WO2020238800A1 - 用于肿瘤的靶向放射性药物及其在影像指导下的靶向放射治疗与免疫治疗的联合疗法 - Google Patents

用于肿瘤的靶向放射性药物及其在影像指导下的靶向放射治疗与免疫治疗的联合疗法 Download PDF

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WO2020238800A1
WO2020238800A1 PCT/CN2020/091861 CN2020091861W WO2020238800A1 WO 2020238800 A1 WO2020238800 A1 WO 2020238800A1 CN 2020091861 W CN2020091861 W CN 2020091861W WO 2020238800 A1 WO2020238800 A1 WO 2020238800A1
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tumor
dota
3prgd
drug
targeted
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PCT/CN2020/091861
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English (en)
French (fr)
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王凡
史继云
高瀚男
贾兵
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北京大学
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Priority claimed from CN201910441556.7A external-priority patent/CN110227169B/zh
Priority claimed from CN202010373843.1A external-priority patent/CN113616818B/zh
Application filed by 北京大学 filed Critical 北京大学
Priority to US17/310,604 priority Critical patent/US20230117927A1/en
Priority to EP20815331.2A priority patent/EP3906947A4/en
Publication of WO2020238800A1 publication Critical patent/WO2020238800A1/zh

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    • 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
    • A61K51/082—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being a RGD-containing peptide
    • 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
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/004—Acyclic, carbocyclic or heterocyclic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen, sulfur, selenium or tellurium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

  • the present invention relates to a targeted radiopharmaceutical modified by RGD polypeptide for tumors, and also relates to a combined therapy with immunotherapy drugs.
  • Integrin is one of the important factors involved in the regulation of tumor angiogenesis and plays an important role in tumor angiogenesis.
  • integrin ⁇ v ⁇ 3 plays the most important role. Integrin ⁇ v ⁇ 3 is highly expressed in tumor neovascular endothelial cells, but not or low in normal cells or mature blood vessels. RGD can specifically bind to integrin ⁇ v ⁇ 3, so RGD molecular probes designed by the specific binding of RGD and integrin ⁇ v ⁇ 3 have been widely studied and used.
  • RGD cyclic RGD
  • modified pharmacokinetic linkers such as glycosylation of RGD
  • linked RGD cyclic peptide monomers into multimers with glutamic acid and used chemical means in the two
  • methods such as inserting Gly3 or PEG4 chains between two RGD monomers in the polymer are used to optimize RGD polypeptides to obtain structurally modified RGD polypeptides.
  • Combination therapy is one of the main methods to improve the effectiveness of PD1/PD-L1 blocking therapy.
  • the anti-tumor immune effect produced by conventional therapy can enhance the effectiveness of immunotherapy. It is generally believed that radiotherapy can promote the release of tumor antigens, enhance the differentiation, proliferation, function and tumor infiltration of effector T cells, and has synergy in the combination of immune checkpoint blocking therapy.
  • radiotherapy combined with immunotherapy is gradually showing advantages in immunotherapy of lung cancer, but its treatment strategy still needs to be studied.
  • Targeted Radiotherapy is a kind of internal radiation therapy based on molecular combination in the body, which has obvious advantages in the treatment of patients with metastases and advanced tumors.
  • Nanobody probes prepared using PD-L1 as a biomarker can perform non-invasive, real-time and dynamic monitoring of the tumor microenvironment, which is helpful to guide the formulation of individualized strategies for targeted radiotherapy combined with immunotherapy, and improve the effectiveness of tumor immunotherapy Sex.
  • RGD polypeptide modified with the following formula:
  • L represents a linker molecule, which has the following structure: Wherein m is an integer of 1-8, for example 2-6, preferably 5;
  • the carboxyl group of the L reacts with the amino group in A through the bond chain, for example, the carboxyl group marked with * in L and the A bond chain;
  • n 0 or 1
  • the RGD polypeptide reacts with the carboxyl group in A through its amino group to react with the bond chain;
  • the RGD polypeptide reacts with another carboxyl group in L through its amino group.
  • the carboxyl group marked * in L is connected to A
  • the carboxyl group marked ** reacts with the RGD polypeptide chain.
  • the RGD polypeptide is an RGD polypeptide selected from the group consisting of c(RGDfV), c(RGDfK), c(RGDfE), c(RGDyk), E[c(RGDyk)] 2 , E[c(RGDfK)] 2 , 3PRGD 2 .
  • the present invention also provides a radionuclide-labeled complex containing the RGD polypeptide modified in the above structure, which has a structure defined as follows:
  • Nu is a radionuclide, such as diagnostic imaging nuclides: 111 In, 64 Cu, 99m Tc, 68 Ga, or therapeutic nuclides: 90 Y, 177 Lu, 89 Sr, 153 Sm, 188 Re;
  • BFC is a bifunctional chelating agent, such as HYNIC (hydrazine amide), MAG 2 (mercaptoacetyl diglycine), MAG 3 (mercaptoacetyl triglycine), DTPA (diethyl triamine pentaacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7 three Carboxylic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11 tetraacetic acid);
  • HYNIC hydrazine amide
  • MAG 2 mercaptoacetyl diglycine
  • MAG 3 mercaptoacetyl triglycine
  • DTPA diethyl triamine pentaacetic acid
  • DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10
  • the bifunctional chelating agent reacts with the -NH 2 in A through the carboxyl group in its structure;
  • the bifunctional chelating agent reacts with the -NH 2 in L through the carboxyl group in its structure.
  • RGD polypeptide is selected from: c(RGDfK), 3PRGD 2 ;
  • Nu is selected from: 90 Y, 177 Lu, 111 In, 64 Cu, 99m Tc;
  • BFC is selected from DTPA, DOTA; when Nu is 111 In, 64 Cu, 68 Ga, 99m Tc, BFC is selected from HYNIC, MAG 2 , MAG 3 , DTPA, DOTA, TETA and NOTA.
  • Nu is 90 Y or 177 Lu and BFC is selected from DTPA and DOTA; when Nu is 111 In, BFC is selected from DTPA and DOTA; when Nu is 64 Cu, BFC is selected from TETA and DOTA; when Nu is 68 Ga , BFC is selected from NOTA, DOTA; when Nu is 99m Tc, BFC is selected from HTNIC, DTPA, MAG 2 , MAG 3 ;
  • the complex formed by the structurally modified polypeptide of the present invention is as follows:
  • a co-ligand when the bifunctional chelating agent as a ligand cannot occupy all the coordination positions of the radionuclide, a co-ligand is also required.
  • the radionuclides and bifunctional chelating agents that require co-ligands in the present invention are well known to those skilled in the art.
  • 99m Tc uses HYNIC as a bifunctional chelating agent, as a co-ligand, it can be the same or different, and they are all known in the prior art.
  • Common co-ligands include water-soluble phosphines (such as triphenylphosphine).
  • Trimetasulfonic acid sodium salt TPPTS Trimetasulfonic acid sodium salt
  • Tricine N-tris(hydroxymethyl)methylglycine
  • Tricine N-bis(hydroxyethyl)glycine
  • gluconate ethylenediamine-N,N'-di Acetate
  • EDDA ethylenediamine-N,N'-di Acetate
  • BP 3-benzoylpyridine
  • PADA pyridine-2-azo-p-xylidine
  • TPPTS and Tricine are used as co-ligands.
  • 177 Lu or 68 Ga uses DOTA as a bifunctional chelating agent, no co-ligand is required to complete the coordination.
  • the present invention provides a complex with the following structure:
  • the present invention also provides a molecular probe with the above complex structure.
  • the present invention also provides a pharmaceutical composition comprising an effective amount of the above-mentioned labeled complex Nu-BFC-A-(L)n-RGD polypeptide.
  • the pharmaceutical composition of the present invention is a diagnostic drug, for example, the drug is used as an imaging agent for the imaging diagnosis of integrin ⁇ v ⁇ 3 positive tumors. It is directly administered to an individual, and a diagnosis is made by detecting the radiation emitted by the compound administered to the subject, and imaging based on the information obtained by the radiation.
  • the diagnostic drug of the present invention is an injectable preparation comprising the above-mentioned labeling complex and an injectable carrier.
  • the imaging agent refers to PET, single photon emission computerized tomography SPECT.
  • the pharmaceutical composition of the present invention is a therapeutic drug used for radiation targeted therapy of integrin ⁇ v ⁇ 3-positive tumors.
  • the drug has specific affinity with integrin ⁇ v ⁇ 3 and is enriched in tumor tissue.
  • Radionuclides emit pure ⁇ -rays or ⁇ -rays accompanied by ⁇ -rays to produce ionizing radiation biological effect destruction Diseased tissue.
  • the therapeutic drug of the present invention is an injectable preparation comprising the above-mentioned labeled complex and an injectable carrier.
  • the pharmaceutical composition of the present invention may also include an immunotherapy drug.
  • the above-mentioned labeled complex of the present invention is used for targeted radiotherapy, combined with immunotherapy drugs, to achieve a synergistic therapeutic effect.
  • the preferred labeling complex is 177 Lu-DOTA-AL-3PRGD 2 .
  • the preferred immunotherapy drugs are PD-1 or PD-L1 immune checkpoint inhibitors. Preferably, it is a PD-1 or PD-L1 monoclonal antibody drug.
  • the PD-1 or PD-L1 monoclonal antibody drugs of the present invention are not particularly limited, and are known in the art to target human or animal PD-1/PD-L1 immune pathways with effective such drugs, such as those that have been marketed
  • a variety of PD-1 monoclonal antibody drugs such as Opdivo (MDX-1106), Keytruda (MK-3475), CT-011, or PD-L1 monoclonal antibody, such as MDX-1105, MPDL3280A, or MEDI4736, or other Known PD-1 or PD-L1 monoclonal antibodies used in clinical trials.
  • the PD-L1 monoclonal antibody used in the examples of the present invention is (10F.9G2).
  • PD-L1 is the ligand of PD-1.
  • the combination of PD-L1 on the cell surface and PD-1 on the surface of lymphocytes can inhibit the function of lymphocytes. Induces the apoptosis of activated lymphocytes, thus playing an important role in autoimmune tolerance and prevention of autoimmune diseases.
  • PD-L1 is overexpressed in tumor tissues, while tumor-infiltrating lymphocytes highly express PD-1.
  • the combination of PD-1 and PD-L1 inhibits the function of lymphocytes and tumor killing effect, and induces lymphocyte regulation. Death weakens the body’s own anti-tumor immune response, and ultimately leads to tumor immune escape.
  • the PD-1 or PD-L1 antibody can block the PD-1/PD-L1 pathway in the body, thereby promoting lymphocyte proliferation, activating the immune system, promoting the body's own anti-tumor response, and further degrading tumors.
  • any PD-1 or PD-L1 immune checkpoint inhibitor can block the PD-1/PD-L1 pathway and realize its own anti-tumor response, thereby treating tumors or cancers. Therefore, the PD-1 or PD-L1 monoclonal antibody drugs of the present invention are not particularly limited, and any known such drugs can be used in the present invention.
  • the combined targeted radiotherapy and immunotherapy pharmaceutical composition according to the present invention further includes a nanobody molecular imaging probe, such as a PD-1 or PD-L1 nanobody molecular imaging probe.
  • a nanobody molecular imaging probe such as a PD-1 or PD-L1 nanobody molecular imaging probe.
  • it is the PD-L1 Nanobody Technetium Marker.
  • the Nanobody molecular imaging probe used in the embodiment of the present invention is 99m Tc-MY1523.
  • the nanobody molecular imaging probe is a PD-L1 nanobody (MY1523) containing an LPETG tag, which can be connected to 99m Tc-HYNIC through Sortase A enzyme -GGGK prepared.
  • the labeled complex and the immunotherapy drug can be administered simultaneously or separately before and after.
  • the immunotherapeutic drug can be administered after the labeled complex.
  • the immunotherapeutic drug is used 3 to 6 days after the labeled complex is used.
  • the nanobody molecular imaging probe is administered after the administration of the labeled complex and before the administration of the immunotherapy drug.
  • the labeled complex, immunotherapeutic drug or nanobody molecular imaging probe of the present invention is an injectable preparation comprising the above-mentioned labeled complex, immunotherapeutic drug or nanobody molecular imaging probe and an injectable carrier.
  • the pharmaceutical composition of the present invention is an intravenous injection, such as a colorless and transparent liquid injection.
  • Excipients suitable for intravenous injection are well-known in the art.
  • the pharmaceutical composition can be formulated in an aqueous solution. If necessary, physiologically compatible buffers, including, for example, phosphate, histidine, citrate, etc., can be used.
  • physiologically compatible buffers including, for example, phosphate, histidine, citrate, etc.
  • tonicity agents such as sodium chloride, sucrose, glucose, etc.
  • co-solvents such as polyethylene glycol
  • low-toxicity surfactants such as polysorbate or poloxamer.
  • the pharmaceutical composition of the present invention further includes an anti-adsorbent, such as physiological saline, 1% cyclodextrin aqueous solution, Tween-20-containing PBS solution (for example, the mass fraction of Tween is 0.01 to 0.1%).
  • the anti-adsorbent-containing pharmaceutical composition of the present invention uses a solution of a surfactant such as Tween-20 (parts by mass, for example, 0.05%), which can effectively avoid the non-specific adsorption of the marker in the infusion solution and significantly prevent the tube
  • the wall's adsorption of radioactive markers realizes the accuracy of the administered dose and avoids the waste of the drug during use.
  • the pharmaceutical composition containing the anti-adsorbent is continuously transferred 4 times in a series of new EP tubes, and the recovery rate of the marker can reach over 97%.
  • kits which respectively contains a drug containing the labeled complex Nu-BFC-A-(L)n-RGD polypeptide of the present invention, an immunotherapeutic drug, and an optional nano Antibody molecular imaging probe.
  • the drug is used to diagnose or treat integrin ⁇ v ⁇ 3 positive tumors
  • the tumors refer to solid tumors, such as in the blood, liver, glands (such as breast, prostate, pancreas), intestine (such as colorectal), Malignant tumors in the kidneys, stomach, spleen, lungs, muscles, bones, etc.
  • the present invention also provides a method for diagnosing or treating hematological system and solid malignant tumors with high expression of integrin ⁇ v ⁇ 3, by administering an effective amount of the aforementioned Nu-BFC-A-(L)n-RGD polypeptide to individuals in need.
  • the individual may be a mammal, such as a human.
  • the present invention also provides a method for treating blood system and solid malignant tumors with high expression of integrin ⁇ v ⁇ 3, by applying an effective amount of labeled complex Nu-BFC-A-(L)n-RGD polypeptide and immunotherapeutic drugs to this Individuals in need.
  • the individual may be a mammal, such as a human.
  • the immunotherapeutic drug is administered after the labeled complex is administered, for example, 3-6 days thereafter.
  • the labeled complex and the immunotherapy drug are administered in the form of injection.
  • the method is performed under the guidance of a nanobody molecular imaging probe.
  • a technetium-labeled nanobody is used to monitor the expression of PD-L1 in the tumor at the in vivo level.
  • the method of the present invention according to the monitoring of PD-L1 expression, it is selected to administer immunotherapeutic drugs when PD-L1 expression increases or peaks.
  • RGD polypeptides all are known in the art.
  • RGD is a small polypeptide containing the amino acid sequence of arginine-glycine-aspartic acid (Arg-Gly-Asp).
  • D-type phenylalanine and valine were added to synthesize RGD cyclic pentapeptide structure-c (RGDfV), where c represents the polypeptide is cyclic, R represents arginine, G represents glycine, D represents aspartic acid, f represents D-phenylglycine and V represents valine.
  • Cyclic pentapeptide structure-c(RGDfV) 5th amino acid is replaced by other amino acids to obtain c(RGDfK), c(RGDfE), c(RGDyk), where K is lysine, E is glutamic acid, and y is D -Tyrosine.
  • c(RGDfK) has the following structure:
  • cyclic peptide structures can form dimers, such as E[c(RGDyk)] 2 and E[c(RGDfK)] 2 , and two RGD cyclic peptides are connected to form a dimer with glutamic acid.
  • 3PRGD 2 refers to a RGD pentacyclic peptide dimer modified with three polyethylene glycols, namely PEG 4 -[PEG 4 -c(RGDXk)] 2 , X is D-phenylglycine, D-tyrosine, etc.
  • its structure diagram is as follows:
  • Bifunctional chelator refers to the metal nuclides that can not only be covalently connected to biomolecules but also can chelate metal nuclides, and its structure can ensure firm binding with metal nuclides, and the introduced metal nuclides Stay away from biomolecules to ensure that their biological activity is not lost, and form a stable nuclide-chelator-biomolecular marker of this type of functional organic material.
  • the bifunctional chelating agents used in the present invention are those known in the prior art in the art, such as HYNIC (hydrazine amide), MAG 2 (mercaptoacetyl diglycine), MAG 3 (mercaptoacetyl triglycine), DTPA ( Diethyltriaminepentaacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid), NOTA (1,4,7-triazacyclic Nonane-1,4,7 tricarboxylic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11 tetraacetic acid), etc.
  • HYNIC hydrazine amide
  • MAG 2 mercaptoacetyl diglycine
  • MAG 3 mercaptoacetyl triglycine
  • DTPA Diethyltriaminepentaacetic acid
  • DOTA 1,4,7,10-tetra
  • treatment and other similar synonyms include alleviation, reduction or amelioration of symptoms of a disease or condition, prevention of other symptoms, amelioration or prevention of the underlying metabolic cause of the symptoms, inhibition of the disease or condition, such as preventing the development of the disease or condition, To alleviate a disease or condition, make the disease or condition better, relieve the symptoms caused by the disease or condition, or stop the symptoms of the disease or condition.
