WO2023245852A1 - 一种cd25靶向多肽、分子探针及应用 - Google Patents

一种cd25靶向多肽、分子探针及应用 Download PDF

Info

Publication number
WO2023245852A1
WO2023245852A1 PCT/CN2022/114251 CN2022114251W WO2023245852A1 WO 2023245852 A1 WO2023245852 A1 WO 2023245852A1 CN 2022114251 W CN2022114251 W CN 2022114251W WO 2023245852 A1 WO2023245852 A1 WO 2023245852A1
Authority
WO
WIPO (PCT)
Prior art keywords
probe
targeting polypeptide
polypeptide
tumor
molecular probe
Prior art date
Application number
PCT/CN2022/114251
Other languages
English (en)
French (fr)
Inventor
杨志
刘福涛
王帅亮
朱华
王培�
王风
Original Assignee
北京肿瘤医院(北京大学肿瘤医院)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京肿瘤医院(北京大学肿瘤医院) filed Critical 北京肿瘤医院(北京大学肿瘤医院)
Publication of WO2023245852A1 publication Critical patent/WO2023245852A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins

Definitions

  • the invention belongs to the field of nuclear medicine imaging agents. Specifically, it relates to a class of polypeptide derivatives and molecular probes that can be used to target the CD25 target of CAR-T cells and their preparation methods and applications.
  • CAR-T Chimeric Antigen Receptor T Cell
  • CAR-T therapy that is, chimeric antigen receptor T cell therapy
  • MSLN Mesothelin
  • HER-2 human epidermal growth factor receptor-2
  • CAR-T cells have limited survival and sustained expansion capabilities, or excessive clonal proliferation, which raises questions about the efficacy and safety evaluation of CAR-T. Huge challenge.
  • the predictive indicators and mechanisms related to the proliferation and good clinical response of CAR-T cells are not yet fully understood, and studies have shown that the efficacy of CAR-T is related to patient age, early treatment, p53 gene mutation, and the number of CAR-T cells. There was no significant correlation with other risk factors.
  • the infusion of CAR-T cells is accompanied by a large number of activation and expansion of CAR-T cells within two weeks.
  • CAR-T therapy includes cytokine release syndrome, nervous system toxicity, and off-target effects, which are all related to the activation and expansion of CAR-T cells in the body. Therefore, evaluating the activation and expansion of CAR-T cells is expected to achieve early prediction of CAR-T efficacy and play a certain role in guiding the development of CAR-T therapy.
  • CAR-T therapy usually uses methods such as complete blood count and bone marrow biopsy, which often causes greater pain to patients and cannot fully evaluate the distribution and activation status of CAR-T cells in the body.
  • methods such as complete blood count and bone marrow biopsy, which often causes greater pain to patients and cannot fully evaluate the distribution and activation status of CAR-T cells in the body.
  • long half-life radionuclides such as 89 Zr, 64 Cu and other labeled antibody molecules are usually used for immunoimaging. The imaging cycle is long, and the signal is lost after the activation and proliferation of CAR-T cells. dilution, thus limiting its application.
  • CD25 is the ⁇ chain of interleukin-2 (IL-2) receptor. It is almost not expressed on the surface of unactivated T cells, but is highly expressed after T cells are activated by antigen stimulation. Therefore, CD25 is an excellent target for specific imaging of activated T cells. In current research, CD25 imaging using various radionuclides such as 68 Ga and 18 F-labeled IL-2 molecules is a relatively mature technology. However, since IL-2 is a biologically active molecule, it will produce certain biological effects after being injected into the human body through intravenous injection, and may cause pain, fever and other discomforts to the patient after injection. Moreover, due to the relatively large molecular weight of IL-2, its clearance rate in the body is relatively slow.
  • IL-2 interleukin-2
  • Small molecule probes have the advantages of rapid metabolic clearance in vivo, low background uptake in organs, high biological safety, easy radioactive labeling, and low cost. Designing CD25-specific small molecule probes is useful for in vivo, non-invasive and quantitative evaluation. The distribution and activation status of T cells in the body are of great significance.
  • the present invention provides a class of CD25-targeting polypeptides and CD25-targeting polypeptides. Peptide imaging agents and their applications.
  • a first aspect of the present invention provides a CD25 targeting polypeptide, the amino acid sequence of the polypeptide is as follows:
  • X1 is arginine, lysine or histidine
  • X2 is phenylalanine, tyrosine, benzyltyrosine or halogenated phenylalanine
  • Histidine is a member of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of phenylalanine and histidine
  • X2 is phenylalanine, tyrosine, benzyltyrosine or halogenated phenylalanine
  • Histidine is phenylalanine, tyrosine, benzyltyrosine or halophenylalanine
  • At least one amino acid in the polypeptide is in D form.
  • the remaining amino acid residues can be L-form, D-form, or a mixture of L-form and D-form.
  • X1 and X5 are in L form, and at least one of the remaining amino acids is in D form.
  • 1-2 amino acids are in the D-form, and the rest are in the L-form.
  • one of X3 and X4 is in D form, and the remaining amino acids are in L form.
  • the label usually refers to a protecting group, preferably a hydroxyl protecting group, such as benzyl.
  • a second aspect of the present invention provides a molecular probe, which is the CD25-targeting polypeptide labeled with a radioactive isotope; the radioactive isotope is directly labeled on the CD25-targeting polypeptide, or through -L -Ch is labeled on the CD25 targeting polypeptide, where L is a bridging group and Ch is a nuclide chelating group. Radioactive isotopes are bound to nuclide chelating groups.
  • the molecular probe can be prepared by a method comprising the following steps: connecting the CD25 targeting polypeptide directly or by covalently linking a radionuclide or a group containing a radionuclide.
  • -L-Ch can be covalently linked to the N-terminus, C-terminus or reactive side chain of the CD25 targeting polypeptide.
  • the reactive side chain refers to the reactive group on the side chain of the CD25 targeting polypeptide, such as amino group and carboxyl group. Amino acids with similar properties such as lysine.
  • X3 is lysine
  • the -L-Ch is covalently connected to X3.
  • the bridging group is derived from an oligopeptide or NH 2 -(PEG) n -COOH. More preferably, the number of amino acids of the oligopeptide is 1-4, and n is 1-4. integer.
  • the nuclide chelating group is usually a group formed by a bifunctional chelating agent.
  • the bifunctional chelating agent can be selected from DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A/NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3 or isonitrile.
  • the radionuclide is preferably a diagnostic radionuclide; specifically, the diagnostic radionuclide is 68 Ga, 64 Cu, 18 F, 86 Y, 90 Y, 89 Zr, 111 In, 99 At least one of mTc, 11 C, 123 I, 125 I and 124 I.
  • the third aspect of the present invention provides the use of the above-mentioned molecular probe in preparing radionuclide imaging diagnostic reagents.
  • the radionuclide imaging diagnostic reagent of the present invention is preferably used as a CAR-T cell efficacy evaluation reagent or treatment tracking reagent with high CD25 expression.
  • the invention provides a CD25-targeting polypeptide and its radionuclide-labeled probe which have high affinity and specific targeting to CD25, high stability in vivo and in vitro, high radionuclide labeling rate and radioactive chemical purity, and rapid clearance. Excellent metabolic properties in the body.
  • the CD25 polypeptide and its radionuclide labeled probe provided by the present invention have obvious cellular uptake in activated human peripheral blood mononuclear cells (hPBMC) with high CD25 expression. In tumor model mice with high CD25 expression, the uptake at the tumor site is high, and the tumor This ratio is clear and effectively improves the signal-to-noise ratio in the imaging diagnosis process. It has imaging guidance significance and application potential in the early prediction of CAR-T efficacy and the development of CAR-T therapy.
  • Figures 1-1 to 1-9 show mass spectra of CD25-targeting polypeptide ligands.
  • Figure 2 shows the results of Radio-HPLC analysis before and after DA-1 probe 68 Ga labeling.
  • Figures 3-1 to 3-3 show the in vivo and in vitro stability results of 68 Ga-DA-3, 68 Ga-DA-4, and 68 Ga-DA-6.
  • Figure 4 shows the immunohistochemical staining results of tumor tissue in B16F10 tumor-bearing mice at different days after vaccination.
  • Figure 5 shows the flow cytometric detection results of CD25 expression in Karpas299 (human large cell lymphoma).
  • Figure 6 shows a comparison of the uptake of 68 Ga-DA-1 probe in hPBMC before and after activation. *p ⁇ 0.05, **p ⁇ 0.01, ****p ⁇ 0.0001.
  • Figure 7 shows the small animal PET/CT dynamic imaging results of the 68 Ga-DA-1 probe applied to the hPBMC mouse model activated by subcutaneous inoculation.
  • the white arrow indicates the hPBMC inoculation site.
  • Figure 8 shows the small animal PET/CT dynamic imaging results of the 68 Ga-DA-1 probe applied to the mouse model of subcutaneous inoculation of unactivated hPBMC.
  • the white arrow indicates the hPBMC inoculation site.
  • Figure 9 shows a comparison of probe uptake changes in small animal PET/CT dynamic imaging of a mouse model inoculated subcutaneously with unactivated hPBMC using the 68 Ga-DA-1 probe.
  • Figure 10 shows a comparison of the uptake of 68 Ga-DA-6 probe in hPBMC before and after activation. *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 11 shows the small animal PET/CT imaging results of 68 Ga-labeled DA series probes applied to B16F10 tumor-bearing mice.
  • the white arrow indicates the tumor location.
  • Figure 12 shows the results of small animal PET/CT imaging of 68 Ga-labeled DA series probes applied to Karpas299 tumor-bearing mice.
  • the white arrow indicates the tumor location.
  • Figure 13 shows the pharmacokinetics of 68 Ga-DA-4, 68 Ga-DA-6, and 68 Ga-DA-7 in the blood of KM mice.
  • Figure 14 shows the biodistribution of 68 Ga-DA-4 and 68 Ga-DA-6 probes in KM mice.
  • Figure 15 shows the small animal PET imaging results of 68 Ga-DA-6 probe in Karpas299 and U87MG tumor-bearing mice.
  • Figure 16 shows the biodistribution of 68 Ga-DA-6 probe in Karpas299 and U87MG tumor-bearing mice. ****p ⁇ 0.0001.
  • the synthesis route of 9 ligands targeting CD25 is as follows: the amino acid sequence is produced through solid-phase synthesis from the C-terminus to the N-terminus. First, the 2-CTC resin is activated, then the first amino acid is connected, the amino acid is deprotected by Fmoc, washed, and condensed. Couple the second amino acid, repeat the above operation to couple different amino acids in the polypeptide chain in sequence, then couple the PEG bridge chain, then couple the chelating agent to the polypeptide, and finally use the lysis solution to couple the constructed CD25-targeting polypeptide
