Radionuclide-labeled pd-l1-targeting nanobodies and methods of making and using the same

Abstract

A novel PD-L1-targeting nanobody, APN09, using a radionuclide ( 99m Tc, 68 Ga、 18 F, 177 The invention discloses an imaging agent labeled with the PD-L1-targeting nanobody, its preparation method, and its application. The imaging agent exhibits excellent affinity and selectivity for tumors. Its labeling method is simple, convenient, time-efficient, and achieves a high labeling rate, making it suitable for detecting PD-L1 expression and monitoring treatment efficacy in malignant tumors.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, radiolabeled radiochemistry, and clinical nuclear medicine, specifically to a series of radiolabeled PD-L1-targeting nanobody APN09 derivative probes and their preparation methods, for the identification, diagnosis, staging, precise localization of lesions, monitoring of treatment efficacy, and radioimmunotherapy of malignant tumors. Background Technology

[0002] With the in-depth development and integration of nuclear medicine and molecular biology, medical imaging technology has entered the era of molecular imaging. Among these advancements, positron emission tomography (PET) and single-photon emission computed tomography (SPECT) enable functional imaging, allowing for the true understanding and diagnosis of diseases at the molecular level, particularly in the diagnosis and treatment of tumors. By tracing receptor changes and abnormalities in cell signal transduction in diseased tissues, these techniques overcome the limitations of modern diagnostic technologies, providing a basis for early tumor diagnosis, clinical staging, and efficacy evaluation, as well as prognostic assessment. They can also be applied to targeted tumor therapy. Tumor radioimmunoassay involves using a diagnostic radionuclide (RTA) to detect specific antibodies or fragments of tumor-associated antigens. 99m Tc, 18 F, 68 After being labeled with radioactive isotopes (such as Ga), the radioactive isotopes are injected into the human body and travel through the bloodstream to the tumor tissue. There, they bind to tumor-related antigens, causing a localized concentration of radioactivity in the tumor tissue that exceeds that of normal tissue. Nuclear medicine imaging equipment is then used to qualitatively and locally visualize the primary lesion and metastases throughout the body, particularly enabling the detection of occult lesions that are difficult to identify using other diagnostic techniques. During decay, alpha or beta radionuclides can release alpha or beta rays, which have a strong ionizing radiation effect and a strong killing effect on tumor cells or abnormally proliferating tissues. Radionuclide therapy refers to the use of radioactive isotopes with therapeutic effects, such as... 131 I, 90 Y, 177 Lu, 225 Ac, 213 Bi and other radionuclides or labeled drugs can be used to precisely kill diseased cells and tissues at close range, thereby achieving the purpose of treatment.

[0003] Currently, immunotherapy blocking the PD-1 / PD-L1 pathway has shown great promise in treating advanced solid tumors, but it has also exhibited certain limitations in practice. It is generally believed that high PD-L1 expression levels are associated with positive response rates to cancer treatment and clinical benefits from anti-PD-1 / anti-PD-L1 therapy. Therefore, screening patients based on PD-L1 expression levels at the lesion site before treatment to identify those who will respond positively to this therapy is of great significance.

[0004] Accurate assessment of PD-L1 status in cancer patients is crucial for improving treatment efficacy. Furthermore, relapse and drug resistance are common issues during targeted therapy; therefore, achieving individualized and precise cancer treatment is a critical concern in targeted cancer diagnosis and treatment. Several studies have shown that PD-L1-targeted PET / SPECT molecular imaging can be used for lesion imaging of tumors and for screening patients responding to anti-PD-1 / anti-PD-L1 immunotherapy. However, due to the large molecular weight (~150 kDa) of traditional monoclonal antibodies, radiolabeled antibodies are slowly cleared from the body, resulting in a high background radiation in normal tissues. This not only significantly reduces the tumor / non-tumor ratio (i.e., imaging contrast) but also increases the likelihood of radiation damage to normal tissues.

[0005] Compared to monoclonal antibodies, nanobodies have gradually attracted widespread attention from researchers in recent years. Nanobodies were first reported by Belgian scientists in the journal *Nature* in 1993. They described a naturally occurring antibody (camel-derived antibody) in the blood of camels, lacking only the light chain and containing only the heavy chain. Importantly, the VHH structure, cloned and expressed as a single heavy chain antibody, possesses structural stability and antigen-binding activity comparable to the original heavy chain antibody, making it the smallest known unit capable of binding to a target antigen. VHH is a single-domain antibody; because its crystal diameter is approximately 2.5 nm, its length approximately 4 nm, and its molecular weight only 15 kDa, it is also called a nanobody (Nb). The main advantages of nanobodies as molecular probes are: (1) They have high affinity and high specificity. Compared with peptides, their affinity is 10 to 100 times higher, which is close to that of monoclonal antibodies; (2) They have very low immunogenicity and toxicity, and they are not as easy to adhere as scFv; (3) They have good tissue penetration and can fully bind to target organs; (4) They have a small relative molecular mass, and the unbound part can be cleared by the kidneys more quickly, reducing background interference in the blood; (5) Using modern genetic engineering antibody technology, high-yield antibodies can be obtained, and their structure can be modified or altered, which is more conducive to molecular-level imaging detection.

