Novel specific targeting CAR-T cell polypeptide compound and probe thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a novel polypeptide compound specifically targeting CAR-T cells and a probe of the novel polypeptide compound, the polypeptide compound is BC-5D3-7, and a solid-phase synthesis method is adopted for preparing the polypeptide compound and comprises the steps that RinkAmideResin serves as a carrier and is sequentially coupled with specific amino acid and a modifying agent, and the polypeptide compound is obtained after deprotection, cleaning, cutting and purification are conducted. The compound can be specifically combined with a BCMA-CD19 antigen on the surface of a CAR-T cell, and the KD value of the compound reaches 10 <-10 > mol / L through SPR (Surface Plasmon Resonance) detection. A probe constructed based on the polypeptide is prepared by coupling the polypeptide with labeled groups such as fluorescent dye and radionuclide, and can be used for CAR-T cell imaging. The polypeptide compound disclosed by the invention is stable in synthesis process and strong in targeting property; the probe can accurately locate the CAR-T cells, provides an effective tool for detection and drug screening of the CAR-T cells, and has important application value in the field of tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a novel class of polypeptide compounds and probes that specifically target CAR-T cells. Background Technology

[0002] CAR-T cells have made significant progress in the treatment of hematological malignancies, but their pharmacokinetic (PK), pharmacodynamic (PD), and safety evaluations face many new challenges. The efficacy and safety of CAR-T cell therapy are highly dependent on their spatiotemporal distribution in vivo, target binding efficiency, and interactions with other microenvironmental factors. On the one hand, insufficient homing efficiency of CAR-T cells is a significant factor limiting efficacy; on the other hand, off-target effects of CAR-T cells can lead to serious adverse reactions. Furthermore, interactions between CAR-T cells and adhesion molecules (such as ICAM-1 and VCAM-1) or immune checkpoints (such as PD-1 / PD-L1) may affect their function. CAR-T cells exhibit proliferative properties; the time, rate, and quantity of this novel "live" drug's proliferation in vivo are closely related to its efficacy and safety, but comprehensive and precise in vivo PK monitoring methods are lacking. Summary of the Invention

[0003] The purpose of this invention is to provide a novel class of polypeptide compounds that specifically target CAR-T cells, including the polypeptide compound BC-5D3-7, which is prepared by the following solid-phase synthesis method: S1: Add 0.1 mmol RinkAmide Resin to the peptide synthesis tube, add 5 ml dichloromethane (DCM), purge with nitrogen (N2) and bubble for 30 min, then filter. Wash with 5 ml N,N-dimethylformamide (DMF), bubble with N2 for 1 min, then filter. Repeat 3 times. S2: Add 5 ml of 20% piperidine, bubble with N2 for 5 min and then filter, repeat 2 times, then wash with 5 ml of DMF, bubble with N2 for 1 min and then filter, repeat 4 times; S3: Add the following in sequence: 5eqFMOC-Lys(Alloc)-OH, 5eqFmoc-8-amino-3,6-dioxaoctanoicacid (twice), 5eqFMOC-β-Ala-OH, 5eqFmoc-Gly-OH, 5eqFmoc-Cys(Trt)-OH, 5eqFMOC-Ala-OH, 5eqFmoc-Gln(Trt)-OH, 5eqFmoc-Phe-OH (twice), 5eqFmoc-Pro-OH, and 5eqFMOC-Ser(tBu)-O H, 5eqFmoc-L-Val-OH, 5eqFmoc-Lys-OH, 5eqFMOC-Ser(tBu)-OH, 5eqFmoc-Phe-OH, 5eqFMOC-D-Tyr(tBu)-OH, each added with a DMF solution of 5eqHATU and 10eqDIPEA, N2 bubble reaction for 30-60 min (Fmoc-Gln(Trt)-OH reacts with Fmoc-L-Val-OH for 60 min, the others for 30 min), after the reaction, wash 3 times with 5 ml DMF, and then repeat the deprotection and washing process of S2; S4: Add 5 eq Chloroacetylchloride DMF solution (containing 5 eq HATU and 10 eq DIPEA), bubble with N2 for 30 min, wash with DMF 3 times, wash with DCM 4 times, add 15 eq phenylsilane N2 and bubble for 5 min, then add 0.25 eq Pd(PPh3)4 N2 and bubble for 40 min, wash with DCM 4 times; S5: Add 5 eq TRI-TERT-BUTYL1,4,7,10-TETRAAZACYCLODOD DMF solution (containing 5 eq HATU and 10 eq DIPEA), bubble with N2 for 30 min, wash with DMF 3 times and DCM 4 times, and then air dry to obtain solid product. S6: The solid-phase product was stirred at room temperature for 2 hours with 5 ml of 95% trifluoroacetic acid (TFA:Tips:H2O=95%:2.5%:2.5%) at room temperature. After filtration, the filtrate was poured into cold diethyl ether, centrifuged at 4000 rpm for 5 minutes, and the precipitate was collected. The product was washed twice with diethyl ether, dried under vacuum, dissolved in 2 ml of dimethyl sulfoxide (DMSO), and the pH was adjusted to 8.0 with triethylamine (Et3N). The reaction was carried out at 25°C for 3 hours. The pH was adjusted to 2.0-3.0 with TFA, filtered through a microporous membrane, and purified by high-performance liquid chromatography (HPLC). The purified product was then lyophilized to obtain BC-5D3-7. BC-5D3-7 specifically binds to the BCMA-CD19 antigen on the surface of CAR-T cells, with a binding affinity KD value of 10⁻. 8 -10⁻¹ 0The concentration was mol / L, and it was detected by surface plasmon resonance (SPR).

