CN116789829A - Radionuclide-labeled PD-L1 targeting nano antibody and preparation method and application thereof - Google Patents
Radionuclide-labeled PD-L1 targeting nano antibody and preparation method and application thereof Download PDFInfo
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Abstract
Novel PD-L1 targeted nano antibody APN09 and radionuclide 99m Tc、 68 Ga、 18 F、 177 Lu) labeling the developing agent of the PD-L1 targeting nano antibody, and a preparation method and application thereof. The imaging agent of the invention has good affinity and selectivity with tumor, and the marking method is simple, convenient to operate,The method has short time consumption and high marking rate, and can be used for detecting the PD-L1 expression of malignant tumor and monitoring the curative effect.
Description
Technical Field
The invention relates to the technical fields of biomedicine, radionuclide-labeled radiochemistry and clinical nuclear medicine, in particular to a series of radionuclide-labeled PD-L1 targeted nanobody APN09 derivative probes and a preparation method thereof, which are used for identifying, diagnosing, staging, accurately positioning focus, monitoring curative effect and radioimmunotherapy of malignant tumors.
Background
With the deep development and fusion of nuclear medicine and molecular biology, medical imaging technology is moving toward the era of molecular imaging, in which positron emission computed tomography (Positron Emission Tomography, PET) and single photon emission computed tomography (Single Photon Emission Computed Tomography, SPECT) are functionally developed, so that people really recognize and diagnose diseases from the molecular level, and particularly, the advantages of the imaging technology are highlighted in the diagnosis and treatment of tumors. The defects in the modern diagnosis technology are overcome by tracing the receptor change of pathological tissues and the abnormality of cell signal transduction, the basis is provided for early diagnosis, clinical staging and curative effect evaluation of tumors, prognosis is evaluated, and the method can be applied to targeted treatment of tumors. The tumor radioimmunoassay imaging is carried out by using specific antibody or its fragment against tumor-associated antigen to diagnose radionuclide 99m Tc、 18 F、 68 Ga, etc.), and then the obtained product is injected into human body, and flows along with blood to reach tumor tissue, and combined with related antigen of tumor so as to make local radioactivity of tumor tissue concentrate to exceed that of normal tissue, then uses nuclear medicine imaging equipment to qualitatively, positionally display primary focus and whole body metastasis focus, in particular, it can find hidden focus which is difficult to make diagnosis. The alpha or beta radionuclide can release alpha rays or beta rays and the like in the decay process, has a strong ionization radiation effect, and has a strong killing effect on tumor cells or abnormal proliferation tissues and the like. Nuclide treatment refers to the use of therapeutically active radionuclides, e.g 131 I， 90 Y， 177 Lu， 225 Ac， 213 Bi and other nuclides or labeled drugs can kill pathological cells and tissues precisely in a short distance, thereby achieving the purpose of treatment.
At present, the immunotherapy for blocking the PD-1/PD-L1 pathway has very good application prospect in the aspect of treating advanced solid tumors, but also has certain limitation in the practical process. It is believed that high PD-L1 expression levels are associated with positive response rates for cancer treatment and clinical benefit from anti-PD-1/anti-PD-L1 treatment. Thus, it is important to screen patients who respond positively to this treatment method by screening them for the expression level of PD-L1 at the lesion site of the patient prior to treatment.
Accurate assessment of the PD-L1 status of tumor patients is critical to improving efficacy. In addition, the problems of recurrence and drug resistance exist in the targeting treatment process, so that the realization of individuation and precision of tumor treatment becomes the most important problem of tumor targeting diagnosis and treatment. There have been several studies showing that PET/SPECT molecular imaging targeting PD-L1 can be used for lesion imaging of tumors and for screening patients for anti-PD-1/anti-PD-L1 immunotherapeutic responses. However, due to the large molecular weight (150 kDa) of conventional monoclonal antibodies, radionuclide-labeled antibodies are cleared slowly in vivo, resulting in a higher radioactive background of normal tissues, which not only significantly reduces the tumor/non-tumor ratio (i.e., imaging contrast), but also increases the likelihood of radiation damage to normal tissues.
