WO2024034759A1 - Immuno-pet using 89zr-labeled anti-cd25 antibodies - Google Patents

Abstract

The present invention relates to: an imaging agent of a PET-based imaging method, the agent comprising 89Zr-labeled anti-CD25 antibodies; and a tumor imaging method or tumor-infiltrating regulatory T cell imaging method using same. The unique 89Zr-labeled anti-CD25 antibodies according to the present invention exhibit a very high uptake rate, specifically in tissue in which CD25 antigens are present, and especially in portions in which lymphomas or regulatory T cells are present, and therefore can be effectively used as an effective non-invasive imaging agent capable of identifying, diagnosing, imaging, and monitoring lymphomas and/or regulatory T cells.

Description

IMMUNO-PET using 89ZR labeled anti-CD25 antibody

The present invention relates to a composition for medical imaging comprising ⁸⁹ Zr labeled anti-CD25 antibody as an active ingredient.

This application claims priority based on Korean Patent Application No. 10-2022-0100554 filed on August 11, 2022, and all contents disclosed in the specification and drawings of the application are incorporated in this application.

Positron emission tomography (PET) uses radioactive pharmaceuticals that emit positrons to image biological changes in the human body caused by disease, and can provide accurate information for early diagnosis of disease and decision-making on treatment methods.

The radiopharmaceutical for cancer diagnosis most commonly used in clinical practice is [ ¹⁸ F]FDG. ¹⁸ F-FDG is a glucose analogue with a radioactive isotope that enters the cell through the glucose transporter (Glut) and is phosphorylated. It is no longer metabolized and stays in the cell, emitting positrons. Cancer cells have an increased number of glucose transporters on the cell surface compared to normal cells, so more ¹⁸ F-FDG flows into cancer cells. Therefore, while existing radiological tests image structures within the human body to find and diagnose anatomical lesions, ¹⁸ F-FDG PET/CT can image biochemical changes before anatomical changes.

Lymphoma is the most common malignant blood cancer that occurs in lymphoid tissue. It is broadly classified into non-Hodgkin's lymphoma and Hodgkin's lymphoma, of which non-Hodgkin's lymphoma, which accounts for the majority, can be classified into B-cell lymphoma and T-cell lymphoma. In the treatment of lymphoma, antibody-based medicines are reported to have a significantly greater effect in eliminating cancer cells through immune responses. For example, in B-cell lymphoma, a monoclonal antibody called rituximab is known to significantly improve the course of lymphoma treatment.

However, the number of patients with refractory lymphoma, including T-cell lymphoma, is significantly increasing, the response to standard anti-cancer treatment is poor, recurrence is common, and antibody-based medicines that are more effective than other cancer types have not yet been developed, making it difficult to overcome the disease. New treatment strategies are urgently needed.

In order to successfully develop antibody treatments for refractory lymphoma, imaging technology is essential to evaluate whether target expression is sufficient in the tumor and monitor whether the treatment reaches the lymphoma. However, biological information cannot be provided by existing ¹⁸ F-FDG PET/CT. (Non-patent Document 1). To overcome this, immuno-PET imaging technology targeting tumor-specific cell-surface antigens is attracting attention. Although immuno-PET imaging technology has the characteristics of shortening the preclinical trial period for new drug development, selecting patients whose lymphoma cells express a sufficient amount of target antigen, and predicting the effect of antibody treatment, it still has the potential to produce new lymphoma-specific antigens. A target is needed.

Currently, clinical trials are underway for immunotherapy that finds and removes only refractory lymphoma cancer cells based on anti-CD25 antibodies. In the development of antibody treatments, immuno-PET is important to show how it moves in the body and how much is delivered to the target cancer, but immuno-PET imaging technology for developing anti-CD25 antibody treatments has not yet been developed. In addition, Treg cells (tumor-infiltrating regulatory T cells) that have infiltrated within the tumor also express CD25 and play an important role in treatment resistance through immune evasion, but there is still no technology to image them. Therefore, the development of CD25-targeted immuno-PET technology will not only allow quantitative evaluation of CD25 expression in lymphoma in the body, but will also contribute to the development of lymphoma treatment by imaging Treg cells infiltrating tumors.

Furthermore, among antibody drugs, antibody-drug conjugates are emerging. Among them, antibody conjugates loaded with beta-emitting isotopes are raising expectations as radioimmunotherapy agents for lymphoma. Currently, radioimmunotherapy uses ¹⁷⁷ Lu. Radioactive isotopes are preferred. Radiation-loaded anti-CD25 antibody radioimmunotherapy for refractory lymphoma is also in the preclinical and clinical development stages, but there is still no immuno-PET imaging technology to support this technology.

Accordingly, the present inventors developed an immuno-PET imaging technology using ^{the 89} Zr-labeled anti-CD25 antibody unique to the present invention and confirmed that it binds with high affinity to lymphoma cells and Treg cells expressing CD25, as well as to The present invention was completed by confirming the high uptake by lymphoma in the body and the excellent target specificity in which uptake was inhibited by non-radioactive anti-CD25 antibodies.

In other words, the purpose of the present invention is to image refractory lymphoma and Treg cells in the body as new treatment targets, so that it can be used to not only contribute to the development of antibody treatments, but also to select treatment targets and predict treatment effects, using mouse and human ⁸⁹ Zr-CD25 The goal is to develop and provide antibodies and immuno-PET imaging technology using them.

Specifically, the purpose of the present invention is to provide an ⁸⁹ Zr labeled anti-CD25 antibody.

Another object of the present invention is to provide a composition for positron emission tomography imaging for tumor diagnosis, comprising ⁸⁹ Zr labeled anti-CD25 antibody as an active ingredient.

Another object of the present invention is to provide a method for preparing ⁸⁹ Zr labeled anti-CD25 antibody comprising the following steps:

(a) reducing the anti-CD25 antibody by reacting it with TCEP (tris-carboxyethylphosphine);

(b) reacting the anti-CD25 antibody reduced in step (a) with deferoxamine-maleimide (DFO-Mal) to produce a DFO-Mal conjugated anti-CD25 antibody; and

(c) reacting the DFO-Mal conjugated anti-CD25 antibody produced in step (b) with ⁸⁹ Zr-oxalate to obtain ^{an 89 Zr} labeled anti-CD25 antibody.

Another object of the present invention is to detect tumor or tumor infiltrating regulatory T cells (regulatory T cells, Tregs); Alternatively, in order to provide information necessary for tumor diagnosis, a method of acquiring a PET image using the ⁸⁹ Zr-labeled anti-CD25 antibody of the present invention as a contrast agent is provided.

Another object of the present invention is to provide a positron emission tomography method for tumor diagnosis, comprising the following steps:

(i) administering ⁸⁹ Zr labeled anti-CD25 antibody to the subject; and

(ii) Obtaining a detection image or video of the object.

