Cyclic peptide molecules specifically targeting cd5 and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a cyclic peptide molecule specifically targeting CD5 and purposes thereof. The surface receptor CD5 of immune cells (T cells, B cells) has been proved to be a potential tumor treatment target, the present application uses the phage display technology widely applied to the development of polypeptide or antibody drugs to carry out multiple rounds of in vitro screening on human CD5 protein, and obtains a cyclic peptide molecule with drug potential and capable of specifically combining with CD5 through second-generation sequencing. The cyclic peptide molecule contains two cysteines, is modified into a ring through a specific chemical crosslinking agent, and the affinity of the cyclic peptide molecule to CD5 is determined to be in the low micromolar level through surface plasmon resonance technology, thereby laying a foundation for the development of a CD5-targeting inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to cyclic peptide molecules that specifically target CD5 and their uses. Background Technology

[0002] Cancer is a widespread, drug-resistant, and adaptable disease that affects people of all ages, genders, and social statuses, posing one of the ultimate challenges to modern medicine. Many cancer patients miss the optimal window for surgery due to a lack of effective methods for early diagnosis of malignant tumors, contributing to persistently high cancer mortality rates. Therefore, the search for novel and effective diagnostic methods and treatment strategies for the early diagnosis, prevention, and personalized treatment of malignant tumors is urgently needed.

[0003] With the deepening research by scholars both domestically and internationally on the relationship between inflammation and the tumor microenvironment, tumor immunotherapy has made significant progress in cancer treatment. Tumor immunotherapy refers to the reactivation of anti-tumor immune responses by regulating the body's own immune system, achieving the effect of specifically killing tumor cells while preserving normal cellular function. Currently, immune checkpoint inhibitors are one of the main strategies in tumor immunotherapy. Compared with antibodies or small molecule inhibitors, peptide inhibitors have the following advantages: 1) strong targeting specificity and high binding affinity; 2) small size, easy production and characterization, and easy introduction of structural modifications; 3) low immunogenicity; 4) good tissue penetration; 5) flexible administration routes, etc. Peptide inhibitors are gradually becoming irreplaceable in the treatment of various diseases and are an ideal and attractive target in the field of international innovative drug research.

[0004] CD5 is a type I transmembrane glycoprotein with a molecular weight of 67 kDa. Its extracellular domain consists of three cysteine-rich domains (D1, D2, D3). Since its discovery, CD5 has been considered a highly specific marker for T cells and B cells. It is expressed in all mature T cells and a small number of B-1a cells, and is overexpressed in regulatory T cells and regulatory B cells. CD5 is a positive and negative regulator of TCR signaling, negatively regulating different signaling pathways mediated by BCR, and has multiple functional activities in the immune system.

[0005] As early as the 1990s, the efficacy of anti-CD5 monoclonal antibodies in patients with chronic lymphocytic leukemia or cutaneous T-cell lymphoma was established. With the continuous development of tumor immunotherapy, antibodies targeting CD5 have shown biological effects in a wider range of malignant tumors, revealing CD5 as an attractive target for tumor immunotherapy. Developing inhibitors targeting CD5 has great application prospects and can be used to enhance T-cell anti-tumor immune responses or regulate autoimmune responses.

[0006] Patent CN117659198A discloses a single-domain antibody targeting CD5, a chimeric antigen receptor, and their applications. The single-domain antibody targeting CD5 comprises CDR1, CDR2, and CDR3, with the specific amino acid sequences being: GYTYSNHC (CDRI), IDSDGST (CDR2), and AAEFGVDCSDYVPFAAGMDY (CDR3). Prior art document 1 also provides the use of the single-domain antibody targeting CD5, the chimeric antigen receptor, and engineered immune effector cells containing this CAR in the preparation of medicaments for the diagnosis, prevention, and / or treatment of diseases or conditions related to CD5 expression.

[0007] To date, there are not many peptide drugs targeting CD5 that have been marketed. Therefore, there is an urgent need to develop cyclic peptide molecules with high affinity, specific targeting of CD5, and more diverse properties to provide more possibilities for targeted therapy of CD5. Summary of the Invention

[0008] This invention provides cyclic peptide molecules that specifically target CD5 and their applications, supplementing new cyclic peptide molecules with high affinity, specific targeting of CD5, and more diverse properties, thus providing more possibilities for targeted therapy of CD5.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] This invention first provides a cyclic peptide molecule that specifically targets CD5, selected from one of the following amino acid sequences:

[0011] (1) Thr-Glu-Cys-Typ-Thr-Thr-Met-Arg-Glu-Cys (as shown in SEQ ID NO.1);

[0012] (2)Met-Cys-Asp-Arg-Leu-Tyr-Typ-Cys-Asp (as shown in SEQ ID NO.2).