[0006] After targeted nanobodies are conjugated with GGGGC sequences or modified with bifunctional chelators (such as Nota, Dota, Resca, and THP), they can be used for diagnostic radionuclide imaging. 99m Tc, 68 Ga、 18 F or therapeutic radionuclide 177 Marked with Lu, etc. 99m Tc is the most widely used single-photon nuclide, derived from... 99 Mo- 99m The Tc generator is used for preparation, and SPECT examination is inexpensive and easy to promote. 68 Ga is a widely used positron-emitting radioactive nuclide. 68 Ga by 68 Ge- 68 Ga generators are readily available. 68 Ga readily coordinates with compounds, making the labeling process relatively simple; compared to cyclotrons, rinsing for labeling is more efficient. 68 Ga has relatively low radioactivity, making it easy to conduct quality control at any time; and 68 Gaussian oxide (Ga) has a short half-life, enabling non-invasive, real-time disease diagnosis and the acquisition of whole-body metabolic images. As an ideal PET radionuclide, it is compatible with... 68 Compared with Ga(T1 / 2=68min), 18 F has a relatively long half-life (T1 / 2 = 109 min). And... 18 F is prepared using an accelerator, making it more suitable for batch preparation of multiple patients, and also more suitable for developing automated synthesis methods.

[0007] 90 Y and 177 Lu is a beta radionuclide. 90 Y has high energy (2280keV) and strong penetrating power (12mm), exhibiting a strong killing effect on tumor cells or abnormally proliferating tissues. 90 Compared to Y, 177 Lu can emit beta rays (0.5 MeV) in a small ion range, which not only ensures that the radiation energy is delivered to the tumor area, but also reduces damage to surrounding normal tissues.

[0008] Therefore, there is an urgent need to develop more PET and SPECT probes that target PD-L1 with high specificity and high sensitivity, which can enable rapid, non-invasive, real-time, and quantitative detection of PD-L1 expression in lesions throughout the body of cancer patients. This would provide in vivo quantitative information that cannot be obtained by conventional laboratory PD-L1 detection, and provide auxiliary detection methods for screening patients and monitoring the efficacy of clinical PD-1 / PD-L1 immunotherapy. Summary of the Invention

[0009] The purpose of this invention is to provide a novel PD-L1-targeting nanobody, APN09, using a radionuclide ( 99m Tc, 68 Ga、 18 F, 177 The invention discloses an imaging agent labeled with the PD-L1-targeting nanobody, its preparation method, and its application. The imaging agent exhibits excellent affinity and selectivity for tumors. Its labeling method is simple, convenient, time-efficient, and achieves a high labeling rate. It can be used for PD-L1 expression detection and efficacy monitoring in malignant tumors, and belongs to the fields of radiopharmaceuticals and nuclear medicine.

[0010] The objective of this invention and the technical problem it solves are achieved by the following technical solutions.

[0011] The complementarity-determining region (CDR) of a PD-L1-targeting nanobody VHH includes the following sequence:

[0012] The amino acid sequence is CDR1 as shown in SEQ ID NO: 4;

[0013] The amino acid sequence is CDR2 as shown in SEQ ID NO: 5; and

[0014] The amino acid sequence is CDR3 as shown in SEQ ID NO: 6.

[0015] A VHH nanobody chain targeting PD-L1 comprises the CDR1, CDR2, and CDR3 sequences as described above. Furthermore, it also includes amino acid sequence fragments of the following framework regions:

[0016] The amino acid sequence is FR1 as shown in SEQ ID NO:7;

[0017] The amino acid sequence is FR2 as shown in SEQ ID NO:8;

[0018] The amino acid sequence is FR3 as shown in SEQ ID NO:9;

[0019] The amino acid sequence is FR4 as shown in SEQ ID NO:10.

[0020] According to one embodiment of the present invention, the amino acid sequence of the VHH chain is shown in SEQ ID NO: 3.

[0021] A nanobody targeting PD-L1 having the VHH chain as described above.

[0022] A conjugate obtained by conjugating the aforementioned nanobody VHH chain with a chelating agent of GGGGC or a radionuclide.

[0023] According to one embodiment of the present invention, the chelating agent for the radionuclide is selected from NOTA, DOTA, RESCA, or THP.

[0024] A radionuclide labeling agent obtained by radionuclide labeling of the above-mentioned conjugate.