  • the target probe was obtained by cutting and purifying with preparative high-performance liquid chromatography, and its structure was identified by mass spectrometry.
  • Figures 1-1 to 1-9 illustrate CD25-targeting polypeptide ligands DA-1, DA-3, DA-4, DA-5, DA-6, DA-7, DA-8, DA-9, DA -10 mass spectrum.
  • -(PEG)2-RFrFY, SEQ ID NO: 3 The preparation of -(PEG)2-RFrFY, SEQ ID NO: 3) is taken as an example.
  • the other peptides can be prepared according to the same method.
  • Resin swelling Put 2-CTC resin (50 mg, 0.015 mmol ⁇ 0.15 mmol) into the reaction tube, add dichloromethane (4 mL), and shake for 30 minutes.
  • the first amino acid connection filter out the solvent through a sand core, add a 2-fold molar excess of N-fluorenylmethoxycarbonyl-L-glutamic acid- ⁇ -tert-butyl ester monohydrate (133 mg, 0.30 mmol), and add Dissolve DMF, then add a 10-fold molar excess of DIEA (261 ⁇ L, 1.50 mmol), shake for 60 min, and block with methanol.
  • Resin swelling Put 2-CTC resin (50 mg, 0.015 mmol ⁇ 0.15 mmol) into the reaction tube, add dichloromethane (4 mL), and shake for 30 minutes.
  • Second Connection of the first amino acid Filter out the solvent through a sand core, add a 2-fold molar excess of Fmoc-O-tert-butyl-L-tyrosine (137.70 mg, 0.30 mmol), add DMF to dissolve, and then add 10 times more A molar excess of DIEA (261 ⁇ L, 1.50 mmol) was added, shaken for 60 min, and blocked with methanol.
  • the 68 Ga labeling of DA series peptide probes uses the following method: dissolve the peptide probe precursor in ultrapure water (1mg/mL), aliquot and store at -20°C. Elute the germanium and gallium generator with 4 mL of 0.05 M HCl. After collecting, 3 mL of the eluate was placed in a 4 mL EP tube, and 185 ⁇ L of NaAc solution (1 M) was added to it. Add 10 ⁇ L of peptide probe precursor into the system, and heat the reaction at 95°C for 15 minutes.
  • FIG. 2 shows the Radio-HPLC analysis results of the DA-1 probe precursor and final product.
  • the retention time of the DA-1 precursor is 13.141 min (blue peak), and the emission peak of 68 Ga-DA-1 is 13.379 min (red peak).
  • the lipid-water partition coefficient was measured using the following method: mix 500 ⁇ L of n-octanol with 490 ⁇ L of PBS solution, add 10 ⁇ L of labeled probe solution, then vortex to mix, centrifuge at 5000 rpm ⁇ 3 min, and take n-octanol and water respectively. Add 10 ⁇ L of phase to a counting tube and measure the radioactivity count. Three samples were taken from each sample, and each probe was measured three times. The lipid-water distribution coefficient was expressed by LogD 7.4 .
  • B16F10 tumor-bearing mice were sacrificed on the 7th, 14th, and 21st days after tumor inoculation.
  • the tumor tissues were removed, dehydrated and fixed with 10% formalin solution, and then immunohistochemistry experiments were performed. The method was as follows: the tumor tissues were dehydrated and then wrapped in paraffin.
  • Buried, paraffin sections were deparaffinized in xylene for 20 min 75% alcohol for 5 minutes, distilled water for 1 minute; soak in 3% H 2 O 2 for 10 minutes; wash with PBS for 5 minutes ⁇ 3 times; microwave antigen retrieval (450W, 10 minutes) in 0.01M citric acid buffer (pH 6.0), time allocation 3 minutes, 3 minutes, 4 minutes, let cool at room temperature; wash with PBS 5min ⁇ 3 times; block with 0.5% skimmed milk powder for 1h; add primary antibody, incubate in a humidified box at 4°C overnight; take out the humidified box the next day, and rewarm at room temperature 20 min, wash with PBS 5 min ⁇ 3 times; add secondary antibody and incubate in a humid box at room temperature for 20 minutes; wash with PBS 5 min ⁇ 3 times; DAB color development, depending on the situation, should not exceed 3-5 minutes.
  • CD25 shows the application value of B16F10 tumor-bearing mice in evaluating the specificity of CD25 peptide probes. Since the tumor load of B16F10 tumor was too large in the late stage of inoculation, the CD25-positive Karpas299 model was used to evaluate the imaging effect of the peptide probe in the imaging experiment.
  • the CD25 expression level of Karpaspas299 cells was determined by flow cytometry.
  • the experimental process is as follows:
  • Cell preparation Centrifuge cells in a 15mL centrifuge tube at 1500r ⁇ 5min, and then wash once with 5mL PBS. The number of cells prepared is 1 ⁇ 10 7 , which is sufficient for use.
  • Cell staining Resuspend the cells in PBS to a suspension of 1 ⁇ 10 7 /mL (do not use a pipette). Take 100 ⁇ L, or 1 ⁇ 10 6 cells, and place them in a flow tube. Divide them into three groups and add them respectively. Mix 5 ⁇ L of PE Anti-Human CD25 antibody, 5 ⁇ L of PE Mouse IgG1k isotype antibody, and blank control, and place at 4°C away from light for 30 minutes.
  • On-machine detection Add 500 ⁇ L of PBS to resuspend the cells, mix well, and then on-machine detection. Note: Non-single suspension cells need to be filtered first.
  • hPBMC human monocyte mononuclear cells
  • PMA 20ng/mL + Ionomycin (1ng/mL)
  • stimulated culture for 8 hours and set unactivated hPBMC as a control.
  • Activated and non-activated hPBMC were mixed with an equal volume of Matrigel (200 ⁇ L, 3 ⁇ 10 6 /mouse) and inoculated subcutaneously into the right shoulder of KM mice. 20 minutes after inoculation, perform isoflurane anesthesia on the animal bed, inject 200 ⁇ Ci of 68 Ga-DA-1 probe through the tail vein, and then perform dynamic PET/CT imaging immediately after the injection to observe the 68 Ga-DA-1 probe. The clustering of needles at the site of vaccination. Outline the ROI and measure the SUVmax of the hPBMC inoculation site and muscle tissue.
  • mice inoculated with activated hPBMC had a continuous increase in target-to-book ratio within 30 minutes of imaging, reaching a maximum of 1.88 at 30 minutes.
  • the highest target-to-book ratio was only 1.11 at 30 minutes ( Figure 9).
  • the B16F10 tumor-bearing mouse model was used to examine the in vivo imaging effects of some peptide probes.
  • the probes 68 Ga-DA-4 and 68 Ga-DA-5 have the highest relative sensitivity in tumors. Uptake, but it contains lysine in its structure, so renal uptake was seen to be significantly higher than other probes.
  • the renal SUVmax of probes such as 68 Ga-DA-6 and 68 Ga-DA-7 decreased significantly, and the muscle background uptake was also relatively low, making 68 Ga-DA -6 and 68 Ga-DA-7 probes can achieve higher tumor-to-tumor ratios during imaging.
  • the Karpas299 tumor-bearing mouse model was used to evaluate the in vivo imaging effects of multiple probes. As shown in Figure 12, significantly reduced renal uptake was also seen with probes 68 Ga-DA-6 and 68 Ga-DA-7, in which lysine was replaced with arginine. It also has relatively lower background uptake and improved target-to-book ratio, making PET/CT imaging quality superior to several other probes.
  • the tumor-to-tumor ratios of 68 Ga-DA-6 and 68 Ga-DA-7 can reach 3.63 and 3.86 respectively (Table 4).
  • the following method was used to study the pharmacokinetics of 68 Ga-labeled peptide probes: 5 female KM mice were taken, and each mouse was injected with 3.7MBq of radiopharmaceuticals through the tail vein at 1 min, 3 min, 5 min, and 10 min after injection. , 15min, 30min, 45min, 1h, 1.5h, 2h, 3h and 4h. Take blood from the orbital vein, weigh it and use a gamma counter to measure its radioactivity count. Set a 1% marker as a reference to calculate the percentage of a single gram of blood. The injection dose rate was recorded as %ID/g, and GraphPad Prism 8.3.0 software was used for nonlinear regression fitting to study its pharmacokinetic characteristics. In the experiment, the pharmacokinetics of a total of three probes, 68 Ga-DA-4, 68 Ga-DA-6 and 68 Ga-DA-7, were investigated.
  • the following method was used to study the biodistribution of 68 Ga-labeled probes in KM mice: 15 female KM mice were taken, and each mouse was injected with 1.11MBq of radiopharmaceuticals through the tail vein at 5min, 30min, 1h, 2h and After 4 hours of sacrifice, the blood, heart, liver, spleen, lungs, kidneys, stomach, small intestine, large intestine, muscles, bones and brain and other organs and tissues were collected, weighed and their radioactivity counts were measured using a gamma counter, and a 1% marker was set as For reference, calculate the percentage injection dose rate of a single gram of tissue, recorded as %ID/g, and examine the distribution characteristics of 68 Ga-DA-4, 68 Ga-DA-6 and probes in KM mice.
  • the metabolic characteristics of the short peptide probes in KM mice are very similar, and both are rapidly cleared through the kidneys.
  • the probes were cleared rapidly in the blood, and the probe uptake in the blood of 68 Ga-DA-4 and 68 Ga-DA-6 dropped by more than 80.4% and 88.6% respectively 30 minutes after injection. And within 30 minutes, the probe has basically reached the equilibrium stage of distribution and clearance, and within 2 hours after injection, the probe uptake level of each organ has basically remained stable.
  • the 68 Ga-DA-6 probe has a relatively significant uptake in the tumor site of Karpas299 tumor-bearing mice ( Figure 15), with an SUVmax of 0.29 and a tumor This ratio T/M was 3.63, and in the tumors of U87MG tumor-bearing mice, probe uptake was significantly reduced, with an SUVmax of 0.16.
  • the probe uptake at the tumor site of Karpas299 tumor-bearing mice also dropped to 0.17.
  • Karpas299 and U87MG tumor-bearing mice were injected with 1.11MBq of 68 Ga-DA-6 probe through the tail vein respectively, and then sacrificed 1 hour after injection.
  • Blood, heart, liver, spleen, lung, kidney, stomach, small intestine, Organs and tissues such as the large intestine, muscles, bones, brain, and tumors are weighed and their radioactivity counts are measured using a gamma counter.
  • a 1% marker is set as a reference to calculate the percent injection dose rate per gram of tissue, recorded as %ID/ g, Examine the distribution characteristics of 68 Ga-DA-6 probe in Karpas299 and U87MG tumor-bearing mice.
  • the 68 Ga-DA-6 probe had a relatively high uptake in tumors of 1.27 ⁇ 0.04% ID/g, while the uptake in U87MG tumors was only 0.54 ⁇ 0.06% ID/g (p ⁇ 0.0001).
  • the uptake of 68 Ga-DA-6 probe in the tumors of Karpas299 tumor-bearing mice was also significantly reduced, only 0.26 ⁇ 0.02% ID. /g(p ⁇ 0.0001).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供一种CD25靶向多肽、分子探针及应用。该多肽的氨基酸序列如下：i)X1X2X3X4X5；或ii)在i)的N端或C端连接标签形成的氨基酸序列；其中，X1为精氨酸、赖氨酸、组氨酸；X2为苯丙氨酸、酪氨酸、苄基酪氨酸、卤代苯丙氨酸；X3为精氨酸、赖氨酸、组氨酸；X4为苯丙氨酸、酪氨酸、苄基酪氨酸、卤代苯丙氨酸；X5为苯丙氨酸、酪氨酸、苄基酪氨酸、卤代苯丙氨酸。