  • the term encompasses the purpose of prevention.
  • the term also includes obtaining therapeutic effects and/or preventive effects. The therapeutic effect refers to curing or improving the underlying disease being treated.
  • the cure or improvement of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect, for example, although the patient may still be affected by the underlying disease, an improvement in the patient's condition is observed.
  • the composition can be administered to patients who are at risk of suffering from a specific disease, or even if a disease diagnosis has not been made, the composition can be administered to patients who have developed one or more physiological symptoms of the disease.
  • the present invention designs and prepares a structurally modified RGD polypeptide, and prepares a series of novel RGD polypeptide molecular probes with improved pharmacokinetic properties from the structurally modified RGD polypeptide.
  • the present invention finds that the RGD polypeptide is structurally modified and engineered, and the molecular probe of the present invention formed by the structure-modified RGD polypeptide through a chelating agent and a radionuclide has higher in vivo and in vitro stability, and is compatible with albumin (albumin).
  • the molecular probe of the present invention has a higher tumor uptake rate, contrast, safety, reduced dosage, reduced side effects, and improved RGD series probes as imaging diagnostic molecules. The imaging effect of the probe in SPECT and/or PET, and the effect of radio-targeted therapy as a therapeutic molecular probe.
  • An example of the novel molecular probe of the present invention 177 Lu-DOTA-AL-3PRGD 2 , has a higher uptake in the blood, thereby increasing the uptake in the tumor, and its cumulative uptake in the blood can reach that of the original probe. About 8 times, the cumulative uptake in the tumor is about 4 times that of the original probe. The tumor uptake reached the highest 4 hours after injection, and its percentage injection dose rate was 26.52 ⁇ 0.58%ID/g, and it was still clearly visible after 48 hours. This property significantly improves the imaging effect of RGD series probes as molecular probes for imaging diagnostics and the effect of radioactive targeted therapy as therapeutic molecular probes.
  • the targeted radiotherapy drug of the present invention can not only be specifically enriched in tumor tissues, and directly act on tumor cells through internal irradiation, but also can activate the body to produce an anti-tumor immune response mediated by T cells.
  • targeted radiotherapy causes remodeling of the tumor immune microenvironment, significantly increases the infiltration of CD4+ and CD8+ T lymphocytes (but not T-reg cells) in tumor tissues, and upregulates myeloid immunity PD-L1 expression level on the surface of cells (not tumor cells).
  • the present invention can effectively increase the activation of initial T cells and the effect stage of effector T cells through the combined use of PD-1/PD-L1 immune checkpoint inhibitors, and play important roles in the early and late stages of tumor immune circulation, respectively.
  • the targeted radiopharmaceutical modified by the RGD polypeptide of the present invention promotes tumor antigen release and immune cell antigen presentation, and plays an important role in the early stage of tumor immune cycle. Therefore, the present invention makes full use of the same and different stages in the tumor immune cycle.
  • the present invention further uses the PD-L1 nanobody molecular imaging probe to monitor the changes in PD-L1 expression in the tumor microenvironment during the treatment process, so as to guide the dosing plan of the combination therapy.
  • the labeled complex of the present invention is an effective targeted radiotherapy drug, which can effectively treat the tumor in the MC-38 syngeneic tumor model with normal immunity.
  • the administration of a labeled complex of 18MBq can completely ablate the tumor through a specific immune response mediated by T cells.
  • the remodeling of the tumor immune microenvironment caused by the targeted radiotherapy of the present invention is mainly reflected in: the internal radiation therapy of the tumor significantly increases the infiltration of CD4 + and CD8 + T lymphocytes (but not T-reg cells) and upregulates the tumor PD-L1 expression on the surface of myeloid immune cells (but not tumor cells).
  • the sixth day of targeted radiotherapy is the most severe immunosuppressive period, when the expression of PD-L1 in the tumor reaches the highest level. Therefore, PD-L1 blockade treatment 3 to 6 days after targeted radiotherapy can obtain the best synergistic effect.
  • the present invention found through experiments that only when PD-L1 is dynamically increased can the PD-L1 monoclonal antibody be administered to obtain a synergistic effect. Therefore, tracking the dynamic expression changes of PD-L1 after targeted radiotherapy has important guiding significance.
  • the application of PD1 or PD-L1 nanobody molecular imaging probes in the present invention can observe the expression of PD-L1 in tumors after targeted radiotherapy, which has guiding significance for the formulation of therapeutic strategies for targeted radiotherapy and immunotherapy. Helps to enhance the effect of combined therapy.
  • Figure 1 Radio HPLC chart of the final product 68 Ga-DOTA-Ac (RGDfk) in Example 1; Figure 1a is before purification, and Figure 1b is after purification;
  • FIG. 1 Biodistribution results of the molecular probe and control probe of Example 1 in mice bearing LLCC57BL/6J;
  • Figure 3 Radio HPLC chart of the final product of Example 3 99m Tc-HYNIC-A-3PRGD 2 ;
  • Figure 5 SPECT/CT imaging of nude mice bearing U87MG in Example 3.
  • Figure 6 Figure A ⁇ B ⁇ C ⁇ D shows the SPECT/CT imaging results of Example 3 and the %ID/g of the control probe in U87 tumor, tumor/kidney, tumor/muscle, tumor/liver, respectively The ratio comparison chart;
  • Figure 7 Figure of the distribution results of the molecular probe of Example 3 in nude mice bearing U87MG;
  • Figure 8 Radio HPLC chart of the final product 177 Lu-DOTA-AL-3PRGD 2 of Example 7;
  • Figure 10 SPECT/CT imaging of U87MG nude mice bearing the molecular probe of Example 7;
  • FIG. 11 Biodistribution map of the molecular probe of Example 7 in U87-bearing mice
  • Figure 12 The trend graph of the tumor volume with time in the molecular probe radiotherapy of Example 7;
  • Figure 13 The biodistribution of the molecular probe of Example 7 in the MC-38 mouse model, the SPECT/CT imaging of MC-38 nude mice, and the tumor volume treated by radiotherapy in the MC-38 mouse model over time Trend chart of change.
  • Figure 14 Treatment experiment of 177 Lu-DOTA-AL-3PRGD 2 in MC-38 tumor-bearing mice.
  • Figure 15 Tumor immune microenvironment on day 6 after targeted radiotherapy.
  • Figure 16 SPECT/CT imaging of 99m Tc-Nanobody.
  • B Using 0, 9 and 18MBq of 177 Lu-DOTA -Imaging on day 6 after AL-3PRGD 2 treatment and background imaging after 18MBq treatment.
  • FIG. 17 Biodistribution of 99m Tc-Nanobody.
  • A On the 6th day after treatment with 0-18MBq 177 Lu-DOTA-AL-3PRGD 2 , the biodistribution of mice 2 hours after injection of 99m Tc-Nanobody.
  • B Linear analysis of PD-L1 expression on myeloid cells in tumor and uptake of tumor 99m Tc-Nanobody.
  • Figure 18 The therapeutic window period of targeted radiation combined with PD-L1 blockade.
  • Figure 19 Synergy verification of targeted radiation combined with PD-L1 blockade.
  • the ethyl acetate layer was dried with anhydrous magnesium sulfate, and was rotary evaporated to dryness under reduced pressure to obtain 26.5 mg of yellow mucus. The yield was 85%. It was confirmed to be the expected product by MALDI-TOF mass spectrometry analysis.
  • the elution gradient is set to 80% A and 20% B at the beginning, 50% A and 50% B at 20 minutes, and 80% A and 20% B at 25 minutes.
  • the fractions with a retention time of 15.7 minutes were collected, and the collections were combined and lyophilized to obtain 3.2 mg of white powder.
  • the yield is 46.3%, and the purity is >95%. It was confirmed to be the expected product by MALDI-TOF mass spectrometry analysis.
  • the 68 Ge- 68 Ga generator was rinsed with 0.05M HCl to obtain 12.4mCi (469.9MBq) 68 Ga liquid. Take 500 ⁇ L of 68 Ga eluent, place it in a clean EP tube, add 12 ⁇ L of 1.25M NaOAc solution, and adjust the pH to 4.0. This 68 Ga solution (4.0mCi, 148MBq) was added to the EP tube containing the peptide. The metal bath was heated to 100°C and reacted for 10 minutes.
  • Purification Purify with Sep-Pak C-18 cartridge, first activate Sep-Pak C-18 column with 10 mL of absolute ethanol, then wash the column with 10 mL of H 2 O. The radioactive sample was passed through a Sep-Pak C-18 column, the column was washed with 10 mL of physiological saline, the free 68 Ga was removed, and finally the column was washed with 0.4 mL of 80% ethanol to collect the radioactive label. Sterilize through 0.22 ⁇ m microporous membrane for later in vivo experiments.
  • Radioactive HPLC Quality control: After the radioactive probe is placed at room temperature for 10 minutes, the purity is monitored by radioactive HPLC.
  • the radioactive HPLC method was used to determine the labeling rate and radiochemical purity.
  • Use HPLC system equipped with radioactivity online detector and Zorbax C18 analytical column (4.6mm x 250mm, pore size), gradient elution for 30 minutes, flow rate 1.0 mL/min, mobile phase A is H 2 O solution, B is acetonitrile (containing 0.05% TFA).
  • the elution gradient is set to 100% A and 0% B at the beginning, 95% A and 5% B at 5 minutes, 78% A and 22% B at 30 minutes, and return to baseline gradient 100% A at 30-35 minutes And 0%B.
  • the water and buffer used are treated with Chelex 100column to remove metal ions.
  • 68 Ga-DOTA-Ac (RGDfk) is labeled with a one-step method, the preparation process is simple and fast, and the labeling rate is 85%. After purification by the C-18Sep-Pak column, the radiochemical purity of the label is greater than 99%. After correction and calculation, the yield of the marker is 80%, as shown in Figure 1.
  • Example 2 In vivo biodistribution experiment of the molecular probe of Example 1
  • Thirty-six tumor-bearing C57BL/6J mice were randomly divided into 9 groups with 4 mice in each group.
  • the four groups were injected with 0.1mL 68 Ga-DOTA-Ac (RGDfk) (about 1.85MBq) in the tail vein, and the four groups were injected with 68 Ga-DOTA-c (RGDfk) (about 1.85MBq) in the tail vein at 0.5h and 1h respectively.
  • 2h, 4h blood samples were taken and then sacrificed.
  • the heart, liver, spleen, lung, kidney, intestine, stomach, bone, sarcoma and tumor were dissected.
  • the probe is metabolized by the kidneys.
  • the biodistribution data is shown in Figure 2.
  • the elution gradient is set to 100% A and 0% B at the beginning, 75% A and 25% B at 5 minutes, 50% A and 50% B at 25 minutes, and return to baseline gradient 100% A and 100% at 25-30 minutes 0%B.
  • the fractions with a retention time of 15.2 minutes were collected, and the collections were combined and lyophilized to obtain 1.95 mg of white powder.
  • the yield is 34.8%, and the purity is >95%. It was confirmed to be the expected product by MALDI-TOF mass spectrometry analysis.
  • 99m Tc-HYNIC-A-3PRGD 2 is prepared by non-SnCl 2 one-step method.
  • the label was analyzed by radio HPLC.
  • the retention time of 99m Tc-HYNIC-A-3PRGD 2 was 11.8 min, and the measured labeling rate was >99%. As shown in Figure 3.
  • Example 4 Molecular probe blood clearance experiment of Example 3
  • mice Take 14 Kunming female 4-5 weeks old mice and randomly divide them into two groups. Each group is injected with 0.1mL 99m Tc-HYNIC-A-3PRGD 2 and 99m Tc-HYNIC-3PRGD 2 (about 1.85MBq). After 1 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20 min, 30 min, 60 min, 90 min, 120 min, blood was taken, and the radioactive cpm count was measured. After decay correction, the percentage injection dose of the two probes in the blood was calculated ( %ID/g).
  • the fast half-life of 99m Tc-HYNIC-A-3PRGD 2 is 4.57 min; the slow half-life is 93.32 min.
  • the 99m Tc-HYNIC-3PRGD 2 (unmodified) drug has a fast half-life of 0.72 min and a slow half-life of 17.91 min.
  • the fast half-life of the drug was increased by 6.3 times, and the slow half-life was increased by 5.2 times.
  • the blood uptake of the unmodified molecular probe was 17.07 ⁇ 11.77% ID/g, while the uptake of 99m Tc-HYNIC-A-3PRGD2 was 44.76 ⁇ 11.83% ID/g, which was about 2.6 times that of the former.
  • the intake value of the former is only 4.39 ⁇ 2.55% ID/g, while the intake of the latter, that is, the molecular probe of the present invention is 33.63 ⁇ 7.83% ID/g, which is 7.6 times that of the former.
  • the former took 1.99 ⁇ 1.54% ID/g, while the latter took 19.06 ⁇ 6.51% ID/g.
  • the intake of the former fell below 0.5%ID/g after 60min.
  • Example 5 SPECT/CT imaging of tumor-bearing nude mice with molecular probes of Example 3
  • 99m Tc-HYNIC-A-3PRGD 2 (hereinafter referred to as the molecular probe of the present invention) was prepared according to Example 3.
  • the 99m Tc-HYNIC-3PRGD 2 (hereinafter referred to as the control probe) was prepared according to the 3PRGD 2 labeling method.
  • radioactive HPLC detection they were diluted with physiological saline to 2mCi/100 ⁇ L, and nude mice bearing U87MG tumor were injected intravenously. Each mouse was injected with 100 ⁇ L of 99m Tc-HYNIC-A-3PRGD 2 via the tail vein. The specific uptake of the drug in various tissues and organs of tumor-bearing mice was verified by a closed experiment.
  • mice in the closed group were injected with 100 ⁇ L of 1mg 3PRGD 2 cold peptide through the tail vein and immediately injected 100 ⁇ L of 99m Tc-HYNIC-A-3PRGD 2 while each drug taken 50 ⁇ Ci quantified.
  • the SPECT/CT images were collected at 0.5, 1, 2, 4, 8, 12, and 24 hours, and the results are shown in Fig. 5 SPECT/CT image of U87MG nude mice.
  • the molecular probe of the present invention can image the tumor well at each collection time point, even 24 hours after injection. This is related to the longer blood retention time of the modified probe, which in turn enhances tumor uptake.
  • the molecular probe of the present invention has a slow clearance from the tumor, and was 11.85 ⁇ 2.18%ID/g, 21.17 ⁇ 0.49%ID/g and 22.27 ⁇ 1.64 at 0.5, 8, and 24h after injection, respectively %ID/g.
  • Figure 6B compared with the control probe, the tumor/kidney ratio of the modified new probe was improved.
  • the tumor-to-kidney ratio of the control probe was 1.14 ⁇ 0.26, and 24h after injection was 1.21 ⁇ 0.17;
  • the corresponding uptake of the molecular probe of the invention is 1.57 ⁇ 0.26. and 1.92 ⁇ 0.17, respectively.
  • the tumor/non-tumor ratio of 3PRGD 2 has also been improved, as shown in Figures 6C and 6D. This shows that the modified molecular probe has better biodistribution and pharmacokinetic properties than before.
  • Example 6 Biodistribution of the molecular probe of Example 3
  • Twenty-four nude mice bearing U87 were randomly divided into 6 groups, 4 in each group, and time points were set according to 1h, 2h, 4h, 8h, and 12h.
  • Each group of nude mice was injected with 20 ⁇ Ci of 99m Tc-HYNIC-A-3PRGD 2 via the tail vein, and one group was injected with 3PRGD 2 as the Block group.
  • the animals were sacrificed, blood and main organs were taken, weighed, and the radioactive cpm count was measured. After decay correction, the percentage injection dose rate per gram of tissue (%ID/g) was calculated.
  • the uptake of imaging agent in U87 tumors first increased and then decreased as time went by, which was at 1h, 2h, 4h, 8h
  • the uptake in the tumor at the four time points was (22.38 ⁇ 3.68%ID/g, 21.71 ⁇ 3.61%ID/g, 25.96 ⁇ 1.69%ID/g, 23.53 ⁇ 3.40%ID/g).
  • the organ with the highest intake is the kidney, which is always above 30% ID/g, so the radioactive probe should be metabolized by the kidney, which is consistent with the results of SPECT/CT imaging.
  • the uptake of the remaining organs after injection is also higher than that of the control probe. The reason is that the modification of the polypeptide structure of the present invention allows it to circulate in the body for a longer time with blood, so that the uptake value of each organ will be increased.
  • the radiochemical purity (RCP) detection of 177 Lu-DOTA-AL-3PRGD2, 177 Lu-3PRGD2 and 177 Lu-DOTA-AL used Agilent HPLC-1260 Infinity liquid chromatography system with radioactivity detector and Agilent ZORBAX Extend-C18( 250x 4.6mm, 5um) chromatographic column (250x 10mm, 5 ⁇ m), the flow rate is 1mL/min.
  • the mobile phase A is water (containing 0.05% TFA)
  • the mobile phase B is acetonitrile (containing 0.05% TFA).
  • the elution gradient and time method are: 0 to 5 minutes 90% mobile phase A and 10% mobile phase B; 25 minutes 60% mobile phase A and 40% mobile phase B; 30 minutes 90% mobile phase A and 10% Mobile phase B.