[0004] Preferably, the HPLC separation and purification in S6 uses a C18 reversed-phase column, the mobile phase is an acetonitrile-water mixture containing 0.1% TFA, and the gradient elution program is: 0-5 min acetonitrile volume fraction 5%-20%, 5-20 min acetonitrile volume fraction 20%-60%, 20-25 min acetonitrile volume fraction 60%-95%, the flow rate is 1.0 ml / min, and the detection wavelength is 220 nm.

[0005] Preferably, the specific conditions for SPR detection are as follows: using a Biacore-1KSPR system (Cytiva, Sweden), the detection buffer is 1×PBS-P+ buffer containing 5% dimethyl sulfoxide (composition: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, 0.05% polysorbate 20, pH 7.4), the temperature is 25℃, BCMA-CD19 is immobilized on the CM5 chip via amine coupling, the peptide compound concentration gradient is 0–3μM, the flow rate is 30μl / min, and the KD value is calculated using a 1:1 combined kinetic model in BiacoreInsightEvaluation software.

[0006] Preferably, the N2 flow rate in S1-5 is 10-20 ml / min, and the N2 is filtered through a 0.2 μm gas filter to remove impurities before being introduced.

[0007] Preferably, the temperature of the cold ether in S6 is -20 to -10°C, and the volume of cold ether added each time is 5 to 8 times the volume of the filtrate; the pore size of the microporous filter membrane is 0.22 μm.

[0008] On the other hand, the present invention provides a probe for targeting CAR-T cells using a polypeptide compound. The probe is composed of a BC-5D3-7 polypeptide compound and a labeling group covalently linked together, wherein the labeling group is a fluorescent dye, a radionuclide, or a quantum dot; wherein the fluorescent dye is selected from FITC, Cy3, or Cy5, and the radionuclide is selected from¹ 8 F, 64 Cu or ¹¹¹In; the linkage site is the lysine side chain amino group or cysteine ​​thiol group in the BC-5D3-7 polypeptide chain. The linkage reaction is carried out by N-hydroxysuccinimide activation method or maleimide coupling method. The reaction temperature is 25-37℃ and the reaction time is 1-2h. This probe can specifically recognize and bind to CAR-T cells, with a targeting binding efficiency of ≥85% and a non-specific binding rate of ≤5%.

[0009] Preferably, when the labeling group is a fluorescent dye, the fluorescence quantum yield of the probe is ≥0.6, the excitation wavelength is 488-650nm, the emission wavelength is 510-670nm, and the fluorescence intensity is stable in a buffer system with pH 6.0-8.0 (coefficient of variation ≤5%).

