LAG-3 targeted diagnosis and treatment integrated molecular imaging probe as well as preparation method and application thereof

Abstract

The invention provides an LAG-3 targeted diagnosis and treatment integrated molecular imaging probe as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) carrying out ultrafiltration on an anti-human LAG-3 monoclonal antibody by using a buffer solution; adding a bifunctional chelating agent for reaction, and performing ultrafiltration purification to obtain a labeled precursor DOTA-alphaLAG-3; (2) mixing the labeled precursor DOTA-alphaLAG-3 with a buffer solution, and then adding radionuclide for reaction; and after the reaction is finished, carrying out ultrafiltration purification on the obtained reaction liquid to obtain the LAG-3 targeted diagnosis and treatment integrated molecular imaging probe. The obtained LAG-3 targeted diagnosis and treatment integrated molecular imaging probe can play a synergistic anti-tumor effect with immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a LAG-3 targeted diagnosis and treatment integrated molecular imaging probe and a preparation method and application thereof. BACKGROUND

[0002] The immune checkpoint inhibitors represented by PD-1 / PD-L1 have prolonged the survival of patients with various cancers. However, the effective rate of single immunotherapy is still low. How to improve the effective rate of immunotherapy for lung cancer patients has become a key clinical problem. Immune combination therapy is one of the main strategies to improve the efficacy of tumor immunotherapy, mainly including combination of immunotherapy or radiotherapy, etc.

[0003] LAG-3, as a new generation of immune checkpoint, is expressed on exhausted T cells, B cells and NK cells, and plays an inhibitory immune effect. Studies have shown that LAG-3 can mediate immunotherapy resistance, and the combination of PD-1 / PD-L1 inhibitors and LAG-3 inhibitors can improve the efficacy of immunotherapy. A variety of drugs targeting LAG-3 have entered the clinical trial stage. Among these drugs, Relatlimab combined with PD-1 inhibitors significantly improved the overall survival of melanoma patients in the 3rd phase clinical trial. However, LAG-3 inhibitors mainly block the binding of MHC class II ligands, and LAG-3 can still mediate immune escape through FGL-1, LSECtin and GAL-3 ligands, leading to LAG-3 inhibitor resistance. Therefore, it has important clinical value to develop more comprehensive combination therapy for LAG-3 highly expressed population to improve the efficacy of immunotherapy.

[0004] Radiotherapy combined with immunotherapy has achieved remarkable efficacy in some tumor patients. The commonly used external beam radiotherapy in clinic has the disadvantages of low tumor lesion irradiation dose, easy damage to normal tissues, influence of irradiation field by respiratory motion, and difficulty in popularization for patients with multiple organ metastases. Internal irradiation radiotherapy can achieve precise targeting of tumor sites, reduce physiological toxicity, and to a great extent overcome the shortcomings of external beam radiotherapy. At present, the nuclide used for internal irradiation radiotherapy in clinic is mainly β nuclide. β nuclide has a deep penetration range (20-130 mm) and is easy to obtain, and is widely used in clinical cancer treatment. Among them 177 Lu (half-life: 6.7d) decays to emit low-energy γ rays, which can be used for real-time monitoring imaging by SPECT; on the other hand, 177 The short-range β particles (average range 0.67 mm) emitted by Lu can achieve tumor killing effect, while causing less damage to adjacent normal tissues, especially bone marrow. Based on 177Lu's targeted radiotherapy has achieved remarkable efficacy in prostate cancer and neuroendocrine tumors, and has been approved by FDA for clinical use. However, there is still a lack of LAG-3-targeted diagnosis and treatment integrated molecular imaging probes, and their anti-tumor effects and immune synergistic effects still need to be studied. SUMMARY

[0005] To solve the above technical problems, the present application provides a LAG-3 targeted diagnosis and treatment integrated molecular imaging probe and a preparation method and application thereof.