Another object of the present invention is to provide a kit for positron emission tomography imaging for tumor diagnosis, including the composition and instructions.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above object, the present invention provides an ⁸⁹ Zr labeled anti-CD25 antibody.

The present invention provides a composition for positron emission tomography imaging for tumor diagnosis, comprising ⁸⁹ Zr-labeled anti-CD25 antibody as an active ingredient.

In one embodiment of the present invention, the composition can detect tumors or tumor-infiltrating regulatory T cells, but is not limited thereto.

In one embodiment of the present invention, the tumor may be lymphoma, but is not limited thereto.

In one embodiment of the present invention, the composition may be for intravenous injection, but is not limited thereto.

The present invention provides a method of making ⁸⁹ Zr labeled anti-CD25 antibody comprising the following steps:

(a) reducing the anti-CD25 antibody by reacting it with TCEP (tris-carboxyethylphosphine);

(b) reacting the anti-CD25 antibody reduced in step (a) with deferoxamine-maleimide (DFO-Mal) to produce a DFO-Mal conjugated anti-CD25 antibody; and

(c) reacting the DFO-Mal conjugated anti-CD25 antibody produced in step (b) with ⁸⁹ Zr-oxalate to obtain ^{an 89 Zr} labeled anti-CD25 antibody.

In one embodiment of the present invention, the antibody may be IgG, but is not limited thereto.

In one embodiment of the present invention, the antibody may have the following characteristics, but is not limited thereto:

⁸⁹ Zr is specifically bound to the disulfide bond site of the anti-CD25 antibody hinge region; and

It is a conjugate form of ⁸⁹ Zr and anti-CD25 antibody.

The present invention provides a positron emission tomography method for tumor diagnosis, comprising the following steps:

(i) administering ⁸⁹ Zr labeled anti-CD25 antibody to the subject; and

(ii) Obtaining a detection image or video of the object.

In one embodiment of the present invention, the method may use a technology selected from the group consisting of PET (Positron Emission Tomography), PET/CT, PET/MR, and immuno-PET, but is not limited thereto. .

The present invention provides a kit for positron emission tomography imaging for tumor diagnosis, including the composition and instructions.

In one embodiment of the invention, the composition is a composition of the invention, and the instructions describe the method as It may be described, but is not limited thereto.

In addition, the present invention provides a composition comprising an ⁸⁹ Zr-labeled anti-CD25 antibody as an active ingredient for use in the diagnosis of tumors; or detection or imaging of a tumor or infiltrating regulatory T cells within a tumor.

Additionally, the present invention relates to diagnosis of tumors; Alternatively, it provides the use of a composition comprising ⁸⁹ Zr-labeled anti-CD25 antibody as an active ingredient for preparing an imaging agent or detection of tumor or intratumoral infiltrating regulatory T cells.

In addition, the present invention relates to ^{the 89} Zr labeled anti-CD25, for diagnosis of tumors; or detection or imaging of a tumor or infiltrating regulatory T cells within a tumor.

Additionally, the present invention relates to diagnosis of tumors; or for preparing an agent for detection ^or imaging of a tumor or infiltrating regulatory T cells within a tumor.

⁸⁹ A medical imaging composition for tumor diagnosis comprising a Zr-labeled anti-CD25 antibody as an active ingredient, and tumor diagnosis using the same; It relates to a method for imaging a tumor or tumor-infiltrating regulatory T cells. The unique ⁸⁹ Zr-labeled anti-CD25 antibody of the present invention is used to detect lymphoma or regulatory T cells among tissues in which the CD25 antigen is present. Since it shows a very high uptake rate specifically in the area where it exists, it can be usefully used as an effective non-invasive PET-related imaging agent that can distinguish, diagnose, image, and monitor lymphoma and/or regulatory T cells.

Figure 1 is an image schematically showing the concept of the development of the ⁸⁹ Zr-CD25 antibody of the present invention targeting regulatory T cells (Treg) in refractory lymphoma and tumor tissue and the development of immuno-PET imaging technology using the same.

Figure 2a schematically shows the process of changes in chemical bonds within the antibody that occur during the labeling process when labeling an antibody with a conventional labeling technology.

Figure 2b schematically shows the binding structure of a labeling substance and an antibody when labeling an antibody using a conventional labeling technology as shown in Figure 2a.

Figure 3a shows the results of confirming the size of the molecule using SDS PAGE after TCEP reduction and DFO-conjugation during the process of specifically labeling the hinge-region cysteine residue of the human anti-CD25 antibody with ⁸⁹ Zr in the present invention. indicates.

Figure 3b shows the results of confirming the size of the molecule using SDS PAGE after TCEP reduction and DFO-conjugation during the process of specifically labeling the hinge-region cysteine residue of the mouse anti-CD25 antibody with ⁸⁹ Zr in the present invention. indicates.

Figure 4 schematically shows the binding structure of the ⁸⁹ Zr-labeled anti-CD25 antibody unique to the present invention.

Figure 5a shows the results of analysis by autoradiography on the human ⁸⁹ Zr-CD25 antibody of the present invention.

Figure 5b shows the results of analysis by autoradiography for the mouse ⁸⁹ Zr-CD25 antibody of the present invention.

Figure 6a shows the results of measuring radioactivity for each fraction separated by size-exclusion chromatography when obtaining the human ⁸⁹ Zr-CD25 antibody of the present invention.

Figure 6b shows the results of measuring radioactivity for each fraction separated by size-exclusion chromatography when obtaining the mouse ⁸⁹ Zr-CD25 antibody of the present invention.

Figure 7a shows the results of evaluating the serum stability of the human ⁸⁹ Zr-CD25 antibody of the present invention after incubation in PBS or FBS for several days and analysis by radio-iTLC.

Figure 7b shows the results of evaluating the serum stability of the mouse ⁸⁹ Zr-CD25 antibody of the present invention after incubation in PBS or FBS for several days and analysis by radio-iTLC.

Figure 8a shows the results of western blotting confirming the expression level of CD25 antigen on the cell surface for human lymphoma cell lines H9, Jurkat, and SUDHL1 and mouse lymphoma cell line EL4.

Figure 8b shows the results of comparing the binding degree of human ⁸⁹ Zr-CD25 IgG of the present invention in SUDHL1 human T cell lymphoma cells with high CD25 expression and human H9 and Jurkat cancer cells with low CD25 expression.

Figure 8c shows the results of confirming the binding specificity of the human ⁸⁹ Zr-CD25 antibody of the present invention using SUDHL1 lymphoma cells with high CD25 expression (the blocking group shows high binding specificity inhibited by 0.5 μM of cold unlabeled antibody). confirmed).

