reported for the first time at home and abroad and has a high degree of innovation.

[0046] (3) The 16 linear peptides or cyclic peptides specifically targeting EphA2 provided by the present invention can be used to prepare tumor targeting screening, diagnosis, tracing, prognosis assessment reagents or therapeutic drugs, and have great potential clinical application prospects.

[0047] (4) The two cyclic peptide-radionuclide conjugates provided by the present invention have shown good therapeutic effects in in vivo and in vitro preclinical studies of EphA2-overexpressing tumors and have the potential for further research. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments so that those skilled in the art can better understand the present invention and implement it.

[0049] Figure 1 :The enrichment degree of EphA2-specific targeting peptides during the screening process was determined based on the phage titer. The phage titer after cyclization was used as the input, and the phage titer on the magnetic beads after strict panning was used as the output. Capture yield represents the output / input ratio. The enrichment degree of phage was determined by comparing the capture yield value.

[0050] Figure 2 :The results of characterizing the binding affinity of 10 cyclic peptide molecules to the target EphA2 using surface plasmon resonance technology.

[0051] Figure 3: The results of characterizing the binding affinity of four cyclic peptide molecules to the target EphA2 using surface plasmon resonance technology.

[0052] Figure 4 :Cyclic peptide-radionuclide conjugate 68 Ga-DOTA-Cyclic E301 and 68 PET / CT images of Ga-DOTA-Cyclic E303 tumor-bearing mice.

[0053] Figure 5 : 177 The therapeutic results of Lu-DOTA-Cyclic E301 on B16F10 subcutaneous xenograft tumors. DETAILED DESCRIPTION

[0054] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0055] The main materials, reagents and formulations in the following embodiments of the present invention are as follows:

[0056] Table 2 Main experimental materials and reagents in the examples

[0057]

[0058]

[0059] The main reagent formulas of the following embodiments of the present invention are as follows:

[0060] 1. 10mg / mL tetracycline (10mL): Weigh 100mg of tetracycline solid and dissolve it in 50% ethanol to make up to 10mL. Filter and sterilize with a 0.22μm filter head, then dispense into 1.5mL centrifuge tubes and store at -20℃ for a long time.

[0061] 2. 2YT medium (1L): 16g / L peptone, 5g / L NaCl, 10g / L yeast extract. Add 15g / L agar to 2YT solid medium. Autoclave at 121℃ for 20min.

[0062] 3. Buffer N (1 L): Weigh 200 g polyethylene glycol 6000 and 146.1 g NaCl, dilute to 1 L with distilled water, and store at 4 °C for a long time.

[0063] 4. Buffer R (1L): Weigh 1.58g NH4HCO3 and 1.86g EDTA, dissolve them, adjust the pH to 8.0, and make up to 1L with distilled water.

[0064] 5. 20 mM TCEP (5 mL): Weigh 28.7 mg TCEP, dilute to 5 mL with distilled water, and store at -20°C for a long time.

[0065] 6. 10 mM chemical cross-linking agent as shown in Table 3.

[0066] Table 3 Formula of chemical crosslinking agent in the embodiment of the present invention

[0067]

[0068]

[0069] 7. Wash Buffer 1 (1 L): Weigh 1.21 g Tris-Cl, 8.77 g NaCl, 2.03 g MgCl2 and 0.11 g CaCl2, dissolve them, adjust the pH to 7.4, and make up to 1 L with distilled water.

[0070] 8. Wash Buffer 2 (10 mL): Take 10 mL of Wash Buffer 1, add 100 mg of BSA and 10 μL of Tween 20, and mix well.

[0071] 9. Wash Buffer 4 (30 mL): Take 30 mL of Wash Buffer 1, add 30 μL of Tween 20, and mix well.

[0072] 10. 0.25M CH3COONa (50mL): Weigh 1.025g CH3COONa, dissolve it, and make up to 50mL with deionized water.

[0073] 11. 0.05M HCl (50mL): Take 264μL of hydrochloric acid, add to deionized water to make up to 50mL, and mix well.

[0074] 12. 60% ethanol solution: Measure 30 mL of anhydrous ethanol and make up to 50 mL with deionized water.

[0075] 13. 70% ethanol solution: Measure 35 mL of anhydrous ethanol and make up to 50 mL with deionized water.