[0013] In one embodiment of the present invention, the CD5-specific cyclic peptide molecule is selected from one of the molecules C101, C202, C301, C501, C601, C701, and C201:

[0014]

[0015]

[0016] The CD5-targeting cyclic peptide molecules provided by this invention all contain two cysteine ​​residues. The thiol group on the side chain of the cysteine ​​residues undergoes an affinity substitution reaction with the active halogen Br on a specific chemical cross-linking agent, thereby modifying it into a ring. The amino acid sequences and binding affinities of C101, C202, C301, C501, C601, C701, and C201 of this invention are specifically shown in Table 1.

[0017] Table 1. Amino acid sequences and binding affinities of cyclic peptides that specifically target CD5.

[0018]

[0019] This invention further provides a method for obtaining cyclic peptide molecules that specifically target CD5: based on phage display technology. Multiple rounds of in vitro screening are conducted using a phage cyclic peptide library targeting the extracellular region of human CD5 protein. Each round of screening follows the steps of "preparing the cyclic peptide library → immobilizing the target protein → blocking → washing → infection and amplification." During the screening process, the stringency of the phage screening conditions is increased based on the degree of phage enrichment. The phage titer after each round of screening is measured primarily by infection counting to track the screening progress. The specifically binding phages enriched in each round of screening are sequenced using the Illumina next-generation sequencing platform, and the next-generation sequencing data is processed using Matlab scripts, including sample sorting, sequence translation, and cluster analysis. Based on the data processing results, the sequence and abundance of the target CD5-specific binding peptide are separated.

[0020] The present invention further provides a method for modifying and purifying the cyclic peptide molecule that specifically targets CD5.

[0021] The method is simple to operate, low in cost, and highly practical, mainly utilizing high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). In one embodiment of the invention, the modification and purification method of the CD5-specific cyclic peptide molecule is as follows:

[0022] The specific modification reaction system was 50% ACN / NH4HCO3 (100mM, pH 8.0), with 0.5mM peptide and 1mM of the corresponding chemical cross-linking agent reacted at 30℃ for 1h. The purified cyclic peptide molecules were obtained by purification in a preparative liquid phase using a mobile phase system of Water (containing 0.1% TFA) and ACN (containing 0.1% TFA). After mass spectrometry verification of the molecular weight, the peptides were lyophilized.

[0023] This invention employs surface plasmon resonance (SPR) technology to characterize the binding affinity of seven cyclic peptide molecules to the target CD5. The results showed that all affinity was in the low micromolar range, providing a promising prospect for the development of CD5-targeting inhibitors.

[0024] The present invention further provides an isolated nucleic acid comprising a nucleic acid sequence encoding a cyclic peptide molecule that specifically targets CD5.

[0025] The present invention further provides a carrier comprising the aforementioned nucleic acid.

[0026] The present invention further provides an engineered immune effector cell comprising the CD5-specific cyclic peptide molecule, the isolated nucleic acid, or the vector.

[0027] The present invention further provides a pharmaceutical composition comprising the CD5-specific cyclic peptide molecule, or the engineered immune effector cells, and one or more pharmaceutically acceptable excipients and / or carriers.

[0028] The present invention further provides the use of the CD5-specific cyclic peptide molecule in the preparation of tumor-targeted screening, diagnostic, tracing or prognostic assessment reagents or medicaments for treating diseases or conditions related to CD5 expression.

[0029] The present invention further provides the use of the engineered immune effector cells or the pharmaceutical composition thereof in the preparation of reagents for tumor-targeted screening, diagnosis, tracing or prognostic assessment or medicaments for treating diseases or conditions related to CD5 expression.

[0030] In one embodiment of the present invention, the disease or condition associated with CD5 expression is cancer.

[0031] In one embodiment of the invention, the diseases or conditions associated with CD5 expression include T-cell leukemia, T-cell lymphoma, or B-cell lymphoma.

[0032] In one embodiment of the present invention, the diseases or conditions associated with CD5 expression include acute T-lymphoblastic leukemia, acute T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, or diffuse large B-cell lymphoma.

