CN111388686A - Nuclide-labeled PD-1 targeted monoclonal antibody and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of nuclear medicine, in particular to a nuclide-labeled PD-1 targeted monoclonal antibody and a preparation method and application thereof. The invention is carried out by mediating PD-1 monoclonal antibody with N-bromosuccinimide or coupling with bifunctional chelating agent deferoxamine124I、64Cu or89And labeling the Zr radionuclide to obtain the nuclide-labeled PD-1 monoclonal antibody. The labeled monoclonal antibody has high labeling rate, high purity and high in vitro stabilityThe composition has the advantages of high affinity, specificity and functional activity to PD-1, good imaging effect, high uptake of PD-1 positive tumor and low liver nonspecific uptake, is beneficial to detection of liver focus, has low whole body background, has good tumor to non-tumor ratio, can be used for diagnosis and treatment process of PD-1 high expression tumor, and realizes individual and precise treatment of targeted tumor medicament.
Description
Technical Field
The invention relates to the technical field of nuclear medicine, in particular to a nuclide-labeled PD-1 targeted monoclonal antibody and a preparation method and application thereof.
Background
The anti-PD-1 or PD-L monoclonal antibody has far-beyond-expected therapeutic effects in patients with various refractory and recurrent tumors in many clinical trials, and therefore, the U.S. FDA rapidly approves the anti-PD-1 monoclonal antibodies pembrolizumab, nivolumab, and the anti-PD-L1 monoclonal antibodies durvalumab, atezolizumab and avelumab, which have become the standard treatments for various tumors.
The research shows that not all tumor patients have curative effect response to the monoclonal antibodies of PD-1 and PD-L channels, the total objective remission rate of the anti-PD-1/PD-L monoclonal antibodies in tumor immunotherapy is lower, the patients with higher expression levels of PD-1 and PD-L I in a tumor microenvironment are more likely to benefit clinically, however, clinical tests show that even PD-L positive patients have no obvious clinical benefit, and the biological behaviors and regulation mechanisms of immune regulation check points are still unclear, therefore, the expression level of PD-L in the tumor microenvironment cannot be used as an ideal biological index for screening positive patients, the research on the biological prediction indexes capable of predicting the safety and the effectiveness of tumor immunotherapy monoclonal antibodies is relatively slow, while the effective biological prediction indexes have important significance for individual therapy of tumor patients, so far, the immunohistochemical method for detecting the expression levels of PD-1 and PD-L is a unique method for detecting the expression levels of PD-1 and PD-L, and the method for detecting PD-1 and PD-4934 tumor metastasis of tumor patients (which is not suitable for detecting tumor patients) by a biopsy method for detecting the tumor-1-PD-1 and PD-3 tumor-mediated immune tumor-mediated tumor metastasis-specific tumor metastasis detection, so that the PD-mediated tumor-receptor immune system can not be used as a method for detecting a tumor-receptor immune biopsy reagent and a tumor-receptor immune biopsy reagent for detecting a tumor-receptor immune biopsy reagent for detecting and a tumor-PD-receptor immune biopsy reagent (PD-receptor immune biopsy reagent, a tumor-PD-receptor immune biopsy reagent for detecting a tumor-PD-receptor immune biopsy reagent for detecting method for detecting a tumor-PD-receptor immune biopsy reagent for detecting a tumor-PD-.
