Radioisotope-labeled Anti-her2 antibody–chelator conjugate, pharmaceutical composition comprising same, and preparation method therefor

Abstract

The present invention provides: a radioisotope-labeled anti-HER2 antibody–chelator conjugate; a diagnostic kit for HER2-expressing cancer or a pharmaceutical composition for diagnosis or treatment of HER2-expressing cancer, both comprising the radioisotope-labeled anti-HER2 antibody–chelator conjugate; and a method for preparing a radioisotope-labeled anti-HER2 antibody–chelator conjugate, the method comprising the steps of reacting trastuzumab or pertuzumab as an anti-HER2 antibody with p-SCN-Bn-PCTA at a molar ratio of 1:10 to 1:20 or 1:5 to 1:15 to form a conjugate, and labeling the conjugate with a radioisotope.

Description

Radioisotope-labeled anti-HER2 antibody-chelator conjugate, pharmaceutical composition containing same, and method for preparing same

[0001] The present invention relates to a radioisotope-labeled anti-HER2 antibody-chelator conjugate.

[0002] The present invention also relates to a diagnostic kit for HER2-expressing cancer, or a pharmaceutical composition for diagnosis or treatment, comprising a radioisotope-labeled anti-HER2 antibody-chelator conjugate.

[0003] The present invention also relates to a method for producing a radioisotope-labeled anti-HER2 antibody-chelator conjugate, comprising the steps of reacting trastuzumab or pertuzumab as an anti-HER2 antibody and p-SCN-Bn-PCTA at a molar ratio of 1:10 to 1:20 or 1:5 to 1:15 to produce a conjugate, and the step of reacting the conjugate with a radioisotope to label it with the radioisotope.

[0004] Conventional anticancer drugs and radiation therapy attack both cancer cells and normal cells, resulting in serious side effects. Therefore, extensive research is being conducted on molecularly targeted cancer therapies, targeting specific molecules based on an understanding of the molecular biological and signaling mechanisms underlying cancer development. Furthermore, research is ongoing on theranosis technology, which enables simultaneous diagnosis and treatment, to provide personalized care by adjusting drug types and dosages based on individual patient characteristics and disease status.

[0005] Breast cancer is the most frequently diagnosed cancer in women worldwide and a leading cause of death. According to breast cancer classification, HER2-positive tumors account for 15-25% of all breast cancer cases.

[0006] Targeted therapies for HER2-expressing breast cancer include Herceptin® or Herzuma®, which contain trastuzumab as the active ingredient, or Perjeta®, which contains pertuzumab. These targeted therapies are administered in combination with chemotherapy.

[0007] In addition, recently, antibody-drug conjugates (ADCs) such as Kadcyla® (ado-trastuzumab emtansine, T-DM1) and Enhertu® (fam-trastuzumab-deruxtecan-nxki), and tyrosine kinase inhibitors (TKIs) for HER2 such as Tykerb® (lapatinib), Nerlynx® (neratinib), and Tukysa® (tucatinib) have been used as therapeutic drugs.

[0008] Although combination therapy with chemotherapy and anti-HER2 antibodies has shown clinical efficacy, most patients with HER2-expressing metastatic breast cancer develop acquired or intrinsic resistance after initial therapeutic response, limiting its effectiveness. Therefore, there is a pressing need to develop new treatments or therapeutics that can demonstrate clinically beneficial outcomes for patients refractory to current combination therapies.

[0009] Recently, antibodies labeled with radioisotopes have been developed as molecularly targeted anticancer agents, but there has been a limitation that they must undergo additional separation and purification processes before they can be used in clinical trials due to low labeling yield.

[0010] Accordingly, the present invention aims to provide a therapeutic agent that can show clinically improving effects on patients who do not respond to combination therapy using radioisotopes, and a method for manufacturing a therapeutic agent with an excellent labeling rate that can be used directly in clinical trials without additional separation and purification processes.

[0011] To solve the above problem, one aspect of the present invention provides a radioisotope-labeled anti-HER2 antibody-chelator conjugate.

[0012] Another aspect of the present invention provides a diagnostic kit for HER2-expressing cancer, or a pharmaceutical composition for diagnosis or treatment, comprising a radioisotope-labeled anti-HER2 antibody-chelator conjugate.

[0013] Another aspect of the present invention provides a method for preparing a radioisotope-labeled anti-HER2 antibody-chelator conjugate, comprising the steps of reacting trastuzumab or pertuzumab as an anti-HER2 antibody and p-SCN-Bn-PCTA at a molar ratio of 1:10 to 1:20 or 1:5 to 1:15 to produce a conjugate, and the step of reacting the conjugate with a radioisotope to label the conjugate with the radioisotope.

[0014] Another aspect of the present invention provides a method for diagnosing or treating HER2-expressing cancer, comprising administering to a subject an anti-HER2 antibody-chelator conjugate according to the present invention.

[0015] Another aspect of the present invention provides a use of an anti-HER2 antibody-chelator conjugate according to the present invention for diagnosing or treating a HER2 expressing cancer.