[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0034] This invention provides cyclic peptide molecules that specifically target CD5 and their uses. CD5, the receptor on the surface of immune cells (T cells, B cells), has been shown to be a potential target for tumor therapy. This invention utilizes phage display technology, widely used in peptide or antibody drug development, to conduct multiple rounds of in vitro screening of human CD5 protein. Next-generation sequencing yielded cyclic peptide molecules with drug potential that specifically bind to CD5. Each cyclic peptide molecule contains two cysteine ​​residues and is modified into a ring using a specific chemical cross-linking agent. Surface plasmon resonance (SPR) technology determined that their affinity for CD5 is in the low micromolar range, laying the foundation for the development of CD5-targeting inhibitors.

[0035] (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.

[0036] (2) The cyclic peptide molecule of the present invention is reported for the first time at home and abroad, and has high innovation.

[0037] (3) The seven CD5-specific cyclic peptides provided by this invention can be used to prepare reagents or therapeutic drugs for tumor-targeted screening, diagnosis, tracing, prognosis assessment, and have great potential clinical application prospects. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, so that those skilled in the art can better understand the present invention and implement it.

[0039] Figure 1 The enrichment level of CD5-specific targeting peptides during the screening process was determined based on phage titers. The phage titer after cyclization was used as the input, and the phage titer on the rigorously washed magnetic beads was used as the output. Capture yield represents the output / input ratio, and the enrichment level of phages was determined by comparing capture yield values.

[0040] Figure 2 The results show the results of characterizing the binding affinity of seven cyclic peptide molecules to the target CD5 using surface plasmon resonance (SPR) technology. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0042] The main materials, reagents, and formulations in the following embodiments of the present invention are as follows:

[0043] Table 2 shows the main experimental materials and reagents used in the examples.

[0044]

[0045]

[0046] The main reagent formulations of the following embodiments of the present invention are as follows:

[0047] 1. 10 mg / mL tetracycline (10 mL): Weigh 100 mg of tetracycline solid, dissolve it in 50% ethanol, and bring the volume to 10 mL. After sterilization by filtration through a 0.22 μm filter, dispense into 1.5 mL centrifuge tubes and store at -20°C for long-term storage.

[0048] 2. 2YT medium (1L): peptone 16g / L, NaCl 5g / L, yeast extract 10g / L. Add 15g / L agar to the 2YT solid medium. Autoclave at 121℃ for 20min.

[0049] 3. Buffer N (1L): Weigh 200g of polyethylene glycol 6000 and 146.1g of NaCl, dilute to 1L with distilled water, and store at 4℃ for a long time.

[0050] 4. Buffer R (1L): Weigh 1.58g NH4HCO3 and 1.86g EDTA, dissolve them, adjust the pH to 8.0, and bring the volume to 1L with distilled water.

[0051] 5. 20mM TCEP (5mL): Weigh 28.7mg TCEP, dilute to 5mL with distilled water, and store at -20℃ for a long time.

[0052] 6. 10mM chemical crosslinking agents are shown in Table 3.

[0053] Table 3 Formulations of chemical crosslinking agents in the embodiments of the present invention

[0054] name Concentration (mM) Mass (mg) Volume (mL) 1,4-Di(bromomethyl)benzene 10 3.3 1.21 (in ACN) 1,3-Di(bromomethyl)benzene 10 2.3 0.85 (in ACN) 2,6-Di(bromomethyl)pyridine 10 2.8 1.04 (in ACN) α,α'-dibromo-o-xylene 10 3.4 1.25 (in ACN) trans-1,4-dibromo-2-butene 10 2.3 1.06 (in ACN) 2,6-Bis(bromomethyl)naphthalene 10 2.7 0.82 (in DMSO) 1,3-Dibromoacetone 10 2.5 1.13 (in ACN)

[0055] 7. Wash Buffer 1 (1L): Weigh 1.21g Tris-Cl, 8.77g NaCl, 2.03g MgCl2 and 0.11g CaCl2, dissolve them and adjust the pH to 7.4, then bring the volume to 1L with distilled water.

[0056] 8. Wash Buffer 2 (10 mL): Measure 10 mL of Wash Buffer 1, add 100 mg of BSA and 10 μL of Wein20, and mix well.

[0057] 9. Wash Buffer 4 (30mL): Measure 30mL of Wash Buffer 1, add 30μL of Tween 20, and mix well.