The monoclonal antibody is only combined with a certain epitope (namely an antigenic determinant) on an antigenic molecule, and can be used as a probe to detect antigenic substances and analyze the relationship between the structure and the function of the antigenic substances on different levels of molecules, cells and organs and clarify the action mechanism of the antigenic substances by utilizing the characteristic that a nuclide-labeled monoclonal antibody is used as the probe, so that the position and the distribution of corresponding biological macromolecules (proteins, nucleic acids, enzymes and the like) combined with the nuclide-labeled monoclonal antibody in vivo can be conveniently determined, and therefore, aiming at a PD-1/PD-L1 signal channel, a molecular probe which has high specificity for diagnosing the channel and evaluating the response of subsequent immune sentinel (immune-checkpoint) antibody drugs is developed and has important significance, a plurality of PD-1/PD-L1 targeted probes are reported in preclinical research at present, and clinical research has been carried out on the predicted curative effect of the PD-1/PD-L1 targeted probe, Fredenike Bensch and the like are used for the first time in 201889The Zr marked atezolizumab carries out PET/CT imaging on a patient to be treated by PD-L1 immunotherapy, and the result shows that the clinical response and SUVmax of the patient have better correlation with immunohistochemistry and RNA sequencing, which indicates that PET molecular imaging can become an important means for evaluating the PD-L1 state and predicting the clinical response of the patient, Adnectin is used by A.N.Niemeijer et al in 201818F-BMS-986192、89PET/CT imaging of 13 NCS L C patients with Zr-Nivolumab, tumor uptake in treatment-responsive patients18F-BMS-986192 and89the SUVpeak of Zr-nivolumab is higher than that of non-reaction patients, which shows that the two probes can be used for long-term and non-invasive quantitative detection of the expression level of PD-1/PD-L1 of immunotherapy patients, and the immune PET imaging is expected to become an important means for accurately predicting the treatment effect of PD-1/PD-L1.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a nuclide-labeled PD-1 targeted monoclonal antibody, and a preparation method and application thereof.
The technical scheme of the invention is as follows:
in a first aspect, the invention provides a radionuclide-labeled PD-1 monoclonal antibody, which is obtained by performing radionuclide labeling after mediating the PD-1 monoclonal antibody with N-bromosuccinimide or coupling with bifunctional chelating agent Deferoxamine (DFO); the radionuclide marked on each mg of the PD-1 monoclonal antibody is 15-60 MBq.
The radionuclide of the present invention is preferably124I、64Cu or89Zr。
124I(T1/2＝4.2d)、64Cu(T1/2＝12.7h)、89Zr (T1/2-78.4 h) is a novel solid target PET nuclide and has wide clinical application prospect. Compared with the PET positive electron nuclide commonly used in clinic at present18F (T1/2 ═ 110min) and11c (T1/2 20.4min),124I、64Cu、89zr has a relatively long half-life period, can be used for long-time imaging research, is more convenient for nuclide transportation, has low liver uptake, and is beneficial to detection of liver lesions.
The PD-1 monoclonal antibody of the invention is preferably Torpialimab.
The anti-PD-1 monoclonal antibody Toriplizumab (also called JS001) has excellent treatment effect in clinical tests of various tumors such as melanoma, nasopharyngeal carcinoma, gastric cancer, urothelial cancer and the like. The invention takes Torpialimab as an example, and uses the next generation nuclides124I、64Cu or89Zr marks Topiramate.
Hair brushIt was found that Topiralimumab was performed using N-bromosuccinimide (NBS) mediated, or coupled with a bifunctional chelating agent DFO124I、64Cu or89The Zr nuclide marker can obviously improve the marking rate, reduce the influence of the nuclide marker on the activity of the antibody, and simultaneously ensure higher yield, purity, functional activity and stability of the marked monoclonal antibody. The animal experiment result shows that the radionuclide-labeled PD-1 targeting monoclonal antibody can be obviously and highly taken in PD-1 high-expression tumors, and the nuclide-labeled PD-1 targeting monoclonal antibody can be used as a probe of a specific targeting PD-1 and is used as an imaging agent for screening tumor immunotherapy patients, monitoring the treatment process and evaluating the curative effect.
In a second aspect, the invention provides an application of the radionuclide-labeled PD-1 monoclonal antibody in preparing a PET/CT molecular diagnostic imaging agent targeting PD-1.
The invention also provides application of the radionuclide-labeled PD-1 monoclonal antibody in preparation of a PD-1-targeted tumor treatment drug.
In a third aspect, the present invention provides a PD-1 targeted tumor diagnostic or therapeutic product comprising a PD-1 monoclonal antibody labeled with said radionuclide.
The product can be a PET/CT molecular diagnosis developer or a tumor targeting drug.
In a fourth aspect, the present invention provides a method for preparing the radionuclide-labeled PD-1 monoclonal antibody, wherein the method can be divided into two parallel technical schemes according to the nuclides used.