[0076] The main primers in the following examples of the present invention are as follows:

[0077] Table 4 Primer list for preparing NGS library in the embodiment of the present invention

[0078]

[0079]

[0080] Example 1

[0081] Screening and identification of EphA2 extracellular domain-targeting random cyclic peptide libraries using phage display:

[0082] The steps of “preparing cyclic peptide library→fixing target protein→blocking→panning→infection amplification” were followed and the screening was repeated for 3 rounds. The screening stringency was gradually increased by reducing the amount of protein used. In order to reduce nonspecific binding during the screening process, Streptavidin magnetic beads and Neutravidin magnetic beads were used alternately in each round of screening, as shown in Table 5. Among them, Neutravidin magnetic beads were prepared according to the instructions of the supplier by mixing 2 mg Neutravidin protein with 3350 μL Dynabeads TM Prepared after incubation with M-280 Tosylactivated (30 mg / mL).

[0083] Table 5 The amount of target protein immobilized and the type of magnetic beads used in each round of phage screening

[0084] Screening rounds Target protein EphA2 fixed amount / library (8 libraries in total) Magnetic bead type Round 1 0.5 ug Streptavidin Magnetic Beads Round 2 0.25 ug Neutravidin Magnetic Beads Round 3 100 ng / 10 ng Streptavidin Magnetic Beads

[0085] The specific method is as follows:

[0086] 1. Preparation of phage linear peptide library

[0087] 1) Take out the E. coli TG1 glycerol bacteria containing the phage display random cyclic peptide library described in Table 2 from the -80°C refrigerator and thaw it at room temperature.

[0088] 2) Take 1 mL of glycerol culture and inoculate it into 250 mL of 2YT medium containing 10 μg / mL tetracycline, at 200 rpm, 30°C, for 16 h.

[0089] 3) Centrifuge the culture at 6500 rpm for 20 min at 4°C. After centrifugation, transfer the supernatant to a new 1L centrifuge cup, add 1 / 4 pre-cooled Buffer N to precipitate the phages, and place on ice for half an hour.

[0090] 4) Centrifuge at 6500 rpm for 45 min at 4°C. After centrifugation, discard the supernatant.

[0091] 5) Dissolve the phage precipitate in 40 mL of degassed Buffer R and transfer to a new 50 mL centrifuge tube. Centrifuge at 4500 rpm for 15 min at 4°C, remove the precipitate, and keep the supernatant.

[0092] 2. Reduction, modification and blocking of phage linear peptide library

[0093] 1) Reduction: Add 2.1 mL of 20 mM reducing agent TCEP to the supernatant of step 1-5) to make the final concentration of TCEP 1.05 mM. Mix well and incubate at room temperature (20-22°C) for 30 min. Add 1 / 4 pre-cooled Buffer N to the mixed system and cool on ice for 10 min. Centrifuge at 4500 rpm for 15 min at 4°C, remove the supernatant and centrifuge again for 2 min to retain the phage precipitate. Dissolve the phage precipitate with 37 mL (4.5 mL × number of chemical cross-linkers) of Buffer R for modification.

[0094] 2) Modification: Divide the reduced phage solution into 8 portions of 4.5 mL each, and mix each portion of phage solution with 0.5 mL of 7 different chemical crosslinkers and 1 control solution (ACN) to make the final concentration of the chemical crosslinker 20 μM. Incubate at 30°C for 1 hour. After modification, obtain phage precipitation according to the operation of 2-1).

[0095] 3) Blocking: Dissolve the 8 chemical crosslinker-modified phage precipitates with 5 mL Wash Buffer 2. Take 50 μL of the precipitates for infection counting. Place the remaining phage solution on a rotating culture mixer and incubate at room temperature at 10 rpm for 30 minutes.

[0096] 3. Solidification and blocking of target protein

[0097] Using a magnetic stand, wash 100 μL of Dynabeads with 1 mL of Wash Buffer 1. TM Wash the M-280 Streptavidin beads twice. Then, resuspend the beads with 100 μL Wash Buffer 1, mix with 4 μg of target protein EphA2, and incubate at room temperature for 10 min at 10 rpm. Wash the beads twice with 1 mL Wash Buffer 1. Finally, resuspend the beads with 410 μL Wash Buffer 2, and incubate at room temperature for 30 min at 10 rpm.

[0098] 4. Phage screening and panning

[0099] 1) Divide the magnetic beads after the rotation incubation in step 3 into 8 portions of 50 μL, mix them with the 8 phage cyclic peptide libraries in steps 2-3) respectively, and rotate and incubate at room temperature for 1 hour at 10 rpm. Remove the supernatant and retain the magnetic beads.