[0016] Another aspect of the present invention provides a use of an anti-HER2 antibody-chelator conjugate of the present invention for preparing a medicament for diagnosing or treating a HER2 expressing cancer.

[0017] In one embodiment of the present invention, in breast cancer and gastric cancer models that are resistant to Herceptin, Herzuma, or Perjeta, which are used for the treatment of HER2-expressing tumors, a radioisotope for PET imaging diagnosis is used. 64 Cu or beta-emitting radioisotope that exhibits radioimmunotherapy effects 177 By labeling Lu with the same conjugate, a conjugate of an anti-HER2 antibody and a bifunctional chelator substance (e.g., 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-trience-3,6,9,-triacetic acid (PCTA) chelator) capable of diagnosis or treatment is prepared, thereby producing a radioisotope. 64 Cu or 177 Lu was labeled with high labeling yield.

[0018] In addition, in one embodiment of the present invention, HER2 expression is noninvasively and quantitatively evaluated through PET imaging in breast cancer and gastric cancer models showing resistance to Herceptin, Perjeta or Herzuma, and beta-ray emitting radioisotope therapy 177 The radioimmunotherapy effect was confirmed by inhibiting tumor growth using a Lu-labeled anti-HER2 antibody-chelator conjugate.

[0019] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

[0020] Figure 1 is 64 Cu / 177 A schematic diagram of the Lu-PCTA-anti-HER2 antibody conjugate is shown.

[0021] Figure 2 shows a MALDI-MS analysis chromatogram of the PCTA-Herceptin antibody conjugate.

[0022] Figure 3 shows a MALDI-MS analysis chromatogram of the PCTA-Perjeta antibody conjugate.

[0023] Figure 4 shows a MALDI-MS analysis chromatogram of the PCTA-Herzuma antibody conjugate.

[0024] Fig. 5 is 64 Cu / 177 Labeling yield analysis of Lu-PCTA-Herceptin antibody conjugates is shown.

[0025] Fig. 6 is 64 Cu / 177 Labeling yield analysis of Lu-PCTA-Perjeta antibody conjugates is shown.

[0026] Figure 7 is 64 Cu / 177 Labeling yield analysis of Lu-PCTA-Herzuma antibody conjugates is shown.

[0027] Figure 8 shows HER2 expression analysis using Western blotting in breast cancer cell lines.

[0028] Figure 9 shows HER2 expression in flow cytometry using Herceptin antibody in breast cancer and gastric cancer cell lines.

[0029] Figure 10 shows HER2 expression in flow cytometry using Perjeta antibody in breast cancer and gastric cancer cell lines.

[0030] Figure 11 shows HER2 expression in flow cytometry using Herzuma antibody in breast cancer and gastric cancer cell lines.

[0031] Figure 12 shows the cytotoxicity evaluation using Herceptin antibody in breast cancer and gastric cancer cell lines.

[0032] Figure 13 shows the cytotoxicity evaluation using Perjeta antibody in breast cancer and gastric cancer cell lines.

[0033] Figure 14 shows the cytotoxicity evaluation using Herzuma antibody in breast cancer and gastric cancer cell lines.

[0034] Figure 15 shows the evaluation of the therapeutic effect of Herceptin antibody in anti-HER2 antibody resistant breast cancer (JIMT-1, Figure 8A) and gastric cancer (NCI-N87, Figure 8B) models.

[0035] Figure 16 shows the evaluation of the therapeutic effect of the Perjeta antibody in anti-HER2 antibody-resistant breast cancer (JIMT-1, Figure 9A) and gastric cancer (NCI-N87, Figure 9B) models.

[0036] Figure 17 shows the evaluation of the therapeutic effect of Herzuma antibody in anti-HER2 antibody resistant breast cancer (JIMT-1, A of Figure 10) and gastric cancer (NCI-N87, B of Figure 10) models.

[0037] Fig. 18 is 64 Cu / 177 Size Exclusion-HPLC chromatogram of Lu-PCTA-Herceptin antibody conjugate is shown.

[0038] Fig. 19 is 64 Cu / 177 Size Exclusion-HPLC chromatogram of Lu-PCTA-Perjeta antibody conjugate is shown.

[0039] Fig. 20 is 64 Cu / 177 Size Exclusion-HPLC chromatogram of Lu-PCTA-Herzuma antibody conjugate is shown.

[0040] Fig. 21 is 64 Cu / 177 In vitro stability analysis of Lu-PCTA-Herceptin antibody conjugate is shown.

[0041] Fig. 22 is 64 Cu / 177 In vitro stability analysis of Lu-PCTA-Perjeta antibody conjugates is shown.

[0042] Fig. 23 is 64 Cu / 177 In vitro stability analysis of Lu-PCTA-Herzuma antibody conjugates is shown.

[0043] Fig. 24 is 64 Cu / 177 In vitro cell binding ability evaluation of Lu-PCTA-Herceptin antibody conjugates is shown.

[0044] Fig. 25 is 64 Cu / 177In vitro cell binding ability evaluation of Lu-PCTA-Perjeta antibody conjugates is shown.

