A PET imaging agent targeting PD-L1, its preparation method and application

Abstract

The present invention discloses a PET imaging agent targeting PD-L1, its preparation method and application. The PET imaging agent of the present invention is obtained by modifying the Durva-F(ab')2 antibody fragment with an acylating agent 3-(4-hydroxyphenyl)propionic acid-N-succinimide ester, and then 124 labeling with 124 I to obtain the I-HPP-Durva-F(ab')2 polypeptide molecular probe. By subjecting the modified Durva-F(ab')2 antibody fragment obtained in the present invention to SHPP amidation modification, the blood clearance rate and labeling specific activity of the probe are improved. The molecular probe of the present invention has a significantly reduced molecular weight, which accelerates the clearance rate of the probe in vivo and is beneficial to clinical translation. The non-specific uptake of this molecular probe in the liver and bone tissues is significantly reduced, which has great clinical significance for patients whose metastases or primary lesions are located in this part.

Description

Technical Field

[0001] The present invention relates to a PET imaging agent targeting PD-L1, a preparation method thereof and an application thereof, belonging to the technical field of radiopharmaceutical chemistry. Background Art

[0002] Anti-PD-1 / PD-L1 therapy refers to blocking the interaction between PD-1 and PD-L1 through antibodies and inhibitors, etc., restoring the activation of T cells, and thus mediating the reactivation of anti-tumor immunity. It occupies an important position in the treatment of various tumors, especially solid tumors. Anti-PD-1 / PD-L1 monoclonal antibodies, such as Pembrolizumab, Nivolumab, Atezolizumab and Durvalumab, have shown clinical application prospects in patients with advanced cancer. Before treatment, patients often detect the expression of PD-L1 in the primary focus or metastatic focus by immunohistochemistry (IHC) to determine whether they are suitable for anti-PD-1 / PD-L1 treatment. In a retrospective study, the results of IHC were only related to the prognosis of less than 29% of the patients. The reasons may include the following points: 1. The spatio-temporal heterogeneity of PD-L1 expression; 2. The limitations of specimen collection; 3. The ability of different tumors to drive immunogenicity may vary. Therefore, there is an urgent need for a method with both high sensitivity and high specificity to detect the expression of PD-L1.

[0003] To solve this problem, medical scientists have conducted many meaningful studies, such as optical imaging, photoacoustic imaging, magnetic resonance imaging and nuclear medicine imaging. Among them, nuclear medicine imaging seems to be the most feasible solution. Immuno-Positron Emission Tomography (immunoPET) refers to obtaining the distribution of a radionuclide-labeled tracer through PET imaging to non-invasively evaluate the expression of the target in the lesion, combining the excellent targeting specificity and affinity of the precursor and the superior imaging sensitivity and resolution of PET. With the application of long half-life radionuclides in clinical practice and their gradual popularization in the development of antibodies, immunoPET imaging has played an increasingly important role in evaluating the expression of biomarkers and predicting the possible efficacy of treatment. In a study on 89 the results of Zr-DFO-Atezolizumab immunoPET imaging of 25 patients with different types of locally advanced or metastatic cancer showed that immunoPET can not only evaluate the expression of PD-L1, but also predict the clinical response of patients to immune blockade treatment. However, due to the labeling method and the characteristics of the antibody itself, it limits 89Application of Zr-labeled Atezolizumab when the lesions are located in the liver, bone and other parts. Anti-PD-L1 immunotherapy is mainly applied to locally advanced or metastatic patients in the field of cancer. The liver and bone are common cancer metastasis sites in these patients. 124 I and 89 Zr both belong to long half-life solid target positron radionuclides. Different from 89 Zr, 124 I can be labeled without a chelating agent and can directly react with tyrosine in protein molecules. Previous studies have shown that 124 the product after 89 I labeling is closer to the biological characteristics of the labeled substance itself. Its uptake in the liver and spleen is significantly lower than that of the product labeled with

[0004] Zr through a chelating agent, and the cell retention and bone adsorption of non-metallic radionuclides are also significantly lower than those of metallic radionuclides. Durvalumab (Durva) is another anti-PD-L1 antibody. Different from Atezolizumab, the liver uptake of this antibody is relatively low.