Description

一种CD25靶向多肽、分子探针及应用 技术领域
本发明属于核医学显像剂领域，具体地，涉及一类可用于靶向CAR-T细胞CD25靶点的多肽类衍生物、分子探针及其制备方法和应用
背景技术
CAR-T(Chimeric Antigen Receptor T Cell)治疗，即嵌合抗原受体T细胞疗法，是目前临床肿瘤治疗的研究前沿热点。据Clinical Trials官网统计，世界范围内有数百项的CAR-T临床试验正在开展之中，其中不仅包括以CD19、CD20等为靶点的血液系统肿瘤CAR-T疗法，还包括靶向间皮素(Mesothelin，MSLN)、人表皮生长因子受体-2(HER-2)等靶点的实体瘤CAR-T疗法。
尽管在临床试验中取得了巨大成功，但在部分患者体内，CAR-T细胞的存活和持续扩增能力有限，或者产生过度的克隆性增殖，这对CAR-T的疗效和安全性评价提出了巨大挑战。目前，与CAR-T细胞的增殖和良好的临床反应相关的预测指标和机制尚未完全清楚，并且有研究表明，CAR-T的疗效与患者年龄、早期治疗、p53基因突变、CAR-T细胞数量和其它风险因素并无显著相关性。但在预后良好的绝大部分患者中，回输CAR-T细胞的两周内伴随着CAR-T细胞大量激活扩增的现象。此外，CAR-T治疗的副作用包括细胞因子释放综合征，神经系统毒性以及脱靶效应等，都与CAR-T细胞在体内的激活扩增有关。因此，评价CAR-T细胞的激活与扩增，有望实现对于CAR-T疗效的早期预测，并在指导CAR-T疗法的开发中起到一定的作用。
现阶段临床评价患者对于CAR-T治疗的响应通常采用全血细胞计数和骨髓穿刺活检等手段，往往给患者带来较大的痛苦，且不能充分评价CAR-T细胞在体内的分布情况和激活状态。目前国内外在评价T细胞激活状态的研究中通常采用长半衰期核素如 89Zr、 64Cu等标记抗体分子进行免疫显像，显像周期较长，且在CAR-T细胞激活增殖后信号被稀释，因此限制了其应用。
CD25是白介素2(interleukin-2，IL-2)受体的α链，其在未激活的T细胞表面几乎没有表达，但在T细胞经抗原刺激激活之后大量表达。因此，CD25是优异的活化T细胞特异性显像靶点。在目前的研究中，利用各种放射性核素如 68Ga、 18F标记IL-2分子进行CD25显像是较为成熟的技术。但是由于IL-2是一种生物活性分子，其经静脉注射进入人体后会产生一定的生物学效应，且在注射后可能会给患者带来疼痛、发热等不适感。且由于IL-2分子量相对较大，其在体内的清除速度相对较为缓慢，利用短半衰期核素标记IL-2分子，难以在短时间内获得优质的显像效果。小分子探针具有体内代谢清 除快、脏器本底摄取低、生物安全性高、易于进行放射性标记、成本低等优点，设计CD25特异性的小分子探针，对于在体、无创和定量评价T细胞的体内分布及激活状态具有重要意义。
发明内容
为了克服现阶段CAR-T显像探针通常为长半衰期核素标记的各类抗体分子，显像周期较长且存在信号稀释的缺陷，本发明提供了一类靶向CD25多肽、靶向CD25多肽显像剂及其应用。
本发明的第一方面提供一种CD25靶向多肽，所述多肽的氨基酸序列如下：
i)X1X2X3X4X5；或
ii)在i)的N端或C端连接标签形成的氨基酸序列；
其中，X1为精氨酸、赖氨酸或组氨酸；X2为苯丙氨酸、酪氨酸、苄基酪氨酸或卤代苯丙氨酸；X3为精氨酸、赖氨酸或组氨酸；X4为苯丙氨酸、酪氨酸、苄基酪氨酸或卤代苯丙氨酸；X5为苯丙氨酸、酪氨酸、苄基酪氨酸或卤代苯丙氨酸。
根据本发明一种优选实施方式，所述多肽中至少有一个氨基酸为D型。其余氨基酸残基可以是L-型，也可以是D-型，或者是L-型、D-型的混合。
更优选地，X1、X5为L型，其余氨基酸中至少有一个为D型。
根据本发明一些实施方式，有1-2个氨基酸为D型，其余均为L型。具体地，X3和X4中之一为D型，其余氨基酸均为L型。
本发明的CD25靶向多肽中，所述标签通常指保护基，优选为羟基保护基，例如苄基。
本发明的第二方面提供一种分子探针，所述分子探针为具有放射性同位素标记的所述CD25靶向多肽；所述放射性同位素直接标记在所述CD25靶向多肽上，或者通过-L-Ch标记在所述CD25靶向多肽上，其中L为桥接基团，Ch为核素螯合基团。放射性同位素与核素螯合基团结合。
所述分子探针可由包括以下步骤的方法制得：使所述CD25靶向多肽直接或通过共价连接放射性核素或含有放射性核素的基团。
具体地，-L-Ch可共价连接在所述CD25靶向多肽的N端、C端或反应性侧链上。所述反应性侧链是指所述CD25靶向多肽的侧链上的反应性基团，如氨基、羧基。具有类似性质的氨基酸如赖氨酸。根据本发明一种具体实施方式，当X3为赖氨酸时，所述-L-Ch共价连接在X3上。
根据本发明一种优选实施方式，所述桥接基团衍生自寡肽或NH 2-(PEG) n-COOH，更优选地，所述寡肽的氨基酸数目为1-4，n为1-4的整数。
所述核素螯合基团通常为双功能螯合剂形成的基团，所述双功能螯合剂可选自DOTA、NOTA、NODA、NODAGA、DOTP、TETA、ATSM、PTSM、EDTA、EC、 HBEDCC、DTPA、SBAD、BAPEN、Df、DFO、TACN、NO2A/NOTAM、CB-DO2A、Cyclen、NOTA-AA、DO3A、DO3AP、HYNIC、MAS3、MAG3或异腈。
根据本发明，所述放射性核素优选为诊断用放射性核素；具体地，所述诊断用放射性核素为 68Ga、 64Cu、 18F、 86Y、 90Y、 89Zr、 111In、 99mTc、 11C、 123I、 125I和 124I中的至少一种。
本发明的第三方面提供上述分子探针在制备核素成像诊断试剂中的应用。
本发明所述核素成像诊断试剂优选作为CD25高表达的CAR-T细胞疗效评价试剂或治疗追踪试剂。
本发明提供的一种靶向CD25多肽及其核素标记探针对CD25具有高亲和力和特异靶向性，体内外具有高稳定性，具有高的核素标记率和放射性化学纯度，具有快速清除的优良体内代谢性质。本发明提供的CD25多肽及其核素标记探针在CD25高表达的激活的人外周血单核细胞(hPBMC)具有明显的细胞摄取，在CD25高表达的肿瘤模型鼠中肿瘤部位摄取高，瘤本比清晰，有效提高了在影像诊断过程中的信噪比，在CAR-T疗效的早期预测，CAR-T疗法的开发中具有影像学指导意义和应用潜力。
本发明的其它特征和优点将在随后具体实施方式部分予以详细说明。
附图说明
通过结合附图对本发明示例性实施方式进行更详细的描述，本发明的上述以及其它目的、特征和优势将变得更加明显。
图1-1至1-9示出了靶向CD25多肽配体的质谱图。
图2示出了DA-1探针 68Ga标记前后的Radio-HPLC分析结果。
图3-1至图3-3示出了 68Ga-DA-3、 68Ga-DA-4、 68Ga-DA-6的体内外稳定性结果图。
图4示出了B16F10荷瘤小鼠在接种不同天数后肿瘤组织免疫组织化学染色结果。
图5示出了Karpas299(人大细胞淋巴瘤)的CD25表达流式细胞术检测结果。
图6示出了 68Ga-DA-1探针在激活前后的hPBMC中的摄取对比。*p<0.05，**p<0.01，****p<0.0001。
图7示出了 68Ga-DA-1探针应用于皮下接种激活hPBMC小鼠模型的小动物PET/CT动态显像结果，白色箭头指示为hPBMC接种部位。
图8示出了 68Ga-DA-1探针应用于皮下接种未激活hPBMC小鼠模型的小动物PET/CT动态显像结果，白色箭头指示为hPBMC接种部位。
图9示出了 68Ga-DA-1探针应用于皮下接种未激活hPBMC小鼠模型的小动物PET/CT动态显像的探针摄取变化对比。
图10示出了 68Ga-DA-6探针在激活前后的hPBMC中的摄取对比。*p<0.05，**p<0.01。
图11示出了 68Ga标记的DA系列探针应用于B16F10荷瘤小鼠的小动物PET/CT 显像结果，白色箭头指示为肿瘤位置。
图12示出了 68Ga标记的DA系列探针应用于Karpas299荷瘤小鼠的小动物PET/CT显像结果，白色箭头指示为肿瘤位置。
图13示出了 68Ga-DA-4、 68Ga-DA-6、 68Ga-DA-7在KM小鼠血液中的药物代谢动力学。
图14示出了 68Ga-DA-4和 68Ga-DA-6探针在KM小鼠体内的生物分布。
图15示出了 68Ga-DA-6探针在Karpas299及U87MG荷瘤鼠的小动物PET显像结果。
图16示出了 68Ga-DA-6探针在Karpas299及U87MG荷瘤鼠的生物分布。****p<0.0001。
具体实施方式
下面将更详细地描述本发明的优选实施方式。虽然以下描述了本发明的优选实施方式，然而应该理解，可以以各种形式实现本发明而不应被这里阐述的实施方式所限制。
实施例中未注明具体条件者，皆按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