[0045] Fig. 26 is 64 Cu / 177 In vitro cell binding ability evaluation of Lu-PCTA-Herzuma antibody conjugates is shown.

[0046] Fig. 27 is 177 In vitro cytotoxicity evaluation of Lu-PCTA-Herceptin antibody conjugates is shown.

[0047] Fig. 28 is 177 In vitro cytotoxicity evaluation of Lu-PCTA-Perjeta antibody conjugates is shown.

[0048] Fig. 29 is 177 In vitro cytotoxicity evaluation of Lu-PCTA-Herzuma antibody conjugates is shown.

[0049] Fig. 30 is 64 Biodistribution evaluation of Cu-PCTA-Herceptin antibody conjugates in antibody-resistant breast cancer (JIMT-1, Figure 30A) and gastric cancer (NCI-N87, Figure 30B) models is shown.

[0050] Fig. 31 is 64 Biodistribution evaluation of Cu-PCTA-Perjeta antibody conjugates in antibody-resistant breast cancer (JIMT-1, Figure 31A) and gastric cancer (NCI-N87, Figure 31B) models is shown.

[0051] Fig. 32 is 64 Biodistribution evaluation of Cu-PCTA-Herzuma antibody conjugates in antibody-resistant breast cancer (JIMT-1, Figure 32A) and gastric cancer (NCI-N87, Figure 32B) models is shown.

[0052] Fig. 33 is 64Immuno-PET images of Cu-PCTA-Herceptin antibody conjugates in HER2-null breast cancer (MDA-MB-231, Figure 33A) and antibody-resistant breast cancer (JIMT-1, Figure 33B) and HER2-null gastric cancer (MKN-45, Figure 33C) and antibody-resistant gastric cancer (NCI-N87, Figure 33D) models are shown.

[0053] Fig. 34 is 64 Immuno-PET images of Cu-PCTA-Perjeta antibody conjugates in HER2-null breast cancer (MDA-MB-231, Figure 34A) and antibody-resistant breast cancer (JIMT-1, Figure 34B) and HER2-null gastric cancer (MKN-45, Figure 34C) and antibody-resistant gastric cancer (NCI-N87, Figure 34D) models are shown.

[0054] Fig. 35 is 64 Immuno-PET images of Cu-PCTA-Herzuma antibody conjugates in HER2-null breast cancer (MDA-MB-231, Figure 35A) and antibody-resistant breast cancer (JIMT-1, Figure 35B) and HER2-null gastric cancer (MKN-45, Figure 35C) and antibody-resistant gastric cancer (NCI-N87, Figure 35D) models are shown.

[0055] Fig. 36 is 177 Biodistribution evaluation of the Lu-PCTA-Herceptin antibody conjugate in antibody-resistant breast cancer (JIMT-1, Figure 36A) and gastric cancer (NCI-N87, Figure 36B) models is shown.

[0056] Fig. 37 is 177 Biodistribution evaluation of the Lu-PCTA-Perjeta antibody conjugate in antibody-resistant breast cancer (JIMT-1, Figure 37A) and gastric cancer (NCI-N87, Figure 37B) models is shown.

[0057] Fig. 38 is 177 Biodistribution evaluation of the Lu-PCTA-Herzuma antibody conjugate in antibody-resistant breast cancer (JIMT-1, Figure 38A) and gastric cancer (NCI-N87, Figure 38B) models is shown.

[0058] Fig. 39 is 177 SPECT / CT images of Lu-PCTA-Herceptin antibody conjugates in antibody-resistant breast cancer (JIMT-1, Figure 39A) and gastric cancer (NCI-N87, Figure 39B) models are shown.

[0059] Fig. 40 is 177 SPECT / CT images of Lu-PCTA-Perjeta antibody conjugates in antibody-resistant breast cancer (JIMT-1, Figure 40A) and gastric cancer (NCI-N87, Figure 40B) models are shown.

[0060] Fig. 41 is 177 SPECT / CT images of the Lu-PCTA-Herzuma antibody conjugate are shown in antibody-resistant breast cancer (JIMT-1, Figure 41A) and gastric cancer (NCI-N87, Figure 41B) models.

[0061] Fig. 42 is 177 Lu-PCTA-Herceptin antibody conjugate exhibits biotherapeutic effects in antibody-resistant breast cancer (JIMT-1, Figure 42A) and gastric cancer (NCI-N87, Figure 42B) models.

[0062] Fig. 43 is 177 Lu-PCTA-Perjeta antibody conjugate exhibits biotherapeutic effects in antibody-resistant breast cancer (JIMT-1, Figure 43A) and gastric cancer (NCI-N87, Figure 43B) models.

[0063] Fig. 44 is 177 Lu-PCTA-Herzuma antibody conjugate exhibits biotherapeutic effects in antibody-resistant breast cancer (JIMT-1, Figure 44A) and gastric cancer (NCI-N87, Figure 44B) models.

[0064] A and B of Fig. 45 177Analysis of the therapeutic effect mechanism of the Lu-PCTA-Herceptin antibody conjugate in the antibody-resistant breast cancer (JIMT-1) model is shown. Figures C, D, and E of Figure 45 show the results of FDG-PET image evaluation and quantitative analysis, and TUNEL and Ki-67 immunohistochemical staining and quantitative analysis.