  • Example 8 Molecular probe blood distribution of Example 7
  • the modified RGD probe of the present invention has a strong binding capacity to MSA, and the probe can circulate in the body for a longer time with the blood, which is caused by the increase of the concentration of the radioactive probe in the blood.
  • the integral of the injection dose rate of the probe per gram of tissue in the blood and the time (area under the curve, AUC) can indicate the effect of the drug in the blood.
  • the AUC (%ID/gh) values of 177 Lu-DOTA-AL-3PRGD 2 and 177 Lu--3PRGD 2 from 0 to 72 hours after administration were 208.9 and 27.0, respectively.
  • Example 9 SPECT/CT imaging of the molecular probe tumor-bearing nude mice of Example 7
  • the SPECT/CT imaging system (Mediso) has 4 probes and parallel hole collimators.
  • U87-MG tumor-bearing mice were injected with 100 ⁇ L (20 MBq) of 177 Lu-DOTA-AL-3PRGD 2 , 177 Lu-3PRGD 2 or 177 Lu-DOTA-AL via the tail vein. Then, SPECT imaging was performed 1, 4, 8, 12, 24, 48 and 72 hours after injection. During the imaging process, the mice were anesthetized with 1.5% isoflurane-oxygen and kept fixed in the prone position on the small animal bed. The mice in the closed group were injected with 1.0 mg Cold peptide.
  • the SPECT image is fused with the CT image, and then the region of interest (ROI) in the SPECT image is outlined in the 3D image, and the percentage injection dose rate (%ID/cc) per gram of tissues and organs is calculated.
  • the imaging and quantitative results include physical half-life and biological half-life, without decay correction. The results are shown in Figure 10.
  • the probe 177 Lu-DOTA-AL-3PRGD 2 of the present invention has appropriate pharmacokinetics, high tumor uptake and tumor contrast in tumors, and more importantly, its uptake in tumors is always significantly higher than Other normal tissues and organs throughout the body.
  • the tumor quantitative results of tumor-bearing mice 1 to 72 hours after injection showed that the cumulative uptake of 177 Lu-DOTA-AL-3PRGD 2 in the tumor was 662.0ID/cc-h; the cumulative uptake of 177 Lu-3PRGD 2 in the tumor The value is 158.7ID/cc-h; the cumulative uptake of 177 Lu-DOTA-AL in the tumor is 266.3ID/cc-h.
  • the molecular probe of the present invention has the highest uptake value at the tumor and the clearance is very slow.
  • the tumor uptake reaches the highest 4 hours after administration of the tumor-bearing mice, and its percentage injection dose rate is 26.52 ⁇ 0.58% ID/g. This indicates that the molecular probe of the present invention has better biodistribution characteristics and pharmacokinetic characteristics than before modification.
  • Example 10 In vivo biodistribution experiment of the molecular probe of Example 7
  • mice were sacrificed 4 hours after injection. Take blood and other major tissues and organs, weigh and measure their radioactivity count (cpm), and calculate the percentage injection dose rate per gram of tissue (%ID/g).
  • the tumor uptake values of the tumor-bearing mice at 1, 4, 24 and 72 hours after administration were 19.93 ⁇ 1.99%ID/g, 28.57 ⁇ 5.27%ID/g, 11.67 ⁇ 2.80%ID/g and 2.66 ⁇ 1.14%ID/g. Except for tumors, the probe has the highest uptake in the kidney, and the radioactive probe should be metabolized by the kidney, which is consistent with the results of SPECT/CT imaging. At 4 hours, the tumor/kidney ratio was more than 2 times (28.57/13.70%ID/g).
  • the tumor uptake value of the closed group 1 hour after administration was 6.41 ⁇ 1.52%ID/g
  • the tumor uptake value of the normal group 1 hour after administration was 19.93 ⁇ 1.98%ID/g.
  • Experimental results showed that the tumor uptake of the blocked group was significantly lower than that of the unblocked group (P ⁇ 0.005), and the uptake of 177Lu-k428-3PRGD2 in U87-MG tumors was specific.
  • Example 11 Radioactive targeted therapy of the molecular probe of Example 7
  • mice in group 1 were injected with 100 ⁇ L (18 MBq) of 177 Lu-DOTA-AL-3PRGD 2 through the tail vein, and mice in group 2 passed 100 ⁇ L (18MBq) of 177 Lu--3PRGD 2 were injected into the tail vein, group 3 mice were injected with 100 ⁇ L (18MBq) of 177 Lu-DOTA-AL through the tail vein; group 4 mice were injected with 100 ⁇ L (9MBq) of 177 Lu-DOTA-AL-3PRGD 2 , group 5 tumor-bearing mice were injected with 100 ⁇ L of PBS as a control. The tumor volume and weight change were monitored every 2 days after the injection, and the tumor volume reached 1000mm 3 as the benevolent endpoint.
  • Figure 12 See Figure 12 for the results.
  • Figure A and Figure C show the molecular probe 177 Lu-DOTA-AL-3PRGD 2 of the present invention and the control group 177 Lu--3PRGD 2 and 177 Lu-DOTA-AL at the same dose (18Mbq) and the molecular probe of the present invention.
  • the tumor volume after 14 days of administration was: 18MBq 177 Lu-DOTA-AL-3PRGD 2
  • the tumor volume of the treatment group was 401.3 ⁇ 195.5mm3
  • the 9MBq 177 Lu-DOTA-AL treatment group was 691.3 ⁇ 195.9mm3
  • 18MBq 177 Lu The tumor volume in the 3PRGD 2 treatment group was 1122.4 ⁇ 189.6 mm3
  • the tumor volume in the 18MBq 177 Lu-DOTA-AL treatment group was 897.4 ⁇ 178.0 mm3
  • the tumor volume in the PBS control group was 1336.3 ⁇ 315.4 mm3.
  • 177 Lu-DOTA-AL-3PRGD 2 can significantly inhibit tumor growth within 10 days after injection after targeted nuclide treatment in U87-MG tumor-bearing mice.
  • the initial volume ratio (V/V0) of the treatment group and the control group on the 10th day was 1.4 and 8.0.
  • Figure B shows the molecular probe of the present invention 177 Lu-DOTA-AL-3PRGD 2 and the control group 177 Lu--3PRGD 2 , 177 Lu-DOTA-AL at the same dose (18Mbq), and the molecular probe of the present invention is decreasing
  • the experimental results showed that BALB/c nude mice were tolerant to the 18MBq dose of 177 Lu-DOTA-AL-3PRGD 2.
  • the body weight and blood routine indexes of the mice in the administration group returned to normal after the decrease. Mice after the 18MBq dose had the largest weight loss on the 6th day, and returned to normal on the 14th day after the administration.
  • the initial weight percentages of the administration group and the control group on the 6th day were 90.8 ⁇ 3.7% and 98.7 ⁇ 5.0%; the initial weight percentages of the administration group on the 14th day were 100.5 ⁇ 4.1% and 100.7 ⁇ 3.4%.
  • the molecular probe of the present invention does not change significantly in the body weight of the mice during 14 days. It can be seen that the molecular probe of the present invention has less side effects and high safety.
  • Example 12 Experiment of the molecular probe of Example 7 in MC-38 mouse model
  • Fig. 13C is a graph showing the trend of tumor size changes over time when the molecular probe 177 Lu-DOTA-AL-3PRGD 2 of the present invention has a dose of 18 Mbq and 9 Mbq. It can be seen from the figure that when the molecular probe of the present invention is dosed at 18 Mbq, the tumor is completely eliminated after the 12th day to the 20th day, which is not the case in Figure 12A of Example 11. Compared with the U87-bearing mouse of Example 18, the MC-38 mouse of this example has autoimmune function, while the latter is an immunodeficient mouse. Therefore, the former is closer to the body state of human patients, which can explain the molecular exploration of the present invention.
  • Fig. 13D is a trend chart of the weight change of mice with time when the molecular probe of the present invention is at doses of 18Mbq and 9Mbq. It can be seen from the figure that the molecular probe of the present invention does not change significantly in the weight of the mouse during 20 days. It can be seen that the molecular probe of the present invention has less side effects and high safety.
  • Example 13 The targeted radiotherapy of Example 7 remodels the tumor immune microenvironment
  • MC-38 mouse colorectal cancer cells
  • DMED high-sugar medium containing 10% heat-inactivated fetal bovine serum
  • a humidity incubator containing 5% CO 2 at 37°C.
  • Female C57/BL6 (4-6 weeks old) mice were purchased from the Laboratory Animal Department of Peking University Health Science Center, and the animal experiments met the regulations and requirements of the Peking University Animal Protection and Use Committee.
  • Tumor volume (mm 3 ) tumor length (mm) x tumor width (mm) x tumor height (mm) x 0.5. When the tumor volume is greater than 1200mm3, the mouse is deemed dead.
  • Tumor tissue was digested with cell digestion solution containing 1 mg/mL collagenase IV (Worthington) and 0.1 mg/mL DNase I (Roche). Stain the surface of T cells at 4°C for 30 minutes, using CD45 (1 ⁇ g/mL, Cat.56-0451-82, eBioscience), CD3e (1 ⁇ g/mL, Cat.25-0031-82, eBioscience) ), CD8a (2.5 ⁇ g/mL, Cat. 11-081-82, eBioscience), CD4 (1 ⁇ g/mL, Cat. 45-0042-82, eBioscience).
  • T cells were stained intranuclearly using Foxp3 stained transcription factor staining buffer kit (Cat.00-523-00, eBioscience) and Foxp3 (1 ⁇ g/mL, Cat. 12-4771-82, eBioscience).
  • Flow cytometry samples were analyzed by Gallios flow cytometer (Beckman Counter), and the experimental data were processed by Flowjo 7.0 software (Tree Star).
  • the results show that targeted radiotherapy can significantly increase the infiltration of CD4 + and CD8 + T lymphocytes (but not T-reg cells) in tumor tissues.
  • the infiltration ratio of CD4 + T lymphocytes (CD45 + CD3e + CD4 + cells) in the tumor was 1.04 ⁇ 0.55, 0.76 ⁇ 0.18, 1.17 ⁇ 0.41, 1.82 ⁇ 0.65, 2.24 ⁇ 0.78 and 4.84 ⁇ 0.85%;
  • the infiltration ratio of CD8 + T lymphocytes (CD45 + CD3e + CD8 + cells) is 1.12 ⁇ 0.51, 1.12 ⁇ 0.48, 1.06 ⁇ 0.31, 2.09 ⁇ 0.58, 2.71 ⁇ 0.53 and 4.09 ⁇ 0.93%;
  • the infiltration ratios of T-reg cells (CD45 + CD3e + CD4 + Foxp-3 + cells) are 0.37 ⁇ 0.27, 0.22 ⁇ 0.08, 0.21 ⁇ 0.07, respectively , 0.28 ⁇ 0.13, 0.31 ⁇ 0.13 and 0.34 ⁇ 0.12%.
  • the results show that targeted radiotherapy can significantly up-regulate the expression of PD-L1 on the surface of myeloid immune cells (not tumor cells) in tumor tissues.
  • the PD-L1 expression (MFI) on tumor cells (CD45 - cells) was 441 ⁇ 68, 511 ⁇ 168, 400 ⁇ 107, 436 ⁇ 64, 659 ⁇ 108 and 556 ⁇ 152;
  • the PD-L1 expression (MFI) of myeloid immune cells (CD45 + CD11b + ) are 1681 ⁇ 144, 2938 ⁇ 588, respectively , 3187 ⁇ 586, 4444 ⁇ 1140, 5085 ⁇ 926 and 6749 ⁇ 533.
  • targeted radiotherapy can significantly increase the infiltration of effector T cells in tumors, and up-regulate PD-L1 in tumors, which has positive significance for anti-PD-1/PD-L1 blockade therapy.
  • Example 14 Targeted radiation combined with PD-L1 blocking therapy under the guidance of images
  • the selected anti-mouse PD-L1 Nanobody (MY1523) is labeled with the LPTEG-His 6 tag at the C-terminus for affinity purification of Ni-Sepharose and the site specificity of the transpeptidase Sortase-A connection.
  • the KD value of the nanobody and the mouse PD-L1 is 49.70 ⁇ 7.90nM, and the IC50 value is 59.23 ⁇ 0.04nM, which has high affinity and specificity.
  • SPECT/CT imaging uses NanoScan SPECT/CT small animal nuclear medicine imaging system, the selected acquisition energy peak is 140keV, the acquired energy peak width is 20%, and the single image acquisition time is 30s.
  • mice were injected with 18MBq 99m Tc-nanobody through the tail vein, and nuclear medicine imaging was performed 2 hours after injection. The mice were anesthetized with isoflurane gas during imaging.
  • SPECT/CT imaging of 99m Tc-nanobody was performed on the 0, 3, and 6 days after targeted radiotherapy, and the tumor uptake was quantified as the percentage injection dose rate (%ID/g).
  • the linear relationship between the tumor's 99m Tc-Nanobody uptake and tumor PD-L1 expression was determined.
  • the uptake of nanobody probes by tumors was measured at the tissue level through biodistribution experiments, and the PD-L1 expression of tumors was measured at the cellular level by flow cytometry after the biodistribution experiments.
  • -3PRGD 2 is used to remodel the tumor immune microenvironment.
  • the biodistribution experiment of 99m Tc-Nanobody was performed.
  • the mice were injected with 720 kBq of 99m Tc-Nanobody through the tail vein, and the mice were sacrificed 2 hours after the administration.
  • the energy peak of the selected ⁇ -count is 135 ⁇ 155keV.
  • the 99m Tc signal collected by the test is not affected by the 177 Lu signal.
  • the tumor tissue is immediately digested to prepare a single cell suspension.
  • tumor cells respectively (CD45 -) PD-L1 expression in myeloid and immune cells (CD45 + CD11b +) were analyzed flow.
  • the uptake of 99m Tc-nanobody by the tumor can accurately reflect the PD-L1 expression level of the tumor.
  • the tumor uptake values of 99m Tc-MY1523 probe were 2.27 ⁇ 0.26, 2.28 ⁇ 0.69, 3.63 ⁇ 0.94, respectively. 4.86 ⁇ 0.58 and 7.66 ⁇ 1.59%ID/g.
  • the uptake of 99m Tc-MY1523 probe in the tumor is significantly positively correlated with the expression of PD-L1 on the infiltrating myeloid immune cells.
  • the 99m Tc signal collected by the test is not Affected by 177 Lu signal.
  • the PD-L1 expression level change of the tumor in the interval of 0-6 days after targeted radiotherapy was measured by 99m Tc-Nanobody SPECT/CT imaging.
  • flow cytometry was used to explore the dynamic changes of PD-L1 in tumors 0-12 days after targeted radiotherapy.
  • the tumor tissue was digested into a single cell suspension, and the dynamic changes of PD-L1 expression levels of tumor cells and myeloid cells were measured by flow cytometry.
  • the expression of PD-L1 in the tumor microenvironment showed a dynamic trend of first high and then low, and reached the highest on the 6th day after administration.
  • Figure 18.A the expression of PD-L1 in the tumor microenvironment continued to be up-regulated 3-6 days after administration, and reached the highest level on the 6th day after administration, followed by 9-12 days after administration in the tumor microenvironment The expression of PD-L1 continued to decrease.
  • a single PD-L1 monoclonal antibody immunotherapy is performed 0-12 days after targeted radiotherapy.
  • the correlation between phases provides a basis for the combined therapy of targeted radiation and PD-L1 blockade under the guidance of imaging.
  • mice in the 6 groups were injected with 100 ⁇ g PD-L1 antibody combination therapy at different time points on the 3rd, 6, 9th, and 12th day.
  • the tumor volume and body weight changes of the mice were monitored every 2 days. When the tumor volume was greater than 1200 mm 3 , the monitoring was terminated and the mice were sacrificed.
  • mice in group 5 were injected with 9MBq of 177 Lu-DOTA-AL-3PRGD 2 via tail vein injection for a single targeted radiotherapy; mice in groups 6-8 were injected with 9MBq of targeted radiation
  • 100 ⁇ g PD-L1 antibody was administered in combination at different time points on the -3, 0, and 3 days. The tumor volume and body weight changes of the mice were monitored every 2 days, and the tumor volume greater than 1200mm 3 was regarded as dead.
  • the therapeutic effect of anti-PD-L1 blockade on day 3 after targeted radiotherapy is significantly better than that of simultaneous administration of targeted radiotherapy and anti-PD-L1 blockade therapy, and is significantly better Anti-PD-L1 treatment was performed on the 3rd day before targeted radiotherapy.
  • Figure 19.C in the combination therapy with 9MBq 177 Lu-DOTA-AL-3PRGD 2 , the 90-day survival rate of the mice in the PD-L1 blocking treatment group at the -3, 0 and 3 day time points The order is 4/7, 4/7 and 7/7.