Figure 8d shows the results confirming that in EL4 lymphoma cells with low CD25 expression, the low binding of mouse ⁸⁹ Zr-CD25 IgG was suppressed only to a small extent by a large amount of cold anti-CD25 IgG (0.5 μM of cold unlabeled antibody in the blocking group). High binding specificity inhibited by was confirmed).

Figure 9a shows the results of FACS analysis performed during the process of separating and purifying CD25(-) T cells and CD25(+) Treg cells from the thymus of a mouse using FACS.

Figure 9b shows the results of hematoxylin & eosin staining of CD25(+) Treg cells and observation with an optical microscope.

Figure 10a shows the results of comparative analysis of the binding rate and specificity of ⁸⁹ Zr-CD25 IgG of the present invention for CD25(-) T cells and CD25(+) Treg cells isolated from the thymus of mice (blocking group includes High binding specificity inhibited by 0.5 μM cold unlabeled antibody was confirmed).

Figure 10b shows the results of confirming the binding rate and specificity of ⁸⁹ Zr-CD25 IgG of the present invention for CD25(+) Treg cells isolated from the spleen of mice (in the blocking group, there was a high level of antibody inhibited by 0.5 μM of cold unlabeled antibody). Binding specificity was confirmed).

Figure 11 shows the results of confirming the body distribution and uptake rate in lymphoma 5 days after administration of ⁸⁹ Zr-CD25 IgG of the present invention in an in vivo mouse lymphoma model (blocking group was treated with 0.8 mg of cold unlabeled antibody) High binding specificity of inhibition was confirmed).

Figure 12 is an image showing the results of PET imaging taken 5 days after administration of ⁸⁹ Zr-CD25 IgG of the present invention in an in vivo mouse lymphoma model, showing high uptake of ⁸⁹ Zr-CD25 IgG by T cell lymphoma.

Figure 13 is an image showing the results of PET imaging on the 5th day of administration of ⁸⁹ Zr-CD25 IgG of the present invention, 1 hour after administration of 0.8 mg of cold unlabeled Ab, in an in vivo mouse lymphoma model (blocking group). . The contrast was found to be reduced by cold unlabeled antibody (blocking) treatment, confirming the high binding specificity of ^{the 89} Zr-CD25 IgG of the present invention.

The present invention developed ⁸⁹ Zr-labeled CD25 IgG, which selectively targets the CD25 antigen, which is expressed in large quantities on the surface of refractory lymphoma and is expected to be a target of therapeutic antibody drugs, and succeeded in developing a new immuno-PET technology using it. This is disclosed for the first time in the present invention.

The present invention provides a composition for positron emission tomography imaging for tumor diagnosis, comprising ⁸⁹ Zr labeled anti-CD25 antibody and ⁹ Zr labeled anti-CD25 antibody as active ingredients.

In the present invention, CD25 corresponds to the alpha subunit of the heterodimeric interleukin 2 receptor (IL2 receptor) (i.e., IL2RA (Interleukin-2 receptor alpha chain)) and plays an important role in the survival and proliferation of lymphoma cells. Therefore, immuno-PET technology targeting CD25 can be used to detect lymphoma foci in the body and non-invasively and quantitatively evaluate CD25 expression. In addition, it provides pharmacokinetic information to evaluate the delivery of anti-CD25 antibody-based medicines to lymphoma, which can be used to select treatment targets and predict treatment effects.

CD25 is rarely or only expressed in small amounts in normal cells, but is expressed abundantly only in activated normal regulatory T cells (Treg cells). These immune cells infiltrate the tumor and suppress the immune response of normal cytotoxic T cells. Contributes to treatment resistance through immune evasion. Therefore, immuno-PET technology targeting CD25 can be used not only for imaging lymphomas that express CD25, but also for imaging Treg cells infiltrated within tumors that interfere with treatment. In this case, ^{the 89} Zr radioisotope was optimized for immuno-PET, which evaluates the in vivo pharmacokinetics of antibodies, due to its advantages such as high in vivo stability and physical half-life of 3.3 days.

For the above reasons, ^{the 89} Zr-labeled anti-CD25 antibody and the immuno-PET imaging technology using it are used to diagnose cancer and predict the effectiveness of anti-CD25 antibody treatment through in-vivo imaging of refractory lymphoma and Treg cells, and to use the ¹⁷⁷ Lu-labeled anti-CD25 antibody. It is important for the success of radioimmunotherapy strategies. In particular, in the case of antibody drugs loaded with therapeutic beta ray emitting ¹⁷⁷ Lu as an antibody-drug conjugate, immuno-PET using an antibody labeled with gamma ray emitting ⁸⁹ Zr is optimal to evaluate the efficiency of delivery to lymphoma.

In the present invention, the 'anti-CD25 antibody' or 'antibody against CD25' refers to a specific protein molecule directed against the antigenic site of CD25. For the purposes of the present invention, the antibody refers to an antibody that specifically binds to the CD25 protein (cell surface antigen), and includes polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. For the purposes of the present invention, it may be preferable for the antibody to be a monoclonal antibody, which is a group of antibodies in which the amino acid sequences of the heavy and light chains of the antibody are substantially identical.

Generating antibodies against CD25 as described above can be easily produced using techniques well known in the art. Polyclonal antibodies can be produced by a method well known in the art, which involves injecting the CD25 protein antigen into an animal and collecting blood from the animal to obtain serum containing the antibody. These polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cows and dogs.

Monoclonal antibodies can be produced using the hybridoma method or phage antibody library technology well known in the art.

Antibodies in the present invention also include functional fragments of the antibody molecule as well as intact forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment that retains at least an antigen-binding function, and preferably, the fragment has at least 50% or 60% of the LRRC15 binding affinity of the parent antibody. Hold 70%, 80%, 90%, 95% or 100% or more. Specifically, in the present invention, the functional fragment of the antibody may preferably include a hinge region, but is not limited thereto, and may be in the form of F(ab')2, F(ab'), etc., for example. F(ab')2 is a fragment produced by hydrolyzing an antibody with pepsin, and consists of two Fabs connected by a disulfide bond at the heavy chain hinge. F(ab') is a monomeric antibody fragment in which a heavy chain hinge is added to Fab obtained by reducing the disulfur bond of the F(ab')2 fragment.

The antibody applied in the present invention is not limited thereto, but may be, for example, selected from the group consisting of IgG, IgA, IgM, IgE, and IgD, and is preferably an IgG antibody. In one embodiment of the present invention, the antibody of the present invention may be selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and preferably may be IgG1 or IgG2. In a specific embodiment of the present invention, the antibody of the present invention may most preferably be IgG2a.

The ⁸⁹ Zr (zirconium) is known as a medical radioisotope capable of emitting positrons. Its physical half-life is 78.41 hours, and upon decay, electron capture (76.6%) and positron emission (22.3%) occur spontaneously.