In recent years, "nanobodies" have received a great deal of attention from researchers as opposed to monoclonal antibodies. Nanobodies were first reported by belgium scientists in the journal of nature in 1993 that there was a heavy chain-only antibody (camel antibody) with a naturally deleted light chain in camelid blood, and importantly, the VHH structure of the heavy chain antibody cloned and expressed alone has structural stability comparable to the original heavy chain antibody and binding activity to the antigen, which is the smallest unit known at present to bind to the antigen of interest. VHH is a single domain antibody, also called Nanobody (Nb) because it has a crystal diameter of about 2.5nm, a length of about 4nm, and a molecular weight of only 15 kDa. The main advantages of the nano antibody as a molecular probe are as follows: (1) The polypeptide has the characteristics of high affinity and high specificity, and compared with the polypeptide, the polypeptide has the affinity of more than 10-100 times and is close to the monoclonal antibody; (2) Very low immunogenicity and toxicity, and not as prone to adhesion as scFv; (3) Has good tissue penetrability and can be fully combined with a target organ; (4) The relative molecular mass is small, and the unbound part can be cleared through the kidney faster, so that the background interference in blood is reduced; (5) By utilizing the modern genetic engineering antibody technology, high-yield antibodies can be obtained, and the structure of the antibodies can be improved or modified, so that the molecular level image detection is facilitated.
The target nanobody can be subjected to GGGGC sequence coupling or modification such as bifunctional chelating agent (for example NOTA, DOTA, RESCA, THP) and the like, and then diagnostic radionuclide can be performed 99m Tc、 68 Ga、 18 F or therapeutic nuclides 177 Lu et al, 99m tc is the most widely used single photon nuclide, consisting of 99 Mo- 99m The Tc generator is prepared, and the SPECT examination cost is low, so that the method is easy to popularize. 68 Ga is a positron radionuclide with wider application, nuclide 68 Ga is composed of 68 Ge- 68 The Ga generator is prepared, the source is convenient, 68 ga is easy to coordinate with the compound, and the marking operation method is simpler; compared to cyclotrons, wash marked 68 The Ga radioactivity is relatively small, so that the quality control can be conveniently performed at any time; and is also provided with 68 The Ga half-life period is short, and the disease can be diagnosed in a noninvasive real-time manner, so that a whole-body metabolic image is obtained. As an ideal PET radionuclide, and 68 compared to Ga (t1/2=68 min), 18 f has a long half-life (t1/2=109 min). And is also provided with 18 F is prepared by an accelerator, is more suitable for batch preparation of a plurality of patients, and is also more suitable for developing an automatic synthesis preparation method.
90 Y and 177 lu is a beta-radionuclide and, 90 the energy of Y is higher (2280 keV), the penetrating power is stronger (12 mm), and the Y has stronger killing effect on tumor cells or abnormal proliferation tissues and the like. And 90 in contrast to the Y-phase of the process, 177 lu emits beta-rays (0.5 MeV) in a small ion range, which not only ensures the radiationEnergy is transmitted to the tumor area, and damage to surrounding normal tissue can be reduced.
Therefore, there is a need to develop more PET and SPECT probes with high specificity and high sensitivity for targeting PD-L1, which can realize rapid, noninvasive, real-time and quantitative detection of the PD-L1 expression condition of the systemic focus in a tumor patient, obtain systemic living body quantitative information which cannot be obtained by the conventional laboratory PD-L1 detection, and provide auxiliary detection means for screening, curative effect monitoring and other aspects of clinical PD-1/PD-L1 immunotherapy patients.
Disclosure of Invention
The invention aims to provide a novel PD-L1 targeted nano antibody APN09, radionuclide 99m Tc、 68 Ga、 18 F、 177 Lu) labeling the developing agent of the PD-L1 targeting nano antibody, and a preparation method and application thereof. The imaging agent has good affinity and selectivity with tumors, and the marking method is simple, convenient to operate, short in time consumption and high in marking rate, can be used for PD-L1 expression detection and curative effect monitoring of malignant tumors, and belongs to the fields of radiopharmaceuticals and nuclear medicine.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
A complementarity determining region (i.e., CDR) of a PD-L1-targeting nanobody VHH comprising the sequence:
the amino acid sequence is shown in SEQ ID NO:4, CDR1;
the amino acid sequence is shown in SEQ ID NO: CDR2 shown in fig. 5; and
the amino acid sequence is shown in SEQ ID NO: CDR3 shown in fig. 6.
A PD-L1-targeting nanobody VHH chain comprising CDR1, CDR2 and CDR3 sequences as described above. Further, it also comprises amino acid sequence fragments of the following framework regions:
FR1 with an amino acid sequence shown as SEQ ID NO. 7;
FR2 with an amino acid sequence shown as SEQ ID NO. 8;
FR3 with the amino acid sequence shown as SEQ ID NO. 9;
FR4 with the amino acid sequence shown as SEQ ID NO. 10.
According to one embodiment of the invention, the amino acid sequence of the VHH chain is as set forth in SEQ ID NO: 3.
A PD-L1-targeting nanobody having a VHH chain as described above.
A conjugate obtained by coupling the nanobody VHH chain described above with a chelating agent for GGGGC or a radionuclide.
According to one embodiment of the invention, the chelator of radionuclides is selected from NOTA, DOTA, RESCA or THP.