[0010] Preferably, when the labeling group is a radionuclide, the specific activity of the probe is 37-74 MBq / μg, the radionuclide labeling rate is ≥95%, and the radiochemical purity is ≥90% after incubation at 37°C for 24 h in phosphate buffered saline (PBS, pH 7.4). Preferably, the binding reagent is used to detect the purity of CAR-T cells in vitro, or to guide CAR-T cells to migrate to the tumor site in vivo.

[0011] Preferably, the imaging reagent is used for in vivo fluorescence imaging, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) to monitor the distribution, survival status and migration trajectory of CAR-T cells in vivo.

[0012] Compared with the prior art, the beneficial effects of the present invention are: 1. The polypeptide probe described in this invention forms a macrocycle through thioether bonds, which has better stability than linear peptides.

[0013] 2. The polypeptide probe described in this invention has a short amino acid sequence, which facilitates chemical synthesis and modification.

[0014] 3. The polypeptide probe described in this invention, by coupling with a radionuclide, enables in vivo visualization monitoring of CAR-T cells using PET probe technology, and achieves quantitative characterization of the dynamic processes and laws of CAR-T cell drug distribution, homing, and proliferation in vivo, breaking through the technical bottleneck of real-time dynamic quantitative monitoring of the in vivo proliferation and transformation of live cell drugs.

[0015] 4. The polypeptide probe described in this invention can achieve covalent coupling with a variety of macrocyclic ligands through a solid-phase polypeptide synthesis method.

[0016] 5. The polypeptide probe described in this invention can be used to label nuclides with different half-lives, such as 68Ga (T1 / 2=68min) or 18F (T1 / 2=110min).

[0017] 6. The polypeptide probe described in this invention targets the scFv of antiBCMA-CD19 in dual CARs, and uses a polypeptide library to screen for high-affinity polypeptide fragments. The obtained polypeptides do not damage the structure of CAR-T cells.