In one embodiment of the present invention, the composition can detect tumors or tumor-infiltrating regulatory T cells, but is not limited thereto.

In the present invention, Treg cells can move within tissues and have the characteristic of influencing resistance to existing tumor treatments through immune evasion when infiltrating into a tumor. In addition, it has been reported to suppress the immune response following the treatment of tumors, for example, lymphoma, and is characterized by abundant expression of CD25. In the present invention, the regulatory T cells (Treg) may preferably be those that abundantly express CD25 on the cell surface (CD25 positive (+) Treg cells). Since the antibody of the present invention has significantly excellent binding affinity and binding specificity for Treg cells, ⁸⁹ Zr-CD25 antibody PET imaging can also be used to identify the role of Treg cells in treatment resistance.

In one embodiment of the present invention, the tumor may be lymphoma, but is not limited thereto.

In the present invention, the tumor includes cancer in the sense commonly known in the art and includes all tumor tissue or tumor cells. In the present invention, if the tumor abundantly expresses CD25 as a cell surface antigen, The type is not particularly limited.

In the present invention, the lymphoma includes non-Hodgkin's lymphoma (NHL), primary, secondary, recurrent lymphoma, indolent lymphoma, aggressive non-Hodgkin's lymphoma (NHL), follicular lymphoma (FL), and chronic lymphocytic lymphoma. It may be leukemia (CLL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), transforming lymphoma (TL), peripheral T-cell lymphoma (PTCL), and is preferably refractory. It may be lymphoma or T-cell lymphoma, but is not limited thereto.

^{The 89} Zr-CD25 antibody of the present invention and the medical imaging method using the same are ¹⁷⁷ Lu-CD25 antibody-based radiation for the treatment of refractory lymphoma and inhibition of recurrence due to the binding affinity and binding specificity for the above-mentioned tumor and intra-tumor infiltrating Treg cells. It plays a role in promoting the development of immunotherapy technology. In addition, the establishment of ⁸⁹ Zr labeling technology for anti-CD25 antibodies can also be used in ¹⁷⁷ Lu labeling technology, contributing to the synthesis of therapeutic ¹⁷⁷ Lu-CD25 antibodies.

The composition of the present invention may contain the function of a contrast agent used in medical imaging. In general, contrast enhancement may mean that a special drug called a contrast agent is injected into the human body to make lesions or blood vessels more visible in many tests performed in radiology departments. In X-ray-based tests such as CT, cerebral angiography, and coronary angiography, contrast agent is required to view blood vessels, but since the 2000s, MRI has been able to view blood vessels without contrast agent. When viewing blood vessels with MRI without a contrast agent, a time of flight method is used, and when contrast agent is administered, a contrast enhanced method is used. Radioactive substances or vasodilators are administered during PET or SPECT examinations, but these are not called contrast agents. However, depending on the general meaning of contrast agents in the art, the composition according to the present invention may include a contrast agent.

In one embodiment of the present invention, the composition may be for intravenous injection, but is not limited thereto. As long as it is a contrast agent administration route known in the art, the specific administration route is not particularly limited.

The composition of the present invention may be used for imaging or monitoring tumors or intra-tumor infiltrating regulatory T cells, but is not limited thereto. Therefore, the present invention provides a method for diagnosing tumors, comprising ^{the 89} Zr labeled anti-CD25 antibody as an active ingredient; A composition for detecting or imaging a tumor or infiltrating regulatory T cells within a tumor is provided.

The present invention provides a method of preparing the ⁸⁹ Zr labeled anti-CD25 antibody comprising the following steps:

(a) reducing the anti-CD25 antibody by reacting it with TCEP (tris-carboxyethylphosphine);

(b) reacting the anti-CD25 antibody reduced in step (a) with deferoxamine-maleimide (DFO-Mal) to produce a DFO-Mal conjugated anti-CD25 antibody; and

(c) reacting the DFO-Mal conjugated anti-CD25 antibody produced in step (b) with ⁸⁹ Zr-oxalate to obtain ^{an 89 Zr} labeled anti-CD25 antibody.

The reaction in step (a) is a reduction reaction, whereby the disulfide bond chain of the antibody hinge region is selectively reduced and the bond is broken. As a result, but not limited thereto, a pair of thiols (reduced disulfide) is preferably generated. In step (a), the anti-CD25 antibody and TCEP (tris-carboxyethylphosphine) are 1: 70 to 130. It is characterized by mixing at a molar concentration ratio of For the purpose of the antibody label structure of the present invention, in step (a), delicate reaction conditions and control that do not break other double sulfide bonds in the antibody other than the hinge region are very important. In a preferred embodiment of the present invention, in step (a), the anti-CD25 antibody and TCEP (tris-carboxyethylphosphine) may be mixed at a molar concentration ratio of 1:100.

In one embodiment of the present invention, the reaction in step (a) may be performed at a temperature of 15 to 25° C., but is not limited thereto. In one embodiment of the present invention, the reaction in step (a) may be preferably performed at a temperature of 21 to 23°C. The time for the reaction in step (a) is not particularly limited, and a person skilled in the art can appropriately select and apply the optimal time depending on the yield of the desired reaction product, but a preferred example in the present invention is 5 to 30 minutes. It may be characterized by reacting, and more preferably, reacting for 10 to 20 minutes. In step (a), delicate reaction conditions and control that do not break other double sulfide bonds in the antibody other than the hinge region are very important.

The reaction in step (b) is preferably a chelation reaction, thereby producing a DFO-Mal conjugated anti-CD25 antibody. Although not limited thereto, the reaction in step (b) may be preferably carried out in the presence of EDTA. In one embodiment of the present invention, the reaction in step (b) may be performed at a temperature of 15 to 25° C., but is not limited thereto. In one embodiment of the present invention, the reaction in step (b) may be preferably performed at a temperature of 21 to 23°C. In step (b), the mixing ratio of each material (reduced anti-CD25 antibody and deferoxamine-maleimide (DFO-Mal) obtained in step (a)) is determined by those skilled in the art to obtain the desired reaction product. A person skilled in the art can appropriately select and apply optimal conditions depending on the yield and speed of acquisition.In addition, a person skilled in the art can appropriately select and apply the optimal time for the reaction in step (b) according to the yield of the desired reaction product. What can be done is not particularly limited, but a preferred example may be reacting for 30 to 120 minutes.

Step (c) is a step for labeling ⁸⁹ Zr as a tracer that can be used in PET (positron emission tomography) and/or medical imaging techniques that can be used in conjunction with PET, and the reaction in step (b) is preferred. It may be a chelation reaction. Through the above reaction, ^{the 89} Zr labeled anti-CD25 antibody of the present invention is finally obtained.