[0004] The F(ab')2 fragment can be produced by enzymatic digestion of the intact antibody, and its molecular weight is reduced from about 150 kDa to about 100 kDa. Compared with the intact antibody, the F(ab')2 fragment shows a faster blood clearance rate while retaining high binding affinity and specificity.

[0005] In previous studies, we successfully synthesized a PET imaging agent targeting PD-L1 124 I-Durva-F(ab')2 ( 124 I-Labeled Monoclonal Antibody and Fragment for the Noninvasive Evaluation of Tumor PD-L1 Expression In Vivo). However, due to its relatively low specific activity and high blood accumulation, its future application is limited. Summary of the Invention

[0006] The object of the present invention is to propose and synthesize a 124 I-labeled molecular probe targeting PD-L1 124 I-HPP-Durva-F(ab')2 to non-invasively evaluate the PD-L1 expression of lesions and guide clinical medication. This molecular probe 124I-HPP-Durva-F(ab')2 has been verified for its targeting to PD-L1 through in vivo and in vitro experiments. The biodistribution and PET / CT imaging results indicate that this probe can highly specifically evaluate the expression of PD-L1. Compared with previous positron drugs, the uptake of this molecular probe in the liver and bones is significantly reduced, and its specific activity and blood activity have also been significantly improved compared with 124 I-Durva-F(ab')2.

[0007] To achieve the above object, the present invention provides a labeling precursor of a PET imaging agent targeting PD-L1, which is obtained by modifying the Durva-F(ab')2 antibody fragment with the acylating agent 3-(4-hydroxyphenyl)propionic acid-N-succinimide ester. Its structural formula is shown in Formula I, wherein the light chain amino acid sequence of the Durva-F(ab')2 antibody fragment is as shown in SEQ ID NO: 1, and the heavy chain amino acid sequence is as shown in SEQ ID NO: 2. The acylating group SHPP is modified on the free amino groups of one or more lysine sites on the light chain and / or heavy chain of the Durva-F(ab')2 antibody fragment,

[0008]

[0009] Light chain amino acid sequence of Durva-F(ab')2 antibody fragment (SEQ ID NO: 1): EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFELTISALEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVPAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0010] Heavy chain amino acid sequence of Durva-F(ab')2 antibody fragment (SEQ ID NO: 2): EVQLVESGGGLVQPGGSLRLSCANSGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT。

[0011] The present invention also provides a PET imaging agent targeting PD-L1, which is obtained by radioactively labeling the above-mentioned labeling precursor with 124 I, wherein 124 I is labeled at the ortho-position of the acyl group and / or the phenolic hydroxyl group on the benzene ring of tyrosine, and one of the ortho-positions of the phenolic hydroxyl group is labeled with 124 I or both ortho-positions are simultaneously labeled with 124 I.

[0012] The present invention also provides the use of the above-mentioned labeling precursor of the PET imaging agent targeting PD-L1 in the preparation of diagnostic reagents / drugs labeled with diagnostic radionuclides and / or therapeutic radionuclides, and / or therapeutic drugs.

[0013] The present invention also provides a preparation method of the above-mentioned PET imaging agent targeting PD-L1, including:

[0014] Step 1: Digest Durvalumab with an enzyme to remove the Fc fragment, and obtain the antibody fragment Durva-F(ab')2 with retained targeting function after purification;

[0015] Step 2: Perform an amidation reaction on the above-obtained antibody fragment with the acylating agent 3-(4-hydroxyphenyl)propionic acid-N-succinimide ester, and obtain the modified product HPP-Durva-F(ab')2 after purification;

[0016] Step 3: Perform 124 I radioactive labeling on the above-obtained modified product, and obtain 124 I-HPP-Durva-F(ab')2 polypeptide molecular probe after purification.

[0017] The present invention also provides the use of the above-mentioned PD-L1-targeted PET imaging agent in the preparation of diagnostic reagents / drugs and / or therapeutic drugs for tumors with high PD-L1 expression, and the tumors include primary tumors or metastatic tumors.

[0018] Preferably, the primary tumors include primary tumors with the lesion location in the liver or bone.