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Abstract

一种结构修饰的RGD多肽，由该多肽和放射性核素形成的配合物，以及包含该配合物的药物组合物，所述药物组合物用于诊断或者治疗整合素αvβ3阳性肿瘤。优选的药物组合物，其还包括免疫治疗药物，以及任选的纳米抗体分子影像探针。在靶向放射性给药治疗后进行PD-L1阻断治疗能够获得最佳的协同疗效。此外，施用PD-1或PD-L1纳米抗体分子影像探针，能够观测到靶向放射治疗后的肿瘤中的PD-1或PD-L1表达，对于靶向放射与免疫联合治疗的治疗策略的制定具有指导意义，有助于提升联合治疗的效果。

Description

用于肿瘤的靶向放射性药物及其在影像指导下的靶向放射治疗与免疫治疗的联合疗法
本申请要求2019年05月24日向中国国家知识产权局提交的专利申请号为201910441556.7，发明名称为“一种结构修饰的RGD多肽的核医学药物”的在先申请的优先权，还要求2020年05月06日向中国国家知识产权局提交的专利申请号为202010373843.1，发明名称为“一种用于肿瘤的靶向放射与免疫联合治疗的药物组合物”的在先申请的优先权，上述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及一种用于肿瘤的RGD多肽修饰的靶向放射性药物，还涉及其与免疫治疗药物的联合疗法。
背景技术
恶性肿瘤的持续生长、侵袭、转移依赖于肿瘤血管生成。整合素是参与调控肿瘤血管增生的重要因子之一，对肿瘤血管生成起着重要作用，整合素中以整合素αvβ3的作用最为重要。整合素αvβ3在肿瘤新生血管内皮细胞高度表达，而在正常细胞或成熟血管不表达或低表达。RGD能够特异性结合整合素αvβ3，因此利用RGD和整合素αvβ3的特异性结合而设计出的RGD类分子探针得到广泛的研究与运用。国内外研究者先后通过将线性RGD改造成环状RGD，药代动力学连接剂修饰例如将RGD糖基化，用谷氨酸将RGD环肽单体联接成多聚体、通过化学手段在二聚体中的两个RGD单体之间插入Gly3或PEG4链等等多种方法来优化RGD多肽，以得到结构修饰的RGD多肽。
然而，很多环状RGD单体肽放射性标记物，肿瘤摄取低，血清除速率快，肾、肝等器官摄取高，这些都限制了环状单体肽作为显像剂的应用；随着多聚化程度提升，放射性RGD多肽在肾、肝、肺等器官摄取也明显增高，而且，多聚化程度越高合成越复杂，造价越高，这些也是RGD探针多聚化发展的制约因素；单纯多聚化或使用大分子量的药代动力学连接剂对RGD探针进行修饰的优势不再明显。
癌症的常规治疗方法(如放疗和化疗)通常作用于肿瘤细胞本身，并可引发大部分患者的反应。尽管这些常规治疗在初期能够产生有效的应答，但在癌症的晚期往往会出现耐药及复发，导致治疗的失败。与上述常规疗法的作用方式不同，免疫疗法通过激活免疫系统，促进机体自身产生抗肿瘤反应，并且不易产生耐药。免疫检查点抑制疗法(immunecheckpoint blockade)作为肿瘤治疗的一大突破，对多种恶性实体肿瘤的免疫治疗具有明显的疗效。细胞程序性死亡受体1(PD1)及其配体-1(PD-L1)是目前研究最普遍的一对免疫检查点，其抑制剂药物在临床上应用广泛。尽管PD1/PD-L1阻断疗法能够引发显著和持续的响应，但其客观有效率只有30％左右，如何提高其有效性仍然是目前肿瘤免疫治疗中的关键性问题。
联合疗法是提高PD1/PD-L1阻断疗法有效性的主要手段之一，利用常规治疗所产生的抗肿瘤免疫效应能够增强免疫治疗的有效性。一般观点认为，放疗可促进肿瘤抗原的释放，增强效应T细胞的分化，增值，功能及肿瘤浸润，并在与免疫检查点阻断治疗的联合中具有协同性。目前，放疗联合免疫治疗在肺癌的免疫治疗中逐渐显现优势，但其治疗策略仍然有待研究。与传统放疗相比，靶向放射治疗(Targeted Radiotherapy)是一种基于体内分子结合的内照射疗法，其在转移瘤和晚期肿瘤患者的治疗中具有明显优势。但是，靶向放射治疗的临床应用时间较短，其所能够引起的免疫学效应尚不明确，因此靶向放射与免疫联合治疗的研究仍然不足。使用PD-L1作为生物标志物制备的纳米抗体探针可对肿瘤微环境进行无创，实时和动态的监测，有利于指导制定靶向放射治疗联合免 疫治疗的个体化策略，提高肿瘤免疫治疗的有效性。
发明内容
为了改善现有技术存在的上述问题，本发明提供如下式结构修饰的RGD多肽：
A-(L)n-RGD多肽
其中A为如下结构：
Figure PCTCN2020091861-appb-000001
L代表连接臂分子，具有如下结构：
Figure PCTCN2020091861-appb-000002
其中m为1-8的整数，例如2-6，优选5；
所述L通过其羧基与A中的氨基反应键链，例如，通过L中标记为*的羧基与A键链；
n为0或1；
当n为0时，所述RGD多肽通过其氨基与A中的羧基反应键链；
当n为1时，所述RGD多肽通过其氨基与L中的另一羧基反应键链，例如当L中标记为*的羧基与A相连时，标记为**的羧基与RGD多肽反应键链；
RGD多肽为选自以下的RGD多肽：c(RGDfV)、c(RGDfK)，c(RGDfE)，c(RGDyk)、E[c(RGDyk)] 2、E[c(RGDfK)] 2、3PRGD 2。
本发明还提供含有如上结构修饰的RGD多肽的放射性核素标记的配合物，其具有如下定义的结构：
Nu-BFC-A-(L)n-RGD多肽
其中：
Nu为放射性核素，例如诊断显像核素： 111In、 64Cu、 99mTc、 68Ga，或者治疗核素： 90Y、 177Lu、 89Sr、 153Sm、 188Re；
BFC为双功能螯合剂(bifunctional chelating agent)，例如HYNIC(联肼尼克酰胺)、MAG 2(巯基乙酰二甘氨酸)、MAG 3(巯基乙酰三甘氨酸)、DTPA(二乙基三胺五乙酸)、DOTA(1,4,7,10-四氮杂环十二烷-1,4,7,10四乙酸)、NOTA(1,4,7-三氮杂环壬烷-1,4,7三羧酸)、TETA(1,4,8,11-四氮杂环十四烷-1,4,8,11四乙酸)；
当n为0时，所述双功能螯合剂通过其结构中具有的羧基与A中的-NH 2反应键链；
当n为1时，所述双功能螯合剂通过其结构中具有的羧基与L中的-NH 2反应键链。
根据本发明，如上结构修饰的多肽RGD和配合物中：
RGD多肽选自：c(RGDfK)、3PRGD 2；
Nu选自： 90Y、 177Lu、 111In、 64Cu、 99mTc；
当Nu为 90Y、 177Lu时，BFC选自DTPA、DOTA；当Nu为 111In、 64Cu、 68Ga、 99mTc时，BFC选自HYNIC、MAG 2、MAG 3、DTPA、DOTA、TETA和NOTA。
根据本发明，Nu为 90Y或 177Lu，BFC选自DTPA、DOTA；Nu为 111In时，BFC选自DTPA、DOTA；Nu为 64Cu时，BFC选自TETA、DOTA；Nu为 68Ga时，BFC选自NOTA、DOTA；Nu为 99mTc时，BFC选自HTNIC、DTPA、MAG 2、MAG 3；
作为实例，由本发明结构修饰的多肽形成的配合物如下所述：
68Ga-DOTA-A-c(RGDfk)；
99mTc-HYNIC-A-3PRGD 2；
177Lu-DOTA-A-L-3PRGD 2。
应当理解，本发明的上述结构修饰的多肽的所有异构体，包括对映异构体、非对映异构体、以及消旋物都属于本发明的范围。本发明包括光学纯形式或混合物的立体异构体，也包括外消旋混合物。例如上述多肽中的结构A或L中的氨基以L-或D-形式存在。
本领域技术人员熟知，如上定义的配合物，当双功能螯合剂作为配体不能占据放射性核素所有配位位置时，还需要协同配体。本发明中需要协同配体的放射性核素和双功能螯合剂是本领域技术人员所熟知的。例如 99mTc以HYNIC作为双功能螯合剂时，作为协同配体，其可以相同或不同，均是现有技术中已知的那些，其中常见的协同配体包括水溶性膦(例如三苯基膦三间磺酸钠盐TPPTS)，N-三(羟甲基)甲基甘氨酸(Tricine)、N-二(羟乙基)甘氨酸、葡庚糖酸盐、乙二胺-N,N’-二乙酸酯(EDDA)、3-苯甲酰基吡啶(BP)、吡啶-2-偶氮-p-二甲苯胺(PADA)等。例如， 99mTc以HYNIC作为双功能螯合剂时，TPPTS和Tricine作为协同配体。而例如 177Lu或者 68Ga以DOTA为双功能螯合剂时，并不需要协同配体完成配位。
作为实例，本发明提供如下结构的配合物：
Figure PCTCN2020091861-appb-000003
本发明还提供一种具有上述配合物结构的分子探针。
本发明还提供一种药物组合物，所述组合物包含有效量的上述标记配合物Nu-BFC-A-(L)n-RGD多肽。
根据本发明，当Nu为诊断核素时，本发明的药物组合物是一种诊断药物，例如所述药物作为显像剂，用于整合素αvβ3阳性肿瘤的显像诊断。将其直接施用于个体，通过检测给受试者施用的该化合物所发射的放射线，基于该放射线获得的信息显像来诊断。优选地，本发明的诊断药物是一种可注射制剂，其包含上述标记配合物和可注射的载体。优选所述显像剂是指作为正电子发射断层扫描PET、单光子发射计算机化断层显像SPECT。
根据本发明，当Nu为治疗核素时，本发明的药物组合物是一种治疗药物，所述药物用于整合素αvβ3阳性肿瘤的放射靶向治疗。将其直接施用于个体，所述药物与整合素αvβ3具有特异性亲和力，富集在肿瘤组织内，放射性核素通过发射纯β-射线或伴有γ射线的β-射线产生电离辐射生物效应破坏病变组织。优选地，本发明的治疗药物是一种可注射制剂，其包含上述标记配合物和可注射的载体。
根据本发明，当Nu为治疗核素时，本发明的药物组合物还可以包括一种免疫治疗药物。此时以本发明的上述标记配合物进行靶向放射治疗，联合免疫治疗药物，达到协同治疗效果。
根据本发明上述的靶向放射治疗和免疫治疗联合的药物组合物，优选的上述标记配合物是 177Lu-DOTA-A-L-3PRGD 2。优选的免疫治疗药物是PD-1或PD-L1免疫检查点抑制剂。优选的，是PD-1或PD-L1单抗药物。本发明的PD-1或PD-L1单抗药物不做特别限定，是本领域已知的靶向人或动物PD-1/PD-L1免疫通路的活性有效的此类药物，例如已上市的多种PD-1单克隆抗体药物，例如Opdivo(MDX-1106),Keytruda(MK-3475),CT-011，或者PD-L1单克隆抗体，例如MDX-1105、MPDL3280A,或MEDI4736，或其他已知的用于临床试验阶段的PD-1或PD-L1单克隆抗体。示例性的，本发明实施例所用的PD-L1单克隆抗体是(10F.9G2)。
本领域技术人员已知，PD-L1是PD-1的配体，在生物体的健康正常状态下，细胞表面的PD-L1与淋巴细胞表面的PD-1结合后，可抑制淋巴细胞功能，诱导活化的淋巴细胞调亡，从而在自身免疫耐受及防止自身免疫性疾病中发挥重要作用。然而，在肿瘤组织中会过度表达PD-L1，而肿瘤浸润淋巴细胞高表达PD-1，PD-1与PD-L1相结合，从而抑制了淋巴细胞的功能及肿瘤杀伤作用，诱导淋巴细胞调亡，消弱了机体本身的抗肿瘤免疫应答，最终导致肿瘤免疫逃逸的发生。而PD-1或PD-L1的抗体能够阻断体内PD-1/PD-L1通路，从而促进淋巴细胞增殖、激活免疫系统，促进机体自身产生抗肿瘤反应，进一步使得肿瘤退化。基于上述机理作用可知，任何的PD-1或PD-L1免疫检查点抑制剂，都可阻断PD-1/PD-L1通路，实现自身抗肿瘤反应，从而治疗肿瘤或癌症。因此本发明的PD-1或PD-L1单抗药物并不做特别限定，任何已知的此类药物都可以用于本发明。
根据本发明的靶向放射治疗和免疫治疗联合的药物组合物，其还进一步包括纳米抗体分子影像探针，例如PD-1或PD-L1纳米抗体分子影像探针。优选的，是PD-L1纳米抗体锝标记物。示例性的，本发明实施例所用的纳米抗体分子影像探针是 99mTc-MY1523。
根据本发明的靶向放射治疗和免疫治疗联合的药物组合物，所述纳米抗体分子影像探针是含有LPETG标签的PD-L1纳米抗体(MY1523)，其可以通过Sortase A酶连接 99mTc-HYNIC-GGGK制备得到。
根据本发明的靶向放射治疗和免疫治疗联合的药物组合物，所述标记配合物与免疫治疗药物可同时或分别前后施用。例如所述免疫治疗药物可在标记配合物后施用，优选的，所述免疫治疗药物在标记配合物使用后3～6天使用。
根据本发明的靶向放射治疗和免疫治疗联合的药物组合物，所述纳米抗体分子影像探针在标记配合物施用之后，而免疫治疗药物施用之前施用。
优选地，本发明的标记配合物、免疫治疗药物或纳米抗体分子影像探针是一种可注射制剂，其包含上述标记配合物、免疫治疗药物或者纳米抗体分子影像探针和可注射的载体。
优选地，本发明的药物组合物是一种静脉注射剂，例如一种无色透明液体针剂。适用于静脉注射剂的辅料是本领域公知的，所述药物组合物可以配置在水溶液中，如果需要，使用生理相容的缓冲剂，包括例如，磷酸盐、组氨酸、柠檬酸盐等，用于调节制剂pH，还可使用张度剂，诸如氯化钠、蔗糖、葡萄糖等，还可使用共溶剂，例如聚乙二醇，低毒性表面活性剂，例如聚山梨酯或泊洛沙姆等。
优选地，本发明的药物组合物还包括抗吸附剂，例如生理盐水，1％环糊精的水溶液，含吐温-20的PBS溶液(如吐温的质量分数为0.01～0.1％)，使用本发明的含有抗吸附剂的药物组合物，使用表面活性剂如吐温-20(质量份数例如0.05％)的溶液，能够有效避免所述标记物在输液中的非特异性吸附，明显防止管壁对放射性标记物的吸附，实现给药剂量的精准化，同时避免了药物在使用过程中的浪费。含有上述抗吸附剂的药物组合物在一系列新的EP管中连续转移4次，标记物的回收率可达97％以上。
根据本发明，还提供一种试剂盒，其分别装有含本发明如上所述的标记配合物Nu-BFC-A-(L)n-RGD多肽的药物、免疫治疗药物，以及任选的纳米抗体分子影像探针。
根据本发明，还提供上述A-(L)n-RGD多肽或者Nu-BFC-A-(L)n-RGD多肽分子探针或者上述药物组合物在制备药物中的用途。根据本发明，所述药物用于诊断或者治疗整合素αvβ3阳性肿瘤，所述肿瘤是指实体肿瘤，例如在血液、肝、腺体(例如乳腺、前列腺、胰腺)、肠(例如结肠直肠)、肾、胃、脾、肺、肌肉、骨头等部位的恶性肿瘤。
本发明还提供一种诊断或治疗整合素αvβ3高表达的血液系统和实体恶性肿瘤的方法，将有效量的上述Nu-BFC-A-(L)n-RGD多肽施用于有此需求的个体。根据本发明，所述个体可以为哺乳动物，如人类。
本发明还提供一种治疗整合素αvβ3高表达的血液系统和实体恶性肿瘤的方法，将有效量的标记配合物Nu-BFC-A-(L)n-RGD多肽和免疫治疗药物施用于有此需求的个体。根据本发明，所述个体可以为哺乳动物，如人类。
根据本发明的方法，所述免疫治疗药物在所述标记配合物施用之后施用，例如在其后3-6天施用。
根据本发明的方法，所述标记配合物和免疫治疗药物以注射形式施用。
根据本发明的方法，所述方法在纳米抗体分子影像探针的指导下进行。例如，在施用标记配合物后，采用锝标纳米抗体对肿瘤中的PD-L1表达情况进行活体水平的监测。
根据本发明的方法，根据对PD-L1表达情况的监测，选择在PD-L1表达增高或顶峰时施用免疫治疗药物。
术语和定义
除非另有定义，否则本文所有科技术语具有的涵义与权利要求主题所属领域技术人员通常理解的涵义相同。除非另有说明，本文引用的所有专利、专利申请、公开材料的全文通过引用方式整体并入本文。
本申请说明书和权利要求书记载的数值范围，当该数值范围被理解为“整数”时，应当理解为记载了该范围的两个端点以及该范围内的每一个整数。例如，“0～10的整数”应当理解为记载了0、1、2、3、4、5、6、7、8、9和10的每一个整数。当该数值范围被理解为“数”时，应当理解为记载了该范围的两个端点以及该范围内的每一个整数以及该范围内的每一个小数。例如，“1～10的数”应当被理解为不仅记载了1、2、3、4、5、6、7、8、9和10的每一个整数，还至少记载了其中每一个整数分别与0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9的和。