[0065] A and B of Fig. 46 177 Analysis of the therapeutic effect mechanism of the Lu-PCTA-Herceptin antibody conjugate in the antibody-resistant gastric cancer (NCI-N87) model is shown. Figures C, D, and E of Figure 46 show the results of FDG-PET image evaluation and quantitative analysis, and TUNEL and Ki-67 immunohistochemical staining and quantitative analysis.

[0066] A and B of Fig. 47 177 Analysis of the therapeutic effect mechanism of the Lu-PCTA-Perjeta antibody conjugate in the antibody-resistant breast cancer (JIMT-1) model is shown. Figures C, D, and E of Figure 47 show the results of FDG-PET image evaluation and quantitative analysis, and TUNEL and Ki-67 immunohistochemical staining and quantitative analysis.

[0067] A and B of Fig. 48 177 Analysis of the therapeutic effect mechanism of the Lu-PCTA-Perjeta antibody conjugate in the antibody-resistant gastric cancer (NCI-N87) model is shown. Figures C, D, and E of 48 show the results of FDG-PET image evaluation and quantitative analysis, and TUNEL and Ki-67 immunohistochemical staining and quantitative analysis.

[0068] A and B of Fig. 49 177 Analysis of the therapeutic effect mechanism of the Lu-PCTA-Herzuma antibody conjugate in the antibody-resistant breast cancer (JIMT-1) model is shown. Figures C, D, and E of 49 show the results of FDG-PET image evaluation and quantitative analysis, and TUNEL and Ki-67 immunohistochemical staining and quantitative analysis.

[0069] A and B of Fig. 50 177Analysis of the therapeutic effect mechanism of the Lu-PCTA-Herzuma antibody conjugate in the antibody-resistant gastric cancer (NCI-N87) model is shown. Figures C, D, and E of Figure 50 show the results of FDG-PET image evaluation and quantitative analysis, and TUNEL and Ki-67 immunohistochemical staining and quantitative analysis.

[0070] Hereinafter, the present invention will be described in detail.

[0071]

[0072] 1. Radioisotope-labeled anti-HER2 antibody-chelator conjugate

[0073] One aspect of the present invention provides a radioisotope-labeled anti-HER2 antibody-chelator conjugate.

[0074] The term "chelator" as used in the present invention refers to a compound composed of a plurality of heteroatoms (typically 0, N, or S) capable of complexing a radioisotope. The chelator may be cyclic or acyclic, and is preferably cyclic. The chelator of the present invention may be appropriately selected depending on the nature and oxidation state of the radioisotope, and may form a stable and inert metal complex depending on the coordination chemistry of the chelator and the radioisotope and the electron donor ability of the chelator.

[0075] 예를 들어서, 본 발명의 킬레이터는 1,4,7,10-tetraazacyclododecane-N, N', N", N'",-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), N"-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)glutaric acid (DOTAGA), 2-(4,7,-bis(carboxymethyl)-1,4,7-triazacyclonon-1-yl)glutaric acid (NOTAGA), 1,4,7-Triazacyclononane phosphinic acid (TRAP), 1,4,7-Triazacyclononane-1- [methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,8,15-tetraazabicyclo[9.3.1]pentadecane-1(15), 11,13-triene-3,6,9-triacetic acid (PCTA), N'-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl) (hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (oxy-Do 3A), p-isothiocyanatobenzyl-DTPA (SCN-Ba-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), It may be, but is not limited to, 1-(2)-methyl-4-isocyanobenzyl-DTPA (MX-DTPA), Hydrazinonicotinamide (HYNIC), 4-amino-1-hydroxybutylidene-1, 1-diphosphonic acid, Ethylenedicysteine ​​(EC) or derivatives thereof.

[0076] For example, in the present invention, the chelator may be a derivative of PCTA. Specifically, the chelator may be p-SCN-Bn-PCTA (3,6,9,15-Tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid; CAS number: 949147-44-8).

[0077] The term “antibody-chelator conjugate” used in the present invention means a form in which an antibody and a chelator are conjugated.

[0078] The term "radioisotope" used in the present invention may mean a diagnostic or therapeutic radioisotope, and may be particularly used in immuno-positron emission tomography (PET; hereinafter referred to as immuno-PET) imaging technology or radioimmunotherapy. The radioisotope may be, for example, 43 / 44 Sc, 47 Sc, 64 Cu, 67 Cu, 68 Ga, 86 Y, 90 Y, 177 Lu, 149 Tb, 151 Tb, 152 Tb 212 Bi, 212 Pb, 213 Bi, 225 Ac, 226 Th or 227 Th may be, and preferably 64 Cu or 177 Lu may be, but is not limited to,

[0079] The term "label" as used in the present invention means that a radioisotope is bound to an antibody-chelate conjugate by a chemical process, and the binding may be achieved by coordinate bonding.