In one embodiment of the present invention, the reaction in step (c) may be performed at a temperature of 15 to 25° C., but is not limited thereto. In one embodiment of the present invention, the reaction in step (c) may be preferably performed at a temperature of 21 to 23°C. In step (c), the mixing ratio of each material ( ⁸⁹ Zr-oxalate and DFO-Mal conjugated anti-CD25 antibody produced in step (b)) is adjusted according to the desired reaction product yield and yield rate by those skilled in the art. A person skilled in the art can appropriately select and apply optimal conditions. In addition, the time for the reaction in step (c) is not particularly limited, and a person skilled in the art can appropriately select the optimal time depending on the yield of the desired reaction product, but a preferred example is reaction for 30 to 120 minutes. It could be.

In step (c), ⁸⁹ Zr-oxalate may be neutralized before being mixed with the DFO-Mal conjugated anti-CD25 ^antibody produced in step (b). . The neutralization (neutralization reaction) is not particularly limited in the type of material (particularly basic material) used as long as it uses an acid and base neutralization reaction known in the art, but for example, it can be mixed with sodium carbonate (Na ₂ CO ₃ ). It may be neutralized. In the neutralization reaction, a person skilled in the art can appropriately select and apply optimal conditions for reaction conditions such as the mixing ratio of each substance, reaction temperature, and reaction time, depending on the desired reaction product yield and acquisition rate. After neutralizing ⁸⁹ Zr-oxalate, DFO-Mal conjugated anti-CD25 antibody is mixed to perform a chelation reaction with ⁸⁹ Zr.

In one embodiment of the present invention, the antibody may be IgG, but is not limited thereto.

In one embodiment of the present invention, the antibody may have the following characteristics, but is not limited thereto:

⁸⁹ Zr is specifically bound to the disulfide bond site of the anti-CD25 antibody hinge region; and

It is a conjugate form of ⁸⁹ Zr and anti-CD25 antibody.

As used herein, the term 'conjugate' includes both conjugates and complexes, and the above terms may be used interchangeably in this specification.

The antibody of the present invention may preferably be characterized in that the disulfide bond in the hinge region is broken and two ⁸⁹ Zr atoms are bonded. Most preferably, the antibody of the present invention may be characterized in that one disulfide bond in the hinge region is broken and two ⁸⁹ Zr atoms are bonded to it (see Figure 4), and it has a structure unique to the present invention. The labeled antibody of the present invention is characterized by high labeling efficiency for target tissues or cells while maintaining high structural stability in vivo.

The present invention provides a positron emission tomography method for tumor diagnosis, comprising the following steps:

(i) administering ⁸⁹ Zr labeled anti-CD25 antibody to the subject; and

(ii) Obtaining a detection image or video of the object.

In the present invention, positron emission tomography (Positron Emission Tomography), abbreviated as PET, is one of the functional imaging techniques used in nuclear medicine, and images the three-dimensional distribution of the degree to which specific metabolic activity is active in the living body. In the industry, when PET is used alone, it is mostly for research purposes, and is generally performed simultaneously with CT (Computed Tomography) or MRI (Magnetic Resonance Imaging), which are anatomical diagnostic methods. It can be used as CT, PET/MRI (PET/MRI), and it is known that immuno-PET (immuno-PET) method can also be used.

In one embodiment of the present invention, the imaging may use a technology selected from the group consisting of PET (Positron Emission Tomography), PET/CT, PET/MR, and immuno-PET, but is not limited thereto. .

The present invention provides a kit for positron emission tomography imaging for tumor diagnosis, including the composition and instructions.

In one embodiment of the invention, the composition is a composition of the invention, and the instructions describe the method as It may be described, but is not limited thereto.

In addition to the above components, the “kit” of the present invention may include other components, devices, materials, etc. commonly required for positron emission tomography and/or methods for imaging such imaging. In addition, all components included in the kit can be used one or more times without limitation, there is no restriction on the order or subsequent use of each substance, and the application of each substance may be carried out simultaneously or in small steps.

The kit of the present invention may include a container in addition to the composition and the instructions. The container may serve to package the component, and may also serve to store and secure the component. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., which may be partially or entirely made of plastic, glass, paper, or foil. , wax, etc. The container may be equipped with a completely or partially removable closure that may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also provide access to the contents by needle. A stopper can be installed. The kit may include an external package, and the external package may include, but is not limited to, instructions for use of the components.

In addition, the present invention provides detection of tumor or/and tumor infiltrating regulatory T cells (regulatory T cells, Tregs); Alternatively, in order to provide information necessary for tumor diagnosis, a method of acquiring a PET image is provided using the ⁸⁹ Zr-labeled anti-CD25 antibody of the present invention as a contrast agent.

In addition, the present invention provides a composition comprising an ⁸⁹ Zr-labeled anti-CD25 antibody as an active ingredient for use in the diagnosis of tumors; or detection or imaging of a tumor or infiltrating regulatory T cells within a tumor.

Additionally, the present invention relates to diagnosis of tumors; Alternatively, it provides the use of a composition comprising a water ⁸⁹ Zr labeled anti-CD25 antibody as an active ingredient for preparing an imaging agent or detection of tumor or intratumoral infiltrating regulatory T cells.

In the present invention, 'individual' refers to an object that requires diagnosis or treatment of a disease, and more specifically, human or non-human mammals (primates), mice, rats, dogs, cats, It may be a mammal such as a horse or a cow, but is not limited thereto.

Below, preferred examples and experimental examples are presented to aid understanding of the present invention. However, the following examples and experimental examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples and experimental examples.

Example 1. ⁸⁹ Preparation of Zr-labeled anti-CD25 antibody

The process of changing the chemical bond within the antibody that occurs during the conventional labeling technology process is schematically shown in Figure 2a, and accordingly, the antibody is labeled with the structure shown in Figure 2b. In the present invention, ^{two 89} Zr atoms are specifically bound to the disulfide bond site of the hinge region of the anti-CD25 antibody in a unique manner, which is different from the conventional techniques of Figures 2a and 2b, and when imaged by immuno-PET, Very excellent images or videos can be obtained. Below, the method for producing ^{the 89} Zr labeled anti-CD25 antibody according to the present invention is described in detail.

1-1. Anti-CD25 antibody reduction and cysteine-specific deferoxamine-maleimide conjugation steps

First, anti-CD25 antibodies derived from humans or mice (InVivoMAb anti-human CD25 (IL-2Rα; Catalog# BE0014) or InVivoMAb anti-mouse CD25 (IL-2Rα; Catalog# BE0012) from BioXCell (West Lebanon, NH); Mouse monoclonal IgG2a anti- CD25 Ab (clone, 　7G7/B6), rat monoclonal IgG1 Ab (clone, 　PC-61.5.3)) position-specifically ( Site-specific deferoxamine-maleimide (deferoxamine[DFO]-maleimide) was conjugated. In this specification, the deferoxamine maleimide conjugated anti-CD25 antibody is referred to as 'DFO-conjugated CD25 IgG', or 'DFO-Mal conjugated anti-CD25 antibody'.