[0006] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0007] The present application provides a preparation method of a LAG-3 targeted diagnosis and treatment integrated molecular imaging probe, which comprises: (1) (1) ultrafiltration of anti-human LAG-3 monoclonal antibody (Relatlimab, aLAG-3) with buffer solution; addition of bifunctional chelating agent reaction, ultrafiltration purification to obtain labeled precursor DOTA-aLAG-3; (2) mixing the labeled precursor DOTA-aLAG-3 with buffer solution, and then adding radionuclide for reaction; after the reaction is completed, the obtained reaction solution is ultrafiltrated to obtain the LAG-3 targeted diagnosis and treatment integrated molecular imaging probe. The obtained LAG-3 targeted diagnosis and treatment integrated molecular imaging probe can exert synergistic anti-tumor effect with immunotherapy.

[0008] Further, in step (1), the bifunctional chelating agent is S-2-(4-isothiocyanate benzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid, denoted as DOTA-p-NCS-Bn.

[0009] Further, in step (1), the removal of excess bifunctional chelating agent is specifically: adding 0.01M PBS buffer solution to ultrafiltrate three times with parameters of 3000g and 10 minutes.

[0010] Further, in step (1), the buffer solution is a sodium carbonate buffer solution with pH 9.0; in step (2), the buffer solution is a sodium acetate buffer solution with pH 5.6.

[0011] Further, step (1) specifically includes the following steps: adding the anti-human LAG-3 monoclonal antibody to a 30kDa ultrafiltration tube, adding 3000g of sodium carbonate buffer at pH 9.0 for ultrafiltration to replace the monoclonal antibody preservation solution with modification buffer; mixing the anti-human LAG-3 monoclonal antibody with 10 times the molar amount of the bifunctional chelating agent S-2-(4-benzyl isothiocyanate)-1,4,7,10-tetraazacyclododecanetetraacetic acid, and gently shaking the mixture in a buffer at pH 9.0 at 37℃ for 2 hours; then removing the excess chelating agent by adding 0.01M PBS to the 30kDa ultrafiltration tube and ultrafiltrationing three times at 3000g for 10 minutes to purify and obtain the labeled precursor DOTA-αLAG-3.

[0012] Furthermore, in step (2), the integrated diagnostic and therapeutic radionuclide is... 177 LuCl3.

[0013] Further, step (2) specifically includes the following steps: mixing the labeled precursor DOTA-αLAG-3 with 0.2M pH 5.6 sodium acetate buffer at a volume ratio of 1:1, and then adding... 177 Mix the LuCl3 solution thoroughly. The dosage is 37 MBq per 300 μg of labeled precursor. 177 LuCl3 was reacted at 37℃ with gentle shaking at 500 rpm for 1 hour. After the reaction, the reaction solution was transferred to a 30 kDa ultrafiltration tube and ultrafiltered three times to remove free nuclides, yielding the purified probe. 177 Lu-DOTA-αLAG-3, also known as the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe.

[0014] The present invention also provides a LAG-3 molecular imaging probe prepared by the above-mentioned method for preparing the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe.

[0015] This invention also provides an application of the above-mentioned LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe in the field of immunotherapy.

[0016] This invention further provides an application of the aforementioned LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe in the field of lung cancer immunotherapy.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0018] The application provides a LAG-3 targeted diagnosis and treatment integrated molecular imaging probe and a preparation method and application thereof, the obtained LAG-3 targeted probe has certain anti-tumor effect, and can exert a synergistic anti-tumor effect with immunotherapy, provides a new strategy for immunotherapy for tumor patients with positive LAG-3 expression, and helps to promote the precision of tumor diagnosis and treatment. BRIEF DESCRIPTION OF DRAWINGS

[0019] One or more embodiments are illustrated by way of example in the figures that form a part of this disclosure and which are shown by way of illustration in the drawings and are not limiting of the embodiments unless otherwise specified herein. The drawings are not necessarily drawn to scale, except as otherwise noted.

[0020] Figure 1 is a cell binding experiment and Kd value provided by the embodiment of the application 177 Radiochemical characteristics of Lu-DOTA-αLAG-3;

[0021] Figure 2 is a cell binding experiment and Kd value provided by the embodiment of the application

[0022] Figure 3 is a cell binding experiment and Kd value provided by the embodiment of the application 177 Lu-DOTA-αLAG-3 and 177 SPECT / CT imaging and quantitative analysis of Lu-DOTA-IgG probe;

[0023] Figure 4 is a cell binding experiment and Kd value provided by the embodiment of the application 177 Therapeutic experiment of Lu-DOTA-αLAG-3 in LAG-3 humanized lung cancer (LLC) mouse model;

[0024] Figure 5 is a cell binding experiment and Kd value provided by the embodiment of the application DETAILED DESCRIPTION

[0025] The application will be described in detail below with reference to specific embodiments.