[0025] According to one embodiment of the present invention, the radionuclide is selected from... 99m Tc, 68 Ga、 18 F, 177 Lu.

[0026] According to one embodiment of the present invention, the radionuclide label is selected from the following molecules:

[0027] 99m Tc-GC-APN09;

[0028] 68 Ga-THP-APN09;

[0029] 18 F-RESCA-APN09;

[0030] 177 Lu-DOTA-APN09;

[0031] Wherein, APN09 represents the VHH chain as shown in SEQ ID NO: 3;

[0032] GC represents GGGGC polypeptide, while THP, RESCA, and DOTA are well-known bifunctional chelating agents in the field.

[0033] A pharmaceutical composition comprising the aforementioned radionuclide label and a pharmaceutically acceptable carrier.

[0034] The use of the complementary determinant region (CDR) of the PD-L1-targeting nanobody VHH, the nanobody VHH chain, the nanobody, the conjugate, the radionuclide label, or the pharmaceutical composition as described above in the preparation of drugs, reagents, detection plates, or kits for diagnosing or treating tumors expressing PD-L1 protein.

[0035] This invention, through extensive and in-depth research and numerous screenings, yielded an anti-PD-L1 nanobody, APN09. This nanobody specifically binds to human PD-L1, obtained through phage display screening using a phage library constructed after immunizing alpacas. The term nanobody has the same meaning as single-domain antibody (VHH), which is the smallest antigen-binding fragment with complete function, consisting of four frame regions (FRs) and three complementarity-determining regions (CDRs) spaced apart. Generally, the frame regions of a nanobody synthesis library are relatively conserved, while the diversity lies in the CDR regions. The CDR regions are the main regions determining affinity and specificity. The nanobody APN09 of this invention exhibits the following CDR regions; therefore, this invention essentially discovers a nanobody with the following CDR regions:

[0036] CDR1:FTFGRFNMKW(SEQ ID NO:4)

[0037] CDR2:SGINSSGSMTDY(SEQ ID NO:5)

[0038] CDR3:RNQWMYGTT(SEQ ID NO:6)

[0039] Furthermore, the framework region FR of the nanobody of the present invention is shown below:

[0040] FR1:MDQVQLVESGGGLVQPGGSLRLSCAASG(SEQ ID NO:7)

[0041] FR2:VRQAPGKEEPEWV(SEQ ID NO:8)

[0042] FR3:ADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCA(SEQ ID NO:9)

[0043] FR4:WYPQSRGTQVTVSS(SEQ ID NO:10)

[0044] By employing the above technical solution, the radionuclide-labeled PD-L1-targeting nanobody imaging agent APN09 designed and synthesized in this invention has at least the following advantages and beneficial effects:

[0045] (1) The APN09 structure preparation process is simple, convenient, time-saving, has a high labeling rate, and the label is stable, which facilitates further application in clinical practice, scientific research and drug development.

[0046] (2) This invention provides a visualization tool for detecting PD-L1 expression in tumors and monitoring treatment efficacy. It can be used for screening PD-L1 positive tumor patients, predicting treatment outcomes, and monitoring treatment efficacy.

[0047] (3) APN09 radiolabeled probe is a labeling compound of nanobodies. It has small molecular weight, low immunogenicity, good tissue penetration ability and high affinity for tumor tissue, and has good application prospects. Attached image description:

[0048] Figure 1 . 99m Determination of the binding affinity of Tc-GC-APN09 to human PD-L1 protein.

[0049] Figure 2 . 68 The radioactivity purity of Ga-THP-APN09 was determined using Radio-TLC and Radio-HPLC.

[0050] Figure 3 . 68 Ga-THP-APN09 was transfected into the human lung adenocarcinoma A549 cell line (A549) and PD-L1 gene. PD-L1 Biodistribution map of tumor-bearing mice at 1 hour.

[0051] Figure 4 . 68 Ga-THP-APN09 in A549 and A549 PD-L1 Imaging of a tumor-bearing mouse model; the location of the tumor is shown by the arrow.

[0052] Figure 5 . 99m Tc-GC-APN09 in A549 and A549 PD-L1 Tumor model imaging in tumor-bearing mice (0.5h, 1h, 2h and 4h).

[0053] Figure 6 . 18 F-RESCA-APN09 on A549 and A549 PD-L1 Tumor imaging in tumor-bearing mouse models (0.5h, 1h, 2h, and 4h). Tumor locations are shown by arrows.

[0054] Figure 7 . 18 F-RESCA-APN09 on A549 and A549 PD-L1 Biodistribution map of tumors in tumor-bearing mouse model (1h). Detailed Implementation

[0055] The following detailed description, in conjunction with specific embodiments, illustrates the nanobody imaging agent of the present invention, its preparation method, and its applications. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. Unless otherwise stated, the raw materials and reagents used in the following embodiments are commercially available products or can be prepared by known methods.