The anti-CD25 antibody was reduced with TCEP (tris-carboxyethylphosphine) at a molar concentration of 70 to 130 times higher than the molar concentration of the above antibody, and then reacted with deferoxamine-maleimide chelator to form a total of two ⁸⁹ Zr at the disulfide bond site in the antibody hinge region. It was possible to specifically bind the deferoxamine-maleimide atom, and the specific method is as follows.

2 mg anti-CD25 antibody was reduced by reacting with 100 mM TCEP (tris(2-carboxyethyl)phosphine, 1:100 molar ratio) at room temperature for 20 minutes. Subsequently, the reduced anti-CD25 antibody was diluted in 0.1 M sodium phosphate solution containing 150mM NaCl and 1mM EDTA (ethylene diamine tetraacetic acid). 56.4 μL of 2mM deferoxamine-maleimide (DFO-Mal) chelator was added and reacted at room temperature for 1 hour to attach to the sulfhydryl group of the cysteine residue in the hinge region of the anti-CD25 antibody. The reaction product was eluted with a PD-10 column to concentrate the DFO-conjugated CD25 antibody fraction, and DFO-conjugated CD25 IgG was obtained from the fraction. The molar ratio of deferoxamine-maleimide:antibody was 60:1.

Using the above method, it was confirmed that two ⁸⁹ Zr atoms were bound to the anti-CD25 antibody. Specifically, when similar antibodies were labeled using the same technique, the mass of the unmodified antibody in the MALDI peak measured by mass spectrometry was found to be 147786 Da. On the other hand, the mass of DFO-conjugated IgG prepared by the method according to the present invention was found to be 75443 Da, which corresponds to 2.18 deferoxamine-maleimide conjugations per antibody, so there are a total of two at the disulfide bond site in the antibody hinge region. It was verified that bonds of ⁸⁹ Zr atoms were formed.

In addition, SDS-PAGE (Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis was performed to confirm whether the DFO-conjugated anti-CD25 antibody (human or mouse antibody) prepared by the above method was in a reduced state. Specifically, 2 μg of DFO-conjugated anti-CD25 antibody (human or mouse antibody) was diluted in water and then mixed with 5x non-reducing sample buffer without dithiothreitol. Afterwards, it was boiled at 95°C for 10 minutes, electrophoresis was performed on an 8% SDS PAGE gel, and stained with 0.5% coomassie blue.

As shown in Figures 3a and 3b, the disulfide bond of human or mouse CD25 IgG antibody was completely reduced by TCEP and it was confirmed that the reduced state was maintained even after DFO conjugation and concentration.

1-2. Deferoxamine-maleimide conjugated anti-CD25 antibody (CD25 Ab-deferoxamine maleimide, also referred to as DFO-conjugated CD25 IgG) ⁸⁹ Zr labeling steps

DFO-conjugated CD25 IgG prepared in Example 1-1 was reacted with ⁸⁹ Zr-oxalate at room temperature to synthesize ⁸⁹ Zr-CD25 IgG with the structure shown in FIG. 4.

Specifically, ⁸⁹ Zr-oxalate (50 μL; Korea Atomic Energy Research Institute) was neutralized with 25 μL of 2 M Na ₂ CO ₃ . DFO-Mal conjugated CD25 IgG (human or mouse antibody) was diluted in 75 μL of 0.5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5), then incubated with ⁸⁹ Zr-oxalate for 1 hour at room temperature. The reaction was incubated and labeled. A 0.25 M sodium acetate solution containing 0.5% gentisic acid was used as an elution buffer, and the reaction product was purified by size-exclusion chromatography with a PD-10 column, and finally, the fraction in which ⁸⁹ Zr-labeled CD25 IgG elutes ( fraction) was separated and purified to obtain ⁸⁹ Zr-labeled CD25 IgG.

An autoradiography experiment was performed to confirm whether ⁸⁹ Zr labeling of the ⁸⁹ Zr labeled CD25 IgG (human or mouse antibody) obtained by the above method was successful. Specifically, for this purpose, ⁸⁹ Zr-CD25 IgG was separated using 8% native PAGE and exposed to a photosensitive plate.

As shown in Figures 5a and 5b, a radioactive IgG band was confirmed at the 170kD position expected for human or mouse ⁸⁹ Zr-CD25 IgG.

In addition, as a result of gamma counting the eluted fractions after separation and purification using PD-10 size-exclusion chromatography, the labeling efficiency of human or mouse ⁸⁹ Zr-CD25 IgG at the first radioactivity peak was 68.7%, respectively. and 67.2%, and the radiochemical purity of the finally obtained ⁸⁹ Zr-CD25 IgG was confirmed to be >98% and the specific radioactivity was confirmed to be >0.8 mCi/mg (see Figures 6a and 6b).

According to these results, ⁸⁹ Zr can be successfully conjugated to CD25 IgG according to Example 1, and the conjugation efficiency is about 70% and the purity is over 98%, making it possible to produce a remarkably excellent ⁸⁹ Zr-labeled CD25 IgG. This has been confirmed.

Example 2. ⁸⁹ Confirmation of stability of Zr-CD25 IgG and labeling in serum

To confirm the serum stability of the ⁸⁹ Zr-labeled CD25 IgG (human or mouse antibody) finally obtained in Example 1-2, radio-instant thin layer chromatography (radio-iTLC) was performed. Specifically, iTLC-SG glass microfiber chromatography paper coated with silica gel was used. In addition, ⁸⁹ Zr-labeled CD25 IgG (human or mouse antibody), respectively, was added to PBS or FBS to serve as an experimental group. Each experimental group was placed in PBS or FBS and reacted at 37°C for 1 to 7 days, and then chromatography was performed using 50 mM EDTA (ethylene diamine tetraacetic acid, pH 5.5) as a solvent. Intact ⁸⁹ Zr-CD25 IgG remains in the basal position, whereas free ⁸⁹ Zr ⁴⁺ ions and ⁸⁹ Zr-EDTA migrate along the solvent front and are separated.

As shown in Figures 7a and 7b, the stability of human or mouse ⁸⁹ Zr-CD25 IgG in each experimental group was >90% (>90%) in PBS and >80% (>80%) in FBS ( n = 2), ⁸⁹ Zr-labeled CD25 IgG was confirmed to be radioactively stable regardless of whether it was a human or mouse antibody.