[0018] 7. The polypeptide probe described in this invention, through structural modification and alteration, obtains a polypeptide ligand structure that specifically binds to BCMA-CD19 dual CAR, exhibiting high specificity and strong affinity for CAR-T cells. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the SPR results of the polypeptide of the present invention; Figure 2 This is a schematic diagram illustrating the response of the polypeptide of the present invention to CAR-T cells; Figure 3 A mutation screening was performed on multiple BC-5D3-9 cells for this invention, and a biotin-linked reaction diagram was also shown at the C-terminus. Figure 4 This is a schematic diagram of the KD value of the polypeptide after mutation A in this invention; Figure 5 The present invention uses flow cytometry to detect the bioactivity of peptide BC-5D-3; Figure 6 This is a schematic diagram illustrating the use of flow cytometry to detect the bioactivity of peptide BC-5D-29 in this invention. Figure 7 This is a schematic diagram illustrating the detection of the bioactivity of peptide BC-5D-1 using flow cytometry according to the present invention. Figure 8 This is a schematic diagram illustrating the detection of the bioactivity of peptide BC-5L-30 using flow cytometry according to the present invention. Figure 9 This is a schematic diagram illustrating the detection of the bioactivity of peptide BC-5L-1 using flow cytometry according to the present invention. Figure 10 This is a schematic diagram of the polypeptide sequence obtained by modifying BC-5D3-9 with NOA in this invention; Figure 11 This represents the KD value of the NOTA-modified peptide of this invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Reference Figure 1 - Figure 11 This invention discloses a novel polypeptide compound that specifically targets CAR-T cells, comprising the polypeptide compound BC-5D3-7, prepared by the following solid-phase synthesis method: S1: Add 0.1 mmol RinkAmide Resin to the peptide synthesis tube, add 5 ml dichloromethane (DCM), purge with nitrogen (N2) and bubble for 30 min, then filter. Wash with 5 ml N,N-dimethylformamide (DMF), bubble with N2 for 1 min, then filter. Repeat 3 times. S2: Add 5 ml of 20% piperidine, bubble with N2 for 5 min and then filter, repeat 2 times, then wash with 5 ml of DMF, bubble with N2 for 1 min and then filter, repeat 4 times; S3: Add the following in sequence: 5eqFMOC-Lys(Alloc)-OH, 5eqFmoc-8-amino-3,6-dioxaoctanoicacid (twice), 5eqFMOC-β-Ala-OH, 5eqFmoc-Gly-OH, 5eqFmoc-Cys(Trt)-OH, 5eqFMOC-Ala-OH, 5eqFmoc-Gln(Trt)-OH, 5eqFmoc-Phe-OH (twice), 5eqFmoc-Pro-OH, and 5eqFMOC-Ser(tBu)-O H, 5eqFmoc-L-Val-OH, 5eqFmoc-Lys-OH, 5eqFMOC-Ser(tBu)-OH, 5eqFmoc-Phe-OH, 5eqFMOC-D-Tyr(tBu)-OH, each added with a DMF solution of 5eqHATU and 10eqDIPEA, N2 bubble reaction for 30-60 min (Fmoc-Gln(Trt)-OH reacts with Fmoc-L-Val-OH for 60 min, the others for 30 min), after the reaction, wash 3 times with 5 ml DMF, and then repeat the deprotection and washing process of S2; S4: Add 5 eq Chloroacetylchloride DMF solution (containing 5 eq HATU and 10 eq DIPEA), bubble with N2 for 30 min, wash with DMF 3 times, wash with DCM 4 times, add 15 eq phenylsilane N2 and bubble for 5 min, then add 0.25 eq Pd(PPh3)4 N2 and bubble for 40 min, wash with DCM 4 times; S5: Add 5 eq TRI-TERT-BUTYL1,4,7,10-TETRAAZACYCLODOD DMF solution (containing 5 eq HATU and 10 eq DIPEA), bubble with N2 for 30 min, wash with DMF 3 times and DCM 4 times, and then air dry to obtain solid product. S6: The solid-phase product was stirred at room temperature for 2 hours with 5 ml of 95% trifluoroacetic acid (TFA:Tips:H2O=95%:2.5%:2.5%) at room temperature. After filtration, the filtrate was poured into cold diethyl ether, centrifuged at 4000 rpm for 5 minutes, and the precipitate was collected. The product was washed twice with diethyl ether, dried under vacuum, dissolved in 2 ml of dimethyl sulfoxide (DMSO), and the pH was adjusted to 8.0 with triethylamine (Et3N). The reaction was carried out at 25°C for 3 hours. The pH was adjusted to 2.0-3.0 with TFA, filtered through a microporous membrane, and purified by high-performance liquid chromatography (HPLC). The purified product was then lyophilized to obtain BC-5D3-7. BC-5D3-7 specifically binds to the BCMA-CD19 antigen on the surface of CAR-T cells, with a binding affinity KD value of 10⁻. 8 -10⁻¹ 0 The concentration was mol / L, and it was detected by surface plasmon resonance (SPR).

[0022] Specifically, the HPLC separation and purification described in S6 uses a C18 reversed-phase column, with an acetonitrile-water mixture containing 0.1% TFA as the mobile phase. The gradient elution program is as follows: 0-5 min acetonitrile volume fraction 5%-20%, 5-20 min acetonitrile volume fraction 20%-60%, 20-25 min acetonitrile volume fraction 60%-95%, flow rate 1.0 ml / min, and detection wavelength 220 nm.

[0023] Specifically, the SPR detection conditions were as follows: a Biacore-1KSPR system (Cytiva, Sweden) was used; the detection buffer was 1×PBS-P+ buffer containing 5% dimethyl sulfoxide (composition: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, 0.05% polysorbate 20, pH 7.4); the temperature was 25℃; BCMA-CD19 was immobilized on the CM5 chip via amine coupling; the peptide compound concentration gradient was 0–3 μM; the flow rate was 30 μl / min; and the KD value was calculated using a 1:1 kinetic model in BiacoreInsightEvaluation software.

[0024] Specifically, the N2 flow rate described in S1-5 is 10-20 ml / min, and the N2 is filtered through a 0.2 μm gas filter to remove impurities before being introduced.

[0025] Specifically, the temperature of the cold ether in S6 is -20 to -10°C, and the volume of cold ether added each time is 5 to 8 times the volume of the filtrate; the pore size of the microporous filter membrane is 0.22 μm.