靶向CD25多肽配体的合成制备。
表1各多肽配体的结构式、化学式和分子量
Figure PCTCN2022114251-appb-000001
Figure PCTCN2022114251-appb-000002
9种靶向CD25的配体合成路线如下：氨基酸序列从C-端到N-端经过固相合成制得，首先将2-CTC树脂活化，然后连接首位氨基酸，氨基脱Fmoc保护，清洗，缩合偶联第二个氨基酸，重复以上操作依次将多肽链中的不同氨基酸进行偶联，然后偶联PEG桥链，随后将螯合剂偶联到多肽，最后通过裂解液将所构建的CD25靶向多肽切割、经制备型高效液相色谱仪纯化制得目标探针，结构经质谱鉴定。图1-1至1-9示出了靶向CD25多肽配体DA-1、DA-3、DA-4、DA-5、DA-6、DA-7、DA-8、DA-9、DA-10的质谱图。
下面，以DA-1(RFK(GGG-DOTA)FY[OBn]，SEQ ID NO：1)；DA-3(DOTA-(PEG)2-RFKFYE，SEQ ID NO：2)；DA-6(DOTA-(PEG)2-RFrFY，SEQ ID NO：3)的制备为例进行说明，其余多肽按照相同方法制备即可。
实施例1
DA-1 RFK(GGG-DOTA)FY[OBn]的制备；
Figure PCTCN2022114251-appb-000003
(一)树脂溶胀，将2-CTC树脂(50mg，0.015mmol～0.15mmol)放入反应管中，加DCM(4mL)，振荡30min。(二)接第一个氨基酸，通过沙芯抽滤掉溶剂，加入2倍摩尔过量的Fmoc-L-Tyr(Bzl)-OH(148mg，0.30mmol)，加入DMF溶解，再加入10倍摩尔过量DIEA(261μL，1.50mmol)，振荡60min。用甲醇封闭。(三)脱保护，去掉甲醇，加20％哌啶DMF溶液(3mL)，5min，去掉再加20％哌啶DMF溶液(3mL)，15min。(四)检测，抽掉哌啶溶液，取十几粒树脂，用乙醇洗三次，加入检测试剂检测，105℃-110℃加热5min，变深蓝色为阳性反应。(五)清洗，DMF(4mL)两次，DCM(4mL)两次，DMF(4mL)两次。(六)缩合，加入下一个Fmoc保护氨基酸Fmoc-L-Phe-OH(116mg，0.30mmol)两倍过量，TBTU(145mg，0.45mmol)三倍过量，DMF(3mL)溶解，加入反应管，立刻加入DIEA(261μL，1.50mmol)十倍过量，反应30min。(七)检测，取十几粒树脂，用乙醇洗三次，加入检测试剂检测，105℃-110℃加热5min，无色为阴性反应。(八)清洗，DMF(10mL/g)一次，DCM(10mL/g)两次，DMF(10mL/g)两次。(九)重复三至八步操作，依次连接序列中氨基酸及螯合剂Fmoc-L-Lys(Dde)-OH，Fmoc-D-Phe-OH，Boc-L-Arg(Pbf)-OH，Fmoc-甘氨酸，Fmoc-甘氨酸，Fmoc-甘氨酸，三叔丁基1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸。(十)抽干，按照下列方法洗树脂。DMF(4mL)两次，甲醇(4mL)两次，DMF(4mL)两次，DCM(4mL)两次，抽干10min。(十一)从树脂上切割多肽，配制切割液(5mL)TFA 95％；水1％；EDT 2％；TIS 2％进行切割。切割时间：120min。(十二)分析提纯：旋干，用高效液相色谱将粗品提纯。(十三)冻干，收集目标多肽溶液放入冻干机中进行浓缩，冻干成白色粉末制得DA-1。MS(m/z)：calc.for C 68H 94N 16O 17[M-H] -1405.7056，found 1405.40。
实施例2
DA-3 DOTA-(PEG)2-RFKFYE的制备
Figure PCTCN2022114251-appb-000004
(一)树脂溶胀：将2-CTC树脂(50mg，0.015mmol～0.15mmol)放入反应管中，加二氯甲烷(4mL)，振荡30min。(二)首位氨基酸连接：通过沙芯抽滤掉溶剂，加入2倍摩尔过量的N-芴甲氧羰基-L-谷氨酸-γ-叔丁酯一水物(133mg，0.30mmol)，加入DMF溶解，再加入10倍摩尔过量DIEA(261μL，1.50mmol)，振荡60min，用甲醇封闭。(三)脱保护：抽去甲醇，加20％哌啶DMF溶液(3mL)，搅拌5min，抽去溶剂后再加20％哌啶DMF溶液(3mL)，搅拌15min。(四)检测：抽掉哌啶溶液，取十几粒树脂，用乙醇洗三次，加入检测试剂检测，105℃-110℃加热5min，变深蓝色为阳性反应。(五)清洗：DMF(4mL)两次，DCM(4mL)两次，DMF(4mL)两次。(六)缩合：加入2倍当量的Fmoc-O-叔丁基-L-酪氨酸(137.70mg，0.30mmol)，3倍当量的TBTU(145mg，0.45mmol)，DMF(3mL)溶解，加入反应管，立刻加入10倍当量的DIEA(261μL，1.50mmol)，反应30min。(七)检测：取十几粒树脂，用乙醇洗三次，加入检测试剂检测，105℃-110℃加热5min，无色为阴性反应。(八)清洗：DMF(10mL/g)一次，DCM(10mL/g)两次，DMF(10mL/g)两次。(九)重复三至八步操作，依次连接序列中氨基酸及螯合剂Fmoc-L-苯丙氨酸，N-芴甲氧羰基-N’-叔丁氧羰基-L-赖氨酸，Fmoc-L-苯丙氨酸，Fmoc-Pbf-L-精氨酸，Fmoc-9-氨基-4,7-二噁壬酸，三叔丁基1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸。(十)抽干，按照下列方法洗树脂。DMF(4mL)两次，甲醇(4mL)两次，DMF(4mL)两次，DCM(4mL)两次，抽干10min。(十一)多肽切割：配制5mL切割液(TFA 95％+水1％+EDT 2％+TIS 2％)进行切割，切割时间为120min。(十二)分析提纯：旋干，用高效液相色谱将粗品提纯。(十三)冻干：收集目标多肽溶液放入冻干机中进行浓缩，冻干成白色粉末，即得探针DA-3，质谱鉴定：MALDI-TOF calc.for C 67H 99N 15O 20[M+H] +1434.726，found 1434.746。
实施例3
DA-6 DOTA-(PEG)2-RFrFY的制备：
Figure PCTCN2022114251-appb-000005
(一)树脂溶胀：将2-CTC树脂(50mg，0.015mmol～0.15mmol)放入反应管中，加二氯甲烷(4mL)，振荡30min。(二)首位氨基酸连接：通过沙芯抽滤掉溶剂，加入2倍摩尔过量的Fmoc-O-叔丁基-L-酪氨酸(137.70mg，0.30mmol)，加入DMF溶解，再加入10倍摩尔过量DIEA(261μL，1.50mmol)，振荡60min，用甲醇封闭。(三)脱保护：抽去甲醇，加20％哌啶DMF溶液(3mL)，搅拌5min，抽去溶剂后再加20％哌啶DMF溶液(3mL)，搅拌15min。(四)检测：抽掉哌啶溶液，取十几粒树脂，用乙醇洗三次，加入检测试剂检测，105℃-110℃加热5min，变深蓝色为阳性反应。(五)清洗：DMF(4mL)两次，DCM(4mL)两次，DMF(4mL)两次。(六)缩合：加入2倍当量Fmoc-L-苯丙氨酸(116mg，0.30mmol)，3倍当量的TBTU(145mg，0.45mmol)，DMF(3mL)溶解，加入反应管，立刻加入10倍当量的DIEA(261μL，1.50mmol)，反应30min。(七)检测：取十几粒树脂，用乙醇洗三次，加入检测试剂检测，105℃-110℃加热5min，无色为阴性反应。(八)清洗：DMF(10mL/g)一次，DCM(10mL/g)两次，DMF(10mL/g)两次。(九)重复三至八步操作，依次连接序列中氨基酸及螯合剂N'-[(2,3-二氢-2,2,4,6,7-五甲基苯并呋喃-5-基)磺酰基]-N-芴甲氧羰基-D-精氨酸，Fmoc-L-苯丙氨酸，Fmoc-Pbf-L-精氨酸，Fmoc-9-氨基-4,7-二噁壬酸，三叔丁基1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸。(十)抽干，按照下列方法洗树脂。DMF(4mL)两次，甲醇(4mL)两次，DMF(4mL)两次，DCM(4mL)两次，抽干10min。(十一)多肽切割：配制5mL切割液(TFA 95％+水1％+EDT 2％+TIS 2％)进行切割，切割时间为120min。(十二)分析提纯：旋干，用高效液相色谱将粗品提纯。(十三)冻干：收集目标多肽溶液放入冻干机中进行浓缩，冻干成白色粉末，即得探针DA-6，质谱鉴定：MALDI-TOF calc.for C 62H 92N 16O 17[M+H] +1333.690，found 1333.695。