A radionuclide label obtained by labeling the conjugate described above with a radionuclide.
According to one embodiment of the invention, the radionuclide is selected from 99m Tc、 68 Ga、 18 F、 177 Lu。
According to one embodiment of the invention, the radionuclide label is selected from the group consisting of:
99m Tc-GC-APN09；
68 Ga-THP-APN09；
18 F-RESCA-APN09；
177 Lu-DOTA-APN09；
wherein APN09 represents the sequence as set forth in SEQ ID NO: 3;
GC represents a GGGGC polypeptide, THP, RESCA, DOTA is a bifunctional chelator well known in the art.
A pharmaceutical composition comprising a radionuclide label as described above and a pharmaceutically acceptable carrier.
Use of a complementarity determining region (i.e. CDR) of a nanobody VHH targeting PD-L1, a nanobody VHH chain, a nanobody, a conjugate, a radionuclide label, or a pharmaceutical composition as described above, in the preparation of a medicament, reagent, assay plate, or kit for diagnosing or treating a tumor that expresses a PD-L1 protein.
Through extensive and intensive research, the invention obtains an anti-PD-L1 nano antibody APN09 through a large number of screening, wherein the anti-PD-L1 nano antibody is a nano antibody which is specifically combined with human PD-L1 and is obtained through phage display screening by using a phage library constructed after immune alpaca. The term nanobody, single domain antibody (VHH), has the same meaning, is the smallest antigen binding fragment with complete function, consisting of 4 Framework Regions (FRs) and 3 Complementarity Determining Regions (CDRs) spaced apart, in general the framework regions of the nanobody synthesis library are more conserved, whereas the diversity is represented by CDR regions, and CDR regions are the main regions determining affinity and specificity, nanobody APN09 of the invention detects the following CDR regions, so the invention is essentially a nanobody found with the following CDR regions:
CDR1：FTFGRFNMKW(SEQ ID NO:4)
CDR2:SGINSSGSMTDY(SEQ ID NO:5)
CDR3:RNQWMYGTT(SEQ ID NO:6)
further, the framework region FR of the nanobody of the invention is as follows:
FR1：MDQVQLVESGGGLVQPGGSLRLSCAASG(SEQ ID NO:7)
FR2：VRQAPGKEPEWV(SEQ ID NO:8)
FR3：ADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCA(SEQ ID NO:9)
FR4：WYPQSRGTQVTVSS(SEQ ID NO:10)
by means of the technical scheme, the radionuclide-labeled PD-L1 targeted nanobody developer APN09 designed and synthesized by the invention has at least the following advantages and beneficial effects:
(1) The APN09 structure has the advantages of simple preparation process, convenient operation, short time consumption, high marking rate, stable marker and convenient further application in clinic, scientific research and drug development.
(2) The invention provides a visualization tool for detecting PD-L1 expression and monitoring curative effect of tumors. Can be used for screening PD-L1 positive tumor patients, and for treatment prognosis and efficacy monitoring.
(3) The APN09 radiolabeled probe belongs to a labeled compound of a nano antibody, has the advantages of small molecular weight, low immunogenicity, good tissue penetrating capacity and high affinity for tumor tissues, and has good application prospect.
Description of the drawings:
FIG. 1. 99m Binding affinity assay of Tc-GC-APN09 to human PD-L1 protein.
Fig. 2. 68 Ga-THP-APN09 was used for determination of radiopurity by Radio-TLC and Radio-HPLC.
Fig. 3. 68 Ga-THP-APN09 transfects A549 cell strain (A549) in human lung adenocarcinoma A549 and PD-L1 genes PD-L1 ) Tumor bearing mice 1h biodistribution profile.
Fig. 4. 68 Ga-THP-APN09 in A549 and A549 PD-L1 Tumor-bearing mice tumor model was visualized and tumor position indicated by arrow.
Fig. 5. 99m Tc-GC-APN09 in A549 and A549 PD-L1 Tumor model imaging of tumor-bearing mice (0.5 h,1h,2h and 4 h).
Fig. 6. 18 F-RESCA-APN09 in A549 and A549 PD-L1 Tumor model imaging of tumor-bearing mice (0.5 h,1h,2h and 4 h). Tumor location is indicated by arrows.
Fig. 7. 18 F-RESCA-APN09 in A549 and A549 PD-L1 Tumor model biodistribution map of tumor-bearing mice (1 h).