[0058] The main primers used in the following embodiments of the present invention are as follows:

[0059] Table 4. Primer list for preparing NGS libraries in the embodiments of the present invention.

[0060]

[0061]

[0062] Example 1

[0063] Screening and identification of random cyclic peptide libraries targeting the CD5 extracellular domain using phage display:

[0064] The screening process followed the steps of "preparing a cyclic peptide library → immobilizing the target protein → blocking → washing → infection and amplification," and was repeated three times. The screening rigor was gradually increased by reducing the amount of protein used. To reduce non-specific binding during the screening process, Streptavidin and Neutravidin magnetic beads were used alternately in each round, as shown in Table 5. Specifically, the Neutravidin magnetic beads were prepared according to the supplier's instructions, with 2 mg of Neutravidin protein mixed with 3350 μL of Dynabeads. TM Prepared after incubation with M-280 Tosylactivated (30 mg / mL).

[0065] Table 5. Target protein fixation amount and magnetic bead type used in each round of phage screening.

[0066] Number of filtering rounds Target protein CD5 immobilization level / library (8 libraries in total) Magnetic bead type Round 1 0.5μg Streptavidin magnetic beads Round 2 0.25μg Neutravidin magnetic beads Round 3 0.1μg Streptavidin magnetic beads

[0067] The specific method is as follows:

[0068] 1. Preparation of a linear phage peptide library

[0069] 1) Remove the E. coli TG1 glycerol tube containing the phage display random cyclic peptide library described in Table 2 from the -80℃ freezer and allow it to thaw at room temperature.

[0070] 2) Take 1 mL of phage display random cyclic peptide library and inoculate it into 250 mL of 2YT medium containing 10 μg / mL tetracycline. Incubate at 200 rpm and 30 °C for 16 h.

[0071] 3) Centrifuge the phage library culture at 6500 rpm for 20 min at 4 °C. After centrifugation, transfer the supernatant to a new 1 L centrifuge cup, add 1 / 4 of pre-chilled Buffer N to precipitate the phage, and place on ice for half an hour.

[0072] 4) Centrifuge at 6500 rpm for 45 min at 4℃. After centrifugation, quickly and carefully discard the supernatant.

[0073] 5) Dissolve the phage pellet in 40 mL of degassed Buffer R and transfer it to a new, sterile 50 mL centrifuge tube. Centrifuge at 4500 rpm for 15 min at 4 °C to remove any residual bacterial cells, retaining the phage supernatant.

[0074] 2. Reduction, modification, and blocking of phage linear peptide libraries

[0075] 1) Reduction: Since the sulfhydryl groups of two cysteine ​​residues are easily spontaneously oxidized to form disulfide bonds, the linear peptide library needs to be reduced before cyclization of the phage mixed library to improve the modification efficiency. TCEP, a reducing agent, has the property of reducing disulfide bonds. Add 2.1 mL of 20 mM TCEP to 40 mL of phage supernatant to make the final concentration of TCEP 1.05 mM. After mixing thoroughly, incubate at room temperature (20–22 °C) for 30 min. Add 1 / 4 of pre-chilled Buffer N to the mixture 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, retaining the phage precipitate. Dissolve the phage precipitate in 37 mL (4.5 mL × number of chemical cross-linking agents) of Buffer R for modification.

[0076] 2) Modification: The reduced phage solution was divided into eight 4.5 mL portions. Each portion was mixed with 0.5 mL of one of seven different chemical cross-linking agents and one control solution (ACN), resulting in a final concentration of 20 μM for the chemical cross-linking agents. After thorough mixing, the mixtures were incubated at 30°C in a three-well electric thermostatic water bath for 1 hour. After modification, phage precipitates were obtained using the method described above.

[0077] 3) Blocking: Dissolve the phage precipitates modified with each of the eight chemical cross-linking agents in 5 mL of Wash Buffer 2. Take 50 μL of each precipitate and store it at 4 °C for infection counting. Place the remaining phage solutions on a rotary culture mixer and incubate at 10 rpm for 30 min at room temperature.