实施例4
多肽探针的 68Ga标记
DA系列多肽探针的 68Ga标记采用如下方法：将多肽探针前体溶解于超纯水(1mg/mL)，分装后置于-20℃保存。以4mL的0.05M的HCl淋洗锗镓发生器，收集后3mL淋洗液置于4mL的EP管中，并向其中补充185μL的NaAc溶液(1M)。取10μL多肽探针前体加入体系中，在95℃下加热反应15min。反应结束后，向体系中补充 2mL纯水并抽出，使用预先活化的Sep-Pak Light C18纯化柱进行纯化，并用5mL纯水冲洗。然后以0.5mL的80％乙醇淋洗C18纯化柱，并向其中补充5mL的生理盐水，并经0.2μm的微孔滤膜过滤后得到可供实验使用的放射性药物。
68Ga标记多肽探针的质控：
68Ga标记配合物的放射化学纯度使用Radio-HPLC分析验证，以 68Ga-DA-1为例，图2为DA-1探针前体及最终产品的Radio-HPLC分析结果。DA-1前体的保留时间为13.141min(蓝色峰)， 68Ga-DA-1的放射峰为13.379min(红色峰)。
结果显示所有探针的标记率均>90％，经C18纯化柱纯化后，所有放射性药物产品的放射性化学纯度>99％。
实施例5
68Ga标记的多肽探针与CD25的亲和力及脂水分配系数测定
多肽探针的亲和力测定利用Karpas299细胞进行饱和实验来获得：将Karpas299细胞计数后放入48孔板(5×10 5/孔)，分别向其中加入固定体积的 68Ga标记探针，活度依次为0.37kBq/孔、1.85kBq/孔、3.7kBq/孔、18.5kBq/孔、37kBq/孔、74kBq/孔、185kBq/孔、370kBq/孔、740kBq/孔、1850kBq/孔、37000kBq/孔、74000kBq/孔(n＝3)，在37℃孵育1h后收集细胞至1.5mL EP管，离心一次后再用1mL预冷的PBS清洗两次，最后用200μL的PBS将细胞重悬后转移至计数管，利用γ计数器测定每孔细胞的放射性计数，利用GraphPad Prism 8.3.0软件进行拟合，计算探针的亲和力Kd值。
脂水分配系数测定采用如下方法：取500μL的正辛醇与490μL的PBS溶液混合，向其中加入10μL标记好的探针溶液，然后涡旋混匀，5000rpm×3min离心，分别取正辛醇和水相10μL，置于计数管，测定其放射性计数。每个样品分别取3个样，每个探针重复测定3次，脂水分配系数采用LogD 7.4来表示。
多肽探针与CD25的亲和力及脂水分配系数测定结果如表2所示，结果显示它们都具有较佳的水溶性，体内主要经由肾脏排泄。此外，亲和力测定结果显示，DA-6探针具有相对较高的亲和力，Kd值约为89.42nM。
表2多肽探针与CD25的亲和力及脂水分配系数测定结果
Figure PCTCN2022114251-appb-000006
实施例6
68Ga标记DA多肽探针体内外稳定性验证
取正常KM小鼠，经尾静脉注射18.5MBq的 68Ga标记的不同多肽探针，在注射后10min或30min时间点按压腹部取尿液，然后断头处死收集血液。尿液直接用纯水稀释后取37kBq进行Radio-HPLC测定。血液在5000rpm离心3min，收集血浆，然后向200μL血浆中加入400μL的无水乙腈，再进行离心后，取上清液进样，进行Radio-HPLC测定。观察探针放射峰的位置，并计算其百分比来评价探针在体内的稳定性。体外稳定性研究主要在生理盐水和5％HSA中进行，将探针与生理盐水和5％HSA分别孵育不同时间。其中，生理盐水组在既定时间可用纯水稀释后直接进行Radio-HPLC分析，而5％HSA组需要先将样品于无水乙腈混合后离心，取上清液进样，进行Radio-HPLC分析。图3-1至图3-3示出了 68Ga-DA-3、 68Ga-DA-4、 68Ga-DA-6的体内外稳定性结果图。实验结果显示探针 68Ga-DA-3注射后30min后可见尿液中游离 68Ga 3+位置的放射峰显著降低，但产品放射峰已消失不见，出现保留时间相对更早的两个放射峰，而血液中放射峰仍然相对较为杂乱，说明 68Ga-DA-3在小鼠体内会发生降解。 68Ga-DA-4和 68Ga-DA-6在小鼠体内的稳定性都较高，在注射后30min，产品放射峰的放射性化学纯度礽保持在93％以上，且Radio-HPLC出峰时间未发生显著变化，说明 68Ga-DA-4和 68Ga-DA-6在小鼠体内未发生明显降解。
实施例7
B16F10荷瘤小鼠免疫组化
B16F10荷瘤鼠分别在接种肿瘤后第7、14、21天处死，取肿瘤组织，用10％的福尔马林溶液脱水固定，然后进行免疫组织化学实验，方法如下：肿瘤组织脱水后石蜡包埋，石蜡切片经二甲苯脱蜡20min×3次；然后将切片在如下梯度浓度的酒精和水中平衡，100％酒精5min×2次，95％酒精5min，90％酒精5min，85％酒精5min，75％酒精5min，蒸馏水1分钟；3％的H 2O 2中浸泡10min；PBS清洗5min×3次；0.01M柠檬酸缓冲液(pH 6.0)中微波抗原修复(450W，10分钟)，时间分配3分钟、3分钟、4分钟，室温下晾凉；PBS清洗5min×3次；利用0.5％脱脂奶粉封闭1h；加入一抗，湿盒孵育4℃过夜；次日拿出湿盒，室温复温20min，PBS清洗5min×3次；加入二抗，室温下湿盒内孵育20分钟；PBS清洗5min×3次；DAB显色，根据情况而定，不要超过3-5分钟。放入PBS中终止，并且用蒸馏水洗一次；苏木素复染1分钟，用清水洗净；盐酸-酒精分色数秒(14mL盐酸/700mL乙醇)；自来水流水返蓝30min；梯度酒精脱水(沿复水反方向走一次，数秒即可)；二甲苯透明；中性树胶封片。免疫组织化学染色结果显示(如图4所示)，B16F10肿瘤内部CD25表达水平随接种时间延长而逐渐提升，接种后第14天可见局部CD25高水平表达，而在接种后21天可见肿瘤 组织高度表达CD25，证明B16F10荷瘤小鼠在评价CD25多肽探针特异性方面的应用价值。由于B16F10肿瘤接种后期肿瘤负荷过大，因此在显像实验中利用CD25阳性表达的Karpas299模型来评价多肽探针的显像效果。
实施例8
CD25在Karpas299细胞中的表达水平测定
Karpaspas299细胞CD25表达水平通过流式细胞术来测定，其实验流程如下：
1)细胞准备：细胞离心，可用15mL离心管，1500r×5min，再用5mL PBS清洗一次。准备1×10 7细胞数量即可满足使用。
2)细胞染色：细胞用PBS重悬为1×10 7/mL的悬液(勿用移液器吹打)，取100μL即1×10 6细胞置于流式管中，分三组，分别加入5μL的PE Anti-Human CD25抗体、5μL的PE Mouse IgG1k同型抗体、以及空白对照，混匀，放置于4℃，避光30min。
3)细胞清洗：加入5mL的PBS重悬细胞，1500r×5min离心，清洗2次。弃上清时直接倒掉即可，勿用移液器吸取。
4)上机检测：加500μL的PBS重悬细胞，混匀即可上机检测。注意：非单悬浮细胞需先过滤网。
流式细胞术实验结果显示，Karpas299细胞系的CD25表达阳性率为79.7％，即接近80％的Karpas299细胞的CD25表达阳性率呈强阳性(图5)。
实施例9
68Ga-DA-1探针在hPBMC中的摄取
DA-1探针实验中hPBMC的激活方式为OKT3+CD28培养基，PMA(20ng/mL)+Ionomycin(1ng/mL)，刺激培养8小时，并设置未激活的hPBMC作为对照。将hPBMC计数后分至小管(100μL PBS，5×10 5/管)，向其中加入70-100kBq的 68Ga-DA-1探针(100μL)，分别孵育30min和60min(n＝3)，然后离心收集hPBMC，并用冷的PBS清洗2次，收集细胞转移至计数管，用γ计数器计数，设置1％的marker为参比，计算每管细胞对于探针的摄取率(AD％/5×10 5cell)。如图6所示， 68Ga-DA-1探针在激活后的hPBMC中的摄取显著高于未激活的hPBMC，在孵育30min后二者间即可观察到显著性差异(p＝0.01)。在孵育1h后，两组间的摄取差异进一步增加(p<0.0001)。此外， 68Ga-DA-1探针在激活后的hPBMC中的摄取水平在孵育后随时间延长而提升(30min vs.60min，p＝0.003)。
实施例10
68Ga-DA-1探针在hPBMC皮下模型中的Micro-PET/CT显像