Detailed Description
The nanobody imaging agent of the invention, and the preparation method and application thereof will be described in further detail with reference to specific examples. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention. Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Example 1 expression and purification of Single Domain antibody APN09
(1) Plasmid Synthesis
The E.coli expression vector used in this study was pET25b. The recombinant plasmid was constructed by inserting the following nucleotide sequence (SEQ ID NO: 1) into two cleavage sites of NcoI and NotI by a conventional method. At the C-terminal of the sequence, a LPETGGHHHHHH sequence was added, wherein the LPETG sequence was used for the enzyme-linked reaction and the hhhhhhhhh tag was used for nickel column protein purification. The nucleotide sequence (SEQ ID NO: 1) is as follows:
CCATGGATCAGGTTCAGCTTGTTGAATCAGGCGGCGGCCTTGTTCAGCCTGGCGGCTCACTTCGCCTTTCATGTGCTGCTTCAGGCTTTACATTTGGCCGCTTTAACATGAAATGGGTTCGCCAGGCTCCTGGCAAAGAACCTGAATGGGTTTCAGGCATTAACTCGTCTGGTTCCATGACCGATTATGCTGATTCAGTTAAAGGCCGCTTTACAATTTCACGCGATAACGCTAAGAATACCCTTTATCTTCAGATGAACTCACTTAAACCTGAAGATACAGCTGTTTATTATTGTGCTCGCAACCAGTGGATGTATGGCACAACATGGTATCCTCAGTCACGCGGCACACAGGTTACAGTTTCCAGTGGATCGTTGCCCGAAACAGGCGGCCATCATCATCATCATCATTGAGCGGCCGC
(2) Plasmid transformation
After the single domain antibody recombinant plasmid is constructed, the full length gene sequencing is verified to be correct, and then the plasmid is transformed into an escherichia coli expression strain BL-21 (DE 3). Competent cells BL-21 (DE 3) were taken at 100. Mu.L and thawed on ice. Dissolving the lyophilized expression plasmid in ddH 2 O was used to give a concentration of 25 ng/. Mu.L. mu.L of plasmid was added to competent cells and left on ice for 30min. And placing competent cells in a 42 ℃ water bath for heat shock for 60-90 s, and rapidly placing the competent cells on ice for 2min. Then 500. Mu.L of LB liquid medium (without resistance) was added to the mixture and shaken for 45min at 37℃with a 220rpm shaker. After the time, 300. Mu.L of the medium was added to a 10cm ampicillin-resistant LB plate, the plate-coated beads were added thereto and shaken 10 times in the cross direction, and the plate-coated beads were poured out. The plates were placed in an incubator at 37℃overnight for incubation.
(3) Inducible expression purification
After the transformation is successful, selecting a monoclonal to carry out protein induction expression, wherein the method comprises the following steps:
1. small shake: picking a monoclonal colony by using a sterilizing gun head, pouring the colony into a shaking tube filled with 5mL of LB culture medium containing ampicillin resistance (100 mug/mL), and shaking at 37 ℃ and 220rpm for overnight culture;
2. large shaking: 5mL of the medium was added to 1L of LB medium containing ampicillin resistance (100. Mu.g/mL). Shaking culture is carried out for 1-2 h at 37 ℃ and 220 rpm. OD600 was measured with Nanodrop every 30min after 1h.
3. Induction: at an OD600 of about 0.6 to 0.9, IPTG inducer was added to the shaking broth to a final concentration of 0.5mmol/L. Shaking culture is carried out for 14 to 16 hours at 16 ℃ and 180 rpm.
4. And (3) collecting thalli: after the induction, the bacterial liquid is poured into a 250mL centrifugal bottle, centrifuged at 9000rpm for 20min, and the supernatant is discarded, thus obtaining bacterial precipitate.
5. Ultrasonic cracking: 50mL of lysis buffer was added to resuspend the cells, protease inhibitor (100. Mu.g/mLPMSF) was added, mixed well on a shaker and poured into a small beaker. After the beaker was fixed in an ice-water mixture at 0 ℃, the cells were crushed using an ultrasonic crusher. The ultrasonic conditions are as follows: the power is 150w, the ultrasonic wave is 6s, the ultrasonic wave is stopped for 6s, and the working time is 1h. After the ultrasonic treatment is finished, the bacterial liquid is collected in a centrifuge tube, centrifuged at 9000rpm for 20min, and the supernatant is collected for standby.
6. Protein hanging column: 1mL of nickel beads are sucked and added into a 10mL of affinity chromatography empty column, ethanol preservation liquid is naturally drained through the action of gravity, and then 10mL of LPBS is added to wash the nickel column so as to ensure the ethanol to be removed. After 1ml of LPBS was added to the suspension of nickel beads, the suspension was added to the sonicated supernatant and the column was spun in a 4℃chromatography cabinet shaker for 2h.