[0100] 2) Wash the magnetic beads 8 times with 200 μL Wash Buffer 4 and then wash twice with 200 μL Wash Buffer 1.

[0101] 5. Phage infection counting and amplification

[0102] 1) The strictly washed magnetic beads described in step 4-2) were mixed with 5 mL of Escherichia coli TG1 bacterial solution in the logarithmic growth phase in a clean bench and cultured at 37° C. for 30 minutes.

[0103] 2) Take out the bacterial solution, centrifuge at 500 rpm for 15 minutes, remove the supernatant, resuspend the cell pellet with 0.5 mL of fresh 2YT medium, and take 10 μL in each tube for screening and enrichment determination.

[0104] 3) The remaining cell culture was spread on 2YT solid medium containing 10 μg / mL tetracycline and cultured overnight at 37°C for 16 h for the next round of screening.

[0105] 4) Take out the phage samples described in step 2-3), take 10 μL of each and add them to 90 μL 2YT medium, dilute them 7 times in sequence, then take 10 μL from each dilution concentration well and add it to 90 μL in the logarithmic growth phase (OD 600 =0.4-0.8) of Escherichia coli TG1, and cultured in a 37°C incubator for 30 min. After infection, 5 μL of each dilution concentration well was spotted on 2YT solid medium containing 10 μg / mL tetracycline, and cultured at 37°C overnight for phage titer counting.

[0106] 5) Take out the phage sample described in step 5-2), take 10 μL of each and add it to 90 μL of 2YT medium, and dilute it 7 times in sequence. Take 5 μL of bacterial solution and spot it on 2YT solid medium containing 10 μg / mL tetracycline, and culture it at 37°C overnight.

[0107] 6) The next day, take out the solid culture medium described in step 5-3), wash off the phage library on the solid culture medium with 4 mL of 2YT culture medium containing 10% glycerol, and store at -20°C for the next round of screening. Take out the solid culture medium used for infection counting and count the number of colonies. The phage titer after circularization is used as the input amount, and the phage titer on the magnetic beads after strict washing is used as the output amount. Capture yield represents the output / input ratio. The enrichment degree of phage is determined by comparing the capture yield value ( Figure 1 ).

[0108] 6. Next-generation sequencing and data processing and analysis

[0109] 1) First step PCR

[0110] Design multiple primers containing different barcodes and sequences complementary to the second-step PCR primers (as listed in Table 4) for the first-step PCR. Correspond different primer pairs to the samples enriched in the first to third rounds of screening (as shown in Table 6) so that the source of the samples can be distinguished according to the barcode when the sequencing data is subsequently processed. Use 2×PCR NG Master Mix (Taq enzyme Mix) for PCR reaction. The concentration of the primer master solution is 10μM, which is first diluted to 1μM. When performing PCR, first add 2×PCR NG Master Mix, water and primers, and finally add the template. The whole process should be performed on ice.

[0111] Table 6 Comparison table of samples enriched in each round of screening and primers in the first round

[0112]

[0113] The PCR reaction system was Template (0.5μL-1μL (glycerol stock.OD around 30)), 2×PCRNG Master Mix (25μL), Forward / Reverse Primer (5μL×1μM), and MQ (14.5μL).

[0114] The PCR program was 95°C × 5 min, (95°C × 30 s, 55°C × 30 s, 72°C × 30 s) × 25, 72°C × 10 min. The PCR product was subjected to DNA electrophoresis in 2.5% agarose gel to confirm the band size.

[0115] 2) Second step PCR

[0116] The product of the first PCR was used as a template for the second PCR. 2×PCR NG Master Mix (Taq enzyme Mix) was used for PCR reaction.

[0117] The PCR reaction system was Template (2μL), 2×PCR NG Master Mix (25μL), Forward / Reverse Primer (1μL×5μM), and MQ (21μL).

[0118] The PCR program was 95°C×5min, (95°C×30s, 55°C×30s, 72°C×30s)×25, 72°C×10min.

[0119] The second step PCR products were mixed and purified using a commercial agarose gel purification kit. Finally, the DNA concentration was measured using a high-sensitivity DNA detection kit and the final concentration was determined using a Qubit instrument, which was then used for next-generation sequencing.