As a first technical solution in parallel, the following steps are carried out124When marking PD-1 monoclonal antibody by I, the marking is realized by using NBS method124I, marking the adjacent para position of benzene ring hydroxyl in the molecular structure of the PD-1 monoclonal antibody. The preparation method comprises the following steps: contacting a PD-1 monoclonal antibody with124Mixing salt I, N-bromosuccinimide and reaction buffer solution to carry out labeling reaction; the PD-1 monoclonal antibody,124The dosage ratio of the salt I and the N-bromosuccinimide is (0.5-10mg): 1 × 107～9×107Bq):(6～120μg)。
Preferably, the reaction buffer solution is 0.05-0.2M PB buffer solution with the pH value of 7.2.
More preferably, the reaction buffer solution accounts for 30-75% of the total volume of the reaction system.
Preferably, the labeling reaction is carried out at 34-37 ℃ for 1-2 min, and the reaction is stopped by 8-12% of human serum albumin.
As a preferable scheme of the invention, the preparation method comprises 25-90 KBq/mu L of Na for every 0.5-1.0 m L124Adding 0.5-1.5M L, 0.1M PB buffer solution with pH of 7.2, 0.5-10mg PD-1 monoclonal antibody and 6-120 mu g N-bromosuccinimide into the solution I, reacting at 37 ℃ for 1-1.5 min, adding 0.05-0.2 ml and 10% human serum albumin into the reaction system to terminate the reaction, purifying the obtained reaction solution by a PD-10 column to obtain the product124I labeled PD-1 monoclonal antibody.
As the second parallel technical scheme, the following64Cu or89When the PD-1 monoclonal antibody is labeled by Zr, the labeling is realized by a bifunctional chelating agent DFO64Cu or89And (3) Zr marked PD-1 targeting monoclonal antibody.
The preparation method comprises the following steps:
(1) mixing a PD-1 monoclonal antibody and DFO for reaction to obtain a labeled precursor, wherein the dosage molar ratio of the PD-1 monoclonal antibody to the DFO is 1 (5-7);
(2) by using64Cu or89Zr carrying out labeling reaction on a labeling precursor, the labeling precursor and64cu or89The dosage ratio of Zr is (15-2000 mu g): (2.5 × 10)7～10×107Bq)。
Preferably, in the step (1), the pH value of the reaction system is adjusted to 8.0-8.5 by deionized 0.05-0.1M sodium bicarbonate solution, and the reaction is carried out for 4-24 h at 4-37 ℃.
In the step (2), the marking precursor is added into a sodium acetate solution of 0.05-0.15M, pH 5.0.0-5.5, and then added64Cu or89Adjusting the pH value of the Zr solution to 7.0-7.2, and incubating at 38-42 ℃ for 25-35 min.
As a preferred embodiment of the present invention, the preparation method comprises:
(1) adding DFO with 6-fold molar equivalent of the PD-1 monoclonal antibody into a PD-1 monoclonal antibody solution with the concentration of 2-5 mg/ml, and adjusting the pH value of a reaction system to 8.5 by using a deionized 0.1M sodium bicarbonate solution; reacting for 4-24 h at 4-37 ℃ to obtain a labeled precursor DFO-PD-1 monoclonal antibody;
(2) taking 15 mu g-2 mg of the marked precursor, adding 0.1-1.0 ml of 0.1M sodium acetate solution with pH of 5.5, and then adding 25-100 MBq of freshly prepared64Cu or89Adjusting the pH value of the Zr solution to 7.0, then controlling the temperature to be 40 ℃, and incubating for 30 min; separating and purifying the obtained reaction solution by a PD-10 column to obtain64Cu or89A Zr-labeled PD-1 monoclonal antibody.
In the preparation method, when the labeling rate is more than 95%, and the target product is purified by using a PD-10 column, the radiochemical purity of the target product is more than 99%.
In the separation and purification of the PD-10 column, the PD-10 column is firstly equilibrated with 0.01M PBS buffer solution with pH7.4, 5M L is added each time, the flow is drained at the flow rate of gravity, the operation is repeated for 5 times, and then the PD-1 monoclonal antibody marked with nuclide is obtained by purification with 0.01M PBS buffer solution with pH 7.4.