RGD多肽：均为本领域已知物质。RGD是含有精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)氨基酸序列的小分子多肽。加入D型苯丙氨酸和缬氨酸，合成了RGD环五肽结构-c(RGDfV)，其中c代表该多肽为环形、R代表精氨酸、G代表甘氨酸、D代表天冬氨酸、f代表D-苯甘氨酸、V代表缬氨酸。环五肽结构-c(RGDfV)的5位氨基酸被其他氨基酸取代后得c(RGDfK)，c(RGDfE)，c(RGDyk)，其中K为赖 氨酸、E为谷氨酸、y为D-酪氨酸。例如c(RGDfK)具有如下结构：
Figure PCTCN2020091861-appb-000004
这些环肽结构可形成二聚体，例如E[c(RGDyk)] 2、E[c(RGDfK)] 2，用谷氨酸将两个RGD环肽连接形成二聚体。3PRGD 2是指含有三个聚乙二醇修饰的RGD五环肽二聚体，即PEG 4-[PEG 4-c(RGDXk)] 2，X为D-苯甘氨酸、D-酪氨酸等。示例性的，其结构示意图如下：
Figure PCTCN2020091861-appb-000005
双功能螯合剂：双功能螯合剂(bifunctional chelator BFC)是指既能与生物分子共价连接又能鳌合金属核素，其结构能保证与金属核素的牢固结合，且引入的金属核素远离生物分子以保证其生物活性不受损失，形成一个稳定的核素-螯合剂-生物分子标记物的这一类功能有机材料。本发明用到的双功能螯合剂是本领域现有技术中已知的那些，例如HYNIC(联肼尼克酰胺)、MAG 2(巯基乙酰二甘氨酸)、MAG 3(巯基乙酰三甘氨酸)、DTPA(二乙基三胺五乙酸)、DOTA(1,4,7,10-四氮杂环十二烷-1,4,7,10四乙酸)、NOTA(1,4,7-三氮杂环壬烷-1,4,7三羧酸)、TETA(1,4,8,11-四氮杂环十四烷-1,4,8,11四乙酸)等。
本文所用的术语“治疗”和其它类似的同义词包括缓解、减轻或改善疾病或病症症状，预防其它症状，改善或预防导致症状的潜在代谢原因，抑制疾病或病症，例如阻止疾病或病症的发展，缓解疾病或病症，使疾病或病症好转，缓解由疾病或病症导致的症状，或者中止疾病或病症的症状，此外，该术语包含预防的目的。该术语还包括获得治疗效果和/或预防效果。所述治疗效果是指治愈或改善所治疗的潜在疾病。此外，对与潜在疾病相关的一种或多种生理症状的治愈或改善也是治疗效果，例如尽管患者可能仍然受到潜在疾病的影响，但观察到患者情况改善。就预防效果而言，可向具有患特定疾病风险的患者施用所述组合物，或者即便尚未做出疾病诊断，但向出现该疾病的一个或多个生理症状的患者施用所述组合物。
有益效果
本发明设计和制备了结构修饰的RGD多肽，以及由该结构修饰的RGD多肽制备得到了药代动力学性质改善的系列新型RGD多肽分子探针。本发明发现，所述RGD多肽通过结构修饰和改造，由该结构修饰的RGD多肽通过螯合剂与放射性核素形成的本发明分子探针具有较高的体内外稳定性、与白蛋白(albumin)的结合率，从而明显延长了半衰期；本发明的分子探针具有较高的肿瘤摄取率、对比度、安全性，降低了使用剂量，减少了副作用，从而改善了RGD系列探针作为显像诊断分子探针在SPECT和/或PET中的显像效果，以及作为治疗分子探针进行放射性靶向性治疗的效果。
本发明新型分子探针的一个实例 177Lu-DOTA-A-L-3PRGD 2，在血液中具有较高摄取，从而提高了在肿瘤中的摄取，其在血液中的累计摄取量可达原探针的8倍左右，在肿瘤中的累积摄取量为原探针的4倍左右。其在注射后4小时，肿瘤摄取达到最高，其百分注射剂量率为26.52±0.58％ID/g，注射后48小时后仍能清晰显像。这一性质明显提高了RGD系列探针作为显像诊断分子探针的显像效果，以及作为治疗分子探针进行放射性靶向性治疗的效果。
本发明所述的靶向放射性治疗药物，不仅能够在肿瘤组织中特异性富集，通过内照射作用直接作用于肿瘤细胞，而且能够激活机体产生由T细胞介导的抗肿瘤免疫反应。在本发明的一个实施例中，靶向放射治疗引起肿瘤免疫微环境的重塑，显著增加肿瘤组织中CD4+和CD8+T淋巴细胞(而非T-reg细胞)的浸润，并上调髓系免疫细胞(而非肿瘤细胞)表面的PD-L1表达水平。
本发明通过联合使用PD-1/PD-L1免疫检查点抑制剂能够有效增加初始T细胞的活化以及效应T细胞的效应阶段，分别在肿瘤免疫循环的前期和后期发挥重要作用。而利用本发明的RGD多肽修饰的靶向放射性药物促进了肿瘤的抗原释放和免疫细胞的抗原呈递，在肿瘤免疫循环的前期发挥重要功能，因此本发明充分利用肿瘤免疫循环中的相同及不同阶段，实现了靶向放射性治疗与免疫治疗的协同性的激活抗肿瘤免疫。此外，本发明还进一步利用PD-L1纳米抗体分子影像探针对治疗过程中肿瘤微环境中的PD-L1表达的变化进行监测，以指导联合治疗的给药方案。
当标记的核素是治疗核素时，本发明的标记配合物是一种有效的靶向放射治疗药物，其在免疫正常的MC-38同系肿瘤模型中，能够有效治疗肿瘤。在本发明的一个实施例中，施用18MBq的标记配合物能够通过T细胞介导的特异性免疫反应使肿瘤完全消融。本发明的靶向放射治疗引起肿瘤免疫微环境的重塑主要体现在：通过肿瘤的内照射治疗，显著增加CD4 +和CD8 +T淋巴细胞(而非T-reg细胞)的浸润，并上调肿瘤中髓系免疫细胞(而非肿瘤细胞)表面的PD-L1表达。靶向放射性给药第6天是最严重的免疫抑制期，此时肿瘤中的PD-L1表达量达到最高。因此在靶向放射治疗后3～6天进行PD-L1阻断治疗能够获得最佳的协同疗效。
另外，本发明通过实验发现，只有在PD-L1动态增高的时间窗口施用PD-L1单克隆抗体才能获得协同疗效，因此跟踪监测PD-L1在靶向放射治疗后的动态表达变化具有重要指导意义，本发明施用PD1或PD-L1纳米抗体分子影像探针，能够观测到靶向放射治疗后的肿瘤中的PD-L1表达，对于靶向放射与免疫联合治疗的治疗策略的制定具有指导意义，有助于提升联合治疗的效果。
附图说明
附图1：实施例1终产物 68Ga-DOTA-A-c(RGDfk)的放射性HPLC图；图1a为纯化前，图1b为纯化后；
附图2：实施例1的分子探针和对照探针在荷LLCC57BL/6J鼠体内生物分布结果；
附图3：实施例3终产物 99mTc-HYNIC-A-3PRGD 2的放射性HPLC图；
附图4：实施例3的血清除实验结果图；
附图5：实施例3的荷U87MG裸鼠SPECT/CT显像图；
附图6：附图A\B\C\D分别为实施例3的SPECT/CT显像结果与对照探针的在U87肿瘤的％ID/g、肿瘤/肾脏，肿瘤/肌肉，肿瘤/肝脏的比值比较图；
附图7：实施例3的分子探针在荷U87MG裸鼠体内分布结果图；
附图8：实施例7的终产物 177Lu-DOTA-A-L-3PRGD 2的放射性HPLC图；
附图9：实施例7的分子探针血清除实验结果图；
附图10：实施例7的分子探针的荷U87MG裸鼠SPECT/CT显像图；
附图11：实施例7的分子探针在荷U87鼠体内生物分布图；
附图12：实施例7的分子探针放射性治疗的肿瘤体积随时间变化的趋势图；
附图13：实施例7的分子探针在MC-38小鼠模型中的生物分布、MC-38裸鼠SPECT/CT显像图和在MC-38小鼠模型中放射性治疗的肿瘤体积随时间变化的趋势图。
附图14： 177Lu-DOTA-A-L-3PRGD 2在MC-38荷瘤小鼠中的治疗实验。(A)不同给药剂量下的靶向放射治疗(n＝7)，(B)治疗过程中小鼠的体重变化(n＝7),(C)靶向放射治疗中使用CD8抗体进行免疫耗竭的治疗实验(n＝7)。
附图15：靶向放射治疗后第6天的肿瘤免疫微环境。(A)经0～18MBq  177Lu-DOTA-A-L-3PRGD 2治疗后，肿瘤组织中T淋巴细胞占肿瘤消化细胞的百分比例(n＝4),(B)肿瘤细胞及髓系细胞上的PD-L1表达情况(n＝4)。细胞分群：CD8 +T细胞(CD45 +CD3e +CD8 +)，CD4 +T细胞(CD45 +CD3e +CD4 +)，T-reg细胞(CD45 +CD3e +CD4 +Foxp3 +)，肿瘤细胞(CD45 -)，髓系细胞(CD45 +CD11b +)。
附图16： 99mTc-纳米抗体的SPECT/CT显像。(A)使用9MBq  177Lu-DOTA-A-L-3PRGD 2治疗后0，3和6天的动态成像(n＝3,*P<0.05)，(B)使用0，9和18MBq的 177Lu-DOTA-A-L-3PRGD 2治疗后第6天的成像及18MBq治疗后的背景显像。
附图17： 99mTc-纳米抗体的生物分布。(A)经0～18MBq  177Lu-DOTA-A-L-3PRGD 2治疗后第6天，小鼠注射 99mTc-纳米抗体后2小时的生物分布。(B)肿瘤内髓系细胞上的PD-L1表达与肿瘤 99mTc-纳米抗体摄取的线性分析。
附图18：靶向放射联合PD-L1阻断的治疗窗口期。(A)经9MBq  177Lu-DOTA-A-L-3PRGD 2治疗3～12天后，肿瘤微环境中肿瘤细胞及髓系细胞上PD-L1的动态变化(n＝4)，(B)靶向放射治疗后3～12天进行的单次抗PD-L1联合治疗(n＝7，**P<0.01)。
附图19：靶向放射联合PD-L1阻断的协同性验证。(A)不同时间点的单一抗PD-L1阻断治疗(n＝7)，(B)靶向放射治疗前，中和后的抗PD-L1阻断联合治疗(n＝7)，(C)联合治疗的小鼠生存率(n＝7)。
具体实施方式
下文通过对本发明实施例的描述，更加详细地对本发明的上述及其他特性和优势进行解释和说明。应当理解，下列实施例旨在对本发明的技术方案进行示例性的说明，而并非旨在对由权利要求及其等价方案所限定的本发明保护范围进行任何限制。
除非另有说明，本文中的材料和试剂均为市售商品，或可由本领域技术人员根据现有技术制备。
本领域技术人员应当理解，下列实施例中的原料、试剂、中间体、目标化合物或反应式均为上文通式化合物或其反应的示例性技术方案，其中的一个或多个具体化合物或具体反应式均可与本发明上述概括性的技术方案结合，且该结合后的技术方案应当被理解为说明书记载的技术方案。
统计学分析
实验结果以平均值±标准偏差(mean±SD)的形式进行表示。组间的差异采用方差分析和t检验对结果进行统计学分析。P<0.05，认为具有统计学差异(*)。
实施例1  68Ga-DOTA-A-c(RGDfk)的制备
DOTA-A-c(RGDfk)的合成路线图如下：
Figure PCTCN2020091861-appb-000006
(1)化合物1的合成
称取4-(4-碘苯基)丁酸(9.8mg，33.8mmol)加入单口瓶，溶解于400μL DMF中，加入NHS(3.9mg，33.9mmol)。再加入5.3μL DIC。30℃恒温搅拌反应2h，TLC(乙酸乙酯：石油醚：乙酸＝100:200:2)监测至原料消失。待反应结束，将反应液溶于乙酸乙酯，用水洗三次，乙酸乙酯相用无水硫酸钠干燥，浓缩后柱层析分离(乙酸乙酯：石油醚：乙酸＝100:200:2)，收集馏分检测，将收集的产品旋干得到白色固体10.2mg，产率78％。经MALDI-TOF质谱分析确认为预期产物。
MS(ESI)：m/z＝387.2137(化学式：C 14H 14INO 4，计算分子量387.00)。取少量固体重新溶解后，经HPLC纯度鉴定纯度＞98％。
(2)化合物2的合成
取化合物1(20mg，51.7mmol)加入装有500μL DMF的圆底烧瓶中溶解，再取Fmoc-D-Lys.HCL(22.8mg，56.3mmol)加入烧瓶，.加入12μL DIEA调节pH＝8.5。30℃恒温搅拌反应0.5h，TLC(乙酸乙酯：石油醚：乙酸＝100:200:1)监测至原料消失。反应结束后，将反应液溶于20mL乙酸乙酯，分别用20mL饱和氯化钠溶液、20mL水洗3次。乙酸乙酯层用无水硫酸镁干燥，减压旋蒸至干，得到黄色粘液26.5mg。产率85％。经MALDI-TOF质谱分析确认为预期产物。
MALDI-TOF-MS：m/z＝1718.33(M+H) +，1741.02(M+Na) +(化学式：C 80H 119N 25O 16S,计算分子量：1719.02Da.)。取少量产物重新溶解后，利用HPLC进行分析，纯度96.7％。
(3)化合物3的合成
称取化合物2(10mg，15.6mmol)加入单口瓶，溶解于500μL DMF中，再加入NHS(1.83mg，15.9mmol)，再向单口瓶中加入DIC 4μL。35℃下搅拌反应35分钟。加热5min时，发现悬浊液变 清。TLC(乙酸乙酯：石油醚：乙酸＝100:200:1)监测至原料消失。反应结束后，将反应液溶于20mL乙酸乙酯，分别用20mL饱和氯化钠溶液、20mL水洗3次。乙酸乙酯层用无水硫酸镁干燥，浓缩后层析分离(乙酸乙酯：石油醚＝1:1)，收集馏分检测，将收集的产品旋干减压旋蒸至干，得到白色固体8.02mg，产率70％。经MALDI-TOF质谱分析确认为预期产物。
MALDI-TOF-MS：m/z＝760.2(M+Na) +(化学式：C 35H 36IN 3O 7,计算分子量：737.16)。
(4)化合物4的合成
取化合物3(11.5mg，15.6mmol)于1mL EP管中，溶解于500μL DMF，加入c(RGDfk)(9.4mg，15.6mmol)，再加入DIEA调节pH＝8.5，30℃反应2小时。使用HPLC监测反应。HPLC(高效液相色谱)方法一：使用安捷伦1100系列HPLC系统配备YMC-Pack ODS-A C18半制备柱(10mm×250mm,
Figure PCTCN2020091861-appb-000007
pore size,颗粒大小为5μm)，梯度淋洗25分钟，流速1mL/min，其中流动相A为H 2O溶液，B为乙腈(含0.05％TFA)。淋洗梯度设定为起始时100％A和0％B，20分钟时0％A和100％B，25分钟时100％A和0％B。产物在14.854min时出峰。
(5)化合物5的合成
向前一步反应EP管中加入125μL哌啶，脱去Fmoc保护基，向反应体系中加入500μL乙醚高速离心沉淀，弃去乙醚，得到产物为白色固体，约为9.55mg，产率为61％。经MALDI-TOF质谱分析确认为预期产物。
MALDI-TOF-MS：m/z[C43H62IN11O9] +(M+H) +，1004.38；实测值1004.88。取少量固体溶解后，HPLC鉴定纯度为94.4％。
(6)化合物6的合成
称取化合物5(5mg，4.98mmol)，DOTA-NHS-ester 3.79mg溶于400μL DMF。加入DIEA调节pH＝8.5，30℃震荡反应30min。半制备HPLC分离纯化，HPLC(高效液相色谱)方法二：使用安捷伦1100系列HPLC系统配备YMC-Pack ODS-A C18半制备柱(10mm×250mm,
Figure PCTCN2020091861-appb-000008
pore size,颗粒大小为5μm)，梯度淋洗25分钟，流速1mL/min，其中流动相A为H 2O溶液，B为乙腈(含0.05％TFA)。淋洗梯度设定为起始时80％A和20％B，20分钟时50％A和50％B，25分钟时80％A和20％B。收集保留时间为15.7分钟时的馏分，合并收集液并冻干，得到白色粉末3.2mg。产率为46.3％，纯度>95％。经MALDI-TOF质谱分析确认为预期产物。
MALDI-TOF-MS：m/z[C55H80IN15O16] +(M+H) +，1390.50；实测值1390.7040。HPLC鉴定纯度为98％。(7) 68Ga-DOTA-A-c(RGDfk)的制备、纯化与质控
准确称取定量DOTA-A-c(RGDfk)，用水重新溶解后，装成20μg/管，置于-80℃冰箱保存。待放射性标记时，取出已经分装好的化合物，室温融化30分钟。
制备：利用0.05M HCl淋洗 68Ge- 68Ga发生器，得到12.4mCi(469.9MBq) 68Ga液。取500μL 68Ga淋洗液，置于洁净EP管中，加入1.25M NaOAc溶液12μL，调节pH值至4.0。将此 68Ga液(4.0mCi，148MBq)加入至装有多肽EP管中。金属浴加热至100℃，反应10分钟。
纯化：用Sep-Pak C-18小柱进行纯化，首先用10mL无水乙醇活化Sep-Pak C-18柱，紧接着用10mL H 2O洗柱。将放射性样品通过Sep-Pak C-18柱，用10mL的生理盐水洗柱，移去游离的 68Ga，最后用0.4mL 80％的乙醇洗柱，收集放射性标记物。过0.22μm微孔滤膜除菌，用于后期的体内实验。
质控：放射性探针室温放置10分钟后，利用放射性HPLC监测纯度。采用放射性HPLC方法测定标记率和放射化学纯度。使用HPLC系统，配备放射性在线检测器和Zorbax C18分析柱(4.6mm x 250mm,