[0120] 3) The second-generation sequencing was completed by GENEWISE Biotechnology Co., Ltd. on the Illumina Novaseq 2×150bp sequencing platform. The sequencing process mainly includes the construction of the sequencing library, bridge PCR amplification and denaturation, and sequencing. After the sequencing is completed, about 10 million sequencing result data with a length of about 240bp are returned for the next step of data analysis.

[0121] 4) Data analysis

[0122] NGS data processing was performed according to the Matlab script described in the paper (Doi: 10.1021 / acschembio.4c00099). The specific method is as follows: including sample sorting, sequence translation, cluster analysis and other processing to determine promising candidate peptide sequences. First, read the second-generation sequencing Fastq data file and divide the data results into different files according to the different barcodes introduced by PCR. Secondly, remove low-quality sequences containing more than 3 Q scores ≤ 18 from the data file according to the set quality standards. The nucleotide sequences starting with ATGGC and ending with GGTTCT (encoded as Met-Ala and Gly-Ser, respectively) were extracted and translated into amino acid sequences. Peptide sequences shorter than 5 amino acids and longer than 14 amino acids were excluded. The remaining sequences were sorted according to abundance. At this point, two steps can be performed: 1 is to use the "looplength.m" script to count the number of cysteines contained in the peptide sequence, and 2 is to use "CommonSeq.m" to compare the same sequences in two different data sets. Next, clustering analysis can be performed using "clustering.m" to compare the abundance and conservation of the top 200 to 1000 peptide sequences. Finally, sequences with a certain conserved motif but low abundance are searched in all data files to identify promising candidate peptide sequences.

[0123] 7. Modification and purification of cyclic peptide molecules

[0124] Order the linear polypeptide sequence described in claim 1 from a polypeptide synthesis company. Use 500 μL of 50% ACN / 50% Water (containing 0.1% TFA) to completely dissolve each 0.5 mg polypeptide. The modified reaction system is 50% ACN / NH4HCO3 (100 mM, pH 8.0), 0.5 mM polypeptide and 1 mM corresponding chemical cross-linking agent for reaction at 30°C for 1 hour. Use Shimadzu UPLC-MS (the chromatographic column is Shim-pack GIST C18 (2.1×50 mm, 2 μm)) to verify the modification of the polypeptide. After confirming that the modification is complete, add acetic acid to quench the reaction. Then, the Shimadzu preparative liquid phase (Hypersil TMThe pure cyclic peptide molecule was purified by PREP HS C18 (10×250mm, 5μm) in a water (containing 0.1% TFA) and ACN (containing 0.1% TFA) mobile phase system, and the purity of the cyclic peptide molecule was verified by Agilent HPLC (chromatographic column: Poroshell 120EC-C18 (4.6×150mm, 4μm)).

[0125] 8. Characterization of the binding between EphA2 and candidate peptide molecules

[0126] In this example, surface plasmon resonance was used to characterize the binding of the target protein EphA2 with 14 candidate polypeptide molecules.

[0127] 1) Immobilization of target protein EphA2

[0128] A streptavidin chip was used to immobilize the biotinylated target protein EphA2. 10 μg / mL of the target protein was captured on the second immobilization channel of the streptavidin chip at a flow rate of 5 μL / min, and the first reference channel was not processed.

[0129] 2) Preparation of cyclic peptide molecule samples

[0130] The candidate peptide was diluted 5 times in a 2-fold or 3-fold serial dilution using running buffer (PBS containing 0.05% Tween 20 and 5% DMSO).

[0131] 3) Specific methods of combined determination

[0132] Binding tests were performed with candidate peptide molecules using a streptavidin chip immobilized with EphA2. Single-cycle or multi-cycle kinetics were used, with a flow rate of 30 μL / min for binding for 120 s and dissociation for 120 s, and a solvent correction was performed after the binding test. After obtaining the surface plasmon resonance data sensorgram, it was opened with Biacore Evaluation Software, and after deducting the reference channel, the data was fitted with a 1:1 binding model to obtain the K D value, association rate constant (Ka), dissociation rate constant (Kd) and chi-square value (Chi 2 ) values ​​and other combined parameters.

[0133] The surface plasmon resonance technique was used to measure that the 14 cyclic peptide molecules specifically targeting EphA2 selected in the present invention all showed good affinity with EphA2. The test results are as follows: Figure 2 The specific affinity values ​​are shown in Table 1.