[0026] The application provides a preparation method of a LAG-3 targeted radioactive imaging probe, and performance testing and verification of the obtained LAG-3 targeted radioactive imaging probe. Specifically, it comprises:

[0027] 1. Antibody conjugation

[0028] Anti-human LAG-3 monoclonal antibody (Selleck, catalog number A2029) or isotype control mouse IgG (Selleck, catalog number A2052) was added to a 30 kDa ultrafiltration tube. 3000 g of sodium carbonate buffer (pH 9.0) was added for ultrafiltration, and the monoclonal antibody preservation solution was replaced with modification buffer. αLAG-3 or IgG was mixed with 10 molar amounts of the bifunctional chelating agent S-2-(4-benzyl isothiocyanate)-1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA-p-NCS-Bn), and reacted with gentle shaking at 37°C, pH 9.0, for 2 hours. Excess chelating agent was removed by ultrafiltration three times with 3000 g of 0.01 M PBS for 10 minutes each to obtain purified labeled precursors DOTA-αLAG-3 and DOTA-IgG.

[0029] 2. Radionuclide labeling

[0030] For marking 177 Lu, the labeled precursors DOTA-αLAG-3 and DOTA-IgG were mixed with 0.2M pH 5.6 sodium acetate buffer at a volume ratio of 1:1, and then added... 177 Mix the LuCl3 solution thoroughly (add 37 MBq per 300 μg of labeled precursor). 177 The reaction mixture was incubated with LuCl3 at 37°C and gently shaken at 500 rpm for 1 hour. After the reaction, the solution was transferred to a 30 kDa ultrafiltration tube and ultrafiltered three times to remove free nuclides, yielding the purified probe. 177 Lu-DOTA-αLAG-3 and its isotype control 177 Lu-DOTA-IgG. Labeling efficiency and radiochemical purity (RCP) were assessed using instantaneous thin-layer chromatography (iTLC).

[0031] 3. In vitro stability

[0032] Labeling rate and RCP were evaluated using iTLC with 1% EDTA as the developing solvent. In the developing solvent, the radiolabeled compound on the silica gel plate remained at the starting point, while the free... 177 Lu migrated to the front end. The RCP was further evaluated using radio-HPLC by detecting the protein UV absorption and radioactivity at 280 nm of the probe. The radiolabeled compound was separated from the free radionuclide using a Biocore SEC-150 column and a 50 mM phosphate mobile phase (containing 150 mM NaCl, flow rate 1 mL / min). The in vitro stability of the probe at 37 °C for 2, 24, 48, 72, 96, and 120 h was evaluated in PBS and 10% FBS.

[0033] 4. Cell binding assay and Kd value

[0034] 74kBq per 177 Lu-DOTA-αLAG-3 was diluted with 200 μL of DMEM complete medium and added to a medium coated with LAG-3. + A549 and A549 cells (10 per well) 5 Cells were incubated in 24-well plates for 0.5, 2, 4, 6, 8, and 24 hours, respectively. The culture medium was then transferred to 5 mL centrifuge tubes, washed twice with PBS, and lysed with 0.5 M NaOH. The cells were gently vortexed for 1 minute, and the cell lysate was transferred to 5 mL centrifuge tubes and washed twice with PBS. Radioactivity in the supernatant and cell lysate was detected using a gamma counter, and cell binding rate was calculated.

[0035] 10 per hole 5 LAG-3 + A549 cells were seeded in 24-well plates and incubated overnight in an incubator. The old culture medium was discarded, and different concentrations of DMEM complete medium diluted in 200 μL were added to each well. 177 Lu-DOTA-αLAG-3 probes (0.01-200 nM) were incubated in an incubator for 4 hours. Afterward, supernatant and cell lysate were collected for gamma counting, and cell binding rates at different probe concentration gradients were calculated. Curves were plotted using Graphpad software for calculation. 177 The affinity constant Kd of the Lu-DOTA-αLAG-3 probe for human LAG-3 protein.