[0078] 3. Immobilization and blocking of target proteins

[0079] Take 100 μL of Dynabeads from a 4°C refrigerator. TMM-280 Streptavidin magnetic beads were used, and the supernatant was removed using a magnetic rack. Next, the beads were washed twice with 1 mL Wash Buffer 1, and the liquid was aspirated. The beads were resuspended in 100 μL Wash Buffer 1 and mixed with 4 μg of the target protein CD5. The mixture was placed on a spin mixer and incubated at 10 rpm for 10 min at room temperature. The beads were washed twice with 1 mL Wash Buffer 1 using a magnetic rack. Finally, the beads were resuspended in 410 μL Wash Buffer 2 and incubated on a spin mixer at 10 rpm for 30 min at room temperature.

[0080] 4. Phage screening and washing

[0081] 1) Close the Dynabeads as described in step 3 TM M-280 Streptavidin magnetic beads were divided into eight 50 μL portions, each mixed with one of eight 5 mL blocked phage cyclic peptide libraries, and incubated at 10 rpm for 1 h at room temperature using a rotary culture mixer. The supernatant was removed, and the magnetic beads were retained.

[0082] 2) Wash the magnetic beads 8 times with 200μL Wash Buffer 4, and then wash them 2 times with 200μL Wash Buffer 1.

[0083] 5. Phage infection counting and amplification

[0084] 1) The magnetic beads that have been thoroughly washed in step 4 are mixed with 5 mL of Escherichia coli TG1 bacterial solution in the logarithmic growth phase in a clean bench and incubated in a 37°C incubator for 30 min to allow the bacteriophages to fully infect TG1.

[0085] 2) Remove the bacterial culture from the incubator, centrifuge at 4500 rpm for 15 min, and remove the supernatant. Resuspend the cell pellet in 0.5 mL of fresh 2YT medium, and take 10 μL from each tube for screening enrichment determination.

[0086] 3) Spread the remaining bacterial culture onto 2YT solid medium containing 10 μg / mL tetracycline and incubate overnight at 37°C for 16 h for the next round of screening.

[0087] 4) Take the phage samples described in steps 2-3), add 10 μL to 90 μL of 2YT medium, and serially dilute 7 times. Then, take 10 μL from each dilution well and add it to 90 μL of medium in the logarithmic growth phase (OD50). 600In a phage-infected E. coli TG1 culture (0.4–0.8 μg / mL), incubate at 37°C for 30 min. After infection, take 5 μL of the phage-infected E. coli culture from each dilution well and spot it onto 2YT solid medium containing 10 μg / mL tetracycline. Incubate overnight at 37°C for phage titer counting.

[0088] 5) Take the phage sample described in step 5-2), add 10 μL to 90 μL of 2YT medium, and serially dilute 7 times. Spot 5 μL of the bacterial culture onto 2YT solid medium containing 10 μg / mL tetracycline and incubate overnight at 37°C.

[0089] 6) The next day, remove the solid culture medium described in steps 5-3) from the incubator, wash the phage library from the solid culture medium with 4 mL of 2YT medium containing 10% glycerol, and store it at -20℃ for the next round of screening. Remove the solid culture medium used for infection counting and count the number of colonies. The phage titer after circularization is used as the input, and the phage titer on the thoroughly washed magnetic beads is used as the output. Capture yield represents the output / input ratio. The enrichment degree of the phage is determined by comparing the capture yield values. Figure 1 ).

[0090] 6. Next-generation sequencing and data processing and analysis

[0091] 1) First step PCR

[0092] Multiple primers containing different barcodes and sequences complementary to the primers used in the second-step PCR step were designed (as listed in Table 4) for the first-step PCR. Different primer pairs were assigned to samples enriched in the first, second, and third rounds of screening (as shown in Table 6) to distinguish sample origins based on barcodes during subsequent sequencing data processing. PCR reactions were performed using 2×PCR NG Master Mix (Taq enzyme Mix). The primer stock solution concentration was 10 μM, diluted to 1 μM beforehand. During the PCR operation, 2×PCR NG Master Mix, water, and primers were added first, followed by the template. The entire process was performed on ice.

[0093] Table 6. Comparison of samples enriched in each round of screening with primers from the first round.

[0094]

[0095] The PCR reaction system consisted of a template (0.5 μL-1 μL (glycerol stock, OD around 30)), 2×PCRNG Master Mix (25 μL), Forward / Reverse Primer (5 μL×1 μM), and MQ (14.5 μL).

[0096] The PCR program was 95℃×5min, (95℃×30s, 55℃×30s, 72℃×30s)×25, 72℃×10min. PCR products were subjected to DNA electrophoresis on a 2.5% agarose gel to confirm band size.