[0056] Example 1: Expression and purification of single-domain antibody APN09

[0057] (1) Plasmid synthesis

[0058] The *E. coli* expression vector used in this study was pET25b. Using standard methods, the following nucleotide sequence (SEQ ID NO:1) was inserted into the NcoI and NotI restriction sites to construct the recombinant plasmid. The LPETGGHHHHHH sequence was added to the C-terminus of the sequence, where the LPETG sequence was used for enzyme-linked reactions and the HHHHHH tag was used for nickel column protein purification. The nucleotide sequence (SEQ ID NO:1) is as follows:

[0059] CCATGGATCAGGTTCAGCTTGTTGAATCAGGCGGCGGCCTTGTTCAGCCTGGCGGCTCACTTCGCCTTTCATGTGCTGCTTCAGGCTTTACATTTGGCCGCTTTAACATGAAATGGGTTCGCCAGGCTCCTGGCAAAGAACCTGAATGGGTTTCAGGCATTAACTCGTCTGGTTCCATGACCGATTATGCTGATTCAGTTAAAGGCCGCT TTACAATTTCACGCGATAACGCTAAGAATACCCTTTATCTTCAGATGAACTCACTTAAACCTGAAGATACAGCTGTTTATTATTGTGCTCGCAACCAGTGGATGTATGGCACAACATGGTATCCTCAGTCACGCGGCACACAGGTTACAGTTTCCAGTGGATCGTTGCCCGAAACAGGCGGCCATCATCATCATCATTGAGCGGCCGC

[0060] (2) Plasmid transformation

[0061] After the single-domain antibody recombinant plasmid was constructed and verified to be correct by full-length gene sequencing, it was transformed into *E. coli* expression strain BL-21(DE3). 100 μL of competent BL-21(DE3) cells were thawed on ice. The lyophilized expression plasmid was dissolved in ddH2O to a concentration of 25 ng / μL. 3 μL of the plasmid was added to the competent cells and incubated on ice for 30 min. The competent cells were then heat-shocked in a 42°C water bath for 60–90 s, and immediately placed on ice for 2 min. 500 μL of LB liquid medium (antibiotic-free) was then added to the mixture, and the mixture was shaken at 37°C and 220 rpm for 45 min. After 45 min, 300 μL of medium was added to a 10 cm ampicillin-resistant LB agar plate, and a plate spreader was added and shaken 10 times in a cross-shaped motion. The plate was then poured out. The plate was inverted and incubated overnight at 37°C.

[0062] (3) Induction of expression and purification

[0063] After successful transformation, select single clones for protein expression induction, as follows:

[0064] 1. Small shake: Pick a single colony with a sterile pipette tip and put it into a shaker tube containing 5 mL of LB medium containing ampicillin resistance (100 μg / mL). Incubate overnight at 37°C and 220 rpm on a shaker.

[0065] 2. Shaking: Add 5 mL of culture medium to 1 L of LB medium containing ampicillin (100 μg / mL). Incubate at 37℃ and 220 rpm for 1–2 h using a shaker. After 1 h, measure OD600 using Nanodrop every 30 min.

[0066] 3. Induction: When the OD600 is approximately 0.6–0.9, add IPTG inducer to the large-scale shake culture to a final concentration of 0.5 mmol / L. Incubate at 16°C and 180 rpm for 14–16 h.

[0067] 4. Collect bacterial cells: After induction, pour the bacterial solution into a 250mL centrifuge bottle, centrifuge at 9000rpm for 20min, discard the supernatant, and obtain the bacterial cell precipitate.

[0068] 5. Sonication lysis: Resuspend the bacterial cells in 50 mL of lysis buffer, then add a protease inhibitor (100 μg / mL PMSF), mix well on a shaker, and pour into a small beaker. Fix the beaker in a 0°C ice-water mixture and use an ultrasonic disruptor to lyse the bacterial cells. The ultrasonic conditions were: 150 W power, 6 seconds of sonication, 6 seconds of pause, and a total operating time of 1 hour. After sonication, collect the bacterial culture in a centrifuge tube, centrifuge at 9000 rpm for 20 minutes, and collect the supernatant for later use.

[0069] 6. Protein column attachment: Add 1 mL of nickel beads to a 10 mL affinity chromatography column. Allow the ethanol preservation solution to drain naturally under gravity. Then, add 10 mL of PBS to wash the nickel column to ensure complete removal of ethanol. Resuspend the nickel beads in 1 mL of PBS and add them to the sonicated lysis supernatant. Rotate the column in a 4°C chromatography cabinet on a shaker for 2 hours to allow the protein to adhere.