The invention has the beneficial effects that:
the invention provides124I/64Cu/89The Zr-labeled PD-1 targeted monoclonal antibody has higher labeling rate and radiochemical purity and better in-vitro stability, and can be used as a novel PET molecular probe;124I/64Cu/89the Zr marked monoclonal antibody has small influence on the activity of the monoclonal antibody, and the marked monoclonal antibody has high affinity, specificity and functional activity to PD-1 and can be specifically combined with the PD-1 on the surface of a tumor cell; the imaging effect is good, the PD-1 positive tumor has higher uptake, and the nonspecific uptake of the liver is lower, thus being beneficial to the detection of the liver focus; the whole body background is low, the ratio of tumor to non-tumor is good, the observation of tumor focus is facilitated, the method can be used for monitoring the PD-1 expression condition of the whole body focus in a real-time and non-invasive manner, monitoring the PD-1 expression heterogeneity of the same focus and different focuses, and observing the change of PD-1 expression in the treatment process, and is a PD-1 high expression tumor treatment process, patient screening, curative effect monitoring and drug resistanceAnd/or early warning of recurrence and metastasis, and realizing individual and accurate treatment of the targeted tumor medicament.
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FIG. 1 shows examples 1 and 2 of the present invention124I, analyzing the physicochemical property and activity of the marked Torpialimab; wherein A is124I, analyzing the molecular weight; b is124I-Torripalimab molecular weight analysis; c is Torpialimab,124I-Torpillamab binding affinity analysis with hPD-1 receptor; d is Topiramate,124I-Torpillamab was analyzed in comparison to hPD-1 receptor binding affinity EC 50; JS001 represents toriplalimab,(nat)I-JS001 represents124I labels Topiramate.
FIG. 2 shows the results of Experimental example 4 of the present invention124I, marking a PET imaging result of Topiramate in an animal body; wherein, A is the imaging result of 4h of injection; b is the imaging result of 24h of injection; c is the imaging result of 60h of injection; d is the statistical result of tumor/muscle signal ratio of the three groups of animals at three time points of 4h, 24h and 60 h;124I-JS001, Blocking and124I-hIGg respectively represent124I-Torpialimab group,124I-Torripalimab block group and124group I-hIgG 1; A. b, C the animals in each figure are sequentially from left to right124I-Torpialimab group,124I-Torripalimab block group and124group I-hIgG 1; arrows in the figure indicate tumor lesions.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Wherein the PD-1 targeting monoclonal antibody Toripalimab is purchased from Shanghai Junshi biological medicine science and technology Co. PD-10 prepacked gel columns were purchased from GE corporation (USA).
EXAMPLE 1 preparation of novel nuclide-labeled PD-1-targeting monoclonal antibody (1)
The present embodiment provides124I label Topiramate: (I)124I-Torpillamab) and a preparation method thereof.124The I-Torpialimab is prepared by NBS reaction, and the specific preparation method is as follows:
to 0.7ml of Na at 45 KBq/. mu. L124Sequentially adding 0.5ml of 0.1M PB buffer (pH7.2), 0.1M L, 10mg/M L Torpialimab monoclonal antibody solution (prepared by water) and 12 mu g N-bromosuccinimide (NBS) into the solution I, reacting at 37 ℃ for 1min, adding 10% Human Serum Albumin (HSA) to terminate the reaction, purifying the reaction solution by a PD-10 pre-gel column to obtain a target product124I-Toripalimab。
The PD-10 pre-packed gel column is subjected to column equilibration with 0.01M PBS buffer solution (pH 7.4) 5M L each time, gravity flow drying, repeating for 5 times, and purifying with 0.01M PBS buffer solution (pH 7.4) to obtain the desired product124I-Toripalimab。
EXAMPLE 2 preparation of novel nuclide-labeled PD-1-targeting monoclonal antibody (2)
The present embodiment provides64Cu-labeled Topiramate: (64Cu-Torpialimab) and a preparation method thereof.64The Cu-Torpialimab is prepared by coupling reaction of bifunctional chelating agents, and the specific preparation method comprises the following steps:
adding DFO with 6 times of molar equivalent to the Torpillamab into 1ml of Torpillamab solution with the concentration of 2mg/ml, and adjusting the pH value of the reaction system to 8.5 by using 0.1M sodium bicarbonate solution subjected to deionization treatment; reacting at 37 ℃ for 24h to obtain a labeled precursor DFO-Torpialimab. 2mg of the labeled precursor was added to 1.0ml of 0.1M sodium acetate pH5.5, followed by 74MBq of freshly prepared64Adjusting the pH value of the Cu solution to 7.0, and incubating for 30min at the temperature of 40 ℃; separating and purifying the obtained reaction solution by a PD-10 column to obtain a target product64Cu-Toripalimab。
The PD-10 pre-packed gel column needs to be filled with 0.01M PBS buffer solution with pH7.4 before useEquilibrating the column, adding 5M L each time, draining at gravity flow rate, repeating for 5 times, and purifying with 0.01M PBS buffer (pH 7.4)64Cu-Toripalimab。
EXAMPLE 3 preparation of novel nuclide-labeled PD-1-targeting monoclonal antibody (3)
The present embodiment provides89Zr labeled Topiramate (R) ((R))89Zr-Torpialimab) and a preparation method thereof.89The Zr-Torpialimab is prepared by coupling reaction of bifunctional chelating agents, and the specific preparation method is as follows:
adding DFO with 6 times of molar equivalent to the Torpillamab into 1ml of Torpillamab solution with the concentration of 2mg/ml, and adjusting the pH value of the reaction system to 8.5 by using 0.1M sodium bicarbonate solution subjected to deionization treatment; reacting at 37 ℃ for 24h to obtain a labeled precursor DFO-Torpialimab. 2mg of the labeled precursor was added to 1.0ml of 0.1M sodium acetate pH5.5, followed by 74MBq of freshly prepared89Adjusting the pH value of the Zr solution to 7.0, and incubating for 30min at the temperature of 40 ℃; separating and purifying the obtained reaction solution by a PD-10 column to obtain a target product89Zr-Toripalimab。
The PD-10 gel column is pre-packed before use by equilibrating the column with 0.01M PBS buffer pH7.4, adding 5M L each time, draining at gravity flow rate, repeating for 5 times, and purifying with 0.01M PBS buffer pH7.4 to obtain the desired product89Zr-Toripalimab。
Experimental example 1 analysis of molecular weight, labeling rate and radiochemical purity of novel nuclide-labeled PD-1-targeted monoclonal antibody
Mass spectrometry of the sample prepared in example 1124The molecular weight of I-Torolitab,124the molecular weight measurement results of I are shown in A of FIG. 1,124the results of the molecular weight measurement of I-Torolitab are shown in B of FIG. 1, and show that,124the molecular weight of I-Topiralizab is increased by 1177Da compared with that of Topiralizab, which indicates that the labeling is successful.
The assay of the samples prepared in example 1 was carried out using the Radio-T L C and Radio-HP L C methods124The specific measurement conditions of the labeling rate and radiochemical purity of I-Torpialimab are as follows:
detecting with Radio-T L C, respectively taking 2 μ L samplesFree Na of 37-74kBq (1-2. mu. Ci) radioactivity124I and after purification124Adding the I-Torpillamab into 20 mu l of saturated EDTA, uniformly mixing, performing radio-T L C analysis, dripping a 2 mu L sample at a position 1cm away from the bottom end of filter paper (Xinhua I), placing the sample in a physiological saline development system, taking out the filter paper after the sample is completely developed, drying the filter paper, and performing radio-T L C detection.
The Radio-HP L C assay was carried out by taking 2. mu. L of free Na containing 37-74kBq (1-2. mu. Ci) radioactivity, respectively124I and after purification124I-Toripalimab was diluted by adding to 50. mu. L0.01.01M PBS pH7.4 and subjected to radio-HP L C analysis under conditions of Agilent Bio SEC-3 gel filtration/size exclusion chromatography at a flow rate of 1M L/min and 0.01M PBS pH7.4 in mobile phase.
The analysis result showed that the product obtained in example 1124The labeling rate of I-Torpillamab is greater than 95%. Purifying by PD-10 pre-packed gel column,124the radiochemical purity of I-Torpilimab is greater than 99%.