7. Purifying a nickel column: the mixture of the cleavage supernatant and the nickel beads is added to an affinity chromatography column, a rubber tube is connected to the liquid outlet of the affinity chromatography column, and the flow is pumped into a centrifuge tube at a constant flow rate by the action of a peristaltic pump. After 3 passes of this flow-through, the supernatant was discarded. After 20mL of the eluent (50 mmol/LTris HCl), 20mL of the eluent containing 20mmol/L imidazole, 10mL of the eluent containing 50mmol/L imidazole and 1mL of the eluent containing 100mmol/L imidazole are sequentially eluted, 4mL of the eluent containing 500mmol/L imidazole is added to nickel beads, uniformly mixed and stood for 5min, and liquid is collected by gravity. This procedure was repeated once and a total of 8mL of eluate was collected. After adding the collected eluate to a 3K dialysis bag, the dialysis bag was placed in a beaker containing PBS solution, a magnetic rotor was added, and dialysis was performed overnight on a magnetic stirrer in a chromatography cabinet at 4 ℃.
8. Purification by size exclusion chromatography: the dialyzed protein solution was put into a ultrafiltration tube having a molecular cut-off of 3K, concentrated by ultrafiltration at 4500rpm and 4℃and purified by Superdex 75 incrustation size exclusion chromatography after centrifugation to a final concentration of 10 mg/mL. An Agilent 1260 high performance liquid chromatography system is adopted, the mobile phase is PBS solution, and the flow rate is 0.8mL/min. The purified protein solution is stored in a refrigerator at the temperature of minus 80 ℃ for standby after the concentration is measured by Nanodrop.
The amino acid protein sequences obtained in this example are shown below:
MDQVQLVESGGGLVQPGGSLRLSCAASGFTFGRFNMKWVRQAPGKEPEWVSGINSSGSMTDYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARNQWMYGTTWYPQSRGTQVTVSSGSLPETGGHHHHHH(SEQ ID NO:2)
removing LPETGGHHHHHH sequence added at the C end of the sequence to obtain the nano antibody APN09 sequence:
MDQVQLVESGGGLVQPGGSLRLSCAASGFTFGRFNMKWVRQAPGKEPEWVSGINSSGSMTDYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARNQWMYGTTWYPQSRGTQVTVSS(SEQ ID NO:3)
mass spectrometry and HPLC characterization of the molecular structure; HPLC analysis conditions: superdex 75Increate 10/300 gel filtration/size exclusion chromatography column, flow rate 0.8mL/min; mobile phase A is 0.1mol/L PBS solution; mobile phase gradient setup: 0.0 to 30.0min of 100 percent of A, and the APN09 structure has good stability, pharmacokinetic property and targeting specificity.
Example 2 68 Leaching of Ga
Leaching the germanium-gallium generator by using 4mL of 0.05mol/L HCl with a syringe at the leaching speed of 1-2 mL/min, discarding the first 1mL of HCl, collecting the rest 3mL of leaching solution, and recording the radioactivity;
example 3 preparation of precursor APN09-GC
Sequentially adding 10mg nanometer antibody APN09 (10 mg/mL), 2mg GGGGC (20 mg/mL) polypeptide and 20 μl CaCl 2 (1 mol/L) and 200 mu L of Sortase enzyme (1 mg/mL) to 1.5mL of a reaction tube, and regulating the pH to 7.5-8.0,4 ℃ by 2mol/L NaOH, and performing a rotary shaking bed reaction for 4h. The reaction solution was centrifuged at 3000rpm for 1min. Sucking the supernatant into the pretreated Ni column, blowing and mixing uniformly, standing for 5min, and receiving a sample. The HPLC system is used for purifying the sample, the mobile phase is PBS, the detection wavelength of the separation and purification column Superdex 75Increate 10/300 is 280nm, the sample injection amount is 500 mu L, and the flow rate is 0.8mL/min.
EXAMPLE 4 preparation of precursor THP-APN09
Concentrating the purified APN09-GC through a 3K ultrafiltration tube until the concentration is 6-8 mg/mL. Mal-THP solid powder was weighed into a 1.5mL EP tube according to a molar ratio of APN09-GC to Mal-THP of 1:5. Adding the concentrated APN09-GC solution, uniformly mixing and vibrating, placing in a metal bath reaction vibrator, and reacting for 1h at 30 ℃. After the reaction, the mixture was filtered through a 0.22 μm aqueous phase needle filter, and purified by an HPLC system, wherein the purification column was Superdex 75 Increate 10/300, the detection wavelength was 280nm, the sample injection amount was 500. Mu.L, and the flow rate was 0.8mL/min, and the THP-APN09 was obtained. Samples were dispensed at 200 μg and stored in a-80℃refrigerator.