[0134] Example 2

[0135] Cyclic peptide-radionuclide conjugates for tumor imaging or therapy:

[0136] 1. 68 Ga-DOTA-Cyclic E301 or 68 Tumor imaging with Ga-DOTA-Cyclic E303

[0137] 1) 68 Ga-DOTA-Cyclic E301 or 68 The preparation method of Ga-DOTA-Cyclic E303 is as follows:

[0138] Dissolve the purified polypeptide in sterile deionized water to 1 mg / mL, take 0.015 mL of polypeptide solution and add appropriate amount of 0.05 M HCl to elute 68 GaCl3 solution (3-4mCi) was added with an appropriate amount of 0.25M CH3COONa to adjust the solution pH to 4.0, and heated at 100°C for 30 minutes. The Sep-Pack C18 column was activated with 70% ethanol and rinsed with saline, and the heated liquid was purified by solid phase extraction (Sep-Pack C18 column), and the radionuclide-labeled peptide product was eluted from the Sep-Pack C18 column with 60% ethanol. 68 Ga-DOTA-Cyclic E301 or 68 Ga-DOTA-Cyclic E303.

[0139] 2) Select B16F10 cells with positive expression of EphA2 to establish a right upper limb tumor-bearing mouse model. The above solution was diluted and injected into the tumor-bearing mice via the tail vein (100 μCi / 0.1-0.2 mL, n=3). MicroPET / CT imaging was performed 1, 2, and 4 hours after injection. Figure 4 As shown, the uptake value of tumor tissue (%ID / g) was calculated, and the results are as follows:

[0140] 1h 2h 4h <![CDATA[ 68 Ga-DOTA-Cyclic E301]]> 4.63 3.93 3.57 <![CDATA[ 68 Ga-DOTA-Cyclic E303]]> 2.13 1.43 0.90

[0141] Micro PET / CT imaging results showed that compared with 68 Ga-DOTA-Cyclic E303 (2.13% ID / g), injected 68 Ga-DOTA-Cyclic E301 showed higher tumor uptake (4.63% ID / g) after 1 hour. And over time, at 2 and 4 hours, 68 Ga-DOTA-Cyclic E301 still maintains a high tumor uptake, and tumor imaging becomes clearer.

[0142] 2. 177Tumor Treatment with Lu-DOTA-Cyclic E301

[0143] 1) 177 Preparation of Lu-DOTA-Cyclic E301

[0144] Take 0.015mL of peptide solution and add appropriate amount of 177 LuCl3 solution (8-10mCi), then add appropriate amount of 0.25MCH3COONa and 0.05M HCl to adjust the solution to pH 4.0, and heat at 100℃ for 30 minutes. After activating the Sep-Pack C18 column with 70% ethanol and washing with saline, the heated liquid was purified by solid phase extraction (Sep-Pack C18 column) column, and eluted from the Sep-Pack C18 column with 60% ethanol to obtain 177 Lu-DOTA-Cyclic E301.

[0145] 2) Select B16F10 cells with positive expression of EphA2 to establish a right upper limb tumor-bearing mouse model. After diluting the above solution, inject it into the body of 3 groups of tumor-bearing mice through the tail vein (100μCi, 200μCi, 400μCi / 0.1-0.2mL, n=5), and set up a PBS group as a control group. Record the weight and tumor volume of the animals every two days to monitor whether the euthanasia endpoint is reached (weight loss exceeds 20% or tumor volume exceeds 2000mm 3 ). Treatment results such as Figure 5 shown.

[0146] go through 177 After treatment with Lu-DOTA-Cyclic E301, the growth of tumors in B16F10 tumor-bearing mice was significantly inhibited. Compared with the PBS control group, the tumor volume of the treated tumor-bearing mice was significantly reduced. In addition, the inhibitory effect on tumors was significantly dose-dependent, that is, with the increase of 177 As the dose of Lu-DOTA-Cyclic E301 increased, the degree of tumor reduction gradually increased.