EXAMPLE 2 affinity analysis of novel nuclide-labeled PD-1-Targeted monoclonal antibody
For the one prepared in example 1124The I-Torpialimab carries out affinity analysis on PD-1 by the following specific method:
torpillamab and multi-continuous-gradient concentration obtained by using direct E L ISA method124The affinity of I-Torpillamab is calculated to obtain the half-maximum effective concentration (EC50) of the two, and the two are analyzed124Affinity of I-Torpillamab to PD-1.
As shown in C and D of FIG. 1, the EC50 of Torpialimab was 0.89. + -. 0.15ng/ml,124the EC50 of I-Torpilimab is 1.03 +/-0.14 ng/m L, the two are not statistically different (P is more than 0.05), and the result shows that,124the affinity of I-Topiramate and Topiramate for PD-1 is consistent.
EXAMPLE 3 in vitro stability analysis of novel nuclide-labeled PD-1-Targeted monoclonal antibodies
For the one prepared in example 1124The in vitro stability analysis of I-Torpialimab was carried out by the following specific method:
will be provided with124I-Torripalimab at pH7.4, respectivelyThe PBS solution and 5% HSA solution were incubated at 4 deg.C for 96h, and the radiochemical purity of the solutions after incubation was calculated and analyzed for in vitro stability.
The results of the in vitro stability analysis show that,124the stability of I-Torpillamab can be kept better in 0.01M PBS solution with pH7.4 and 5% HSA solution after incubation for 96 hours at 4 ℃: at the time of 96h, the reaction kettle is,124the radiochemical purity of I-Torpillamab in 0.01M PBS solution at pH7.4 and 5% HSA solution was 95.70 + -0.05% and 96.0 + -0.20%, respectively.
EXAMPLE 4 PET imaging study of novel nuclide-labeled PD-1-targeted monoclonal antibody in animals
Humanized PD-1 was transferred into S180 sarcoma mouse model, 9S 180 sarcoma mouse models transferred into humanized PD-1 were taken, and randomly divided into 3 groups:124I-Torpialimab group,124I-Torpilimab block group,124Group I-hIgG 1.12418.5MBq by caudal vein injection of I-Torpialimab group124I-Toripalimab；124The I-Torritalimab block group is firstly injected with Torritalimab for blocking and then injected with 18.5MBq via tail vein124I-Toripalimab；124I-hIgG1 group was injected via tail vein with 18.5MBq124I-hIgG 1. Each group was anesthetized under isoflurane (2% isoflurane-30% oxygen/air) at 4h, 24h, and 60h post-injection, respectively, and imaging studies were performed on small animal Micro-PET.
The Micro-PET imaging result is shown in FIG. 2, and at 4h, the tumor site begins to be enriched124I-Torpillamab; after the injection was carried out for 24 hours,124the I-Torpillamab is obviously enriched at the tumor site, and the metabolic enrichment in the heart and the liver is reduced; at the time of 60h, the reaction kettle is,124the non-specific metabolism of the I-Torpillamab in the animal body is gradually eliminated, a small amount of radioactivity residue is visible in the heart, the intake in the liver is obviously reduced,124the I-Torpillamab is mainly enriched at a tumor part, and the signal ratio of tumor muscles can reach (3-9): 1. Comparison of124Group I-Torpialimab and124I-hIgG1 group and124the imaging result of the I-Torpialimab block group,124I-hIgG1 group and124the I-Torpialimab block group is at 4h and 24h, and the probes are mainly distributed in the heartAnd no obvious metabolic enrichment is seen in tumor parts in the liver, and after 60 hours of injection, the metabolic enrichment is reduced in the heart and the liver, but no obvious uptake is seen in the tumor. The above results show that it is possible to obtain,124the I-Torpialimab probe can realize high-sensitivity, specificity and noninvasive detection of the expression condition of the tumor PD-1.