EXAMPLE 5 preparation of precursor RESCA-APN09
And weighing Mal-RESCA solid powder into a 1.5mL EP tube according to the mol ratio of APN09-GC to Mal-RESCA of 1:10, sucking the concentrated APN09-GC solution by a pipette, adding the concentrated APN09-GC solution into a reaction tube, vibrating and mixing uniformly, and placing the mixture in a multifunctional constant-temperature mixing metal bath for reaction for 1-2 h at 37 ℃. After the reaction was completed, the reaction solution was filtered through a 0.22 μm aqueous phase pin filter, and purified by an HPLC system to obtain a precursor. The purification chromatographic column is Superdex 75Increatse 10/300, the detection wavelength is 210nm and 280nm, the sample injection amount is 500 mu L, and the flow rate is 0.8mL/min. The molecular weight of APN09-RESCA was detected using MALDI-TOF. The precursor samples were dispensed at 200. Mu.g and stored in a-80℃refrigerator.
The precursor DOTA/NOTA-aPN09 can also be prepared according to the method of example 4.
Example 6 68 Preparation of Ga-THP-APN09
Taking out split-packed precursor THP-APN09 (200 mug) with concentration of 2-3 mg/mL from a refrigerator at-80 ℃, adding 300 mug of 2.0mol/L sodium acetate solution after rapid dissolution, and adding 1mL 68 Fresh rinsed in Ga (259-740 MBq) step (1) 68 Ga, pH=6.0-6.5, mixing, and reacting at normal temperature (20-30 deg.c) for 10min. Adding 0.22 μm microporous membrane, adding 2.0mL physiological saline into the system, filtering the product into sterile vacuum bottle, and standing to obtain radioactivity of the product 68 Ga-THP-APN09。
Example 7 quality control
Tlc: mu.L of saturated EDTA-2Na solution was pipetted into a 0.5mLEP tube, 1. Mu.L of the reaction solution was added, mixed well, spotted on iTLC-SG strips, developed with physiological saline and subjected to scanning analysis on Radio-TLC. To be used for 68 1. Mu.L of Ga ion solution was mixed with 20. Mu.L of saturated EDTA-2Na solution and spotted on iTLC-SG strips as a control. The results are shown in FIG. 1. A. The radiopurity of the radiolabeled probe was determined by HPLC, HPLC analysis conditions: mobile phase PBS, separation and purification column Superdex 75Increatase 10/300, sample injection amount 37KBq, flow rate 0.8mL/min, and sample injection time 30min. 68 GaCl 3 Solution 37KBq served as control. The results are shown in FIG. 1. B.
Example 8 99m Preparation of Tc-GC-APN09
Na of 370MBq 99m TcO 4 Adding into a sodium glucoheptonate kit (Beijing Shi Hong pharmaceutical development center), shaking to dissolve the kit, reacting at room temperature for 5min, adding 100 μg GC-APN09, 100 μg 0.2mol/L succinic acid buffer (pH=4.7) and 10 μl 10mol/L EDTA-2Na, and reacting at 37deg.C in water bath for 1 hr. The radiolabeled single domain antibody solution was purified on a NAP-5 gel exclusion column and filtered through a disposable 0.22 μm filter. 99m The radiochemical purity of Tc-GC-APN09 was measured by an on-line thin layer chromatography-silica gel method, developed with physiological saline, and then detected by a radiation TLC imaging scanner.
Example 9 177 Preparation of Lu-DOTA-APN09
Radioactivity (radioactivity) 177 Lu-marked PD-L1 targeted nano antibody imaging agent 177 The preparation method of Lu-DOTA-APN09 comprises the following steps:
to the labeling precursor, 100. Mu.L of NaAc/HAc (pH=5.5) solution, 370MBq, was added sequentially 177 LuCl 3 Reacting the solution at 37deg.C for 30min to obtain 177 The radiochemical purity of Lu-DOTA/NOTA-aPN09 is more than 80 percent. And (3) separating and purifying by using PD-10, adding 1-2 mL of physiological saline into the purified product, uniformly mixing, and filtering to a sterile vacuum bottle by using a sterile needle filter with the size of 0.22 mu m to obtain the product.
Example 10 18 Preparation of F-RESCA-APN09
Taking 100 mu L of physiological saline 18 F leacheate (about 1110 MBq) was added 4. Mu.L 2mmol/L AlCl 3 The solution is reacted for 5min, 110 mu L of NaAc/HAc (pH=4.1) solution and 100 mu L of RESCA-APN09 solution are added, the pH of the system is=4.6-4.8, and the reaction is carried out for 12min at normal temperature. Purifying with a molecular exclusion column PD-10 pretreated by PBS solution, adding 1-2 mL of physiological saline into the purified product, uniformly mixing, and filtering to a sterile vacuum bottle by a sterile needle filter of 0.22 mu m to obtain the product.