Figure PCTCN2020091861-appb-000009
pore size)，梯度淋洗30分钟，流速1.0mL/min，其中流动相A为H 2O溶液，B为乙腈(含0.05％TFA)。淋洗梯度设定为起始时100％A和0％B，5分钟时95％A和5％B，30分钟时78％A和22％B，30-35分钟回到基线梯度100％A和0％B。
在进行标记前，水和所用的缓冲液都用Chelex 100column处理，去除金属离子。 68Ga-DOTA-A-c(RGDfk)用一步法标记，制备过程简单快速，标记率在85％，经C-18Sep-Pak柱纯化后，标记物的放化纯均大于99％。经校正计算后，标记物产率在80％，见图1。
实施例2：实施例1的分子探针体内生物分布实验
取荷瘤C57BL/6J小鼠36只，随机分为9组，每组4只。其中四组尾静脉各注射0.1mL  68Ga-DOTA-A-c(RGDfk)(约1.85MBq)，四组尾静脉注射 68Ga-DOTA-c(RGDfk)(约1.85MBq)，分别于0.5h、1h、2h、4h取血后处死，解剖取心、肝、脾、肺、肾、肠、胃、骨、肉、瘤。测质量和放射性计数，称重并测量放射性cpm计数，经衰变校正后计算每克组织的百分注射剂量(％ID/g)。剩余一组尾静脉同时注射0.1mL  68Ga-DOTA-A-c(RGDfk)和0.05mL c(RGDfk)溶液(0.5mg)，一小时后处死，取器官称重并测量放射性cpm计数，经衰变校正后计算每克组织的百分注射剂量(％ID/g)。
注射 68Ga-DOTA-A-c(RGDfk)0.5h后在血液里摄取达到了33.32±5.49％ID/g，肿瘤摄取在7.52±0.99％ID/g，而与之对比， 68Ga-DOTA-c(RGDfk)0.5h后血液摄取值在1.49±0.49％ID/g，在肿瘤内的摄取值为2.74±0.73％ID/g。在随后的时间点里， 68Ga-DOTA-A-c(RGDfk)肿瘤摄取始终高于 68Ga-DOTA-c(RGDfk)，注射4h后 68Ga-DOTA-A-c(RGDfk)肿瘤摄取值为6.57±0.89％ID/g，是 68Ga-DOTA-c(RGDfk)的3.5倍，说明，新型探针能有效地和血清蛋白结合，提高探针的血液半衰期，有效提高了肿瘤摄取值(P<0.01，n＝4)。该探针经肾脏代谢。生物分布数据见图2。
实施例3： 99mTc-HYNIC-A-3PRGD 2的制备
HYNIC-A-3PRGD 2合成路线示意图如下：
Figure PCTCN2020091861-appb-000010
R＝3PRGD 2
(1)化合物1、2、3的合成
化合物的合成方法参考实施例1。
(2)化合物7的合成
称取化合物3(5.64mg，7.65mmol)于1mL EP管中，溶解于500μL DMF，加入3PRGD 2(15.75mg，7.8mmol)，再加入DIEA调节pH＝8.5，室温反应过夜。使用HPLC监测反应。采用HPLC(高效液相色谱)方法一，产物在13.17min时出峰。HPLC收集，经质谱鉴定，确定为预期产物。
MALDI-TOF-MS：m/z＝2680.89(化学式：C 123H 181IN 24O 35,计算分子量：2681.22Da.)。
(3)化合物8的合成
向前一步反应EP管中加入125μL哌啶，脱去Fmoc保护基，再向反应体系中加入500μL乙醚高速离心沉淀，弃去乙醚，得到产物为白色固体，约为10.6mg，产率为56％。经MALDI-TOF质谱分析确认为预期产物。
MALDI-TOF-MS：m/z＝2460.85(M+H) +，2482.87(M+Na) +(化学式：C 108H 171IN 24O 33,计算分子量：2459.15Da.)。
取少量固体重新溶解后，利用HPLC进行纯度分析，纯度92.5％。
(4)化合物9的合成
称取化合物8(5mg，2.03mmol)，SBZH-HYNIC 0.9mg溶于500μL DMF中。加入DIEA调节pH＝8.5，30℃震荡反应10分钟。半制备HPLC分离纯化，HPLC(高效液相色谱)方法三：使用安捷伦1100系列HPLC系统配备YMC-Pack ODS-A C18半制备柱(10mm×250mm,
Figure PCTCN2020091861-appb-000011
pore size,颗粒大小为5μm)，梯度淋洗30分钟，流速1mL/min，其中流动相A为H 2O溶液，B为乙腈(含0.05％TFA)。淋洗梯度设定为起始时100％A和0％B，5分钟时75％A和25％B，25分钟时50％A和50％B，25-30分钟返回基线梯度100％A和0％B。收集保留时间为15.2分钟时的馏分，合并收集液并冻干，得到白色粉末1.95mg。产率为34.8％，纯度>95％。经MALDI-TOF质谱分析确认为预期产物。
MALDI-TOF-MS：m/z＝2763.07(M+H) +，2785.21(M+Na) +(化学式：C 121H 180IN 27O 37S,计算分子量：2762.18Da.)。
取少量固体溶解后经HPLC分析，纯度大于99％。
(5) 99mTc-HYNIC-A-3PRGD 2的制备与质控
在EP管中依次加入20μL HYNIC-A-3PRGD 2(1mg/mL溶于纯水)，100μL tricine溶液(100mg/mL溶于25mM琥珀酸盐缓冲液,pH 5.0),100μL TPPTS(60mg/mL溶于25mM琥珀酸盐缓冲液,pH 5.0)，100μL Na 99mTcO 4(10mCi)。混匀后，置100℃水浴加热25min。标记产物冷却后，用HPLC(HP1100高效液相色谱，配有LB-509放射性检测器)分析其标记率和放射化学纯度。
99mTc-HYNIC-A-3PRGD 2采用非SnCl 2一步法制备。放射性HPLC对标记物进行分析， 99mTc-HYNIC-A-3PRGD 2保留时间为11.8min，测得的标记率＞99％。如图3所示。
实施例4：实施例3的分子探针血清除实验
取14只昆明雌性4-5周龄小白鼠，随机分为两组，每一组分别注射0.1mL 99mTc-HYNIC-A-3PRGD 2， 99mTc-HYNIC-3PRGD 2(约1.85MBq)，注射后分别于1min、3min、5min、7min、10min、15min、20min、30min、60min、90min、120min取血，测量放射性cpm计数，经衰变校正后计算两种探针在血液里的百分注射剂量(％ID/g)。
通过血清除实验，我们可以看到结构改造后的3PRGD 2探针，其体内性质和原探针相比发生了明显改变。 99mTc-HYNIC-A-3PRGD 2的药物快速半衰期为4.57min；慢速半衰期为93.32min。而 99mTc-HYNIC-3PRGD 2(未结构修饰)的药物快速半衰期为0.72min；慢速半衰期为17.91min。药物快速半衰期提高了6.3倍，慢速半衰期提高了5.2倍。注射1min，未修饰的分子探针血液摄取值为17.07±11.77％ID/g，而 99mTc-HYNIC-A-3PRGD2摄取为44.76±11.83％ID/g，是前者的约2.6倍。在5min时，前者摄取值只有4.39±2.55％ID/g，而后者即本发明分子探针摄取为33.63±7.83％ID/g，是前 者的7.6倍。10min时，前者摄取1.99±1.54％ID/g，而后者摄取为19.06±6.51％ID/g。前者摄取在60min后，降到0.5％ID/g以下，在240min时，，摄取值为0.34±0.18％ID/g；与之对比，后者的摄取为2.81±0.83％ID/g，是原探针的8倍。可见，结构修饰极大的延长了3PRGD 2的血液滞留时间(P<0.01，n＝7)，这样就有利于增加探针在肿瘤部位的摄取值。血清除结果见图4。
实施例5：实施例3的分子探针荷瘤裸鼠SPECT/CT显像
99mTc-HYNIC-A-3PRGD 2(下称本发明分子探针)按实施例3制备。同时按照3PRGD 2标记方法进行 99mTc-HYNIC-3PRGD 2(下称对照探针)的制备。进行放射性HPLC检测后，用生理盐水分别稀释至2mCi/100μL，取荷U87MG瘤裸鼠分别进行静脉注射，每只小鼠经由尾静脉注射100μL的 99mTc-HYNIC-A-3PRGD 2。该药物在荷瘤鼠各组织和器官中的特异性摄取通过封闭实验进行验证，封闭组小鼠通过尾静脉注射100μL的1mg 3PRGD 2冷肽，并立即注射100μL的 99mTc-HYNIC-A-3PRGD 2同时每种药取50μCi进行定量。分别在0.5、1、2、4、8、12、24h进行SPECT/CT图像采集，结果见图5荷U87MG裸鼠SPECT/CT显像图。
图6A为 99mTc-HYNIC-A-3PRGD 2和 99mTc-HYNIC-3PRGD 2在U87肿瘤的％ID/g；6B、6C、6D分别为 99mTc-HYNIC-A-3PRGD 2和 99mTc-HYNIC-3PRGD 2肿瘤/肾脏，肿瘤/肌肉，肿瘤/肝脏的比值比较。结果表示为means±SD,(n＝3)。
本发明分子探针在各个采集时间点，肿瘤都能很好的成像，甚至是在注射24小时后。这与改造后探针有更长的血液滞留时间，进而增强了肿瘤摄取有关。如图6A定量分析所示，本发明分子探针在肿瘤处的清除很缓慢，在注射后0.5、8、24h分别为11.85±2.18％ID/g，21.17±0.49％ID/g和22.27±1.64％ID/g。如图6B所示，与对照探针相比，修饰后的新探针瘤/肾比得到提高，注射后1h，对照探针瘤肾比为1.14±0.26,注射后24h为1.21±0.17；而本发明分子探针相应摄取分别为1.57±0.26.,1.92±0.17。改造后3PRGD 2的瘤/非瘤比也得到了改善，如图6C、6D所示。这表明，改造后的分子探针与改造前相比，有着更好的生物分布特性、药代动力学特性。
实施例6：实施例3的分子探针生物分布
将24只荷U87裸鼠随机分成6组，每组4只，按照1h、2h、4h、8h、12h设置时间点。每组裸鼠分别经尾静脉注射20μCi的 99mTc-HYNIC-A-3PRGD 2，一组注射3PRGD 2用作Block组。并于注射后将动物处死，取血液及主要脏器，称重并测量放射性cpm计数。经衰变校正后计算每克组织的百分注射剂量率(％ID/g)。
生物分布标样制备：以注射器将100μL用于生物分布实验的标记物溶液加入100mL容量瓶中，加水定容到100mL，充分混匀，以移液器准确取出1mL用γ计数器测量放射性计数，此计数放大100倍，作为标准注射剂量，制备三个平行样，取平均值。
如图7所示，尾静脉注射 99mTc--HYNIC-A-3PRGD 2后，随着时间的延长，显像剂在U87肿瘤中的摄取先升后降，其在1h、2h、4h、8h四个时间点的肿瘤中的摄取分别为(22.38±3.68％ID/g，21.71±3.61％ID/g，25.96±1.69％ID/g，23.53±3.40％ID/g)。摄取最高的器官是肾脏，一直在30％ID/g以上，所以放射性探针应该是经过肾脏代谢，这和SPECT/CT显像结果是一致的。注射后其余各器官摄取也都较对照探针高，原因在于本发明的多肽结构修饰使其随血液在体内循环时间更久，这样，各器官的摄取值都会有所提高。
实施例7： 177Lu-DOTA-A-L-3PRGD 2的制备
Figure PCTCN2020091861-appb-000012
(1)化合物2的制备同实施例1
(2)化合物11的制备
称取100.0mg(250μmol)化合物10，76.4mg(400μmol)EDC.HCL和46.0mg(400μmol)NHS溶解于5mL二氯甲烷，室温搅拌过夜。混合物通过硅胶层析进行分离和纯化，洗脱液为含有2％甲醇的二氯甲烷。减压蒸馏除去溶剂，得白色粉末状固体85.2mg。HPLC梯度和时间方法为：0分钟 50％流动相A和50％流动相B；25分钟时10％流动相A和90％流动相B；30分钟时50％流动相A和50％流动相B。取少量产物用HPLC检验纯度(保留时间为17.7分钟)，然后用MALDI-TOF质谱鉴定。
(3)化合物12的制备
称取85.2mg(147μmol)Fmoc-Cys(tBu)-NHS化合物11，61.0mg(145μmol)化合物2，加入400μL DMF和20μL DIEA，水浴超声形成悬液。加入400μL纯水使溶液中的固体完全溶解，室温搅拌过夜。混合物经HPLC方法进行分离和纯化，收集保留时间为20.9分钟的洗脱峰。HPLC梯度和时间方法为：0分钟50％流动相A和50％流动相B；25分钟时10％流动相A和90％流动相B；30分钟时50％流动相A和50％流动相B。冷冻干燥，得白色粉末状固体67.2mg。取少量产物用HPLC方法验证纯度，然后用MALDI-TOF质谱鉴定。
(4)化合物13的制备
称取67.2mg(84.1μmol)化合物12，加入20％哌啶-DMF溶解后室温反应10分钟。混合物经HPLC方法进行分离纯化，收集保留时间为24.1分钟的洗脱峰。HPLC梯度和时间方法为：0分钟85％流动相A和15％流动相B；25分钟时45％流动相A和55％流动相B；30分钟时85％流动相A和15％流动相B。冷冻干燥，得白色粉末状固体27.8mg。取少量产物用HPLC方法验证纯度，然后用MALDI-TOF质谱鉴定。
(5)化合物14的制备
称取10.0mg(12.5μmol)化合物13，10mg(13.1μmol)DOTA-NHS溶于200μL DMF。加入10μLDIEA，将不溶物超声打散成悬液。加入200μL纯水使溶液中的固体完全溶解，室温搅拌过夜。混合物通过HPLC方法进行分离纯化，收集保留时间为21.7分钟的洗脱峰。HPLC梯度和时间方法为：0分钟85％流动相A和15％流动相B；25分钟时45％流动相A和55％流动相B；30分钟时85％流动相A和15％流动相B。冷冻干燥，得白色粉末状固体8.5mg。产物用HPLC方法验证纯度，然后用MALDI-TOF质谱鉴定。
(6)化合物15的制备
称取8.5mg(8.8μmol)化合物14，加入200μL 20％TFMSA-TFA反应30秒，立即加入400μL DMF防止产物酸解。混合物经HPLC方法进行分离纯化，收集保留时间为26.6和26.9分钟的洗脱峰。HPLC梯度和时间方法为：0分钟85％流动相A和15％流动相B；25分钟时45％流动相A和55％流动相B；30分钟时85％流动相A和15％流动相B。冷冻干燥，得白色粉末状固体1.2mg。产物用HPLC方法验证纯度，然后用MALDI-TOF质谱对其进行鉴定。
(7)MAL-3PRGD 2(L7)的制备
称取10mg(4.8μmol)3PRGD2，2.0mg(6.5μmol)Mal-NHS，加入200μLDMF和10μLDIEA。混合液在室温下搅拌过夜。混合物经HPLC方法进行分离纯化，收集保留时间为22.1分钟的洗脱峰。HPLC梯度和时间方法为：0分钟90％流动相A和10％流动相B；25分钟时60％流动相A和40％流动相B；30分钟时90％流动相A和10％流动相B。冷冻干燥，得白色粉末状固体6.7mg。取少量产物用HPLC方法验证纯度，然后用MALDI-TOF质谱对其进行鉴定。
(8)化合物16的制备
称取0.7mg(0.8μmol)化合物15，加入1.8mg(0.8μmol)MAL-3PRGD2，加入0.1M磷酸缓冲液(pH＝7.0)，室温震荡反应过夜。产物经HPLC方法进行分离纯化，收集保留时间为25.6分钟的洗脱峰。HPLC梯度和时间方法为：0分钟90％流动相A和10％流动相B；25分钟时60％流动相A和40％流动相B；30分钟时90％流动相A和10％流动相B。合并产物峰洗脱液进行冷冻干燥，得白色粉末状固体1.2mg。取少量产物用HPLC检测纯度，然后用MALDI-TOF质谱鉴定。HPLC分析产物纯度>98％，MALDI-TOF质谱结果显示m/z＝3160.84。实验结果表明[M+H]+与C137H215IN30O45S的理论分子量M＝3161.36相符。
(9) 177Lu-DOTA-A-L-3PRGD 2的制备、纯化与质控
称取20μg化合物16、DOTA-3PRGD 2或DOTA-A-L，然后加入200μL醋酸铵缓冲液(0.1M，pH＝4.8)和5～25mCi 177LuCl 3。将混合液置于99℃空气浴加热器中反应20分钟，自然冷却后即得。为了防止标记物发生辐射自分解，加入200μL龙胆酸水溶液(1mg/mL)。标记物能够保持6小时以上的室温放置稳定性。