[0026] On the other hand, the present invention provides a probe for targeting CAR-T cells with a polypeptide compound, comprising a probe consisting of a BC-5D3-7 polypeptide compound and a labeling group covalently linked together, wherein the labeling group is a fluorescent dye, a radionuclide, or a quantum dot; wherein the fluorescent dye is selected from FITC, Cy3, or Cy5, and the radionuclide is selected from¹ 8 F, 64 Cu or ¹¹¹In; the linkage site is the lysine side chain amino group or cysteine ​​thiol group in the BC-5D3-7 polypeptide chain. The linkage reaction is carried out by N-hydroxysuccinimide activation method or maleimide coupling method. The reaction temperature is 25-37℃ and the reaction time is 1-2h. This probe can specifically recognize and bind to CAR-T cells, with a targeting binding efficiency of ≥85% and a non-specific binding rate of ≤5%.

[0027] Specifically, when the labeling group is a fluorescent dye, the fluorescence quantum yield of the probe is ≥0.6, the excitation wavelength is 488-650nm, the emission wavelength is 510-670nm, and the fluorescence intensity is stable in a buffer system with pH 6.0-8.0 (coefficient of variation ≤5%).

[0028] Specifically, when the labeling group is a radionuclide, the specific activity of the probe is 37-74 MBq / μg, the radionuclide labeling rate is ≥95%, and the radiochemical purity is ≥90% after incubation at 37°C for 24 hours in phosphate buffered saline (PBS, pH 7.4). Specifically, the binding reagent is used to detect the purity of CAR-T cells in vitro, or to guide CAR-T cells to migrate to the tumor site in vivo.

[0029] Specifically, the imaging reagents are used for in vivo fluorescence imaging, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) to monitor the distribution, survival status and migration trajectory of CAR-T cells in vivo.