[0097] 2) Second step PCR

[0098] The products from the first PCR step were mixed in proportion according to the band brightness and used as templates for the second PCR step. PCR reactions were performed using 2×PCR NG Master Mix (Taq enzyme Mix).

[0099] The PCR reaction system consisted of a template (2 μL), 2×PCR NG Master Mix (25 μL), Forward / Reverse Primer (1 μL × 5 μM), and MQ (21 μL).

[0100] The PCR program was 95℃×5min, (95℃×30s, 55℃×30s, 72℃×30s)×25, 72℃×10min.

[0101] The remaining PCR products from the second step 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, ready for next-generation sequencing.

[0102] 3) Next-generation sequencing was performed by Genewiz Biotechnology Co., Ltd., using the Illumina Novaseq 2×150bp sequencing platform. The sequencing process mainly included library construction, bridge PCR amplification and denaturation, and sequencing. After sequencing, approximately 10 million sequencing data points of about 240bp in length were returned for further data analysis.

[0103] 4) Data Analysis

[0104] NGS data processing was performed using the Matlab script described in the paper (Doi:10.1021 / acschembio.4c00099). The specific methods included sample sorting, sequence translation, and cluster analysis to identify promising candidate peptide sequences. First, the Fastq sequencing data file was read, and the data results were distributed to different files based on the different barcodes introduced by PCR. Second, low-quality sequences containing more than three Q scores ≤18 were removed from the data files according to the established quality criteria. Nucleotide sequences starting with ATGGC and ending with GGTTCT (encoding Met-Ala and Gly-Ser, respectively) were extracted and translated into amino acid sequences. Peptide sequences shorter than 7 amino acids and longer than 16 amino acids were excluded. The remaining sequences were sorted according to abundance. At this point, two steps could be performed: 1) using the "looplength.m" script to count the number of cysteine ​​residues in the peptide sequence; 2) using "CommonSeq.m" to compare identical sequences from two different datasets. 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.

[0105] 7. Modification and purification of cyclic peptide molecules

[0106] Two linear polypeptide sequences containing two cysteine ​​residues, as described in Table 1, were ordered from a polypeptide synthesis company. Each 0.5 mg polypeptide was completely dissolved in 500 μL of 50% ACN / 50% Water (containing 0.1% TFA). The modification reaction system was 50% ACN / NH4HCO3 (100 mM, pH 8.0), with 0.5 mM polypeptide and 1 mM of the corresponding chemical cross-linking agent reacted at 30°C for 1 h. The modification of the polypeptide was verified using Shimadzu UPLC-MS (Shim-pack GIST C18 column (2.1 × 50 mm, 2 μm)). After confirming complete modification, the reaction was quenched with acetic acid. Subsequently, the polypeptide was processed by Shimadzu preparative liquid chromatography (Hypersil). TM The cyclic peptide molecules were purified by using PREP HS C18 (10×250mm, 5μm) in a mobile phase system of water (containing 0.1% TFA) and ACN (containing 0.1% TFA). The purity of the cyclic peptide molecules was verified by Agilent HPLC (Poroshell 120EC-C18 column (4.6×150mm, 4μm)).

[0107] 8. Characterization of the binding of CD5 to 7 cyclic peptide molecules

[0108] In this embodiment, the surface plasmon resonance method was used to characterize the binding of the target protein CD5 to the seven cyclic peptide molecules.

[0109] 1) Immobilization of the target protein CD5

[0110] Since the target protein CD5 carries a biotin tag, it can bind to streptavidin. Therefore, a streptavidin chip (Lot.number: 10315802) was selected when measuring the affinity of the target protein and the cyclic peptide. The CD5 stock solution was diluted with 1×PBS buffer to a working concentration of 10 μg / mL, and the target protein was captured onto the second immobilization channel of the streptavidin chip at a flow rate of 5 μL / min. The first reference channel was left untreated.

[0111] 2) Preparation of cyclic peptide molecular samples

[0112] All seven cyclic peptide molecules were dissolved in 100% DMSO. The peptide stock solution was first diluted with 1.05×PBST (0.05% Tween 20) buffer to obtain the initial concentration of peptides in 1.05×PBST containing 5% DMSO. Subsequently, the peptides were serially diluted 2-fold or 3-fold with 1.05×PBST (containing 5% DMSO) to five concentration gradients.