177Lu-DOTA-A-L-3PRGD2、 177Lu-3PRGD2和 177Lu-DOTA-A-L的放射化学纯度(RCP)检测使用带有放射性检测器的Agilent HPLC-1260Infinity液相色谱系统和Agilent ZORBAX Extend-C18(250x 4.6mm，5um)色谱柱(250x 10mm，5μm)，流速为1mL/min。流动相A为水(含0.05％TFA)，流动相B为乙腈(含0.05％TFA)。淋洗梯度和时间方法为：0～5分钟90％流动相A和10％流动相B；25分钟时60％流动相A和40％流动相B；30分钟时90％流动相A和10％流动相B。
所述终产物 177Lu-DOTA-A-L-3PRGD 2的放射性HPLC图见图8
实施例8：实施例7的分子探针血液分布
取10只昆明小白鼠，分为两组，分别注射0.1mL，取15只昆明小鼠随机分为三组(n＝5)，通过尾静脉注射100μL(740KBq)的本发明实施例14的 177Lu-DOTA-A-L-3PRGD 2，以及作为对照的 177Lu-3PRGD2或 177Lu-DOTA-A-L。小鼠注射后按时间点从内眦取适量血液，称重并测量放射性计数cpm。计算放射性药物在血液中的每克组织的百分注射剂量率(％ID/g)。实验结果用GraphPad Prism 7.0软件进行非线性回归分析，计算血液代谢双室模型(Two phase decay)中的快速半衰期和慢速半衰期。
根据实验结果，结果见图9： 177Lu-DOTA-A-L-3PRGD 2的快速半衰期和慢速半衰期分别为6.909min和77.15min，而 177Lu--3PRGD 2的快速半衰期和慢速半衰期分别为1.231min和20.83min，可见本发明的 177Lu-DOTA-A-L-3PRGD 2与 177Lu--3PRGD 2相比，血液滞留时间有着明显延长，证明本发明对多肽的结构修饰确实能延长c(RGDfk)在血液里的滞留时间。这说明本发明改造后的RGD探针与MSA结合能力强，探针能随着血液在体内循环更长时间，使血液里的放射性探针浓度增加所致。探针在血液的每克组织的百分注射剂量率与时间的积分(曲线下面积，AUC)能够表示药物在血液中的作用效果。 177Lu-DOTA-A-L-3PRGD 2、 177Lu--3PRGD 2在给药后0～72小时的AUC(％ID/g-h)值依次为208.9、27.0。实验结果表明，本发明新型 177Lu-DOTA-A-L-3PRGD 2具有比 177Lu--3PRGD 2高7.7倍的血液药代动力学。虽然 177Lu-DOTA-A-L在血液内的最慢慢速半衰期比本发明的分子探针还要长，但其作为分子探针的其他性质，例如在肿瘤中的滞留和摄取要比本发明的分子探针差很多，具体参见下述实施例。
实施例9：实施例7的分子探针荷瘤裸鼠SPECT/CT显像
SPECT/CT显像系统(Mediso公司)具有4个探头及平行孔准直器。U87-MG荷瘤鼠经尾静脉注射100μL(20MBq)的 177Lu-DOTA-A-L-3PRGD 2、 177Lu-3PRGD 2或 177Lu-DOTA-A-L。然后注射后1、4、8、12、24、48和72小时进行SPECT显像。小鼠在显像过程中使用1.5％异氟烷-氧气进行麻醉，保持其在小动物床上的俯卧位的固定。封闭组小鼠在注射标记物的同时，混入了1.0mg的
Figure PCTCN2020091861-appb-000013
冷肽。将SPECT图像与CT图像融合，然后在3D显像图中勾勒出SPECT图像中的感兴趣区域(ROI)，计算组织和器官的每克组织的百分注射剂量率(％ID/cc)。显像及定量结果包括物理半衰期和生物半衰期，不进行衰变矫正。结果见图10。
我们发现本发明的探针 177Lu-DOTA-A-L-3PRGD 2具有适当的药代动力学，在肿瘤中具有高肿瘤摄取和肿瘤对比度，更重要的是，其在肿瘤中的摄取始终显著高于全身其他正常组织和器官。荷瘤鼠注射后1～72小时的肿瘤定量结果表明， 177Lu-DOTA-A-L-3PRGD 2在肿瘤中的累计摄取值为662.0ID/cc-h； 177Lu-3PRGD 2在肿瘤中的累计摄取值为158.7ID/cc-h； 177Lu-DOTA-A-L在肿瘤中的累计摄 取值为266.3ID/cc-h。 177Lu-DOTA-A-L-3PRGD 2在各个采集时间点，肿瘤都能很好的成像，甚至是在注射48小时后肿瘤处成像清晰，而对照探针本底高、对比度低，在肿瘤处富集少。如图10定量分析所示，本发明分子探针在肿瘤处的摄取值最高、清除很缓慢，荷瘤鼠给药后4小时，肿瘤摄取达到最高，其百分注射剂量率为26.52±0.58％ID/g。这表明，本发明的分子探针与改造前相比，有着更好的生物分布特性、药代动力学特性。
实施例10：实施例7的分子探针体内生物分布实验
取16只U87-MG荷瘤鼠，经尾静脉注射100μL(0.74MBq)的 177Lu-DOTA-A-L-3PRGD 2。注射后1、4、24和72小时处死小鼠(n＝4)；取4只荷瘤小鼠，通过尾静脉注射100μL混有740KBq的 177Lu-DOTA-A-L-3PRGD 2和0.5mg的3PRGD 2冷肽，注射后1小时处死小鼠；取12只荷瘤小鼠随机分成3组(n＝4)，经尾静脉注射100μL(0.74MBq)的 177Lu-DOTA-A-L-
3PRGD 2、 177Lu-3PRGD2或 177Lu-DOTA-A-L，注射后4小时处死小鼠。取血液和其他主要组织和器官，称重并测量其放射性计数(cpm)，计算每克组织的百分注射剂量率(％ID/g)。
如图11a所示，注射后4小时， 177Lu-DOTA-A-L-3PRGD 2的肿瘤摄取值为26.52±0.58％ID/g，177Lu-3PRGD2的肿瘤摄取值为4.91±0.92％ID/g， 177Lu-DOTA-A-L的肿瘤摄取值为4.80±1.19％ID/g。177Lu-k428-3PRGD2在肿瘤中的摄取显著高于177Lu-3PRGD2(P<0.0001)和177Lu-k428(P<0.0001)。
如图b所示，荷瘤鼠给药后1、4、24和72小时的肿瘤摄取值分别为19.93±1.99％ID/g，28.57±5.27％ID/g，11.67±2.80％ID/g和2.66±1.14％ID/g。除肿瘤之外，该探针在肾脏中的摄取最高，放射性探针应该是经过肾脏代谢，这和SPECT/CT显像结果是一致的。在4小时，肿瘤/肾脏比为2倍以上(28.57/13.70％ID/g)。
如图c所示，封闭组给药后1小时的肿瘤摄取值为6.41±1.52％ID/g，正常组给药后1小时的肿瘤摄取值为19.93±1.98％ID/g。实验结果表明，封闭组的肿瘤摄取显著低于未封闭组(P<0.005)，177Lu-k428-3PRGD2在U87-MG肿瘤中的摄取具有特异性。
实施例11：实施例7的分子探针的放射性靶向治疗
取35只U87-MG荷瘤小鼠随机分成5组(n＝7)：第1组小鼠通过尾静脉注射100μL(18MBq)的 177Lu-DOTA-A-L-3PRGD 2，第2组小鼠通过尾静脉注射100μL(18MBq)的 177Lu--3PRGD 2，第3组小鼠通过尾静脉注射100μL(18MBq)的 177Lu-DOTA-A-L；第4组小鼠通过尾静脉注射100μL(9MBq)的 177Lu-DOTA-A-L-3PRGD 2，第5组荷瘤小鼠注射100μL的PBS作为对照。注射后每2天监测肿瘤体积和体重变化，肿瘤体积达到1000mm 3设定为仁慈终点。
结果参见附图12。图A和图C为本发明的分子探针 177Lu-DOTA-A-L-3PRGD 2与对照组 177Lu--3PRGD 2、 177Lu-DOTA-A-L在相同剂量(18Mbq)时、以及本发明分子探针在减半剂量(9Mbq)时的肿瘤体积随时间变化的趋势图。
给药14天后肿瘤体积为：18MBq 177Lu-DOTA-A-L-3PRGD 2治疗组的肿瘤体积为401.3±195.5mm3，9MBq  177Lu-DOTA-A-L治疗组的肿瘤体积为691.3±195.9mm3，18MBq 177Lu--3PRGD 2治疗组的肿瘤体积为1122.4±189.6mm3，18MBq  177Lu-DOTA-A-L治疗组的肿瘤体积为897.4±178.0mm3，PBS对照组的肿瘤体积为1336.3±315.4mm3。治疗实验结果表明，在注射剂量为18MBq的条件下， 177Lu-DOTA-A-L-3PRGD 2治疗组的治疗效果显著优于 177Lu-3PRGD 2治疗组(p<0.0001)和 177Lu-DOTA-A-L治疗组(p<0.05)。在治疗过程中，以上三组小鼠的体重变化及生存指标均处于正常范围。同时注射三种探针的小鼠体重变化比例均在正常范围之内，没有出现显著的急毒性。更重要的是，减少 177Lu-DOTA-A-L-3PRGD 2的给药剂量至9MBq，其治疗效果仍然显著高于双倍剂量的 177Lu-3PRGD 2(p<0.01)。实验结果表明，与传统 177Lu- 3PRGD 2相比， 177Lu-DOTA-A-L-3PRGD 2具有更好的治疗效果。实验证明， 177Lu-DOTA-A-L-3PRGD 2在U87-MG荷瘤鼠中的靶向核素治疗后能够显著抑制注射后10天内的肿瘤生长。治疗组和对照组第10天的初始体积比(V/V0)为1.4和8.0。
图B为本发明的分子探针 177Lu-DOTA-A-L-3PRGD 2与对照组 177Lu--3PRGD 2、 177Lu-DOTA-A-L在相同剂量(18Mbq)时、以及本发明分子探针在减半剂量(9Mbq)时的小鼠体重随时间变化的趋势图。实验结果表明，BALB/c裸鼠对18MBq给药剂量的 177Lu-DOTA-A-L-3PRGD 2耐受，给药组的小鼠体重和血常规指标下降后恢复正常。18MBq剂量给药后的小鼠在第6天的体重下降比例最大，并在给药后第14天恢复正常。第6天给药组和对照组的初始体重百分数为90.8±3.7％和98.7±5.0％；第14天给药组的初始体重百分数为100.5±4.1％和100.7±3.4％。与对照组相比，本发明的分子探针在14天期间，小鼠体重变化不明显，可见本发明的分子探针副作用较小，安全性高。
实施例12：实施例7的分子探针在MC-38小鼠模型中的试验
(1)实施例7的分子探针体内生物分布实验
结果参见图13A。本发明的分子探针在1、4、24、72小时的分布情况。从结果可知，本发明的分子探针随着时间的延长，显像剂在MC-38肿瘤中的摄取先升后降，其在4小时达到最高摄取量，其百分注射剂量率为32.51±4.95％ID/g。，且为所有器官中最高。可见，本发明的分子探针具有很高的肿瘤/其它器官的摄取比。
(2)实施例7的分子探针MC-38裸鼠SPECT/CT显像
结果参见图13B。本发明的探针在1、4、24小时采集时间点，肿瘤都能很好的成像，肿瘤处成像清晰、几乎无本底、对比度高。这表明，本发明的分子探针与改造前相比，有着更好的生物分布特性、药代动力学特性。
(3)注射实施例7的分子探针后，肿瘤体积和小鼠体重变化
结果参见图13C和13D。图13C为本发明的分子探针 177Lu-DOTA-A-L-3PRGD 2在剂量18Mbq、9Mbq时，肿瘤尺寸随时间变化的趋势图。由图可见，本发明的分子探针在18Mbq剂量时，第12天后至20天，肿瘤完全被消除，而实施例11的图12A却并非如此。本实施例的MC-38鼠与实施例18的荷U87鼠相比，前者具有自身免疫功能，而后者是免疫缺陷鼠，因此前者更接近人类患者的机体状态，这可以说明本发明的分子探针在治疗肿瘤可激发治疗者自身的免疫机能，使得治疗效果更好，能够高效抑制和消除肿瘤。图13D为本发明的分子探针在剂量18Mbq、9Mbq时，小鼠体重随时间变化的趋势图。由图可见，本发明的分子探针在20天期间，小鼠体重变化不明显，可见本发明的分子探针副作用较小，安全性高。
实施例13实施例7的靶向放射治疗对肿瘤免疫微环境的重塑
(1)动物模型
MC-38(小鼠结直肠癌细胞)细胞由中国科学院生物物理研究所感染与免疫院重点研究所组提供。细胞培养在含10％热灭活胎牛血清的DMED高糖培养基中，并于37℃在含有5％CO 2的湿度培养箱中进行培养。雌性C57/BL6(4～6周龄)小鼠购自北京大学医学部实验动物部，动物实验符合北京大学动物保护和使用委员会的规定和要求。为了制备荷瘤小鼠模型，在正常小鼠的右腋处皮下注射100μL(数量为1 x 10 6)的MC-38单细胞悬液，约1周后即可成瘤。
(2)靶向放射治疗
取21只MC-38荷瘤小鼠随机分成3组(n＝7)，分别通过静脉注射100μL不同放射剂量(0，9或18MBq)的 177Lu-DOTA-A-L-3PRGD 2，给药后每2天监测肿瘤的体积和体重变化，比较各组之 间的疗效差异。肿瘤体积(mm 3)＝肿瘤长度(mm)x肿瘤宽度(mm)x肿瘤高度(mm)x 0.5。当肿瘤体积大于1200mm3时判定小鼠为死亡。
结果显示： 177Lu-DOTA-A-L-3PRGD 2的靶向放射治疗效果明显。如图14.A所示，在使用18MBq 177Lu-DOTA-A-L-3PRGD 2治疗的荷瘤小鼠中，肿瘤在给药12天后完全消融，并在治愈后的1个月没有复发。使用9MBq剂量治疗的小鼠肿瘤生长受到明显的抑制，但使用9MBq低剂量治疗不能长期抑制肿瘤的生长。
结果显示： 177Lu-DOTA-A-L-3PRGD 2的靶向放射治疗具有良好的有效性和安全性。如图14.B所示，虽然治疗组(9和18MBq)小鼠的体重在治疗后的2～4天有较为明显的下降，但在治疗结束后均能够恢复到与对照组的同等水平。
(3)效应T细胞介导的抗肿瘤免疫反应
为了探究 177Lu-DOTA-A-L-3PRGD 2靶向放射治疗中，免疫系统所起到的抗肿瘤作用，我们使用抗CD8抗体耗竭靶向放射治疗过程中小鼠体内的效应T细胞，并比较耗竭小鼠及正常小鼠中的肿瘤治疗效果之间的差异。取21只MC-38荷瘤小鼠随机分成3组(n＝7)：第1组小鼠通过尾静脉注射磷酸盐缓冲液作为实验对照；第2组小鼠通过尾静脉注射18MBq  177Lu-DOTA-A-L-3PRGD 2进行常规靶向放射治疗；第3组小鼠在经过18MBq的靶向放射治疗外，在给药后0～6天，每2天通过尾静脉注射200μg CD8抗体耗竭小鼠体内的效应T细胞。给药后每2天监测肿瘤的体积变化。
结果显示：效应T细胞在靶向放射治疗中发挥重要抗肿瘤功能。如图14.C所示，在18MBq剂量下，与在免疫正常小鼠中的治疗效果相比，CD8抗体耗竭小鼠的靶向放射治疗效果明显受损，由此可见，CD8 +T细胞在靶向放射治疗中发挥重要作用。 177Lu-DOTA-A-L-3PRGD 2除了对肿瘤细胞的直接杀伤作用外，还能够引起机体产生显著的抗肿瘤免疫作用，并对抑制肿瘤的生长起到关键性作用。
(4)肿瘤免疫微环境的变化
取20只MC-38荷瘤鼠随机分为5组(n＝4)，分别通过静脉注射100μL不同放射剂量(0，6，9，12和18MBq)的 177Lu-DOTA-A-L-3PRGD 2，用于重塑肿瘤的免疫微环境。在给药后第6天，处死小鼠，取肿瘤组织制备单细胞消化悬液，并通过流式细胞术分析瘤体内的T-淋巴细胞浸润情况，并探究肿瘤细胞及免疫细胞上的PD-L1表达水平变化。肿瘤组织消化使用含有1mg/mL胶原酶IV(Worthington)和0.1mg/mL DNA酶I(Roche)的细胞消化液。在4℃下对T细胞表面进行染色，染色30分钟，染色抗体使用CD45(1μg/mL，Cat.56-0451-82，eBioscience)，CD3e(1μg/mL，Cat.25-0031-82，eBioscience)，CD8a(2.5μg/mL，Cat.11-081-82，eBioscience)，CD4(1μg/mL，Cat.45-0042-82，eBioscience)。随后，使用Foxp3染色的转录因子染色缓冲液试剂盒(Cat.00-523-00，eBioscience)和Foxp3(1μg/mL，Cat.12-4771-82，eBioscience)对T细胞进行核内染色。在4℃下对肿瘤细胞和髓系免疫细胞进行表面染色，染色30分钟，抗体使用CD45(1μg/mL，Cat.56-0451-82，eBioscience)，CD11b(1μg/mL，Cat.11-0112-82，eBioscience)和CD274(1μg/mL，Cat.12-5982-82，eBioscience)在4℃下染色30分钟。流式样品使用Gallios流式细胞仪(Beckman Counter)进行分析，并使用Flowjo 7.0软件(Tree Star)处理实验数据。