[0030] The working principle of this invention is as follows: Add 0.1 mmol RinkAmideResin to a peptide synthesis tube, add 5 ml DCM, bubble with N2 for 30 min, and then filter to remove the filtrate. Add 5 ml DMF to the synthesis tube, bubble with N2 for 1 min, and then filter. Repeat this process three times. Add 5 ml 20% Piperidine, bubble with N2 for 5 min, and then filter to remove the filtrate. Repeat this process twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat this process four times. Add 5 eq FMOC-Lys(Alloc)-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as the solvent) to the synthesis tube, and bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat this process three times. Add 5 ml 20% Piperidine, bubble with N2 for 5 min, and then filter to remove the filtrate. Repeat this process twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat this process four times. Add 5 eq Fmoc-8-amino-3,6-dioxaoctanoicacid, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to a synthesis tube. Bubble with N2 for 30 min, then remove the filtrate. Repeat twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and remove the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat four times. Add 5 eq Fmoc-8-amino-3,6-dioxaoctanoicacid, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to a synthesis tube. Bubble with N2 for 30 min, then remove the filtrate. Repeat twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and remove the filtrate. After repeating twice, wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat four times. Add 5 eq FMOC-β-Ala-OH, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, and bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, and then filter to remove the filtrate. After repeating twice, wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat four times. Add 5 eq FMOC-β-Ala-OH, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, and bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, and then filter to remove the filtrate. After repeating twice, clean the solid resin with DMF, bubble with N2 for 1 minute, and repeat four times.Add 5 eq Fmoc-Cys(Trt)-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent) to a synthesis tube and bubble with N2 for 30 min. Wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, and then filter to remove the filtrate. Repeat twice, then wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating four times. Add 5 eq FMOC-Ala-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent) to a synthesis tube and bubble with N2 for 30 min. Wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, and then filter to remove the filtrate. Repeat twice, then wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating four times. Add 5 eq Fmoc-Gln(Trt)-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent) to a synthesis tube and bubble with N2 for 60 min. Wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating three times. Add 5 ml of 20% Piperidine and bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating four times. Add 5 eq Fmoc-Phe-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent) to a synthesis tube and bubble with N2 for 30 min. Wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating three times. Add 5 ml of 20% Piperidine and bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating four times. Add 5 eq Fmoc-Phe-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent) to a synthesis tube and bubble with N2 for 30 min. Wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, and then filter to remove the filtrate. Repeat twice, then wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating four times. Add 5 eq Fmoc-Pro-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent) to a synthesis tube and bubble with N2 for 30 min. Wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, and then filter to remove the filtrate. Repeat twice, then wash the solid-phase resin with DMF and bubble with N2 for 1 min, repeating four times. Add 5 eq FMOC-Ser(tBu)-OH, 5 eq HATU, 10 eq DIPEA, and DMF as solvent to the synthesis tube and bubble with N2 for 30 min.Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat four times. Add 5 eq Fmoc-L-Val-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent) to the synthesis tube, bubble with N2 for 60 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat four times. Add 5 eq Fmoc-Lys-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent) to the synthesis tube, bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat four times. Add 5 eq FMOC-Ser(tBu)-OH, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat four times. Add 5 eq FMOC-Phe-OH, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat four times. Add 5 eq FMOC-D-Tyr(tBu)-OH, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat four times. Add 5 eq Chloroacetylchloride, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat three times.Add DCM for cleaning, bubble with N2 for 1 min, then dry. Repeat 4 times. Add 15 eq phenylsilane, bubble with N2 for 5 min. Add 0.25 eq Pd(PPh3)4, bubble with N2 for 40 min. Add DCM for cleaning, bubble with N2 for 1 min, then dry. Repeat 4 times. Clean the solid resin with DMF. Add 5 eq TRI-TERT-BUTYL1,4,7,10-TETRAAZACYCLODOD, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, bubble with N2 for 30 min. Clean the solid resin with DMF, bubble with N2 for 1 min, repeat 3 times. Add DCM for cleaning, bubble with N2 for 1 min, then dry. Repeat 4 times. Pour out and air dry. The solid product was obtained and poured into a 25 mL flask. 5 mL of 95% TFA (TFA:Tips:H2O = 95%:2.5%:2.5%) was added. After stirring at room temperature for 2 h, the mixture was filtered. The filtrate was poured into cold diethyl ether and centrifuged at 4000 rpm for 5 min. The supernatant was discarded, and the precipitate was retained. Cold diethyl ether was added again, and the precipitate was shaken thoroughly. After centrifugation at 4000 rpm for 5 min, the supernatant was discarded, and the precipitate was retained. This process was repeated twice. The precipitate was dried under vacuum to obtain a solid powder. The solid was dissolved in 2 mL of DMSO, and the pH was adjusted to 8.0 with Et3N. The reaction was carried out at 25 °C for 3 h. After the reaction, TFA was added to adjust the pH to 2.0-3.0. The mixture was filtered through a microporous membrane and purified by HPLC. The product was analyzed by mass spectrometry and then lyophilized to obtain compound BC-5D3-7.

[0031] When using surface plasmon resonance (SPR) to detect the KD value between peptides and receptors, the binding affinity of the peptide compound to BCMA-CD19 was determined by surface plasmon resonance (SPR). SPR experiments were performed using a Biacore-1KSPR system (Cytiva, Sweden). Experiments were conducted in 1×PBS-P+ buffer containing 5% dimethyl sulfoxide at 25°C. The conjugation amount (RL) of BCMA-CD19 was calculated using the following formula: RL = MW ligand / (MW analyte * Sm) * Rmax (where Rmax is the maximum binding amount on the chip surface, set to 100 RU; Sm is the ligand-analyte binding ratio). A quantified amount of BCMA-CD19 was immobilized on a CM5 chip via amine coupling, and then a diluted peptide compound (0–3 μM) was passed through the chip at a flow rate of 30 μl / min. Solvent correction was performed simultaneously. The affinity constant KD value was calculated using a 1:1 binding kinetic model in BiacoreInsightEvaluation software.