[0070] 7. Nickel Column Purification: Add the mixture of lysate supernatant and nickel beads to an empty affinity chromatography column. Connect a rubber tubing to the liquid outlet of the affinity chromatography column and use a peristaltic pump to pump the flow-through solution into a centrifuge tube at a constant flow rate. After three flow-throughs, discard the supernatant. Elute sequentially with 20 mL of elution buffer (50 mmol / L Tris HCl), 20 mL of elution buffer containing 20 mmol / L imidazole, 10 mL of elution buffer containing 50 mmol / L imidazole, and 1 mL of elution buffer containing 100 mmol / L imidazole. Add 4 mL of elution buffer containing 500 mmol / L imidazole to the nickel beads, mix well, and let stand for 5 min. Collect the liquid by gravity. Repeat this process once, collecting a total of 8 mL of eluent. After adding the collected eluent to a 3K dialysis bag, place the dialysis bag in a beaker containing PBS solution, add a magnetic rotor, and dialyze overnight on a magnetic stirrer in a chromatography cabinet at 4°C.

[0071] 8. Size Exclusion Chromatography Purification: The dialyzed protein solution was added to an ultrafiltration tube with a molecular cutoff of 3K and concentrated by ultrafiltration at 4500 rpm and 4°C. After centrifugation to a final concentration of 10 mg / mL, purification was performed using Superdex 75 increase molecular size exclusion chromatography. An Agilent 1260 high-performance liquid chromatography system was used, with PBS solution as the mobile phase and a flow rate of 0.8 mL / min. The purified protein solution was then measured for concentration using Nanodrop and stored at -80°C for later use.

[0072] The amino acid protein sequence obtained in this embodiment is shown below:

[0073] MDQVQLVESGGGLVQPGGSLRLSCAASGFTFGRFNMKWVRQAPGKEPEWVSGINSSGSMTDYADSVKGRFTISRDDNAKNTLYLQMNSLKPEDTAVYYCARNQWMYGTTWYPQSRGTQVTVSSGSLPETGGHHHHHH(SEQ IDNO:2)

[0074] After removing the LPETGGHHHHHH sequence added to the C-terminus of the sequence, the APN09 nanobody sequence of the present invention is obtained:

[0075] MDQVQLVESGGGLVQPGGSLRLSCAASGFTFGRFNMKWVRQAPGKEPEWVSGINSSGSMTDYADSVGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARNQWMYGTTWYPQSRGTQVTVSS(SEQ ID NO:3)

[0076] The molecular structure was characterized by mass spectrometry and HPLC. HPLC analysis conditions: Superdex 75 Increase 10 / 300 gel filtration / size exclusion column, flow rate 0.8 mL / min; mobile phase A was 0.1 mol / L PBS solution; mobile phase gradient setting: 0.0–30.0 min 100% A. The APN09 structure exhibits good stability, pharmacokinetic properties, and target specificity.

[0077] Example 2 68 Ga rinsing

[0078] Use a syringe to take 4 mL of 0.05 mol / L HCl to rinse the germanium-gallium generator at a rate of 1-2 mL / min. Discard the first 1 mL of HCl and collect the remaining 3 mL of rinsing solution. Record the radioactivity.

[0079] Example 3: Preparation of precursor APN09-GC

[0080] 10 mg of APN09 nanobody (10 mg / mL), 2 mg of GGGGC (20 mg / mL) peptide, 20 μL of CaCl2 (1 mol / L), and 200 μL of Sortase enzyme (1 mg / mL) were added sequentially to a 1.5 mL reaction tube. The pH was adjusted to 7.5–8.0 with 2 mol / L NaOH, and the reaction was carried out at 4 °C on a rotating shaker for 4 h. The reaction solution was centrifuged at 3000 rpm for 1 min. The supernatant was transferred to a pretreated Ni column, mixed thoroughly by pipetting, and allowed to stand for 5 min before collecting the sample. The sample was purified using an HPLC system with PBS as the mobile phase, a Superdex 75 Increase 10 / 300 column, a detection wavelength of 280 nm, an injection volume of 500 μL, and a flow rate of 0.8 mL / min.

[0081] Example 4: Preparation of precursor THP-APN09

[0082] The purified APN09-GC was concentrated using a 3K ultrafiltration tube to a concentration of 6–8 mg / mL. Mal-THP solid powder was weighed into a 1.5 mL EP tube according to a molar ratio of APN09-GC to Mal-THP of 1:5. The concentrated APN09-GC solution was added, and the mixture was shaken thoroughly. The tube was then placed in a metal bath shaker and reacted at 30 °C for 1 h. After the reaction, the mixture was filtered through a 0.22 μm aqueous syringe filter and purified by HPLC using a Superdex 75 Increase 10 / 300 column, a detection wavelength of 280 nm, an injection volume of 500 μL, and a flow rate of 0.8 mL / min to obtain THP-APN09. The sample was aliquoted into 200 μg portions and stored at -80 °C.