[0080] The term "radiochemical purity" as used in the present invention means the percentage of radioactivity present in the desired chemical form among the total radioactivity of a specific radiopharmaceutical. In the present invention, among the total radioactivity, 64 Cu / 177 It refers to the percentage of radioactivity generated from the Lu-PCTA-anti-HER2 antibody conjugate. In the radioisotope-labeled anti-HER2 antibody-chelator conjugate of the present invention, the radioisotope 64 Cu and 177 It may be selected from Lu. 177Lu is reported to be a promising therapeutic radioisotope because it has low-energy beta rays and thus low tissue penetration, minimizing radiation transmission to surrounding normal tissues when targeted to tumor tissue and enabling localization of cytotoxic radiation to the target area. Additionally, it emits low-energy gamma rays, enabling single-photon emission computed tomography (SPECT) imaging, which is useful for determining whether the radiation is localized to the target area when used as a therapeutic agent.

[0081] HER2-expressing tumors can be noninvasively and quantitatively assessed using immuno-positron emission tomography (PET), which visualizes the accessibility of targeting agents. Furthermore, the therapeutic dose, treatment cycle, and therapeutic effect of targeted drug therapies can be assessed imaging-wise, and the therapeutic efficacy of targeted therapies exhibiting cytotoxicity can be predicted before administration. In antibody-resistant HER2-expressing tumors, immuno-PET can be used to preemptively assess HER2 expression and tumor accessibility, which can be useful in predicting the therapeutic efficacy of therapeutic drugs conjugated with therapeutic radioisotopes.

[0082] Immuno-PET, by combining antibodies with radioisotopes capable of positron emission tomography, allows for noninvasive, quantitative acquisition of whole-body images. Furthermore, it can efficiently assess target expression in primary and metastatic tumors in patients. Furthermore, assessing target expression within tumor tissue using conventional immunohistochemical staining can help predict tumor heterogeneity and identify patients likely to respond appropriately to targeted therapies.

[0083] The above-mentioned radioimmunotherapy is a treatment method that utilizes monoclonal antibodies against tumor-specific antigens labeled with therapeutic radioisotopes to selectively deliver radiation to tumors, resulting in therapeutic effects. Radioimmunotherapy effectively delivers therapeutic radiation to diseased sites using antibodies as carriers. Furthermore, the cross-fire effect allows for the delivery of radiation across diseased sites, even when the expression of the target antigen is low, thereby enhancing therapeutic effects.

[0084] In the radioisotope-labeled anti-HER2 antibody-chelator conjugate of the present invention, the anti-HER2 antibody may be selected from, but is not limited to, Trastuzumab and Pertuzumab.

[0085] Specifically, the above trastuzumab or pertuzumab may refer to a product or biosimilar product containing trastuzumab or pertuzumab as an active ingredient, respectively. For example, trastuzumab may be Herceptin® or Herzuma® containing it as an active ingredient, and pertuzumab may be Perjeta® containing it as an active ingredient, but is not limited thereto, and may also refer to other biosimilar products containing trastuzumab or pertuzumab as an active ingredient.

[0086] Additionally, in the above anti-HER2 antibody-chelator conjugate, the anti-HER2 antibody means an antigen-binding fragment of the antibody, unless otherwise specified.

[0087] HER2 is a transmembrane tyrosine kinase receptor protein known as a key factor in initiating signal transduction systems such as tumor cell growth, survival, and invasiveness. It has been reported that HER2 mediates signal transduction by forming homodimers between membrane proteins or heterodimers with membrane proteins such as HER1, HER3, and HER4 belonging to other HER families.

[0088] Trastuzumab and pertuzumab are recombinant humanized monoclonal antibodies that bind to the IV and II extracellular domains of HER2, respectively, and inhibit signal transduction pathways. Herceptin® (trastuzumab) is known to bind to HER2, mediate receptor endocytosis and degradation, inhibit the secreted form of HER2, and mediate antibody-dependent cytotoxicity, resulting in its therapeutic effects. Meanwhile, Perjeta® (pertuzumab) inhibits HER2 heterodimerization, and has been shown in nonclinical and clinical studies to be effective when combined with chemotherapy and Herceptin®. Additionally, the antibody-drug conjugate Kecyla®, which is combined with a microtubule formation inhibitor, has been reported to be effective in patients with HER2-positive metastatic breast cancer.

[0089] In the anti-HER2 antibody-chelator conjugate of the present invention, the binding ratio of the chelator per molecule of the anti-HER2 antibody may be, but is not limited to, 1.0 to 5.0, 1.2 to 5.0, 1.4 to 5.0, 1.6 to 5.0, 1.8 to 5.0, 2.0 to 5.0, 1.0 to 4.0, 1.2 to 4.0, 1.4 to 4.0, 1.6 to 4.0, 1.8 to 4.0, 2.0 to 4.0, 1.0 to 3.0, 1.2 to 3.0, 1.4 to 3.0, 1.6 to 3.0, 1.8 to 3.0, or 2.0 to 3.0. In the present invention, when the binding ratio of the antibody and the chelator is within the above range, an excellent labeling yield of the radioisotope can be exhibited.