[0032] The binding rate of peptides to CART cells was detected by flow cytometry. The NOTA-labeled peptides were detected using a direct labeling method, with a sample size of 1 × 10⁻⁶. 6BCMA-CD19 CAR-T cells were washed with 1 ml of pre-chilled 1×PBS, centrifuged, and the supernatant was discarded. Different concentrations of fluorescein-labeled peptides were added, and the cells were incubated on ice in the dark for 30 min, inverting and mixing every 15 min. The supernatant was discarded after centrifugation. Cells were washed twice with 1 ml of pre-chilled 2% BSA / PBS, and finally resuspended in 400 μl of pre-chilled 1×PBS for flow cytometry analysis. The indirect labeling method was used to detect the NOTA-labeled peptides. 1×10⁻⁶ NOTA-labeled peptides were used. 6 Centrifuge BCMA-CD19 CAR-T cells, discard the supernatant, add 1 ml of pre-chilled 1×PBS to wash the cells, add different concentrations of biotin-labeled peptides (positive control: add Protein L-Biotein to 1 μg / ml), incubate on ice for 1 h, invert and mix once every 15 min, centrifuge and discard the supernatant; add 1 ml of pre-chilled 2% BSA / PBS to wash the cells twice, finally resuspend the cells in 50 μl of 2% BSA / PBS, add 0.5 μl of Streptavidin-APC antibody (Biolgend 405207), incubate at room temperature in the dark for 20 min, invert and mix once every 8 min, wash the cells twice again with 1 ml of pre-chilled 2% BSA / PBS, resuspend the cell pellet in 400 μl of pre-chilled 1×PBS, and perform flow cytometry analysis.

[0033] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A polypeptide compound that specifically targets CAR-T cells, characterized in that: The polypeptide compound BC-5D3-7 was prepared by the following solid-phase synthesis method: S1: Add 0.1 mmol RinkAmide Resin to the peptide synthesis tube, add 5 ml dichloromethane (DCM), purge with nitrogen (N2) and bubble for 30 min, then filter. Wash with 5 ml N,N-dimethylformamide (DMF), bubble with N2 for 1 min, then filter. Repeat 3 times. S2: Add 5 ml of 20% piperidine, bubble with N2 for 5 min and then filter, repeat 2 times, then wash with 5 ml of DMF, bubble with N2 for 1 min and then filter, repeat 4 times; S3: Add the following in sequence: 5eqFMOC-Lys(Alloc)-OH, 5eqFmoc-8-amino-3,6-dioxaoctanoicacid (twice), 5eqFMOC-β-Ala-OH, 5eqFmoc-Gly-OH, 5eqFmoc-Cys(Trt)-OH, 5eqFMOC-Ala-OH, 5eqFmoc-Gln(Trt)-OH, 5eqFmoc-Phe-OH (twice), 5eqFmoc-Pro-OH, and 5eqFMOC-Ser(tBu)-O H, 5eqFmoc-L-Val-OH, 5eqFmoc-Lys-OH, 5eqFMOC-Ser(tBu)-OH, 5eqFmoc-Phe-OH, 5eqFMOC-D-Tyr(tBu)-OH, each added with a DMF solution of 5eqHATU and 10eqDIPEA, N2 bubble reaction for 30-60 min (Fmoc-Gln(Trt)-OH reacts with Fmoc-L-Val-OH for 60 min, the others for 30 min), after the reaction, wash 3 times with 5 ml DMF, and then repeat the deprotection and washing process of S2; S4: Add 5 eq Chloroacetylchloride DMF solution (containing 5 eq HATU and 10 eq DIPEA), bubble with N2 for 30 min, wash with DMF 3 times, wash with DCM 4 times, add 15 eq phenylsilane N2 and bubble for 5 min, then add 0.25 eq Pd(PPh3)4 N2 and bubble for 40 min, wash with DCM 4 times; S5: Add 5 eq TRI-TERT-BUTYL1,4,7,10-TETRAAZACYCLODOD DMF solution (containing 5 eq HATU and 10 eq DIPEA), bubble with N2 for 30 min, wash with DMF 3 times and DCM 4 times, and then air dry to obtain solid product. S6: The solid product was stirred at room temperature for 2 hours with 5 ml of 95% trifluoroacetic acid (TFA:Tips:H2O=95%:2.5%:2.5%). After filtration, the filtrate was poured into cold diethyl ether, centrifuged at 4000 rpm for 5 minutes, and the precipitate was retained. The product was washed twice with diethyl ether, dried under vacuum, dissolved in 2 ml of dimethyl sulfoxide (DMSO), and the pH was adjusted to 8.0 with triethylamine (Et3N). The reaction was carried out at 25°C for 3 hours. The pH was adjusted to 2.0-3.0 with TFA. After filtration through a microporous membrane, the product was separated and purified by high performance liquid chromatography (HPLC) and lyophilized to obtain BC-5D3-7. The BC-5D3-7 can specifically bind to the BCMA-CD19 antigen on the surface of CAR-T cells, with a binding affinity KD value of 10⁻. 8 -10⁻¹ 0 The concentration was mol / L, and it was detected by surface plasmon resonance (SPR).

