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WO2024034759A1 - Immuno-tep utilisant des anticorps anti-cd25 marqués au 89zr - Google Patents

Immuno-tep utilisant des anticorps anti-cd25 marqués au 89zr Download PDF

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Publication number
WO2024034759A1
WO2024034759A1 PCT/KR2023/002337 KR2023002337W WO2024034759A1 WO 2024034759 A1 WO2024034759 A1 WO 2024034759A1 KR 2023002337 W KR2023002337 W KR 2023002337W WO 2024034759 A1 WO2024034759 A1 WO 2024034759A1
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Prior art keywords
antibody
tumor
present
cells
pet
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Korean (ko)
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이경한
정경호
이진희
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Sungkyunkwan University
Samsung Life Public Welfare Foundation
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Sungkyunkwan University
Samsung Life Public Welfare Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody

Definitions

  • the present invention relates to a composition for medical imaging comprising 89 Zr labeled anti-CD25 antibody as an active ingredient.
  • Positron emission tomography 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.
  • Non-patent Document 1 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.
  • 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.
  • 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.
  • antibody-drug conjugates are emerging.
  • antibody conjugates loaded with beta-emitting isotopes are raising expectations as radioimmunotherapy agents for lymphoma.
  • 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.
  • 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.
  • 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.
  • 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:
  • step (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
  • DFO-Mal deferoxamine-maleimide
  • step (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);
  • 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:
  • Another object of the present invention is to provide a kit for positron emission tomography imaging for tumor diagnosis, including the composition and instructions.
  • 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.
  • the composition can detect tumors or tumor-infiltrating regulatory T cells, but is not limited thereto.
  • the tumor may be lymphoma, but is not limited thereto.
  • 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:
  • step (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
  • DFO-Mal deferoxamine-maleimide
  • step (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 antibody may be IgG, but is not limited thereto.
  • 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
  • the present invention provides a positron emission tomography method for tumor diagnosis, comprising the following steps:
  • 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.
  • the composition is a composition of the invention, and the instructions describe the method as It may be described, but is not limited thereto.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Reg regulatory T cells
  • 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 ⁇ M 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 ⁇ M 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 ⁇ M 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 ⁇ M 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.
  • 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.
  • IL2 receptor heterodimeric interleukin 2 receptor
  • IL2RA Interleukin-2 receptor alpha chain
  • 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.
  • 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.
  • immuno-PET using an antibody labeled with gamma ray emitting 89 Zr is optimal to evaluate the efficiency of delivery to lymphoma.
  • the 'anti-CD25 antibody' or 'antibody against CD25' refers to a specific protein molecule directed against the antigenic site of CD25.
  • 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.
  • the antibody 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.
  • 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.
  • 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.
  • 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.
  • the composition can detect tumors or tumor-infiltrating regulatory T cells, but is not limited thereto.
  • 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.
  • 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.
  • the regulatory T cells 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.
  • the tumor may be lymphoma, but is not limited thereto.
  • the tumor includes cancer in the sense commonly known in the art and includes all tumor tissue or tumor cells.
  • the tumor if the tumor abundantly expresses CD25 as a cell surface antigen, The type is not particularly limited.
  • 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.
  • NDL leukemia
  • MZL marginal zone lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lymphoma
  • TL transforming lymphoma
  • PTCL peripheral T-cell lymphoma
  • 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.
  • 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.
  • 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.
  • contrast agent is required to view blood vessels, but since the 2000s, MRI has been able to view blood vessels without contrast agent.
  • a contrast enhanced method is used. Radioactive substances or vasodilators are administered during PET or SPECT examinations, but these are not called contrast agents.
  • the composition according to the present invention may include a contrast agent.
  • the composition may be for intravenous injection, but is not limited thereto.
  • 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:
  • step (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
  • DFO-Mal deferoxamine-maleimide
  • step (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.
  • a pair of thiols reduced disulfide
  • the anti-CD25 antibody and TCEP tris-carboxyethylphosphine
  • the anti-CD25 antibody and TCEP tris-carboxyethylphosphine
  • the anti-CD25 antibody and TCEP tris-carboxyethylphosphine
  • the anti-CD25 antibody and TCEP tris-carboxyethylphosphine
  • the anti-CD25 antibody and TCEP tris-carboxyethylphosphine
  • 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.
  • the reaction in step (b) may be preferably carried out in the presence of EDTA.
  • the reaction in step (b) may be performed at a temperature of 15 to 25° C., but is not limited thereto.
  • the reaction in step (b) may be preferably performed at a temperature of 21 to 23°C.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DFO-Mal conjugated anti-CD25 antibody is mixed to perform a chelation reaction with 89 Zr.
  • the antibody may be IgG, but is not limited thereto.
  • 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
  • conjugates 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:
  • PET positron emission tomography
  • CT Computed Tomography
  • MRI Magnetic Resonance Imaging
  • 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.
  • the composition is a composition of the invention, and the instructions describe the method as It may be described, but is not limited thereto.
  • 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.
  • 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.
  • the present invention provides detection of tumor or/and tumor infiltrating regulatory T cells (regulatory T cells, Tregs);
  • 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.
  • 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.
  • 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.
  • '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.
  • FIG. 2a 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.
  • 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.
  • the method for producing the 89 Zr labeled anti-CD25 antibody according to the present invention is described in detail.
  • anti-CD25 antibodies derived from humans or mice InVivoMAb anti-human CD25 (IL-2R ⁇ ; Catalog# BE0014) or InVivoMAb anti-mouse CD25 (IL-2R ⁇ ; Catalog# BE0012) from BioXCell (West Riverside, 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.
  • 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 ⁇ L 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.
  • DFO-Mal deferoxamine-maleimide
  • 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.
  • 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 ⁇ g 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.
  • CD25 Ab-deferoxamine maleimide also referred to as DFO-conjugated CD25 IgG
  • 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.
  • 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.
  • mM EDTA ethylene diamine tetraacetic acid
  • lymphoma cells with the highest CD25 expression level were selected.
  • the expression levels of the CD25 marker were examined in human lymphoma cell lines H9, Jurkat, and SUDHL1 and mouse lymphoma cell line EL4.
  • the cancer cells were lysed, the proteins were separated and quantified, and 15 ⁇ g 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.
  • 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.
  • FACS analysis fluorescence-activated cell sorting analysis
  • 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.
  • FACS buffer 500 mL of FACS buffer was added again, and flow cytometry was performed with FACSCalibur (BE Biosciences) using CellQuest software.
  • 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.
  • binding was inhibited with a large amount of cold anti-CD25 antibody (binding inhibition experiment) and binding specificity was quantified as % blocking.
  • the EL4 cell line was used as a comparison group.
  • 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.
  • 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.
  • the high 89 Zr-CD25 IgG binding to CD25(+) Treg cells is inhibited by 72.1% in the presence of 0.5 ⁇ M of cold unlabeled antibody, so the 89 Zr-CD25 IgG according to the present invention has excellent binding ability.
  • Example 5 In vivo mouse lymphoma model 89 Analysis of body distribution of Zr-CD25 IgG
  • 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.
  • the 89 Zr-CD25 IgG antibody of the present invention has excellent tumor-specific targeting and binding effects even in vivo.
  • 89 PET image analysis was performed to confirm the imaging (imaging, contrast) effect of Zr-CD25 IgG.
  • 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.
  • 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.
  • ROIs regions-of-interest
  • micro-PET/CT imaging results showed high 89 Zr-CD25 IgG binding (uptake) with excellent contrast in the tumor area.
  • 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). .
  • 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.
  • 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 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.
  • lymphoma particularly refractory and/or T-cell lymphoma
  • diagnosis of lymphoma 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.