[0090] Specifically, the reaction molar ratio of the antibody and the chelator can be set differently depending on the type of antibody. For example, when trastuzumab or pertuzumab is reacted with p-SCN-Bn-PCTA at a molar ratio of 1:15 or 1:10, respectively, to produce a conjugate, the binding ratio of the antibody and the chelator in the produced conjugate is within the above range, and at this time, an excellent labeling yield of the radioisotope for the antibody can be exhibited.

[0091]

[0092] 2. A diagnostic kit for HER2-expressing cancer, or a pharmaceutical composition for diagnosis or treatment, comprising a radioisotope-labeled anti-HER2 antibody-chelator conjugate.

[0093] One aspect of the present invention provides a kit for diagnosing HER2-expressing cancer. The kit for diagnosing HER2-expressing cancer comprises a radioisotope-labeled anti-HER2 antibody-chelator conjugate.

[0094] Additionally, the kit of the present invention may further comprise one or more other components, solutions, or devices suitable for diagnosing cancer, and may further comprise packaging.

[0095] One aspect of the present invention provides a pharmaceutical composition for diagnosing or treating HER2-expressing cancer. The pharmaceutical composition for diagnosing or treating HER2-expressing cancer comprises a radioisotope-labeled anti-HER2 antibody-chelator conjugate.

[0096] In the above kit or pharmaceutical composition, the HER2-expressing cancer may be a cancer resistant to anti-HER2 antibody treatment, and the HER2-expressing cancer may be a cancer resistant to combination therapy with an anti-HER2 antibody and a chemotherapy drug. For example, the HER2-expressing cancer may be breast cancer or stomach cancer. The chemotherapy drug may be, but is not limited to, paclitaxel for breast cancer and cisplatin or capecitabine for stomach cancer.

[0097] In addition, the pharmaceutical composition of the present invention may further include an appropriate carrier, excipient, or diluent according to a conventional method. Specifically, when formulating, it may be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Preparations for administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0098] In addition, the pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. The administration method may be, for example, administered via skin, rectum, vein, intraperitoneal, intramuscular, subcutaneous, intrauterine epidural, or intracerebroventricular injection, but is not limited thereto.

[0099] In addition, the pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar function. For administration, it may additionally contain one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, the compound according to the present invention is easy to prepare in various formulations, and for example, it may be prepared in the form of injection formulations such as aqueous solutions, suspensions, and emulsions, powders, tablets, capsules, pills, granules, or injection solutions by additionally adding diluents, dispersants, surfactants, binders, and lubricants.

[0100] The contents described in the above “1. Radioisotope-labeled anti-HER2 antibody-chelator conjugate” can be equally applied to the kit and pharmaceutical composition of the present invention.

[0101] Another aspect of the present invention provides a method for diagnosing or treating HER2-expressing cancer, comprising administering to a subject an anti-HER2 antibody-chelator conjugate according to the present invention.

[0102] Another aspect of the present invention provides a use of an anti-HER2 antibody-chelator conjugate according to the present invention for diagnosing or treating a HER2 expressing cancer.

[0103] Another aspect of the present invention provides a use of an anti-HER2 antibody-chelator conjugate of the present invention for preparing a medicament for diagnosing or treating a HER2 expressing cancer.

[0104] In addition, in the present invention, the same contents as those applied to pharmaceutical compositions can be applied to the diagnostic or therapeutic methods and uses.

[0105]

[0106] 3. Method for producing a radioisotope-labeled anti-HER2 antibody-chelator conjugate and an antibody-chelator conjugate produced by the method

[0107] One aspect of the present invention is:

[0108] (a) a step of reacting an anti-HER2 antibody and p-SCN-Bn-PCTA to produce a conjugate; and

[0109] (b) Provided is a method for producing a radioisotope-labeled anti-HER2 antibody-chelator conjugate, comprising a step of reacting the conjugate with a radioisotope to label the conjugate with the radioisotope.

[0110] In the above step (a), the reaction molar ratio of the anti-HER2 antibody and the chelator may be set differently depending on the type of anti-HER2 antibody. For example, the reaction may be performed at a molar ratio of 1:5 to 1:25, 1:5 to 1:20, 1:5 to 1:15, 1:5 to 1:10, 1:10 to 1:25, 1:10 to 1:20, 1:10 to 1:15, 1:11 to 1:25, 1:11 to 1:20, or 1:11 to 1:15.

[0111] Specifically, when the anti-HER2 antibody is trastuzumab, the reaction may be performed at a molar ratio of 1:15, and when it is pertuzumab, the reaction may be performed at a molar ratio of 1:10. When the reaction molar ratio of the antibody and the chelator satisfies this value, an excellent radioisotope labeling yield may be exhibited.

[0112] More specifically, one aspect of the present invention is:

[0113] (a) a step of reacting an anti-HER2 antibody and p-SCN-Bn-PCTA at a molar ratio of 1:10 to 1:20 to produce a conjugate; and

[0114] (b) comprising a step of labeling the conjugate with a radioisotope by reacting the conjugate with the radioisotope,

[0115] The above anti-HER2 antibody is trastuzumab,

[0116] A method for preparing a radioisotope-labeled anti-HER2 antibody-chelator conjugate is provided.