[0036] 5. Lung Cancer Model Construction and Immunotherapy

[0037] 1×10⁻⁶ mg / L was subcutaneously injected into the right hind limb of a 6-week-old male LAG-3 humanized mouse. 6 LLC cells were used to construct a lung cancer model. When the tumor volume reached 50-100 mm... 3 Treatment was initiated at that time. All experimental mice were divided into four groups: control group, αPD-L1 immunotherapy monotherapy group, 177 Lu-DOTA-αLAG-3 internal radiation therapy group and αPD-L1+ 177 Lu-DOTA-αLAG-3 combined treatment group. Mice in the internal irradiation group received a single injection of approximately 11.1 MBq via the tail vein. 177 Lu-DOTA-αLAG-3 probe. The immunotherapy group received intraperitoneal injections of αPD-L1 every three days, starting on day two, for a total of three administrations. The internal irradiation-immunotherapy combined group received... 177 Lu-DOTA-αLAG-3 probe therapy and immunotherapy (every other day). Weight and tumor volume were monitored every other day for 2 weeks.

[0038] 6. SPECT / CT Imaging and Data Processing

[0039] 7.4 MBq 177 Lu-DOTA-αLAG-3 probe or 177 Lu-DOTA-IgG was injected via tail vein into LAG-3 human mice carrying LLC, followed by SPECT / CT imaging using isoflurane anesthesia. SPECT scans were performed at 2, 24, 48, 72, 96, and 120 hours post-injection, with each scan lasting 10 minutes. Subsequently, CT scans were performed in "normal full" mode for attenuation correction. Regions of interest (ROIs) including the tumor, blood pool, and contralateral muscle were delineated using PMOD software at each time point, and calculations were performed accordingly. 177 Lu-DOTA-αLAG-3 probe and 177 The tumor / muscle and tumor / blood pool signal-to-noise ratios of Lu-DOTA-IgG were used to assess the imaging capabilities of the probe.

[0040] 7. Biosafety assessment

[0041] For evaluation 177 The biocompatibility of the Lu-DOTA-αLAG-3 probe was assessed by enucleating the eyeballs of mice at the end of treatment for complete blood count, liver function, and kidney function tests. Additionally, the heart, liver, spleen, lungs, and kidneys were stained with hematoxylin and eosin (HE) to observe morphological changes at the organ level.

[0042] result:

[0043] 1. 177 Preparation and characterization of Lu-DOTA-αLAG-3

[0044] HPLC chromatograms showed that the chemical purity of both αLAG-3 and DOTA-αLAG-3 exceeded 95%. Radioactive HPLC chromatogram results showed... 177 The labeling efficiency of Lu-DOTA-αLAG-3 exceeds 95%, and the probe specific activity is approximately 18.5 MBq / nmol, making it suitable for direct use in imaging or therapy. Furthermore, 177 Lu-DOTA-αLAG-3 maintained an RCP of over 95% in PBS and FBS for 120 h, indicating good in vitro stability. Figure 1 As shown. Figure 1 Section a shows the HPLC chromatograms of αLAG-3 and DOTA-αLAG-3, indicating that their chemical purity both exceed 95%; section b shows... 177 The Radio-HPLC chromatogram of the Lu-DOTA-αLAG-3 probe indicates a probe labeling rate exceeding 95%; point c is... 177The in vitro stability of Lu-DOTA-αLAG-3 showed that the radiochemical purity of the probe was more than 95% in PBS and FBS within 120 h, and the probe had good in vitro stability.