[0019] The present invention also provides the use of the above-mentioned PD-L1-targeted PET imaging agent in the preparation of a diagnostic and therapeutic kit for tumors with high PD-L1 expression.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) In view of the relatively high uptake of the PD-L1-targeted imaging agents reported in the current literature in the liver and bones, which is not conducive to the evaluation of this part of patients with metastases or primary lesions located in the liver and bone; the present invention utilizes the characteristic that durvalumab binds to PD-L1 with high affinity and specificity, and modifies the structure of durvalumab. The active region F(ab')2 is retained, and the heavy chain constant region Fc is removed. While retaining the molecular specificity, the molecular weight of the probe is reduced. After the obtained Durva-F(ab')2 antibody fragment is modified by SHPP amidation, the blood clearance rate and labeling specific activity of the probe are improved, overcoming the defects that the application of PET probes prepared by the direct labeling method is limited by their relatively low specific activity and high blood accumulation;

[0022] (2) The PD-L1-targeted PET imaging agent of the present invention retains the functional region F(ab')2 of the monoclonal antibody Durvalumab, and the molecular weight is significantly reduced, from about 146.3 kDa of the whole antibody to about 100 kDa. The clearance rate of the probe in the body is accelerated, which is beneficial to clinical translation. Diagnostic images can be obtained in 4 hours, and the imaging effect is the best at 24 hours, while the complete antibody can provide diagnostic images at 24 hours, and the best imaging time is about 96 hours;

[0023] (3) The non-specific uptake of the PET molecular probe of the present invention in the liver and bone tissues is significantly reduced, and these two parts are the parts where primary or metastatic cancer foci are more likely to appear. Therefore, the characteristics of this imaging agent have great clinical significance for meeting the needs of patients with metastases or primary lesions located in this part; compared with the probes prepared by direct labeling, the PET molecular probe of the present invention has higher tumor contrast and labeling specific activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the molecular probe of the present invention 124Synthesis route diagram of I-HPP-Durva-F(ab')2;

[0025] Figure 2 SDS-PAGE analysis of Durva-F(ab')2 solution: Lane M: Marker protein in kilodaltons; Lane 1: Non-reduced Durva-F(ab')2; Lane 2: Reduced Durva-F(ab')2;

[0026] Figure 3 Mass spectrometry results of Durva-F(ab')2;

[0027] Figure 4 HPLC analysis results of Durva-F(ab')2 24 hours after purification;

[0028] Figure 5 Results of the number of SHPPs linked to HPP-Durva-F(ab')2 generated after modification with SHPP by mass spectrometry analysis;

[0029] Figure 6 For 124 Results of radiochemical purity analysis (A), stability analysis (B - E), and specific activity comparison (F) of I-HPP-Durva-F(ab')2; Among them, 124 The stability analysis of I-HPP-Durva-F(ab')2 is respectively the analysis results (B) of incubation in PBS for 24 hours, the analysis results (D) of incubation in PBS for 72 hours, the analysis results (C) of incubation in FBS for 24 hours, and the analysis results (E) of incubation in FBS for 72 hours;

[0030] Figure 7 For 124 Biodistribution map of I-HPP-Durva-F(ab')2;

[0031] Figure 8 For tumor-bearing mice injected with 124 MIP (left) and PET / CT (right) images 48 hours after injection of I-HPP-Durva-F(ab')2. Detailed implementation manners

[0032] To make the present invention more obvious and understandable, preferred embodiments are hereby described in detail in conjunction with the accompanying drawings as follows.

[0033] Example 1

[0034] 124 Preparation of I-HPP-Durva-F(ab')2:

[0035] 200 μg of intact Durva was incubated with 200 U of IdeS protease in digestion buffer (20 mM sodium phosphate, 10 mM sodium chloride, pH = 6.5) at 37 °C for 120 minutes. After the digestion product was incubated with protein A magnetic beads at room temperature for 2 hours, it was placed in a magnetic rack for 1 minute to separate the beads attached with Fc fragment and the supernatant containing F(ab')2 fragment, and the purified F(ab')2 fragment was obtained. The obtained purified F(ab')2 fragment was mixed with SHPP reagent and incubated at 35 - 40 °C for 80 - 160 min to obtain a reaction product; the reaction product was separated and purified by a 7k centrifugal column to obtain HPP-Durva-F(ab')2. 124 I was produced by 124 Te(p,n) 124 reaction in a Sumitomo HM-20 cyclotron. The product was heated and collected in a pre-prepared NaOH solution (10 mM, pH = 12). Before labeling, HCl (0.1 M, pH = 1) was added to the radioactive iodine solution to neutralize NaOH. Subsequently, 10×PBS (0.1 M, pH = 7.4), Durva-F(ab')2 fragment solution (~2 mg / mL) and 124 I solution (111 MBq / mL, pH 7.0 - 7.2) were added to an iodination tube pre-coated with 100 μg Iodogen. Incubate at room temperature for 15 minutes, shaking gently every 5 minutes. 124 125I-HPP-Durva-F(ab')2 was purified by a pre-equilibrated PD-10 desalting column and eluted with PBS into centrifuge tubes. 0.5 mL of eluate was collected in each centrifuge tube in turn, and the radioactivity of each tube was measured separately by a gamma counter to determine the location of the product (usually in the 4th to 6th tubes).

[0036] Example 2

[0037] 124 Quality control and stability test of 125I-HPP-Durva-F(ab')2:

[0038] SDS-PAGE was performed using a 12% non-reducing gel at a voltage of 120 V for 1 hour. The sample loading order for each lane was as Figure 2 shown, and then stained with Coomassie Brilliant Blue at room temperature (RT). HPLC was performed on an Agilent 1260 InfinityII system with a column model: Tosoh Bioscience TSKgel G3000SWXL (7.8 mm x 30 cm). Mass spectrometry analysis was performed using a Waters Bioaccord mass spectrometer.

[0039] SDS-PAGE results showed that the intact Durva antibody was located at approximately 150 kDa, which was very close to the theoretical molecular weight. After purification, a single band of approximately 100 kDa was observed in the lane where Durva-F(ab')2 was added ( Figure 2 ). Subsequently, mass spectrometry results confirmed that the molecular weight of Durva-F(ab')2 was 98.544 kDa ( Figure 3 ). SEC-HPLC analysis showed that the retention time of Durva-F(ab')2 was the main peak at 8.0 to 10.0 minutes ( Figure 4 ). The number of SHPPs linked to HPP-Durva-F(ab')2 generated after SHPP modification was analyzed by mass spectrometry ( Figure 5 ). The radiochemical purity ( 124 A), labeling stability ( Figure 6 B–6E), and specific activity ( Figure 6 F) of labeled Figure 6 I-HPP-Durva-F(ab')2 were analyzed using iTLC. The labeled product had a high radiochemical purity (>98%) and a specific activity of approximately 3.0 GBq / μmol. To demonstrate the stability of 124 I-HPP-Durva-F(ab')2, the probe was incubated in PBS or FBS at 37 °C for 24 hours or 72 hours. The results of iTLC showed that even after incubation for 72 h, the radiochemical purity of 124 I-HPP-Durva-F(ab')2 was still >93%. Compared with 124 I-Durva-F(ab')2 generated by the direct labeling method, the specific activity of 124 I-HPP-Durva-F(ab')2 labeled after SHPP modification was significantly increased.

[0040] Example 3

[0041] 124 Biodistribution experiment of

[0042] I-HPP-Durva-F(ab')2: 124 Biodistribution experiments were performed at 4 h, 12 h, 24 h, 48 h, and 72 h after injection of Figure 7 I-labeled HPP-Durva-F(ab')2. After anesthetizing the mice, the eyeballs were removed to collect blood, and then the mice were sacrificed. The main tissues and organs (heart, lung, liver, spleen, stomach, intestine, kidney, brain, muscle, tumor) were collected. After weighing the removed tissues and organs and performing radioactive counting, the radioactivity of each organ or tissue was expressed as %ID / g. The results are shown in Table 1 and

[0043] As can be seen from Table 1 and Figure 7 For124 The uptake of I-HPP-Durva-F(ab')2 reached a peak of approximately 1.57% ID / g in tumors 12 hours after injection and then remained at a relatively high radioactivity level. Among normal organ tissues, blood had the highest activity level, which gradually decreased over time, indicating that the radionuclide molecular probe had good blood stability. Besides blood, the radioactive uptake in the liver and kidneys was the highest among various organs, suggesting that the radionuclide molecular probe was metabolized in the liver and excreted through the kidneys. At 48 hours, the contrast of the tumor was obvious. Except for the dim liver and bladder images, only bright tumor uptake could be seen on the image.