[0083] Example 5: Preparation of the precursor RESCA-APN09

[0084] Weigh Mal-RESCA solid powder into a 1.5 mL EP tube according to a molar ratio of APN09-GC to Mal-RESCA of 1:10. Add the concentrated APN09-GC solution to the reaction tube using a pipette, vortex to mix, and place in a multifunctional thermostatic mixing bath at 37℃ for 1–2 h. After the reaction, filter the reaction solution through a 0.22 μm aqueous syringe filter and purify the precursor using an HPLC system. The purification column was a Superdex 75 Increase 10 / 300, with detection wavelengths of 210 nm and 280 nm, an injection volume of 500 μL, and a flow rate of 0.8 mL / min. The molecular weight of APN09-RESCA was determined using MALDI-TOF. The precursor sample was aliquoted into 200 μg portions and stored at -80℃.

[0085] The precursor DOTA / NOTA-APN09 can also be prepared according to the method in Example 4.

[0086] Example 6 68 Preparation of Ga-THP-APN09

[0087] Remove the aliquoted precursor THP-APN09 (200 μg) from the -80℃ freezer. The concentration is 2–3 mg / mL. Dissolve it rapidly and add 300 μL of 2.0 mol / L sodium acetate solution, then add 1 mL of [the solution is missing from the original text]. 68 Ga(259-740MBq) fresh rinse in step (1) 68 Ga, the reaction system pH = 6.0-6.5, was inverted and mixed thoroughly, and reacted at room temperature (20-30℃) for 10 min. A 0.22 μm microporous membrane was then added, followed by 2.0 mL of physiological saline. The product was filtered into a sterile vacuum bottle for later use, yielding the radioactive product. 68Ga-THP-APN09.

[0088] Example 7 Quality Control

[0089] iTLC: Pipette 20 μL of saturated EDTA-2Na solution into a 0.5 mL EP tube, add 1 μL of reaction solution, mix well, spot the sample onto an iTLC-SG strip, develop with physiological saline, and perform scanning analysis on Radio-TLC. 68 1 μL of Ga ion solution was mixed with 20 μL of saturated EDTA-2Na solution and spotted onto an iTLC-SG strip as a control. Results are shown below. Figure 1 As shown in Figure A. The radioactivity purity of the radiolabeled probe was determined by HPLC. The HPLC analysis conditions were: mobile phase PBS, separation and purification column Superdex 75 Increase 10 / 300, injection volume 37 KBq, flow rate 0.8 mL / min, and injection time 30 min. 68 37 KBq of GaCl3 solution was used as a control. Results are shown below. Figure 1 As shown in .B.

[0090] Example 8 99m Preparation of Tc-GC-APN09

[0091] 370 MBq of Na 99m TcO4 was added to a sodium gluconate kit (Beijing Shihong Pharmaceutical Research Center), and the kit was shaken to dissolve. After reacting at room temperature for 5 min, 100 μg of GC-APN09, 100 μL of 0.2 mol / L succinate buffer (pH = 4.7), and 10 μL of 10 mol / L EDTA-2Na were added, and the mixture was reacted in a water bath at 37 °C for 1 h. The radiolabeled single-domain antibody solution was purified on a NAP-5 gel exclusion column and filtered through a disposable 0.22 μm filter membrane. 99m The radiochemical purity of Tc-GC-APN09 was determined by instantaneous thin-layer chromatography-silica gel method. After development with physiological saline, it was detected by a radiometric TLC imaging scanner.

[0092] Example 9 177 Preparation of Lu-DOTA-APN09

[0093] radioactivity 177 Lu-labeled PD-L1 targeted nanobody imaging agent 177 The preparation method of Lu-DOTA-APN09 is as follows:

[0094] 100 μL of NaAc / HAc (pH = 5.5) solution was added sequentially to the labeled precursor, followed by 370 MBq of... 177LuCl3 solution, reacted at 37℃ for 30 min, yielded 177 Lu-DOTA / NOTA-APN09 has a radiochemical purity greater than 80%. It is purified using PD-10. The purified product is then added to 1–2 mL of physiological saline, mixed well, and filtered through a 0.22 μm sterile needle filter into a sterile vacuum bottle to obtain the final product.

[0095] Example 10 18 Preparation of F-RESCA-APN09

[0096] Take 100 μL of physiological saline 18 Add F eluent (approximately 1110 MBq) to 4 μL of 2 mmol / L AlCl3 solution and react for 5 min. Then add 110 μL of NaAc / HAc (pH = 4.1) solution and 100 μL of RESCA-APN09 solution, maintaining the system pH at 4.6–4.8. React at room temperature for 12 min. Purify using a size exclusion column (PD-10) pretreated with PBS. Add 1–2 mL of physiological saline to the purified product, mix well, and filter through a 0.22 μm sterile syringe filter into a sterile vacuum bottle to obtain the final product.