Example 3. In vitro lymphoma cell model ⁸⁹ Confirmed excellent binding affinity and specificity of Zr-CD25 IgG

3-1. Confirmation of CD25 expression level by lymphoma cell type and preparation of lymphoma cell model

⁸⁹ To analyze the binding affinity and specificity of Zr-CD25 IgG, lymphoma cells with the highest CD25 expression level were selected.

Specifically, Through western blotting analysis The expression levels of the CD25 marker were examined in human lymphoma cell lines H9, Jurkat, and SUDHL1 and mouse lymphoma cell line EL4. First, the cancer cells were lysed, the proteins were separated and quantified, and 15 μg of each protein was electrophoresed using a 10% gel and transferred to a membrane, followed by rabbit-anti-CD25 primary antibody (abcam #ab231441; 1:1000 dilution). Reacted overnight at 4°C. After washing with TBST buffer three times for 10 minutes each, it was reacted with HRP-conjugated anti-rabbit secondary antibody (Cell Signaling #7074S; 1:2000 dilution) for 1 hour at room temperature. After washing with TBST buffer three times for 10 minutes each, the membrane was reacted with an enhanced chemiluminescence substrate and the film was exposed to detect and quantify the band intensities of CD25 protein.

Additionally, for quantitative analysis of the expression level of the CD25 marker on the cell surface, cancer cells were stained by attaching a FITC or phycoerythrin (PE) fluorescence signal to anti-CD25 IgG, and then FACS analysis (fluorescence-activated cell sorting analysis) was performed. First, the cells were harvested with a nonenzymatic cell-dissociation solution, washed, and incubated with phycoerythrin for 30 minutes at room temperature (RT) in a phosphate-buffered saline solution containing 5% fetal bovine serum and 0.2% bovine serum albumin. Incubation was performed with antibody. After washing with FACS buffer, 500 mL of FACS buffer was added again, and flow cytometry was performed with FACSCalibur (BE Biosciences) using CellQuest software.

As a result of the experiment, as shown in Figure 8a, it was confirmed that the expression level of CD25 was significantly high in the SUDHL1 human T lymphoma cancer cell line, and the SUDHL1 cell line was used as a representative CD25-overexpressing lymphoma cell in the following experiments.

3-2. In an in vitro SUDHL1 lymphoma cell model ⁸⁹ Confirmed excellent binding affinity and specificity of Zr-CD25 IgG

The binding affinity and specificity of ⁸⁹ Zr-CD25 IgG prepared according to Example 1 were confirmed in an in vitro SUDHL1 lymphoma cell model.

Specifically, the cells were cultured and reacted with ⁸⁹ Zr-CD25 IgG (human), the cells were washed, and the uptake rate was measured in %ID by a conventional method using a gamma counter. At this time, H9 and Jurkat cell lines were set as comparison groups.

As shown in Figure 8b, as a result of comparing the degree of binding to human ⁸⁹ Zr-CD25 IgG in SUDHL1 human T cell lymphoma cells with high CD25 expression and human H9 and Jurkat cancer cells with low CD25 expression, dozens of binding was found in SUDHL1 lymphoma cells. A fold higher binding level was confirmed (n = 6, both P <0.001).

In addition, binding was inhibited with a large amount of cold anti-CD25 antibody (binding inhibition experiment) and binding specificity was quantified as % blocking. At this time, the EL4 cell line was used as a comparison group.

As shown in Figure 8c, as a result of a binding inhibition experiment using a large amount of cold anti-CD25 antibody to measure binding specificity in SUDHL1 lymphoma cells with high CD25 expression, ⁸⁹ Zr-CD25 IgG showed an inhibition rate of more than 97%. It was shown to have binding specificity (n = 6, P <0.001), and it was confirmed that the binding of human ⁸⁹ Zr-CD25 IgG according to the present invention was almost completely inhibited. In EL4 lymphoma cells with low CD25 expression, the low binding of mouse ⁸⁹ Zr-CD25 IgG was only slightly inhibited by a large amount of cold anti-CD25 antibody (Figure 8d).

Example 4. In Treg immune cells ⁸⁹ Confirmed excellent binding affinity and specificity of Zr-CD25 IgG

The binding affinity and specificity of ⁸⁹ Zr-CD25 IgG prepared according to Example 1 to Tregs were analyzed.

First, CD25(+) Treg cells were purified and isolated from the thymus or spleen of normal mice using the Easy prep kit (Stem Cell Tech) according to the manufacturer's protocol. Specifically, CD25(-) T cells and CD25(+) Treg cells were isolated and purified from the thymus of mice using FACS (see Figure 9a), and the Treg cells were stained with hematoxylin and eosin (see Figure 9b). Additionally, the number of CD25(+) Treg cells was expanded for 2 weeks using anti-CD3 antibody and IL2. Afterwards, the degree of binding of ⁸⁹ Zr-CD25 IgG to CD25(-) T cells and CD25(+) Treg cells was analyzed in the same manner as in Example 3-2.

As shown in FIG. 10A, the binding of ⁸⁹ Zr-CD25 IgG to CD25(+) Treg cells was confirmed to be significantly higher at 391.2 ± 135.2% compared to CD25(-) T cells isolated from the mouse thymus. In particular, the high ⁸⁹ Zr-CD25 IgG binding to CD25(+) Treg cells is inhibited by 72.1% in the presence of 0.5 μM of cold unlabeled antibody, so ^{the 89} Zr-CD25 IgG according to the present invention has excellent binding ability. In addition, target specificity was confirmed to be significantly excellent (n =6, P <0.001, see Figure 9a).

In addition, as shown in Figure 10b, the same binding affinity and specificity as for CD25(+) Treg cells isolated from the spleen of mice were confirmed. Specifically, ⁸⁹ Zr-CD25 IgG formed a high level of binding to CD25(+) Treg cells, and the above results were confirmed to be inhibited by 87.0 ± 0.7% under the condition of 0.5 μM cold unlabeled antibody (n = 3). , P < 0.01, see Figure 9b).

These results suggest that ⁸⁹ Zr-CD25 IgG according to the present invention has both high binding affinity and target specificity for CD25(+) Treg cells, regardless of the origin of the Treg cells.

Example 5. In vivo mouse lymphoma model ⁸⁹ Analysis of body distribution of Zr-CD25 IgG

⁸⁹ To analyze the body distribution of Zr-CD25 IgG, an in vivo mouse lymphoma model was first prepared. Specifically, SUDHL1 human T cell lymphoma cells were cultured and 1 x 10 ⁷ SUDHL1 cells were subcutaneously injected into the shoulder area of immunodeficient balb/C nude mice. About 21 days after transplantation, ⁸⁹ Zr-CD25 IgG (human) was injected into the tail vein of mice (n = 6) in which 1 cm tumors were confirmed to have formed. After 5 days, they were sacrificed by oral dislocation, and the tumor, blood, and major organs were removed and weighed, and the radioactivity coefficient was measured with a high-energy gamma counter to determine the intake rate as % injected-dose per gram (%ID/g). ) was calculated in units.