Example 11 68 Biodistribution of Ga-THP-APN09 in mouse tumor model
Nude mice were inoculated with A549 cells subcutaneously on the left side of the anterior axilla and inoculated with A549 cells on the right side PD-L1 Cells, tumor growth to about 100mm after 6-8 days 3 (n=4). Intravenous injection of 0.2mL (7.4 MBq) into the tail of a mouse 68 Ga-THP-APN09, mice were sacrificed at 1h, blood, brain, heart, liver, spleen, lung, kidney, stomach, intestine, pancreas, meat, tumor were collected, radioactivity was counted by gamma counter after weighing, and intake (% ID/g) of each organ and tissue was calculated, and experimental results were expressed as mean+ -SD. Competing for binding to the blocking group, a total of 50nmol nanobodies were injected. Injection of 68 The radioactive uptake of each organ at different time points after Ga-THP-APN09 is shown in figure 2, 68 Ga-THP-APN09 is mainly metabolized by kidneys, the uptake in positive tumors is higher, the uptake in negative tumors is low, the uptake in competitive binding blocking group imaging positive tumors is obviously reduced, and the targeting of the probe to PD-L1 is proved.
Example 12 68 Ga-THP-APN09 mouse tumor model imaging
Nude mice were inoculated with A549 cells subcutaneously on the left side of the anterior axilla and inoculated with A549 cells on the right side PD-L1 Cells, tumor growth to about 100mm after 6-8 days 3 (n=4). Intravenous injection of 0.2mL (7.4 MBq) into the tail of a mouse 68 Ga-THP-APN09, micro-PET scanning imaging was performed after 1h. Image reconstruction was performed on the whole-body decay corrected coronal region of interest (ROI) obtained from the Micro-PET scan. The results are shown in FIG. 3, where the positive tumor tissue of the mice had high uptake, the negative tumor tissue had low uptake, and the competitive binding blocking group had low uptake. And the probe is fast in metabolism and high in target cost, so that the marker has good specificity and affinity, and is a tumor imaging agent with potential.
Example 13 99m Binding affinity assay for Tc-GC-APN 09.
According to the radioactivity 99m Tc-marked PD-L1 targeted imaging agent 99m The preparation was carried out by the method of Tc-GC-APN 09. 99m Quantitative evaluation of Tc-GC-APN09 affinity for PD-L1 protein was performed on 96-well high adsorption removable ELISA plates coated with 0.2. Mu.g/well PD-L1. In the saturation binding assay, the specific activity of the marker is adjusted to 12mci/mg, and thenDilution by excess ratio, gradient 99m Tc-GC-APN09 (0.8 to 355 nmol/L) was added to the plate 100uL per well and incubated for 1h at 37 ℃. There were 4 replicates per concentration group and 1 standard was left. After 1h, the 96-well plates used in the saturation binding experiments were washed 3 times with PBST, and the wells of the detachable plates were collected and counted by gamma counter. Based on the measured radioactivity count/radioactivity count of standard x addition 99m Tc-GC-APN09 concentration to obtain the combined compound 99m Tc-GC-APN 09-hPD-L1). To add into 99m Tc-GC-APN09 concentration is the abscissa, combined compound [ ] 99m Tc-GC-APN 09-hPD-L1) concentration as ordinate, equilibrium dissociation constants (KD) were analyzed using Prism V.7.0. 99m The binding affinity assay results for Tc-GC-APN09 are shown in FIG. 4.
Example 14 99m Tc-GC-APN09 mouse tumor model imaging
Nude mice were inoculated with A549 cells subcutaneously on the left side of the anterior axilla and inoculated with A549 cells on the right side PD L1 cells, tumor growth to 100mm after 6-8 days 3 (n=3). Injection of tracers 99m After Tc-GC-APN09, at the time points of 0.5h,1h,2h and 4h, the mice are anesthetized by medical oxygen containing 2% isoflurane, and after anesthesia is completed, the mice are placed on an acquisition bed for imaging. The raw SPECT data is reconstructed in the whole-body region, and then SPECT and CT images are fused by nucleoine v 2.01 (Mediso inc.) software to give a maximum signal intensity projection (MIP) of the whole-body imaging through the back view. 99m The binding specificity of Tc-GC-APN09 in vivo was determined by A549 which was simultaneously positive for PD-L1 PD-L1 And negative A549 tumors are verified by imaging experiments. 99m The results of Tc-GC-APN09 are shown in FIG. 5, in which the positive tumor tissue of the mice had high uptake and the negative tumor tissue had low uptake. And the renal uptake decreased significantly over time, with better tumor/kidney ratio.