[0044] Table 1 4 - 72 hours 124 Table of biodistribution changes of I-HPP-Durva-F(ab')2 (% ID / g)

[0045] organ 4 hours 12 hours 24 hours 48 hours 72 hours blood 5.44 2.49 0.6 0.21 0.16 heart 0.72 0.35 0.05 0.11 0.05 lung 0.77 0.2 0.04 0.14 0.12 liver 2.68 2.01 0.94 0.44 0.38 stomach 0.51 0.31 0.08 0.07 0.04 intestine 0.5 0.5 0.06 -0.02 0.04 spleen 1.26 1.05 0.45 0.21 0.17 kidney 3.81 2.05 0.74 0.37 0.32 muscle 0.23 0.21 0.07 0.02 0.03 brain 0.18 0.11 -0.02 0.07 0.01 tumor 1.25 1.57 1.21 1.11 0.95

[0046] Example 4

[0047] 124 PET imaging of I-HPP-Durva-F(ab')2 in tumor-bearing mice:

[0048] For tumor-bearing mice, 1.11 MBq of I-HPP-Durva-F(ab')2 was injected via the tail vein. 124 PET imaging was performed at predetermined time points after injection. The mice were intraperitoneally anesthetized 5 minutes before imaging. When imaging, the localization image of the mice was first obtained. After ensuring the accurate position, the original PET images were collected and exported after being processed by the onboard software Inveon Research Workspace. The PET imaging results at 48 h are as Figure 8 shown.

[0049] The above embodiments are only the preferred embodiments of the present invention and do not limit the present invention in any formal or substantial way. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A labeling precursor of a PD-L1-targeted PET imaging agent, characterized in that, It is obtained by modifying the Durva-F(ab')2 antibody fragment with the acylating agent 3-(4-hydroxyphenyl)propionic acid N-succinimide ester, and its structural formula is shown in Formula I. Among them, the light chain amino acid sequence of the Durva-F(ab')2 antibody fragment is shown in SEQ ID NO: 1, and the heavy chain amino acid sequence is shown in SEQ ID NO:

2. The acylating group SHPP is modified on the free amino groups of one or more lysine sites on the light chain and / or heavy chain of the Durva-F(ab')2 antibody fragment.

2. A PET imaging agent targeting PD-L1, characterized in that, Obtained by radioactively chemically labeling the labeling precursor according to claim 1, wherein 124 I is labeled at the ortho-position of the acyl group and / or the phenolic hydroxyl group on tyrosine, wherein one of the ortho-positions of the phenolic hydroxyl group is labeled with 124 I or both ortho-positions are simultaneously labeled with 124 I. 124 I.

3. Use of the labeling precursor of the PD-L1-targeted PET imaging agent according to claim 1 in the preparation of a diagnostic reagent / drug labeled with a diagnostic radionuclide and / or a therapeutic radionuclide, and / or a therapeutic drug.

4. The preparation method of the PD-L1-targeted PET imaging agent according to claim 2, characterized in that, Including: Step 1: Digest Durvalumab with an enzyme to remove the Fc fragment, and obtain the antibody fragment Durva-F(ab')2 with retained targeting function after purification; Step 2: Perform an amidation reaction on the obtained antibody fragment with the acylating agent 3-(4-hydroxyphenyl)propionic acid N-succinimide ester, and obtain the modified product SHPP-Durva-F(ab')2 after purification; Step 3: Perform 124 I radioactive labeling on the above-obtained modified product, and after purification, obtain 124 I-HPP-Durva-F(ab')2 polypeptide molecular probe.

5. Use of the PD-L1-targeted PET imaging agent according to claim 2 in the preparation of a diagnostic reagent / drug and / or therapeutic drug for tumors with high PD-L1 expression, characterized in that, The tumor includes a primary tumor or a metastatic tumor.

6. Use of the PD-L1-targeted PET imaging agent according to claim 2 in the preparation of a diagnostic and therapeutic kit for tumors with high PD-L1 expression.

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