[0117] In addition, one aspect of the present invention is

[0118] (a) a step of reacting an anti-HER2 antibody and p-SCN-Bn-PCTA at a molar ratio of 1:5 to 1:15 to produce a conjugate; and

[0119] (b) comprising a step of labeling the conjugate with a radioisotope by reacting the conjugate with the radioisotope,

[0120] The above anti-HER2 antibody is pertuzumab,

[0121] A method for preparing a radioisotope-labeled anti-HER2 antibody-chelator conjugate is provided.

[0122] Another aspect of the present invention provides a radioisotope-labeled anti-HER2 antibody-chelator conjugate prepared by the above method.

[0123] The radioisotope of the above step (b) is 43 / 44 Sc, 47 Sc, 64 Cu, 67 Cu, 68 Ga, 86 Y, 90 Y, 177 Lu, 149 Tb, 151 Tb, 152 Tb 212 Bi, 212 Pb, 213 Bi, 225 Ac, 226 Th or 227 Th It can be, and preferably 64 Cu or 177Lu may be, but is not limited to, the radioisotope of step (b) above. 64 Cu and 177 It may be selected from Lu.

[0124] The above step (b) may be performed at a pH of 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8 or 6 to 7, and preferably may be performed at pH 6.5.

[0125] The above step (b) may be carried out in a sodium acetate buffer, wherein the concentration of the buffer may be 500 to 600 mM, 500 to 580 mM, 500 to 560 mM, 500 to 540 mM or 500 to 520 mM, and preferably 510 mM.

[0126] The temperature at which step (b) is performed may be appropriately selected depending on the type of radioisotope being labeled. For example, step (b) may be performed at a temperature of 20 to 40°C.

[0127] Specifically, radioisotopes 64 When labeling Cu, it can be performed at a temperature of 23 to 27°C, 24 to 26°C or preferably 25°C, and the radioisotope 177 When labeling Lu, it can be performed at a temperature of 35 to 40°C, 36 to 39°C, or preferably 37°C.

[0128] Additionally, the above step (b) may be performed for 50 to 70 minutes or 50 to 60 minutes, and preferably may be performed for 60 minutes.

[0129] Specifically, the step (b) may be performed at a temperature of 20 to 40°C for 50 to 70 minutes in a sodium acetate buffer solution having a concentration of 500 to 600 mM at pH 6.5.

[0130] For example, radioactive isotopes 64 In case of labeling Cu, it may be performed for 60 minutes under conditions of sodium acetate buffer concentration of 510 mM and pH 6.5 at 25°C.

[0131] For example, radioactive isotopes 177 When labeling Lu, it can be performed for 60 minutes under conditions of sodium acetate buffer concentration of 510 mM and pH 6.5 at 37°C.

[0132] One aspect of the present invention relates to a radioisotope-labeled anti-HER2 antibody-chelator conjugate manufactured by the above manufacturing method. The contents described in the above section "1. Radioisotope-labeled anti-HER2 antibody-chelator conjugate" can be equally applied to the method for manufacturing the anti-HER antibody-chelator conjugate of the present invention and the antibody-chelator conjugate manufactured by the above manufacturing method.

[0133] According to one embodiment of the present invention, a radioisotope for PET imaging diagnosis 64 By quantitatively evaluating the level of HER2 expression in target tumors refractory to existing anti-HER2 antibody treatments with various resistance mechanisms using Cu-labeled anti-HER2 antibody (Herceptin, Perjeta and Herzuma)-chelator conjugates, a beta-emitting radioisotope for therapeutic use in the future will be used. 177 The therapeutic potential of Lu-labeled anti-HER2 antibody (e.g., Herceptin, Perjeta, Herzuma)-chelator conjugates can be assessed by imaging.

[0134] Radioisotope for beta-ray therapy 177Lu-labeled anti-HER2 antibody (e.g., Herceptin, Perjeta, Herzuma)-chelator conjugates can be used as a new treatment strategy for tumors resistant to existing anti-HER2 antibodies, as a substance that can overcome resistance mechanisms by delivering radiation of beta-emitting radioisotopes to HER2-expressing tumors with various resistance mechanisms.

[0135] Meanwhile, all numerical values ​​used in the present invention are intended to include the corresponding numerical value and adjacent numerical values ​​within an equivalent / similar range. Furthermore, unless otherwise specified, all numerical values ​​used in the present invention are intended to include a range that can be modified by the term "about."

[0136] Hereinafter, manufacturing examples and examples of the present invention are described, but the present invention is not limited thereto.