2. The polypeptide compound that specifically targets CAR-T cells according to claim 1, characterized in that: The HPLC separation and purification described in S6 uses a C18 reversed-phase column, with an acetonitrile-water mixture containing 0.1% TFA as the mobile phase. The gradient elution program is as follows: 0-5 min acetonitrile volume fraction 5%-20%, 5-20 min acetonitrile volume fraction 20%-60%, 20-25 min acetonitrile volume fraction 60%-95%, flow rate 1.0 ml / min, and detection wavelength 220 nm.

3. The polypeptide compound that specifically targets CAR-T cells according to claim 1, characterized in that: The specific conditions for SPR detection were as follows: a Biacore-1KSPR system (Cytiva, Sweden) was used; the detection buffer was 1×PBS-P+ buffer containing 5% dimethyl sulfoxide (composition: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, 0.05% polysorbate 20, pH 7.4); the temperature was 25℃; BCMA-CD19 was immobilized on the CM5 chip via amine coupling; the peptide compound concentration gradient was 0–3μM; the flow rate was 30μl / min; and the KD value was calculated using a 1:1 kinetic model in BiacoreInsightEvaluation software.

4. The polypeptide compound that specifically targets CAR-T cells according to claim 1, characterized in that: The N2 flow rate described in S1-5 is 10-20 ml / min, and the N2 is filtered through a 0.2 μm gas filter to remove impurities before being introduced.

5. The polypeptide compound that specifically targets CAR-T cells according to claim 1, characterized in that: The temperature of the cold ether in S6 is -20 to -10°C, and the volume of cold ether added each time is 5 to 8 times the volume of the filtrate; the pore size of the microporous filter membrane is 0.22 μm.

6. A probe that specifically targets CAR-T cells, characterized in that: The probe is composed of a BC-5D3-7 polypeptide compound and a labeling group covalently linked together. The labeling group is a fluorescent dye, a radionuclide, or a quantum dot; wherein the fluorescent dye is selected from FITC, Cy3, or Cy5, and the radionuclide is selected from¹ 8 F, 64 Cu or ¹¹¹In; the linkage site is the lysine side chain amino group or cysteine ​​thiol group in the BC-5D3-7 polypeptide chain. The linkage reaction is carried out by N-hydroxysuccinimide activation method or maleimide coupling method. The reaction temperature is 25-37℃ and the reaction time is 1-2h. This probe can specifically recognize and bind to CAR-T cells, with a targeting binding efficiency of ≥85% and a non-specific binding rate of ≤5%.

7. The probe specifically targeting CAR-T cells according to claim 6, characterized in that: When the labeling group is a fluorescent dye, the fluorescence quantum yield of the probe is ≥0.6, the excitation wavelength is 488-650nm, the emission wavelength is 510-670nm, and the fluorescence intensity is stable in a buffer system with pH 6.0-8.0 (coefficient of variation ≤5%).

8. The probe specifically targeting CAR-T cells according to claim 6, characterized in that: When the labeling group is a radionuclide, the specific activity of the probe is 37-74 MBq / μg, the radionuclide labeling rate is ≥95%, and the radiochemical purity is ≥90% after incubation at 37°C for 24 h in phosphate buffered saline (PBS, pH 7.4).

9. The probe specifically targeting CAR-T cells according to claim 6, characterized in that: The binding reagent is used to detect the purity of CAR-T cells in vitro, or to guide CAR-T cells to migrate to the tumor site in vivo.

10. The application of the probe according to claim 6 in the preparation of CAR-T cell imaging reagents, characterized in that: The imaging reagents are used for in vivo fluorescence imaging, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) to monitor the distribution, survival status and migration trajectory of CAR-T cells in vivo.

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