[0045] 2. Cell binding experiment and Kd value

[0046] Within 24 hours, 177 The binding rate of Lu-DOTA-αLAG-3 probe to LAG-3 + A549 cells gradually increased with time, and was significantly higher than that of LAG-3 - A549 cells at 0.5, 2, 4, 6, 8 and 24 h (all p<0.0001; Figure 2 ). The incubation of multiple concentration gradients of the probe with LAG-3 + A549 cells for 4 h, it was found that the number of cell binding gradually increased with the increase of the concentration of the probe, and the concentration corresponding to half of the maximum binding capacity was 8.3 nM, i.e. the Kd value was 8.3 nM, as shown in Figure 2 . Figure 2 The cell binding experiment is shown in a, which shows that 177 The cell binding rate of Lu-DOTA-αLAG-3 probe to LAG-3 + A549 cells was significantly higher than that of LAG-3-A549 cells; the Kd value is shown in b, which is 8.3 nM. These results show that 177 The Lu-DOTA-αLAG-3 probe can specifically bind to LAG-3 and has a nanomolar level of affinity.

[0047] 3. SPECT / CT imaging and biodistribution

[0048] The LAG-3 humanized mouse model bearing LLC was divided into 177 Lu-DOTA-αLAG-3 probe group and 177 Lu-DOTA-IgG control group, and the SPECT / CT imaging images at 2, 8, 24, 48, 72, 96 and 120 h after administration showed that from 24 h, the tumor site of the mice showed obvious 177 Lu-DOTA-αLAG-3 probe signal (12.7±2.6%ID / g), which gradually increased thereafter, reaching a peak at 96 h (24.3±4.5%ID / g), and there was still obvious uptake at 120 h. While 177 Lu-DOTA-IgG showed obvious signal (13.9±3.1%ID / g) from 48 h after injection, and reached a peak at 72 h (16.8±2.2%ID / g), as shown in Figure 3 .

[0049] The quantitative analysis results showed that 177The T / M and T / B ratios of the Lu-DOTA-αLAG-3 probe were significantly higher at 96 h than those of the previous probe. 177 Lu-DOTA-IgG control probe (T / M: 18.6 vs. 7.0, p<0.01; T / B: 2.3 vs. 1.2, p<0.05), such as Figure 3 As shown. Figure 3 In the middle, point a is 177 Lu-DOTA-αLAG-3 and 177 SPECT / CT imaging of the Lu-DOTA-IgG probe; point b is... 177 Lu-DOTA-αLAG-3 and 177 Quantitative analysis of SPECT / CT images of the Lu-DOTA-IgG probe. The above results indicate that... 177 The Lu-DOTA-αLAG-3 probe achieved long-term enrichment at the tumor site, highlighting its therapeutic potential.

[0050] 4. 177 Treatment trials of Lu-DOTA-αLAG-3 in lung cancer models

[0051] In order to investigate 177 The therapeutic potential of Lu-DOTA-αLAG-3 and its ability to synergistically fight tumors with immunotherapy were investigated. Targeted radiotherapy experiments were conducted in LAG-3-bearing humanized mice with LLC lung cancer, divided into four groups: 11.1 MBq, 11.1 MBq + αPD-L1, αPD-L1, and a PBS control group. Figure 4 As shown. Figure 4 In the diagram, section a represents the treatment timeline for LLC LAG-3-bearing humanized mice; section b represents αPD-L1, 177 Images of mouse tumors at the end of treatment in the Lu-DOTA-αLAG-3 group, the combined treatment group, and the control group; point c is a summary of tumor growth curves for the four groups of mice; point d is the tumor growth curve within each of the four groups of mice; point e is the tumor weight of the four groups of mice after treatment; point f is the weight change curve of the four groups of mice during treatment.

[0052] Treatment results showed that the 11.1 MBq single-drug probe group had slower tumor growth and lighter tumor weight compared to the control group, demonstrating a certain anti-tumor effect (p<0.05). Furthermore, the single-drug αPD-L1 immunotherapy group had no anti-tumor effect compared to the control group (p>0.05), but the internal radiation therapy combined with αPD-L1 immunotherapy group showed significantly slower tumor growth and lighter tumor weight compared to the control group, demonstrating a significant tumor suppression effect (p<0.05). This suggests that although single-drug immunotherapy does not have a significant therapeutic effect, 177The Lu-DOTA-αLAG-3 probe can exert a synergistic anti-tumor effect with immunotherapy.