将激活和未激活的hPBMC分别与等体积的基质胶混合(200μL，3×10 6/只)，接 种至KM小鼠右侧肩部皮下。在接种后20min时在动物床上进行异氟烷麻醉，经尾静脉注射200μCi的 68Ga-DA-1探针，然后在注射后立刻进行动态PET/CT显像，观察 68Ga-DA-1探针在接种部位的聚集状态。勾画ROI，测量hPBMC接种部位及肌肉组织的SUVmax。如图7所示，在皮下接种激活hPBMC的小鼠PET/CT显像时，从显像开始30min内能够看到接种部位显著的探针摄取，注射后30min时接种部位的SUVmax为0.58，且在60min内接种部位的SUVmax高于肌肉摄取。而在接种未激活hPBMC的小鼠体内(图8)，接种部位的SUVmax在注射后60min内与肌肉摄取基本处于相同水平，在注射探针后30min，接种部位的SUVmax仅为0.35。此外，接种激活hPBMC的小鼠在显像30min内靶本比持续增加，在30min时最高可达1.88。而接种未激活hPBMC的小鼠显像中30min时靶本比最高仅为1.11(图9)。
实施例11
68Ga-DA-6探针在hPBMC中的摄取
hPBMC利用含5μg/mL的PHA-P的RPMI-1640培养液培养48h刺激激活，并设置未激活的hPBMC作为对照。将hPBMC计数后分至小管(100μL PBS，5×10 5/管)，分别向其中加入74kBq的 68Ga-DA-6探针(100μL)，分别孵育30min和60min(n＝3)，然后离心收集hPBMC，并用冷的PBS清洗2次，收集细胞转移至计数管，用γ计数器计数，设置1％的marker为参比，计算每管细胞对于探针的摄取率(AD％/5×10 5cell)。 68Ga-DA-6探针在激活后的hPBMC中孵育2h后可观察到高于未激活hPBMC的摄取(0.60±0.12AD％vs.0.35±0.04AD％，p＝0.02)。此外， 68Ga-DA-6探针在激活后的hPBMC中的摄取水平在孵育后随时间延长而提升(1h为0.22±0.07AD％，p＝0.008)(图10)。
实施例12
68Ga标记的多肽探针在B16F10及Karpas299荷瘤小鼠的Micro-PET/CT显像
取 68Ga标记的多肽探针，每只B16F10或Karpas299荷瘤鼠经尾静脉注射7.4MBq的放射性药物，然后在注射后1h经异氟烷麻醉后行静态PET/CT显像，观察探针在肿瘤部位的聚集情况。勾画ROI，测定肿瘤、肌肉及主要脏器的SUVmax。
利用B16F10荷瘤鼠模型考察了部分多肽探针的体内显像效果，从图11和表3可以看出，探针 68Ga-DA-4、 68Ga-DA-5在肿瘤中具有相对最高的摄取，但是其结构中含有赖氨酸，因此可见肾脏摄取显著高于其它探针。但是在将赖氨酸替换为精氨酸后，如 68Ga-DA-6和 68Ga-DA-7探针的肾脏SUVmax显著下降，肌肉本底摄取也相对较低，从而使得 68Ga-DA-6和 68Ga-DA-7探针显像时能获得较高的瘤本比。
表3  68Ga标记的DA系列探针在B16F10荷瘤小鼠的脏器摄取及瘤本比
Figure PCTCN2022114251-appb-000007
进一步地，利用Karpas299荷瘤鼠模型评价多个探针的体内显像效果。如图12所示，将赖氨酸替换为精氨酸的探针 68Ga-DA-6和 68Ga-DA-7同样可见显著降低的肾脏摄取。并且具有相对更低的本底摄取，靶本比提升，使得PET/CT显像质量优于其他数个探针。 68Ga-DA-6和 68Ga-DA-7的瘤本比分别可达3.63和3.86(表4)。
表4  68Ga标记的DA系列探针在Karpas299荷瘤小鼠的脏器摄取及瘤本比
Figure PCTCN2022114251-appb-000008
实施例13
68Ga标记的多肽探针药物代谢动力学
68Ga标记的多肽探针药物代谢动力学研究均采用如下方法：取雌性KM小鼠5只，每只小鼠经尾静脉注射3.7MBq的放射性药物，分别在注射后1min，3min，5min，10min，15min，30min，45min，1h，1.5h，2h，3h和4h经眼眶静脉取血，称重并利用γ计数器测定其放射性计数，设置1％的marker作为参比，计算单克血液的百分注射剂量率，记为％ID/g，利用GraphPad Prism 8.3.0软件作非线性回归拟合，研究其药物代谢动力学特点。实验中共考察了 68Ga-DA-4、 68Ga-DA-6和 68Ga-DA-7共计3个探针的药物代谢动力学。
选取了3个短肽探针DA-4、DA-6、DA-7来考察两种系列探针的血液药物代谢动力学。药物代谢动力学研究表明，3个探针的体内代谢均符合二室模型，如图13所示。通过GraphPad 8.3.0软件进行拟合，得到 68Ga-DA-4探针的药物代谢动力学二室模型公式如下：Ct＝1.74+12.26×e -1.01t+10.63×e -0.03t，其分布相和清除相的半衰期分别为0.69min和19.91min。 68Ga-DA-6探针的药物代谢动力学二室模型公式如下：Ct＝2.83+14.51×e -0.55t+6.65×e -0.04t，其分布相和清除相的半衰期分别为1.26min和15.64min。 68Ga-DA-7探针的药物代谢动力学二室模型公式如下：Ct＝1.64+12.82×e -0.44t+8.01×e -0.03t，其分布相和清除相的半衰期分别为1.58min和20.69min。药物代谢动力学研究表明， 68Ga-DA-4、 68Ga-DA-6和 68Ga-DA-7探针在体内的分布和清除都非常迅速，符合小分子显像探针的药物代谢动力学特点。
实施例14
68Ga标记的多肽探针在KM小鼠的生物分布
68Ga标记探针在KM鼠的生物分布研究采用如下方法：取雌性KM小鼠15只，每只小鼠经尾静脉注射1.11MBq的放射性药物，分别在注射后5min，30min，1h，2h及4h处死，取血液、心脏、肝脏、脾脏、肺、肾脏、胃、小肠、大肠、肌肉、骨及脑等脏器和组织，称重并利用γ计数器测定其放射性计数，设置1％的marker作为参比，计算单克组织百分注射剂量率，记为％ID/g，考察 68Ga-DA-4和 68Ga-DA-6和探针在KM小鼠体内的分布特点。
此外，从探针 68Ga-DA-4和 68Ga-DA-6探针在2h内的分布结果来看，短肽探针在KM小鼠体内的代谢特点十分相似，均为经由肾脏快速清除。如图14，探针在血液中清除迅速， 68Ga-DA-4和 68Ga-DA-6在注射后30min血液中的探针摄取即分别下降超过80.4％和88.6％。且在30min内探针已基本达到分布和清除的平衡阶段，至注射后2h内各脏器探针摄取水平已基本保持稳定。
实施例15
68Ga-DA-6在阳性模型Karpas299及隐性模型U87MG荷瘤小鼠的Micro-PET/CT显像
取Karpas299及U87MG荷瘤小鼠，分别经尾静脉注射7.4MBq的 68Ga-DA-6探针，然后在注射后1h按压小鼠腹部促进排尿，再利用异氟烷麻醉小鼠后行小动物PET/CT显像，利用CT-AC重建方法获得PET图像，勾画感兴趣区，测定肿瘤及肌肉组织的SUVmax等数据，计算瘤本比(tumor-to-muscle ratio，T/M)。Karpas299阻断组小鼠在注射探针前24h，需预先经尾静脉注射1mg的Basiliximab。
从注射后1h时的小动物PET/CT显像视觉分析来看， 68Ga-DA-6探针在Karpas299荷瘤小鼠的肿瘤部位具有较为显著的摄取(图15)，SUVmax为0.29，瘤本比T/M为3.63，而在U87MG荷瘤小鼠的肿瘤中，探针摄取显著降低，SUVmax为0.16。此外，在提前注射Basiliximab进行阻断后，Karpas299荷瘤小鼠肿瘤部位的探针摄取也下降至0.17。
实施例16
68Ga-DA-6探针在Karpas299及U87MG荷瘤小鼠的生物分布
取Karpas299及U87MG荷瘤小鼠，分别经尾静脉注射1.11MBq的 68Ga-DA-6探针，然后在注射后1h处死，取血液、心脏、肝脏、脾脏、肺、肾脏、胃、小肠、大肠、肌肉、骨、脑及肿瘤等脏器和组织，称重并利用γ计数器测定其放射性计数，设置1％的marker作为参比，计算单克组织百分注射剂量率，记为％ID/g，考察 68Ga-DA-6探针在Karpas299和U87MG荷瘤小鼠体内的分布特点。
如图16所示，在Karpas299荷瘤小鼠体内， 68Ga-DA-6探针在肿瘤中具有相对较高的摄取，为1.27±0.04％ID/g，而在U87MG肿瘤中的摄取仅为0.54±0.06％ID/g(p<0.0001)，此外，利用Basiliximab进行阻断后，Karpas299荷瘤小鼠肿瘤中 68Ga-DA-6探针的摄取也显著下降，仅为0.26±0.02％ID/g(p<0.0001)。
以上已经描述了本发明的各实施例，上述说明是示例性的，并非穷尽性的，并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下，对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。