As shown in FIG. 11, the uptake of ⁸⁹ Zr-CD25 IgG by SUDHL1 lymphoma tumor in the mouse body was confirmed to be 9.4 ± 2.3 %ID/g. This is 10 times more than that in muscle, and the uptake rate in the heart, lungs, liver, spleen, and kidneys was also found to be only about 30 to 40% of that in tumors.

In addition, when 0.8 mg of cold unlabeled antibody was first injected into the mouse model and then ⁸⁹ Zr-CD25 IgG was injected, the uptake of ⁸⁹ Zr-CD25 IgG in SUDHL1 lymphoma tumor was significantly reduced to 3.8 ± 0.8 %ID/g. appear. This is a 60% reduction compared to the previous experiment result, suggesting the high binding specificity of ⁸⁹ Zr-CD25 IgG (n = 6, P < 0.001, see Figure 11). On the other hand, there was no change in the uptake of ⁸⁹ Zr-CD25 IgG in the blood and other organs by cold unlabeled antibody.

Accordingly, it was verified that ^{the 89} Zr-CD25 IgG antibody of the present invention has excellent tumor-specific targeting and binding effects even in vivo.

Example 6. In vivo mouse lymphoma model ⁸⁹ Confirm the excellent imaging effect of Zr-CD25 IgG

⁸⁹ PET image analysis was performed to confirm the imaging (imaging, contrast) effect of Zr-CD25 IgG. First, SUDHL1 human T cell lymphoma cells were cultured and 1 x 10 ⁷ SUDHL1 cells were subcutaneously injected into the shoulder area of immunodeficient balb/C nude mice. About 21 days after transplantation, 0.2 mCi ⁸⁹ Zr-CD25 IgG (human) was injected into the tail vein of mice (n = 6) with 1 cm tumors formed, and 5 days later, small animal-specific Inveon micro PET/CT scanner (Siemens, PET/CT images were acquired using USA). At this time, some mice were pre-injected with cold antibodies at a 5:1 molar ratio 1 hr before to analyze the tumor binding (tumor uptake) specificity of ⁸⁹ Zr-CD25 IgG. PET-based tissue radioactivity was analyzed by applying regions-of-interest (ROIs) in non-attenuation corrected images, including blood pool, major organs, and tumor.

As shown in Figure 12, micro-PET/CT imaging results showed high ⁸⁹ Zr-CD25 IgG binding (uptake) with excellent contrast in the tumor area. In addition, when 0.8 mg of cold unlabeled antibody was first injected into the same mouse model and then ⁸⁹ Zr-CD25 IgG was injected, it was confirmed that the tumor's ⁸⁹ Zr-CD25 IgG uptake was significantly reduced and the contrast decreased (see Figure 13). .

According to these results, it was confirmed that ⁸⁹ Zr-CD25 IgG prepared according to the method of the present invention has stability in serum and has excellent binding ability to tumors and Tregs infiltrated within the tumor, as well as specificity for this binding ability. It has been done. In addition, it has been confirmed that excellent quality tumor-specific PET images can be obtained through these characteristics, and it can be used in a variety of ways as a composition and method for diagnosis, imaging, and confirmation of treatment effects for lymphomas, including intractable lymphomas. This has been confirmed.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

The present invention relates to an imaging agent for a PET-based imaging method comprising an ⁸⁹ Zr-labeled anti-CD25 antibody, and a method for imaging a tumor or a method for imaging tumor-infiltrating regulatory T cells using the same. . ^{The 89} Zr-labeled anti-CD25 antibody prepared by the unique method of the present invention is stably maintained in serum, and has a specific high concentration in tissues where CD25 antigen is present, tumors, especially lymphomas, or areas where a large number of regulatory T cells are present. It exhibits an uptake rate and is characterized by significantly excellent binding affinity and binding specificity. Accordingly, diagnosis of lymphoma, particularly refractory and/or T-cell lymphoma; Alternatively, it can be used for detecting, imaging, and monitoring lymphoma or infiltrating regulatory T cells within lymphoma, and is expected to be useful as a diagnostic, detection, monitoring, and imaging agent for effective non-invasive medical imaging.

Claims (13)

⁸⁹ A composition for positron emission tomography imaging for tumor diagnosis, comprising a Zr-labeled anti-CD25 antibody as an active ingredient. According to paragraph 1, The composition is characterized in that it detects a tumor or tumor-infiltrating regulatory T cells. According to paragraph 1, Composition, wherein the tumor is lymphoma. According to paragraph 1, The composition is characterized in that it is for intravenous injection. A method of producing ^{the 89} Zr labeled anti-CD25 antibody of claim 1 comprising the following steps: (a) reducing the anti-CD25 antibody by reacting it with TCEP (tris-carboxyethylphosphine); (b) reacting the anti-CD25 antibody reduced in step (a) with deferoxamine-maleimide (DFO-Mal) to produce a DFO-Mal conjugated anti-CD25 antibody; and (c) reacting the DFO-Mal conjugated anti-CD25 antibody produced in step (b) with ⁸⁹ Zr-oxalate to obtain ^{an 89 Zr} labeled anti-CD25 antibody. According to clause 5, A method, characterized in that the antibody is IgG. According to clause 5, A method wherein the antibody has the following characteristics: ⁸⁹ Zr is specifically bound to the disulfide bond site of the anti-CD25 antibody hinge region; and It is a conjugate form of ⁸⁹ Zr and anti-CD25 antibody. A positron emission tomography method for tumor diagnosis comprising the following steps: (i) administering ⁸⁹ Zr labeled anti-CD25 antibody to the subject; and (ii) Obtaining a detection image or video of the object. According to clause 8, The method, wherein the imaging uses a technology selected from the group consisting of PET (Positron Emission Tomography), PET/CT, PET/MR, and immuno-PET. ⁸⁹ A kit for positron emission tomography imaging for tumor diagnosis, comprising a composition comprising a Zr-labeled anti-CD25 antibody as an active ingredient, and instructions. According to clause 10, The composition is the composition of claim 1, and the instructions are the methods of claims 5 and 8. A kit, characterized as described. ⁸⁹ Diagnosis of a tumor of a composition comprising a Zr-labeled anti-CD25 antibody as an active ingredient; or for the detection or imaging of tumors or infiltrating regulatory T cells within tumors. Diagnosis of tumors; or use of a composition comprising ⁸⁹ Zr-labeled anti-CD25 antibody as an active ingredient for preparing an imaging agent or detection of tumor or intratumoral infiltrating regulatory T cells.

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