[0097] Example 11 68 Biodistribution of Ga-THP-APN09 in mouse tumor models

[0098] Nude mice were subcutaneously inoculated with A549 cells on the left side of the forelimb axilla and on the right side. PD-L1 Cells, the tumor grew to about 100 mm after 6-8 days. 3 (n=4). 0.2 mL (7.4 MBq) was injected into the tail vein of mice. 68 Mice were sacrificed after 1 hour using Ga-THP-APN09. Blood, brain, heart, liver, spleen, lung, kidney, stomach, intestine, pancreas, flesh, and tumor tissues were collected. After weighing, radioactivity was measured using a gamma counter, and the uptake (%ID / g) of each organ and tissue was calculated. Experimental results are expressed as mean ± SD. In the competitive binding blocking group, a total of 50 nmol of nanobody was injected. 68 Radioactive uptake of various organs at different time points after Ga-THP-APN09 is shown in the figure. Figure 2 , 68 Ga-THP-APN09 is mainly metabolized by the kidneys. It is uptaken in high-positive tumors and low in negative tumors. The uptake in positive tumors was significantly reduced in the competitive binding blocking group, which proves the targeting of the probe to PD-L1.

[0099] Example 12 68 Imaging of Ga-THP-APN09 mouse tumor model

[0100] Nude mice were subcutaneously inoculated with A549 cells on the left side of the forelimb axilla and on the right side. PD-L1 Cells, the tumor grew to about 100 mm after 6-8 days. 3 (n=4). 0.2 mL (7.4 MBq) was injected into the tail vein of mice. 68 Ga-THP-APN09 was subjected to Micro-PET scanning 1 hour later. Image reconstruction was performed on the regions of interest (ROIs) of the whole-body decay-corrected coronal maps obtained from the Micro-PET scans. Results are as follows: Figure 3 As shown, positive tumor tissues in mice exhibited high uptake, while negative tumor tissues showed low uptake. The competitive binding blocking group also showed low uptake in positive tumor tissues. Furthermore, the probe was metabolized rapidly, resulting in a high target-to-probe ratio, indicating that this marker possesses good specificity and affinity, making it a promising tumor imaging agent.

[0101] Example 13 99m Binding affinity determination of Tc-GC-APN09.

[0102] According to the radioactivity 99m Tc-labeled PD-L1 targeted imaging agent 99m The Tc-GC-APN09 was prepared using the method described above. 99m Quantitative assessment of the affinity of Tc-GC-APN09 for PD-L1 protein was performed on a 96-well highly adsorbed removable ELISA plate coated with 0.2 μg / well of PD-L1. In the saturation binding assay, the specific activity of the label was adjusted to 12 mci / mg, and then serially diluted. 99m Tc-GC-APN09 (0.8 to 355 nmol / L) was added to each well at 100 μL and incubated at 37 °C for 1 h. Four replicates were prepared for each concentration group, with one standard sample reserved. After 1 h, the 96-well plates used in the saturation binding experiment were washed three times with PBST, and the wells of the removable plates were collected and counted using a gamma counter. The radioactivity count was calculated as: (Measured radioactivity count / Standard radioactivity count) × Added 99m The concentration of Tc-GC-APN09 can be used to obtain the binding complex ( 99m The concentration of Tc-GC-APN09-hPD-L1. To add 99m The concentration of Tc-GC-APN09 is plotted on the x-axis, along with the binding complex ( 99m The equilibrium dissociation constant (KD) was analyzed using Prism V.7.0 with the concentration of Tc-GC-APN09-hPD-L1 as the ordinate. 99m The binding affinity assay results for Tc-GC-APN09 are as follows: Figure 4 As shown.

[0103] Example 14 99mImaging of Tc-GC-APN09 mouse tumor model

[0104] Nude mice were subcutaneously inoculated with A549 cells on the left side of the forelimb axilla and on the right side. PD -L1 cells, the tumor grew to 100 mm in 6-8 days. 3 (n=3). Injection tracer 99m Following Tc-GC-APN09, mice were anesthetized with medical oxygen containing 2% isoflurane at time points of 0.5h, 1h, 2h, and 4h. After anesthesia, they were placed on the acquisition bed for imaging. The original SPECT data were reconstructed from the whole body, and then the SPECT and CT images were fused using Nucline v 2.01 (Mediso Inc.) software. The maximum signal intensity projection (MIP) of the whole body imaging was given through the rear view. 99m The binding specificity of Tc-GC-APN09 in vivo is achieved by simultaneously targeting PD-L1-positive A549. PD-L1 This was confirmed by imaging experiments on negative A549 tumors. 99m The results of Tc-GC-APN09 are as follows: Figure 5 As shown, mouse tumor-positive tissues exhibited high uptake, while tumor-negative tissues showed low uptake. Furthermore, renal uptake decreased significantly over time, resulting in a better tumor / kidney ratio.