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Abstract

La présente invention concerne : un agent de contraste d'un procédé d'imagerie basée sur une TEP, l'agent comprenant des anticorps anti-CD25 marqués au 89Zr ; et un procédé d'imagerie tumorale ou un procédé d'imagerie de cellules T régulatrices à infiltration tumorale l'utilisant. Les anticorps anti-CD25 marqués au 89Zr uniques selon la présente invention présentent un taux d'absorption très élevé, plus précisément dans un tissu dans lequel des antigènes CD25 sont présents, et en particulier dans des parties dans lesquelles des lymphomes ou des cellules T régulatrices sont présents. Par conséquent, ils peuvent être efficacement utilisés en tant qu'agent d'imagerie non invasive efficace pouvant identifier, diagnostiquer, imager et surveiller des lymphomes et/ou des cellules T régulatrices.
PCT/KR2023/002337 2022-08-11 2023-02-17 Immuno-tep utilisant des anticorps anti-cd25 marqués au 89zr Ceased WO2024034759A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
WO2004110390A2 (fr) * 2003-06-17 2004-12-23 Immunomedics, Inc. Immunoconjugues anti-cd74 et procedes associes
WO2015153772A2 (fr) * 2014-04-01 2015-10-08 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Complexe zirconium 89-oxine utilisé en tant qu'agent de marquage de cellule pour tomographie par émission de positrons
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WO2021113450A2 (fr) * 2019-12-05 2021-06-10 Imaginab, Inc. Procédés d'imagerie utilisant de multiples agents d'imagerie
KR20220093103A (ko) * 2019-09-13 2022-07-05 어드메어 테라퓨틱스 소사이어티 항종양용해 바이러스 항원 항체 및 이의 사용 방법

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WO2004110390A2 (fr) * 2003-06-17 2004-12-23 Immunomedics, Inc. Immunoconjugues anti-cd74 et procedes associes
WO2015153772A2 (fr) * 2014-04-01 2015-10-08 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Complexe zirconium 89-oxine utilisé en tant qu'agent de marquage de cellule pour tomographie par émission de positrons
US10905784B2 (en) * 2017-02-10 2021-02-02 Regeneron Pharmaceuticals, Inc. Radiolabeled anti-LAG3 antibodies for immuno-PET imaging
KR20220093103A (ko) * 2019-09-13 2022-07-05 어드메어 테라퓨틱스 소사이어티 항종양용해 바이러스 항원 항체 및 이의 사용 방법
WO2021113450A2 (fr) * 2019-12-05 2021-06-10 Imaginab, Inc. Procédés d'imagerie utilisant de multiples agents d'imagerie

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