[0147] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A linear peptide or cyclic peptide molecule specifically targeting EphA2, characterized in that: Selected from one of the molecules E001, E002, E101, E102, E201, E202, E301, E302, E303, E304, E401, E402, E501, E502, E301-DOTA or E303-DOTA, the amino acid sequence of E001, E002, E101, E102, E201, E202, E301, E302, E303, E304, E401, E402, E501, E502, E301-DOTA or E303-DOTA is as follows: (1)E001:Phe-Arg-Phe-Glu-Ala-Tyr-Pro-Asp-Ala-Gln-Arg-Leu; (2)E002:Ser-Val-Met-Tyr-Arg-Ser-Leu-Pro-Leu-Glu-Phe-Phe-Phe-Thr; (3)E101:Phe-Cys-Ala-Tyr-Pro-Cys-Asn-His; (4)E102:Lys-Arg-Trp-Cys-Ala-Phe-Pro-Cys-Leu; (5)E201:Cys-Pro-Trp-Asp-Phe-Cys-Trp; (6)E202:Ile-Thr-Leu-Thr-Cys-Pro-Cys-Thr-Leu-Gly-Ser-Trp; (7)E301 / E501:Phe-Leu-Ala-Cys-Pro-Glu-Thr-His-Cys-Thr; (8)E302:Phe-Gln-Ala-Cys-Pro-Met-Asn-Cys-Arg-Gly-Ser-Trp; (9)E303 / E502:Phe-Arg-Ala-Cys-Pro-Phe-Asp-Cys-Thr-Gly-Ser-Trp; (10)E304:Gln-Phe-Leu-Ala-Cys-Pro-Met-Thr-Ser-Cys-Gly-Ser-Trp; (11)E401:Leu-Arg-Phe-Cys-Asp-Tyr-Ile-Gly-Cys-Thr; (12)E402:Ser-Phe-Cys-Ala-Trp-Ile-Gly-Cys-Thr; (13)E301-DOTA: Phe-Leu-Ala-Cys-Pro-Glu-Thr-His-Cys-Thr-Gly-Ser-Trp-Lys(DOTA); (14)E303-DOTA: Phe-Arg-Ala-Cys-Pro-Phe-Asp-Cys-Thr-Gly-Ser-Tyr-His-Lys(DOTA).

2. The linear peptide or cyclic peptide molecule specifically targeting EphA2 according to claim 1, characterized in that: The molecules E001, E002, E101, E102, E201, E202, E301, E302, E303, E304, E401, E402, E501, E502, E301-DOTA or E303-DOTA are selected from one of the following structures:

3. A polymer, characterized in that The polymer includes at least two of the linear peptide or cyclic peptide molecules that specifically target EphA2 selected from claim 1 or 2, and the linear peptide or cyclic peptide molecules that specifically target EphA2 are selected from molecules E001, E002, E101, E102, E201, E202, E301, E302, E303, E304, E401, E402, E501, E502, E301-DOTA or E303-DOTA.

4. An isolated nucleic acid, characterized in that Encoding the linear peptide or cyclic peptide molecule specifically targeting EphA2 as claimed in claim 1 or 2.

5. A carrier, characterized in that The vector comprises the multimer of claim 3 or the nucleic acid of claim 4.

6. An engineered immune effector cell, characterized in that: It comprises the linear peptide or cyclic peptide molecule specifically targeting EphA2 according to claim 1 or 2, the multimer according to claim 3, the isolated nucleic acid according to claim 4, or the vector according to claim 5.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the linear peptide or cyclic peptide molecule specifically targeting EphA2 according to claim 1 or 2, or the engineered immune effector cell according to claim 6, and one or more pharmaceutically acceptable excipients and / or carriers.

8. The pharmaceutical composition according to claim 7, characterized in that Radioactive metal nuclides 68 Ga, 177 Lu-labeled EphA2-targeting cyclic peptide-radionuclide conjugate 68 Ga / 177 Lu-DOTA-Cyclic E301 and 68 Ga-DOTA-Cyclic E303.

9. Use of the linear peptide or cyclic peptide molecule specifically targeting EphA2 according to claim 1 or 2, the multimer according to claim 3, the engineered immune effector cell according to claim 6 or the pharmaceutical composition according to claim 7 in the preparation of a tumor targeted screening, diagnosis, tracing or prognosis assessment reagent or a drug for treating a disease or condition associated with EphA2 expression.

10. The use according to claim 8, characterized in that ， The linear peptide or cyclic peptide molecule specifically targeting EphA2 according to claim 1 or 2, and the use of the polymer according to claim 3 in the preparation of tumor diagnostic imaging agents or tumor radionuclide therapy; preferably, the use in the preparation of radionuclide imaging agents or therapeutic agents, wherein the radionuclide is selected from 68 Ga, 177 Lu; The diseases or conditions associated with EphA2 expression include liver cancer, lung cancer, colorectal cancer, glioma cancer, pancreatic cancer, prostate cancer, ovarian cancer, uterine cancer, gastric cancer, melanoma or breast cancer.

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