[0105] Example 15 18 F-RESCA-APN09 mouse tumor model imaging

[0106] According to the radioactivity 18 F-labeled PD-L1 targeted imaging agent 18 The F-RESCA-APN09 method was used to prepare it.

[0107] Nude mice were subcutaneously inoculated with A549 cells on the left side of the forelimb axilla and on the right side. PD-L1 Cells, the tumor grew to 100mm in 6-8 days. 3 (n=3). 0.2 mL (7.4 MBq) was injected into the tail vein of mice. 18 F-RESCA-APN09 was subjected to Micro-PET imaging 1 hour later. Image reconstruction was performed on the regions of interest (ROIs) of the whole-body decay-corrected coronal images obtained from the Micro-PET scans. Results are as follows: Figure 6 As shown, positive tumor tissues in mice exhibited high uptake, while negative tumor tissues showed low uptake. The positive tumor tissues in the competitive binding blocking group also showed low uptake.

[0108] Example 16 18 Biodistribution of the F-RESCA-APN09 mouse tumor model

[0109] According to the radioactivity 18 F-labeled PD-L1 targeted imaging agent 18 The F-RESCA-APN09 method was used to prepare it.

[0110] Nude mice were subcutaneously inoculated with A549 cells on the left side of the forelimb axilla and on the right side. PD-L1 Cells, the tumor grew to 100mm in 6-8 days. 3 (n=4). 0.2 mL (0.74 MBq) was injected into the tail vein of mice. 18 Mice were sacrificed at 1 hour using F-RESCA-APN09. Blood, brain, heart, liver, spleen, lung, kidney, stomach, intestine, bone, flesh, and tumors were collected. After weighing, radioactivity was measured using a gamma counter, and the uptake (%ID / g) of each organ and tissue was calculated. Experimental results are expressed as mean ± SD. In the competitive binding blocking group, a total of 50 nmol of nanobody was injected. 18 Radioactive uptake of various organs at different time points after F-RESCA-APN09 is shown in the figure. Figure 7 , 18 F-RESCA-APN09 is primarily metabolized by the kidneys and gallbladder, exhibiting high uptake in positive tumors and low uptake in negative tumors. In the competitive binding blocking group, positive tumors show low uptake. This imaging agent demonstrates good in vitro and in vivo stability, excellent imaging performance, and good affinity and specificity for PD-L1, making it suitable for in vivo monitoring of PD-L1 expression levels.

[0111] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanobody targeting PD-L1, wherein the VHH chain comprises an amino acid sequence fragment of the complementarity-determining region: The amino acid sequence is CDR1 as shown in SEQ ID NO: 4; The amino acid sequence is CDR2 as shown in SEQ ID NO: 5; and The amino acid sequence is CDR3 as shown in SEQ ID NO:

6.

2. The PD-L1-targeting nanobody according to claim 1, wherein the VHH chain further comprises amino acid sequence fragments of the following framework region: The amino acid sequence is FR1 as shown in SEQ ID NO: 7; The amino acid sequence is FR2 as shown in SEQ ID NO: 8; The amino acid sequence is FR3 as shown in SEQ ID NO: 9; and The amino acid sequence is FR4 as shown in SEQ ID NO:

10.

3. The PD-L1-targeting nanobody according to claim 1 or 2, wherein the amino acid sequence of its VHH chain is shown in SEQ ID NO:

3.

4. A conjugate obtained by conjugating the VHH chain of the nanobody according to any one of claims 1-3 with a chelating agent of GGGGC or a radionuclide.

5. The conjugate according to claim 4, wherein the chelating agent for the radionuclide is selected from NOA, DOTA, RESCA, or THP.

6. A radionuclide label obtained by radionuclide labeling of the conjugate according to any one of claims 4-5.

7. The radionuclide label according to claim 6, wherein the radionuclide is selected from... 99m Tc, 68 Ga、 18 F, 177 Lu.

8. The radionuclide label according to claim 7, wherein the radionuclide label is selected from the following molecules: 99m Tc-GC-APN09; 68 Ga-THP-APN09; 18 F-RESCA-APN09; 177 Lu-DOTA-APN09; in, APN09 represents the VHH chain as shown in SEQ ID NO: 3; GC represents GGGGC polypeptide, and THP, RESCA, and DOTA are bifunctional chelating agents.

9. A pharmaceutical composition comprising the radionuclide label as described in any one of claims 6-8 and a pharmaceutically acceptable carrier.

10. The use of the PD-L1-targeting nanobody according to any one of claims 1-3, the conjugate according to any one of claims 4-5, the radionuclide label according to any one of claims 6-8, or the pharmaceutical composition according to claim 9 in the preparation of a medicament, reagent, detection plate, or kit for diagnosing tumors expressing PD-L1 protein.

Images