Example 15 18 F-RESCA-APN09 mouse tumor model imaging
According to the radioactivity 18 F-labeled PD-L1 targeted imaging agent 18 F-RESCA-APN 09.
Nude mice were inoculated with A549 fines subcutaneously on the left side of the forelimb axillaThe right side of the cell is inoculated with A549 PD-L1 Cells, tumor growth to 100mm after 6-8 days 3 (n=3). Intravenous injection of 0.2mL (7.4 MBq) into the tail of a mouse 18 F-RESCA-APN09 was subjected to Micro-PET scanning imaging after 1h. Image reconstruction was performed on the whole-body decay corrected coronal region of interest (ROI) obtained from the Micro-PET scan. The results are shown in FIG. 6, where the positive tumor tissue of the mice had high uptake, the negative tumor tissue had low uptake, and the competitive binding blocking group positive tumor had low uptake.
Example 16 18 Biodistribution of F-RESCA-APN09 mouse tumor model
According to the radioactivity 18 F-labeled PD-L1 targeted imaging agent 18 F-RESCA-APN 09.
Nude mice were inoculated with A549 cells subcutaneously on the left side of the anterior axilla and inoculated with A549 cells on the right side PD-L1 Cells, tumor growth to 100mm after 6-8 days 3 (n=4). Intravenous injection of 0.2mL (0.74 MBq) into the tail of a mouse 18 F-RESCA-APN09, mice were sacrificed at 1h, blood, brain, heart, liver, spleen, lung, kidney, stomach, intestine, bone, meat, tumor were collected, radioactivity was counted by a gamma counter after weighing, and the uptake (% ID/g) of each organ and tissue was calculated, and the experimental results were expressed as mean.+ -. SD. Competing for binding to the blocking group, a total of 50nmol nanobodies were injected. Injection of 18 The radioactive uptake of each organ at various time points after F-RESCA-APN09 is shown in FIG. 7, 18 F-RESCA-APN09 is metabolized mainly by the kidneys and gall bladder, with high uptake in positive tumors, low uptake in negative tumors, and low uptake in competitive binding blocking groups imaging positive tumors. The imaging agent has good in-vivo and in-vitro stability and good imaging effect, has good affinity and specificity to PD-L1, and can be used for realizing in-vivo monitoring on PD-L1 expression level.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1.A complementarity determining region (i.e., CDR) of a PD-L1-targeting nanobody VHH chain comprising the sequence:
the amino acid sequence is shown in SEQ ID NO:4, CDR1;
the amino acid sequence is shown in SEQ ID NO: CDR2 shown in fig. 5; and
the amino acid sequence is shown in SEQ ID NO: CDR3 shown in fig. 6.
2. A PD-L1-targeting nanobody VHH chain comprising CDR1, CDR2 and CDR3 sequences as described above. Further, it also comprises amino acid sequence fragments of the following framework regions:
FR1 with an amino acid sequence shown as SEQ ID NO. 7;
FR2 with an amino acid sequence shown as SEQ ID NO. 8;
FR3 with the amino acid sequence shown as SEQ ID NO. 9;
FR4 with the amino acid sequence shown as SEQ ID NO. 10.
Preferably, the amino acid sequence of the VHH chain is as set forth in SEQ ID NO: 3.
3. A PD-L1-targeting nanobody having a VHH chain according to claim 2.
4. A conjugate derived from the nanobody VHH chain of claim 2 coupled to a chelating agent for GGGGC or a radionuclide.
Preferably, the chelator of radionuclides is selected from NOTA, DOTA, RESCA or THP.
5. A radionuclide label derived from radionuclide labeling the conjugate of claim 4.
6. The radionuclide label according to claim 5, said radionuclide being selected from the group consisting of 99m Tc、 68 Ga、 18 F、 177 Lu。
Preferably, the radionuclide label is selected from the group consisting of:
99m Tc-GC-APN09；
68 Ga-THP-APN09；
18 F-RESCA-APN09；
177 Lu-DOTA-APN09；
wherein APN09 represents the sequence as set forth in SEQ ID NO: 3;
GC represents a GGGGC polypeptide, THP, RESCA, DOTA is a bifunctional chelator well known in the art.
7. A pharmaceutical composition comprising the radionuclide label of claim 5 and a pharmaceutically acceptable carrier.
8. Use of the complementarity determining region (i.e. CDR) of the nanobody VHH targeting PD-L1 of claim 1, the nanobody VHH chain of claim 2, the nanobody of claim 3, the conjugate of claim 4, the radionuclide label of claim 5, or the pharmaceutical composition of claim 7 for the preparation of a medicament, reagent, assay plate, or kit for diagnosing or treating a tumor that expresses PD-L1 protein.
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