[0113] 3) Combined with the specific measurement method

[0114] Binding assays were performed using a streptavidin chip with immobilized CD5 and seven cyclic peptide molecules. Single-cycle or multi-cycle kinetic methods were primarily employed. The running buffer was PBST containing 5% DMSO. Binding time was 120 s, dissociation time was 120 s, and the flow rate was 30 μL / min. Solvent correction was performed after the binding assay. After acquiring the surface plasmon resonance data sensor images, they were opened with Biacore Evaluation Software. After subtracting the reference channel, the data were fitted using a 1:1 binding model to obtain the KD value, binding rate constant (Ka), dissociation rate constant (Kd), and chi-square value (Chi). 2 The value is combined with parameters such as )

[0115] Surface plasmon resonance (SPR) technology showed that the seven CD5-specific cyclic peptide molecules selected in this invention all exhibited good affinity for CD5, as indicated by the test results. Figure 2 As shown in the figure. Among them, the affinities of C101, C202, C301, C501, C601, C701, and C201 with the target protein CD5 are 4±1 μM, 3.06 μM, 2±1 μM, 6.37 μM, 9.63 μM, 6±4 μM, and 21±5 μM, respectively.

[0116] Example 2

[0117] CD5 is a highly promising tumor target molecule, and in-depth research on peptides targeting CD5 has significant potential and value. Seven cyclic peptide molecules specifically targeting CD5 can be synthesized in vitro and used in combination with various therapeutic technologies. For example, CD5-targeting peptides can be conjugated with drugs to form PDC drugs, driving the enrichment of toxic payloads at tumor sites or cells, limiting non-targeted cytotoxicity, and inducing tumor cell apoptosis. Secondly, CD5-targeting peptides can also serve as nanomedicine delivery carriers. Compared with other nanomedicine building materials, peptides can self-assemble into fine nanostructures, are easy to target and modify, and have advantages such as high drug loading capacity, small molecular weight, low immunogenicity, and low preparation cost. In addition, CD5-targeting peptides can be labeled with radioactive elements for intraoperative imaging, highly sensitively labeling tiny tumor lesions invisible to the naked eye, providing doctors with a clear view to reduce postoperative recurrence rates. Alternatively, CD5-targeting peptides can be conjugated with siRNA, combining the specificity of the targeting peptide with the therapeutic effect of siRNA to increase siRNA accumulation at tumor sites, thereby enhancing the therapeutic effect on tumors. In summary, the combination of CD5-targeting peptides with high-efficiency drug platforms can be applied to the preparation of reagents or therapeutic drugs for tumor targeted screening, diagnosis, tracing, prognostic assessment, and treatment, showing promising application prospects.

[0118] The above description of the embodiments is intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be easily made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any 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 invention should be within the protection scope of the present invention.

Claims

1. A cyclic peptide molecule specifically targeting CD5, characterized in that, the amino acid sequence of which is selected from any one of the following: (1) Thr-Glu-Cys-Typ-Thr-Thr-Met-Arg-Glu-Cys; (2) Met-Cys-Asp-Arg-Leu-Tyr-Typ-Cys-Asp; The cyclic peptide molecule specifically targeting CD5 contains two cysteines, and the side chain mercapto groups of the cysteines will undergo affinity substitution reaction with active halogen Br on a specific chemical cross-linking agent, thereby being modified into a ring.

2. The cyclic peptide molecule specifically targeting CD5 according to claim 1, characterized in that, The cyclic peptide molecule specifically targeting CD5 is selected from one of the following molecules: C101, C202, C301, C501, C601, C701, C201: 。 3. An engineered immune effector cell, characterized in that, The pharmaceutical composition comprises the cyclic peptide molecule specifically targeting CD5 according to claim 1 or 2, or the engineered immune effector cell according to claim 3, and one or more pharmaceutically acceptable excipients and / or carriers.

4. A pharmaceutical composition, characterized by, 5. Use of the cyclic peptide molecule specifically targeting CD5 according to claim 1 or 2 in the preparation of a reagent for screening, diagnosis, tracing or prognosis evaluation of T cell lymphoma, or a drug for treating T cell lymphoma.

6. Use of the engineered immune effector cell according to claim 3 or the pharmaceutical composition according to claim 4 in the preparation of a reagent for screening, diagnosis, tracing or prognosis evaluation of T cell lymphoma, or a drug for treating T cell lymphoma. ​

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