Claims (10)

  1. 一种CD25靶向多肽，其特征在于，所述多肽的氨基酸序列如下：
    i)X1X2X3X4X5；或
    ii)在i)的N端或C端连接标签形成的氨基酸序列；
    其中，X1为精氨酸、赖氨酸或组氨酸；X2为苯丙氨酸、酪氨酸、苄基酪氨酸或卤代苯丙氨酸；X3为精氨酸、赖氨酸或组氨酸；X4为苯丙氨酸、酪氨酸、苄基酪氨酸或卤代苯丙氨酸；X5为苯丙氨酸、酪氨酸、苄基酪氨酸或卤代苯丙氨酸。
  2. 根据权利要求1所述的CD25靶向多肽，其中，所述多肽中至少有一个氨基酸为D型。
  3. 根据权利要求2所述的CD25靶向多肽，其中，X1、X5为L型，其余氨基酸中至少有一个为D型，优选地，X3和X4中之一为D型，其余氨基酸均为L型。
  4. 根据权利要求1所述的CD25靶向多肽，其中，所述标签为保护基。
  5. 根据权利要求4所述的CD25靶向多肽，其中，所述标签为羟基保护基。
  6. 一种分子探针，其特征在于，所述分子探针为具有放射性同位素标记的权利要求1-5中任意一项所述的CD25靶向多肽；所述放射性同位素直接标记在所述CD25靶向多肽上，或者通过-L-Ch标记在所述CD25靶向多肽上，其中L为桥接基团，Ch为核素螯合基团。
  7. 根据权利要求6所述的分子探针，其中，-L-Ch共价连接在所述CD25靶向多肽的N端、C端或反应性侧链上。
  8. 根据权利要求6或7所述的分子探针，其中，所述桥接基团衍生自寡肽或NH 2-(PEG) n-COOH，所述寡肽的氨基酸数目为1-4，n为1-4的整数；
    所述核素螯合基团为双功能螯合剂形成的基团，所述双功能螯合剂选自DOTA、NOTA、NODA、NODAGA、DOTP、TETA、ATSM、PTSM、EDTA、EC、HBEDCC、DTPA、SBAD、BAPEN、Df、DFO、TACN、NO2A/NOTAM、CB-DO2A、Cyclen、NOTA-AA、DO3A、DO3AP、HYNIC、MAS3、MAG3或异腈；所述放射性核素为诊断用放射性核素；所述诊断用放射性核素为 68Ga、 64Cu、 18F、 86Y、 90Y、 89Zr、 111In、 99mTc、 11C、 123I、 125I和 124I中的至少一种。
  9. 权利要求6-8中任意一项所述的分子探针在制备核素成像诊断试剂中的应用。
  10. 根据权利要求9所述的应用，其中，所述核素成像诊断试剂为CD25高表达的CAR-T细胞疗效评价试剂或治疗追踪试剂。
PCT/CN2022/114251 2022-06-23 2022-08-23 一种cd25靶向多肽、分子探针及应用 WO2023245852A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210714305.3A CN114773433B (zh) 2022-06-23 2022-06-23 一种cd25靶向多肽、分子探针及应用
CN202210714305.3 2022-06-23