[0053] 5. 177 Biological safety of Lu-DOTA-αLAG-3

[0054] In order to evaluate 177 the biological safety of Lu-DOTA-αLAG-3, blood routine, liver and kidney function indexes, and heart, liver, spleen, lung and kidney HE staining results were compared among the four groups. The results showed that the common blood routine and liver and kidney function indexes of the probe single-drug treatment group and the combined treatment group had no obvious changes compared with the control group, and the heart, liver, spleen, lung and kidney HE staining had no obvious morphological changes, such as Figure 5 as shown in the figure. Figure 5 Among them, a is the result of hematological test; b is HE staining. This indicates that the probe has no obvious systemic toxicity and has certain biological safety at this treatment dose.

[0055] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in actual application, various changes can be made in form and details without departing from the spirit and scope of the present application. Any person skilled in the art can make respective changes and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be limited by the scope defined by the claims.

Claims

1. A method for preparing a LAG-3-targeted therapeutic molecular imaging probe, the method comprising: (1) Ultrafiltration of anti-human LAG-3 monoclonal antibody with buffer; A bifunctional chelating agent was added to carry out the reaction, and the excess bifunctional chelating agent was removed by ultrafiltration purification to obtain the labeled precursor DOTA-αLAG-3. (2) The labeled precursor DOTA-αLAG-3 was mixed with a buffer solution, and then a radionuclide was added to react. After the reaction was completed, the resulting reaction solution was ultrafiltered to purify and obtain the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe.

2. The method for preparing the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe according to claim 1, characterized in that, In step (1), the bifunctional chelating agent is S-2-(4-benzyl isothiocyanate)-1,4,7,10-tetraazacyclododecanetetraacetic acid, denoted as DOTA-p-NCS-Bn.

3. The method for preparing the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe according to claim 1, characterized in that, In step (1), the ultrafiltration purification to remove excess of the bifunctional chelating agent specifically involves adding 0.01M PBS and ultrafiltration three times at a rate of 3000g and 10 minutes.

4. The method for preparing the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe according to claim 1, characterized in that, In step (1), the buffer solution is a sodium carbonate buffer solution with pH 9.0; in step (2), the buffer solution is a sodium acetate buffer solution with pH 5.

6.

5. The method for preparing the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe according to claim 1, characterized in that, Step (1) specifically includes the following steps: adding anti-human LAG-3 monoclonal antibody to a 30kDa ultrafiltration tube, adding 3000g of sodium carbonate buffer at pH 9.0 for ultrafiltration to replace the monoclonal antibody preservation solution with modification buffer; mixing anti-human LAG-3 monoclonal antibody with 10 times the molar amount of bifunctional chelating agent S-2-(4-benzyl isothiocyanate)-1,4,7,10-tetraazacyclododecanetetraacetic acid, and gently shaking the mixture in a buffer at pH 9.0 at 37℃ for 2 hours; then removing excess chelating agent by adding 0.01M PBS to the 30kDa ultrafiltration tube and ultrafiltration three times at 3000g for 10 minutes to purify and obtain the labeled precursor DOTA-αLAG-3.

6. The method for preparing the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe according to claim 1, characterized in that, In step (2), the radioactive nuclide is 177 LuCl3.

7. The method for preparing the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe according to claim 1, characterized in that, Step (2) specifically includes the following steps: mixing the labeled precursor DOTA-αLAG-3 with 0.2M pH 5.6 sodium acetate buffer at a volume ratio of 1:1, and then adding... 177 Mix the LuCl3 solution thoroughly. The dosage is 37 MBq per 300 μg of labeled precursor. 177 LuCl3 was reacted at 37℃ with gentle shaking at 500 rpm for 1 hour. After the reaction, the reaction solution was transferred to a 30 kDa ultrafiltration tube and ultrafiltered three times to remove free nuclides, yielding the purified probe. 177 Lu-DOTA-αLAG-3, also known as the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe.

8. The LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe prepared by the method of any one of claims 1 to 7.

9. The application of the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe according to claim 8 in the field of immunotherapy.

10. The application of the LAG-3 targeted diagnostic and therapeutic integrated molecular imaging probe according to claim 8 in the field of lung cancer immunotherapy.

Images