Prepared by the invention124The I-toripilimuab probe was approved by the ethical committee of the beijing oncology hospital (2019KT67) and was filed by the chinese clinical trials registry [ world health organization clinical trials registration platform (WHO ICTRP) first-level registration agency ], approval number: ChiCTR 1900027666. Clinical studies began since 11 months 2019, and preliminary studies showed:124the I-Torpillamab probe has good safety in a human body, is mainly taken in a heart, a blood pool and a spleen, is taken in organs such as a brain, muscles and a lung, is taken in a lower degree, and is taken in each organ gradually reduced along with time, and can be used for effectively and specifically imaging PD-1 high-expression tumor focuses.
In conclusion, the novel nuclide provided by the invention124I/64Cu/89The Zr-marked PD-1 targeted monoclonal antibody has higher marking rate and radiochemical purity, higher affinity and functional activity to PD-1, better in vitro stability and good imaging effect, and the PD-1 positive tumor pair124I/64Cu/89The Zr marked PD-1 targeted monoclonal antibody has higher uptake, is beneficial to realizing the diagnosis and accurate staging of PD-1 positive tumor lesions, and is expected to become a targeted PD-1 imaging agent with good application prospect through further animal level before clinical study.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The PD-1 monoclonal antibody labeled by the radionuclide is characterized in that the PD-1 monoclonal antibody is mediated by N-bromosuccinimide or coupled with a bifunctional chelating agent deferoxamine, and then the PD-1 monoclonal antibody is labeled by the radionuclide; the radionuclide marked on each mg of the PD-1 monoclonal antibody is 15-60 MBq.
2. The radionuclide-labeled PD-1 monoclonal antibody according to claim 1, characterized in that the radionuclide is124I、64Cu or89Zr；
Preferably, the PD-1 monoclonal antibody is Torpialimab.
3. Use of the radionuclide-labeled PD-1 monoclonal antibody according to claim 1 or 2 for the preparation of a PD-1-targeted PET/CT molecular diagnostic imaging agent.
4. Use of the radionuclide-labeled PD-1 monoclonal antibody according to claim 1 or 2 for the preparation of a medicament for the treatment of tumors targeting PD-1.
5. A PD-1-targeted tumor diagnostic or therapeutic product comprising the radionuclide-labeled PD-1 monoclonal antibody according to claim 1 or 2.
6. The method for preparing the radionuclide-labeled PD-1 monoclonal antibody according to claim 1 or 2, characterized in that when employed, the antibody is124When I marks, the preparation method comprises the following steps: contacting a PD-1 monoclonal antibody with124Mixing salt I, N-bromosuccinimide and reaction buffer solution to carry out labeling reaction; the PD-1 monoclonal antibody,124The dosage ratio of the salt I and the N-bromosuccinimide is (0.5-10mg): 1 × 107～9×107Bq):(6～120μg)。
7. The method for preparing the radionuclide-labeled PD-1 monoclonal antibody according to claim 1 or 2, characterized in that when employed, the antibody is64Cu or89When Zr marks, the preparationThe method comprises the following steps:
(1) mixing a PD-1 monoclonal antibody and deferoxamine for reaction to obtain a labeled precursor, wherein the dosage molar ratio of the PD-1 monoclonal antibody to the deferoxamine is 1: (5-7);
(2) by using64Cu or89Zr carrying out a labeling reaction on the labeling precursor, the labeling precursor and64cu or89The dosage ratio of Zr is (15-2000 mu g): (2.5 × 10)7～10×107Bq)。
8. The method according to claim 6, wherein the reaction buffer is 0.05 to 0.2M PB buffer having a pH of 7.2;
preferably, the reaction buffer solution accounts for 30-75% of the total volume of the reaction system.
9. The method according to claim 6 or 8, wherein the labeling reaction is carried out at 34-37 ℃ for 1-2 min, and the reaction is terminated with 8-12% human serum albumin.
10. The preparation method according to claim 7, wherein in the step (1), the pH value of the reaction system is adjusted to 8.0-8.5 by deionized 0.05-0.1M sodium bicarbonate solution, and the reaction is carried out at 4-37 ℃ for 4-24 h;
in the step (2), the marking precursor is added into a sodium acetate solution of 0.05-0.15M, pH 5.0.0-5.5, and then added64Cu or89Adjusting the pH value of the Zr solution to 7.0-7.2, and incubating at 38-42 ℃ for 25-35 min.
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