Publications (1)

Publication Number Publication Date
WO2023245852A1 true WO2023245852A1 (zh) 2023-12-28

Family

ID=82422365

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/114251 WO2023245852A1 (zh) 2022-06-23 2022-08-23 一种cd25靶向多肽、分子探针及应用

Country Status (2)

Country Link
CN (1) CN114773433B (zh)
WO (1) WO2023245852A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114773433B (zh) * 2022-06-23 2022-09-06 北京肿瘤医院(北京大学肿瘤医院) 一种cd25靶向多肽、分子探针及应用
CN117645652A (zh) * 2022-09-02 2024-03-05 核欣(苏州)医药科技有限公司 环肽及其制备方法、包括其的复合物、及其用途
WO2024055075A1 (en) * 2022-09-14 2024-03-21 Biosceptre (Aust) Pty Ltd In vivo detection of immune cells

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1478098A (zh) * 2000-09-14 2004-02-25 ��˹��ɫ�л���ҽ���������޹�˾ Il-2-和il-15-介导的t细胞应答的调节
WO2016077061A2 (en) * 2014-11-12 2016-05-19 Immunomedics, Inc. Methods and compositions for improved labeling of targeting peptides
US20160199524A1 (en) * 2015-01-09 2016-07-14 Immunomedics, Inc. Radiosensitivity of fluorophores and use of radioprotective agents for dual-modality imaging
CN111212661A (zh) * 2018-09-11 2020-05-29 润俊(中国)有限公司 白介素-2多肽偶联物及其用途
WO2021183832A1 (en) * 2020-03-11 2021-09-16 Ambrx, Inc. Interleukin-2 polypeptide conjugates and methods of use thereof
CN114773433A (zh) * 2022-06-23 2022-07-22 北京肿瘤医院(北京大学肿瘤医院) 一种cd25靶向多肽、分子探针及应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7550439B2 (en) * 2004-01-23 2009-06-23 Cornell Research Foundation, Inc. Methods for reducing oxidative damage
JP7174757B2 (ja) * 2017-07-04 2022-11-17 イントゥーセル，インコーポレーティッド 切断可能なリンカーを含む化合物及びその使用
BR112020025287A2 (pt) * 2018-06-25 2021-03-09 University Of Washington Projeto de novo de miméticos de interleucina potentes e seletivos
CN112472802B (zh) * 2020-12-04 2022-04-26 国家纳米科学中心 一种细菌外膜囊泡、包含该细菌外膜囊泡的通用纳米疫苗及其制备方法和应用
WO2021205196A1 (en) * 2020-12-30 2021-10-14 Muhammad Taliah Compositions and prevention and intervention methods for covid-19 with divine ayats' fitra30 covid-19 protocol

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1478098A (zh) * 2000-09-14 2004-02-25 ��˹��ɫ�л���ҽ���������޹�˾ Il-2-和il-15-介导的t细胞应答的调节
WO2016077061A2 (en) * 2014-11-12 2016-05-19 Immunomedics, Inc. Methods and compositions for improved labeling of targeting peptides
US20160199524A1 (en) * 2015-01-09 2016-07-14 Immunomedics, Inc. Radiosensitivity of fluorophores and use of radioprotective agents for dual-modality imaging
CN111212661A (zh) * 2018-09-11 2020-05-29 润俊(中国)有限公司 白介素-2多肽偶联物及其用途
WO2021183832A1 (en) * 2020-03-11 2021-09-16 Ambrx, Inc. Interleukin-2 polypeptide conjugates and methods of use thereof
CN114773433A (zh) * 2022-06-23 2022-07-22 北京肿瘤医院(北京大学肿瘤医院) 一种cd25靶向多肽、分子探针及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DAMOISEAUX JAN: "The IL-2 – IL-2 receptor pathway in health and disease: The role of the soluble IL-2 receptor", CLINICAL IMMUNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 218, 1 July 2020 (2020-07-01), AMSTERDAM, NL , XP086241545, ISSN: 1521-6616, DOI: 10.1016/j.clim.2020.108515 *
DATABASE REGISTRY 30 September 2021 (2021-09-30), ANONYMOUS: "INDEX NAME NOT YET A SSIGNED ", XP093118748, retrieved from STN Database accession no. 2703745-80-4 *

Also Published As

Publication number Publication date
CN114773433A (zh) 2022-07-22
CN114773433B (zh) 2022-09-06

Similar Documents

Publication Publication Date Title
WO2023245852A1 (zh) 一种cd25靶向多肽、分子探针及应用
US9550001B2 (en) Compositions, methods of synthesis and use of carbohydrate targeted agents
US5230883A (en) Method for localization and treatment of tumors using polylysine complexes
CN112043839A (zh) 靶向转铁蛋白受体的放射性同位素标记多肽显像剂及其应用
CN110227169A (zh) 一种结构修饰的rgd多肽的核医学药物
CN110496233B (zh) 一种spect显像剂及其标记前体及其制备方法、组合物和用途
CN113444146A (zh) 靶向成纤维细胞活化蛋白探针、制备方法及其在制备pet显像剂中的应用
CN110357945B (zh) 一种靶向肿瘤的柯萨奇病毒/腺病毒的模拟肽及其应用
CN115651063A (zh) 放射性核素标记的ptp多肽及其应用
CN116751258B (zh) Mdm2/mdmx靶向多肽及其应用
TW201511774A (zh) 放射性標誌之主動標靶性醫藥組合物及其用途
CN116832180A (zh) 一种靶向乳腺癌的多肽pet分子探针及其制备方法和应用
WO2023184839A1 (zh) 一种新型靶向肿瘤PD-L1的探针 99mTc标记亲合体的制备
KR20240105380A (ko) Psma 항원을 표적으로 하는 리간드 화합물, 이의 킬레이트 및 전립선암의 진단 및 치료를 위한 이의 응용
Prasanphanich et al. The effects of linking substituents on the in vivo behavior of site-directed, peptide-based, diagnostic radiopharmaceuticals
CN106492237B (zh) 一种isoDGR多肽放射性药物及其制备方法和应用
WO2022032844A1 (zh) 一种肿瘤间质显像剂及其制备方法
CN107674117B (zh) Cu-64 标记的Dimer-San A环肽衍生物胰腺癌分子探针的制备方法
Mohammadgholi et al. Human fibronectin extra-domain B (EDB)-specific aptide (APTEDB) radiolabelling with technetium-99m as a potent targeted tumour-imaging agent
CN107586321B (zh) F-18标记修饰Dimer-San A探针的制备方法
CN108440665A (zh) 一种用于肺癌诊断的99mTC标记的生长抑素类似物及其制备方法
Chen et al. 68Ga-labeled TMTP1 radiotracer for PET imaging of cervical cancer
CN117777296B (zh) B7h3亲和体及其诊疗核素标记物的制备方法与应用
CN113372413B (zh) 一种靶向CD11b受体的PET显像剂及其标记前体及其制备方法、组合物和用途
US9234002B2 (en) Small peptide specific for lung cancer and application thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22947593

Country of ref document: EP

Kind code of ref document: A1