结果显示：靶向放射治疗能够显著增加肿瘤组织中CD4 +和CD8 +T淋巴细胞(而非T-reg细胞)的浸润。如图15.A所示，不同给药剂量(0、6、9、12和18MBq)的刺激下，肿瘤中CD4 +T淋巴细胞(CD45 +CD3e +CD4 +细胞)的浸润比例依次为1.04±0.55，0.76±0.18，1.17±0.41，1.82±0.65，2.24±0.78和4.84±0.85％；CD8 +T淋巴细胞(CD45 +CD3e +CD8 +细胞)的浸润比例依次为1.12±0.51，1.12±0.48，1.06±0.31，2.09±0.58，2.71±0.53和4.09±0.93％；T-reg细胞(CD45 +CD3e +CD4 +Foxp-3 +细胞)的浸润比例依次为0.37±0.27，0.22±0.08，0.21± 0.07，0.28±0.13，0.31±0.13和0.34±0.12％。
结果显示：靶向放射治疗能够显著上调肿瘤组织中髓系免疫细胞(而非肿瘤细胞)表面的PD-L1表达水平。如图15.B所示，不同给药剂量(0、3、6、9、12和18MBq)的刺激下，肿瘤细胞(CD45 -细胞)上的PD-L1表达量(MFI)依次为441±68，511±168，400±107，436±64，659±108和556±152；髓系免疫细胞(CD45 +CD11b +)的PD-L1表达量(MFI)依次为1681±144，2938±588，3187±586，4444±1140，5085±926和6749±533。
综上，靶向放射治疗能过够显著增加肿瘤中效应T细胞的浸润，并上调肿瘤中PD-L1，对抗PD-1/PD-L1阻断治疗具有积极意义。
实施例14影像指导下的靶向放射联合PD-L1阻断治疗
(1)PD-L1纳米抗体探针 9mTc-纳米抗体的制备：
以PD-L1为生物标志物制备 9mTc-纳米抗体。所选抗小鼠PD-L1的纳米抗体(MY1523)在C末端使用LPTEG-His 6标签进行标记，用于Ni-琼脂糖凝胶的亲和纯化和转肽酶Sortase-A的位点特异性连接。该纳米抗体与鼠PD-L1的KD值为49.70±7.90nM，IC50值为59.23±0.04nM，具有高亲和力与特异性。
99mTc-纳米抗体采用两步法进行标记。首先，制备 99mTc-HYNIC-G 4K(HYNIC＝6-hydrazinonicotinyl，G4K＝Gly-Gly-Gly-Gly-Lys)。在200μL琥珀酸缓冲液(250mM，pH＝4.8)中加入3μg HYNIC-G 4K(NH 2-G 4K(HYNIC)-OH)、5mg TPPTS(三苯基膦-3,3′,3〃-三磺酸钠)、6.5mg tricine(N-三(羟甲基)甲基甘氨酸)和74～96MBq Na 99mTcO 4淋洗液，混合后于99℃下反应15min。反应后自然冷却，然后用2M NaOH调节混合液的pH至7～8。其次，我们用 99mTc-HYNIC-G 4K标记MY1523，制备 99mTc-MY1523。将74MBq  99mTc-HYNIC-G 4K，100μg MY1523，50μg Sortase-A的混合溶液加入10μL(1M)CaCl 2，混匀后将反应液于室温下反应20min。以含有0.1％吐温-20(pH＝7.4)的磷酸盐缓冲液为洗脱剂，使用高效排阻色谱柱(SuperoseTM 12，GE healthcare)对产物进行纯化。以生理盐水为展开剂,使用浸渍硅胶的玻璃微纤维色谱纸(ITLC-SG)进行薄层层析测定产物的放射化学纯度(Na 99mTcO 4和 99mTc-HYNIC-G 4K的Rf值为0.7～1， 99mTc-MY1523的Rf值为0～0.3)。所制备 9mTc-纳米抗体的放射化学纯度>95％，比活度为18.5～37MBq/nmol。
(2)SPECT/CT显像
通过 99mTc-纳米抗体的SPECT/CT显像对肿瘤的PD-L1进行实时，无创和动态分析。SPECT/CT显像使用NanoScan SPECT/CT小动物核医学成像系统，所选采集能量峰为140keV，所采集的能峰宽度为20％，单张图像采集时间为30s。显像时，通过尾静脉给小鼠注射18MBq  99mTc-纳米抗体，并于注射后2小时进行核医学成像，小鼠在显像过程中使用异氟烷气体进行麻醉。取9只MC-38荷瘤鼠分为3组(n＝3)，分别通过尾静脉注射100μL的磷酸盐缓冲液，9或18MBq的 177Lu-DOTA-A-L-3PRGD 2。分别在靶向放射治疗后的第0，3和6天，进行 99mTc-纳米抗体的SPECT/CT显像，定量肿瘤摄取为百分注射剂量率(％ID/g)。
结果显示：靶向放射治疗后肿瘤对抗PD-L1纳米抗体探针的摄取有显著的增加。如图16.A所示，与对照组(PBS)相比，低剂量治疗组(9MBq)在给药后第0～6天，肿瘤的 99mTc-纳米抗体摄取不断增加，治疗后第6天的肿瘤摄取最高。随后，我们比较了不同给药剂量组(0，9和18MBq)在靶向放射治疗后的第6天的肿瘤摄取差异。如图16.B所示，9或18MBq靶向放射治疗后的肿瘤 99mTc-纳米抗体摄取明显高于对照组，经测试所采集的 99mTc信号不受 177Lu信号的影响。
(3)生物分布
为了验证 99mTc-纳米抗体活体监测肿瘤PD-L1表达的准确性，测定肿瘤的 99mTc-纳米抗体摄取与肿瘤PD-L1表达的线性关系。肿瘤的纳米抗体探针摄取通过生物分布实验进行组织水平测定，肿瘤的PD-L1表达情况在生物分布实验后通过流式细胞术进行细胞水平测定。取20只MC-38荷瘤鼠随机分为5组(n＝4)，每组小鼠通过脉注射100μL不同给药剂量(0，6，9，12和18MBq)的 177Lu-DOTA-A-L-3PRGD 2用于肿瘤免疫微环境的重塑。靶向放射治疗第6天后，进行 99mTc-纳米抗体的生物分布实验。小鼠通过尾静脉注射720kBq的 99mTc-纳米抗体，并于给药后2小时处死小鼠。取血液，肿瘤组织及其它主要组织或器官称重，并测量其放射性计数，计算各个组织和器官的百分注射剂量率(％ID/g)。所选γ-计数的能峰为135～155keV，经测试所采集的 99mTc信号不受 177Lu信号的影响，在测量肿瘤的放射性计数后，立刻对肿瘤组织进行消化，制备单细胞悬液，分别对肿瘤细胞(CD45 -)及髓系免疫细胞(CD45 +CD11b +)的PD-L1表达进行流式分析。
结果显示：通过测定肿瘤的 99mTc-纳米抗体摄取能够准确的反应肿瘤的PD-L1表达水平。如图17.A所示，不同治疗剂量(0、6、9、12和18MBq)治疗后，肿瘤对 99mTc-MY1523探针的摄取值依次为2.27±0.26，2.28±0.69，3.63±0.94，4.86±0.58和7.66±1.59％ID/g。如图17.B所示，肿瘤组织中的 99mTc-纳米抗体摄取与肿瘤中PD-L1表达有良好的线性关系(R 2＝0.80)。生物分布结果与显像结果相一致，同时， 99mTc-MY1523探针在肿瘤中的摄取与浸润髓系免疫细胞上PD-L1的表达呈现明显的正相关，经测试所采集的 99mTc信号不受 177Lu信号的影响。
(4)PD-L1动态变化窗口期
靶向放射治疗后0～6天区间内肿瘤的PD-L1表达水平变化，即PD-L1的表达窗口期，通过 99mTc-纳米抗体的SPECT/CT显像进行测定。为了进一步探究完整的PD-L1表达窗口期，通过流式细胞术探究靶向放射治疗后0～12天肿瘤的PD-L1的动态变化。取32只MC-38荷瘤鼠随机分为2组(n＝16)，分别注射100μL的磷酸盐缓冲液或9MBq的 177Lu-DOTA-A-L-3PRGD 2，并在给药后3，6，9和12天处死小鼠。取肿瘤组织消化成单细胞悬液，并通过流式细胞术测定肿瘤细胞及髓系细胞的PD-L1表达水平的动态变化。
结果显示：靶向放射治疗后，肿瘤微环境中的PD-L1表达呈现先高后低的动态趋势，并于给药后第6天达到最高。如图18.A所示，给药后3～6天肿瘤微环境中的PD-L1表达持续上调，并在给药后第6天达到最高，随后给药后9～12天肿瘤微环境中的PD-L1表达持续降低。
(5)PD-L1阻断治疗窗口期
靶向放射治疗后0～12天进行单次PD-L1单克隆抗体的免疫治疗，以PD-L1的动态变化为依据，探究PD-L1阻断治疗的窗口期与PD-L1动态变化的窗口期的相关性，为影像指导下的靶向放射与PD-L1阻断的联合治疗提供依据。取49只MC-38荷瘤鼠(60～80mm 3)随机分为7组(n＝7)，将进行靶向放射治疗的当日定义为第0天。第1组小鼠通过尾静脉注射100μL的磷酸盐缓冲液作为实验对照；第2组小鼠注射通过尾静脉注射9MBq的 177Lu-DOTA-A-L-3PRGD 2进行单一的靶向放射治疗；第3～6组小鼠注射除了进行9MBq的靶向放射治疗外，分别在第3，6，9，12天的不同时间点进行100μg PD-L1抗体的联合治疗。每2天监测小鼠的肿瘤体积和体重变化，肿瘤体积大于1200mm 3时结束监测并处死小鼠。
结果显示：给药后3～6天是PD-L1单克隆抗体的有效治疗窗口，在肿瘤PD-L1增高表达的窗口期内进行PD-1/PD-L1抑制剂的联合用药有利于增强免疫治疗的有效性。如图18.B所示，靶向放射治疗后3～6天进行PD-L1抗体阻断联合治疗的效果要显著优于单纯靶向放射治疗组，但是靶向放射治疗后9～12天进行PD-L1抗体阻断的联合治疗效果不明显。
(6)联合治疗的协同性
由于在肿瘤发展的早期进行PD-L1阻断治疗的效果通常优于肿瘤晚期，因此在联合治疗中免疫治疗的效果可受到肿瘤大小的影响，联合治理协同性需要进一步得到证实。为了验证靶向放射治疗与PD-L1免疫检查点阻断治疗的协同性，我们比较了靶向放射治疗前后(即窗口期内外)进行PD-L1阻断治疗的联合疗效。取56只MC-38荷瘤鼠(60～80mm 3)随机分为8组(n＝7)。将进行靶向放射治疗的当日定义为第0天，实验开始的当日为第-3天。第1组小鼠通过尾静脉注射100μL的磷酸盐缓冲液作为实验对照；第2～4组小鼠分别在第-3，0，3天的不同时间点通过尾静脉注射100μg的PD-L1抗体进行单一的免疫治疗；第5组小鼠注射通过尾静脉注射9MBq的 177Lu-DOTA-A-L-3PRGD 2进行单一的靶向放射治疗；第6～8组小鼠注射除了进行9MBq的靶向放射治疗外，分别在第-3，0，3天的不同时间点进行100μg PD-L1抗体的联合给药。每2天监测小鼠的肿瘤体积和体重变化，肿瘤体积大于1200mm 3视为死亡。
结果显示：与提前给药和共同给药策略相比，靶向放射治疗后进行抗PD-L1阻断效果更为显著。在PD-L1增高的时间窗口内进行PD-L1免疫检查阻断治疗能够显著抑制肿瘤的生长，延长小鼠的生存期，联合治疗表现出显著的协同性。如图19.A所示，第-3天的单一PD-L1阻断治疗治疗效果显著优于第0或3天的治疗效果，PD-L1阻断治疗的早期给药要显著由于晚期治疗。如图19.B所示，在靶向放射治疗后第3天进行抗PD-L1阻断的治疗效果著优于靶向放射治疗与抗PD-L1阻断治疗的同时给药，且显著优于靶向放射治疗前第3天进行抗PD-L1治疗。如图19.C所示，在与9MBq 177Lu-DOTA-A-L-3PRGD 2的联合治疗中，第-3，0和3天时间点进行PD-L1阻断治疗组中小鼠的90天生存率依次为4/7，4/7和7/7。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种如下式结构定义的RGD多肽：
    A-(L)n-RGD多肽
    其中A为如下结构：
    Figure PCTCN2020091861-appb-100001
    L代表连接臂分子，具有如下结构：
    Figure PCTCN2020091861-appb-100002
    其中m为1-8的整数，例如2-6，优选5；
    所述L通过其羧基与A中的氨基反应键链，例如，通过L中标记为*的羧基与A键链；
    n为0或1；
    当n为0时，所述RGD多肽通过其氨基与A中的羧基反应键链；
    当n为1时，所述RGD多肽通过其氨基与L中的另一羧基反应键链，例如当L中标记为*的羧基与A相连时，标记为**的羧基与RGD多肽反应键链；
    RGD多肽为选自以下的RGD多肽：c(RGDfV)、c(RGDfK)，c(RGDfE)，c(RGDyk)、E[c(RGDyk)] 2、E[c(RGDfK)] 2、3PRGD 2。
  2. 含有如权利要求1的RGD多肽的放射性核素标记的配合物，其具有如下定义的结构：
    Nu-BFC-A-(L)n-RGD多肽
    其中：
    Nu为放射性核素，例如诊断显像核素： 111In、 64Cu、 99mTc、 68Ga，或者治疗核素： 90Y、 177Lu、 89Sr、 153Sm、 188Re；
    BFC为双功能螯合剂(bifunctional chelating agent)，例如HYNIC(联肼尼克酰胺)、MAG 2(巯基乙酰二甘氨酸)、MAG 3(巯基乙酰三甘氨酸)、DTPA(二乙基三胺五乙酸)、DOTA(1,4,7,10-四氮杂环十二烷-1,4,7,10四乙酸)、NOTA(1,4,7-三氮杂环壬烷-1,4,7三羧酸)、TETA(1,4,8,11-四氮杂环十四烷-1,4,8,11四乙酸)；
    当n为0时，所述双功能螯合剂通过其结构中具有的羧基与A中的-NH 2反应键链；
    当n为1时，所述双功能螯合剂通过其结构中具有的羧基与L中的-NH 2反应键链。
  3. 如权利要求2所述的配合物，Nu为 90Y或 177Lu时，BFC选自DTPA、DOTA；Nu为 111In时，BFC选自DTPA、DOTA；Nu为 64Cu时，BFC选自TETA、DOTA；Nu为 68Ga时，BFC选自NOTA、DOTA；Nu为 99mTc时，BFC选自HTNIC、DTPA、MAG 2、MAG 3；
  4. 如权利要求2所述的配合物，其如下所述：
    68Ga-DOTA-A-c(RGDfk)；
    99mTc-HYNIC-A-3PRGD 2；
    177Lu-DOTA-A-L-3PRGD 2；
    优选的，其具有如下结构：
    Figure PCTCN2020091861-appb-100003
  5. 一种药物组合物，其包含有效量的权利要求2的标记配合物Nu-BFC-A-(L)n-RGD多肽。优选地，所述药物组合物是一种静脉注射剂，例如一种无色透明液体针剂。优选地，所述药物组合物还包括抗吸附剂，例如生理盐水，1％环糊精的水溶液，和/或含吐温-20的PBS溶液(如吐温的质量分数为0.01～0.1％)。
  6. 根据权利要求5的药物组合物，其中Nu为 90Y、 177Lu，所述药物用于整合素αvβ3阳性肿瘤的靶向放射性治疗。
  7. 一种药物组合物，其包括权利要求6的靶向放射性治疗药物和免疫治疗药物，和任选的纳米抗体分子影像探针。优选的所述标记配合物是 177Lu-DOTA-A-L-3PRGD 2。优选的免疫治疗药物是PD-1或PD-L1免疫检查点抑制剂，例如是PD-1或PD-L1单抗药物。优选的纳米抗体分子影像探针是PD-1或PD-L1纳米抗体分子影像探针。例如 99mTc-MY1523。
  8. 根据权利要求7的药物组合物，所述靶向放射性治疗药物与免疫治疗药物可同时或分别前后施用。例如所述免疫治疗药物可在靶向放射性治疗药物后施用，优选的，所述免疫治疗药物在靶向放射性治疗药物使用后3～6天使用。优选的，所述纳米抗体分子影像探针在靶向放射性治疗药物施用之后，而免疫治疗药物施用之前施用。
  9. 一种试剂盒，其分开装有权利要求6的靶向放射性治疗药物，免疫治疗药物和任选的纳米抗体分子影像探针。
  10. 权利要求1的多肽或权利要求2的配合物或权利要求5或7的药物组合物在制备药物中的用途，所述药物用于诊断或者治疗整合素αvβ3阳性肿瘤。优选的，所述肿瘤是指实体肿瘤，例如在血液、肝、腺体(例如乳腺、前列腺、胰腺)、肠(例如结肠直肠)、肾、胃、脾、肺、肌肉、骨头等部位的恶性肿瘤。
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