[0137] [Manufacturing example]

[0138] 64 Cu / 177 Preparation of Lu-PCTA-anti-HER2 antibody (Herceptin, Perjeta, Herzuma) conjugates

[0139]

[0140] Manufacturing Example 1. Manufacturing of PCTA-anti-HER2 antibody (Herceptin, Perjeta, Herzuma) conjugate

[0141] 20 mg of anti-HER2 antibodies (Herceptin (Roche Korea), Perjeta (Roche Korea), Herzuma (Celltrion)) were reacted with the bifunctional chelator p-SCN-Bn-PCTA (Macrocyclics, Dallas, TX, USA) in 100 mM sodium bicarbonate buffer at pH 8.5 at room temperature for 2 hours, and then further reacted overnight at 4°C. For Herceptin and Herzuma, the reaction was performed at a molar ratio of antibody to bifunctional chelator of 1:15, and for Perjeta, the reaction was performed at a molar ratio of 1:10 to prepare the conjugate. Unconjugated chelator was then removed by dialysis and ultrafiltration.

[0142] Finally, the conjugate was concentrated to 2–5 mg / mL by treating with 20 mM sodium acetate buffer, pH 6.5. To measure the number of chelators per antibody, mass spectrometry was performed using a MALDI mass spectrometer (Voyager-DE STR, PerSpective Biosystems Inc.), and the conjugation ratio of bifunctional chelators per antibody molecule was analyzed.

[0143] In the case of Herceptin, 2.41 ± 0.22 bifunctional chelators were conjugated per antibody molecule, in the case of Perjeta, 1.92 ± 0.01 bifunctional chelators were conjugated per antibody molecule, and in the case of Herzuma, 2.92 ± 0.69 bifunctional chelators were conjugated per antibody molecule (see Figures 2 to 4).

[0144]

[0145] Manufacturing Example 2. Manufacturing of PCTA-anti-HER2 antibody (Herceptin, Perjeta, Herzuma) conjugate

[0146] An antibody chelator conjugate was prepared in the same manner as in Manufacturing Example 1, except that the molar ratio of Herceptin and chelator was set to 1:10.

[0147]

[0148] Example. 64 Cu / 177 Preparation of Lu-PCTA-anti-HER2 antibody (Herceptin, Perjeta, Herzuma) conjugates

[0149] 64 Cu was produced using a 50 Mev cyclotron at the Korea Institute of Radiological & Medical Sciences. 177 Lu was purchased and used from ITM AG.

[0150] 1 mg of the PCTA-anti-HER2 antibody conjugate of the above manufacturing example 64 For Cu, use 1–2 mCi (37–74 MBq). 177For Lu, 2–20 mCi (74–740 MBq) was used and added to the PCTA-anti-HER2 antibody conjugate solution. 64 In the case of Cu, it reacts at room temperature (25 ℃). 177 In the case of Lu, the reaction was carried out by shaking at ×1,000 rpm per minute using a stirrer at 37 ℃ for 1 hour.

[0151] The above reaction mixture was reacted in a 500 mM to 1,000 mM sodium acetate buffer solution (pH 6.5). The radiolabeled product and purity were immediately measured using instant thin-layer chromatography silica gel (ITLC-sg) (Pall Corp.) as the stationary phase and 20 mM citric acid buffer solution at pH 5 with 50 mM EDTA. At this time, all showed a labeling yield of 98% or more (see Figures 5 to 7).

[0152] Radiochemical purity was confirmed by size-exclusion high-performance liquid chromatography. To minimize antibody damage, the reaction was performed at room temperature, yielding the results described above. Therefore, the labeling step at elevated temperatures was not performed.

[0153] Conjugate antibody: Chelating agent molar yield Example 164Cu / 177Lu-PCTA-anti-HER2 antibody (Herceptin) 1:1598% or more Example 264Cu / 177Lu-PCTA-anti-HER2 antibody (Herzuma) 1:1598% or more Example 364Cu / 177Lu-PCTA-anti-HER2 antibody (Perjeta) 1:1098% or more Example 464Cu / 177Lu-PCTA-anti-HER2 antibody (Herceptin) 1:1077.5% ~ 95%

[0154]

[0155] [Experimental Example]

[0156] Experimental Example 1. Evaluation of HER2 expression in breast and stomach cancer cell lines.

[0157] SK-BR-3 (Korea Cell Line Bank), BT-474 (American Type Culture Collection; ATCC), JIMT-1 (AddexBio), and KPL-4 (Yamashida, Nagasaki University) were selected as HER2-expressing breast cancer cell lines, and MDA-MB-231 (ATCC) was selected as a control cell line. NCI-N87 (Korea Cell Line Bank) was selected as a HER2-expressing gastric cancer cell line, and MKN-45 (Korea Cell Line Bank) was selected as a control cell line.

[0158] For HER2 expression western blot analysis of HER2-expressing cell lines, proteins were extracted from each cell line, quantified, electrophoresis was performed, and western blot analysis was performed using anti-HER2 / ErbB2 antibody (#2165, Cell signaling) and anti-β-actin antibody (#4967, Cell signaling). As a result, expression of HER2 protein of approximately 185 kDa was confirmed in the order of BT-474, SK-BR-3, and JIMT-1 cell lines.

[0159] As a result of performing flow cytometry analysis by reacting each cell line with 2 ug of Herceptin, Perjeta, and Herzuma as primary antibodies and treating with 2 ug of anti-human IgG Fc specific FITC conjugated antibody as secondary antibody, HER2 expression was high in the order of BT-474, KPL-4, SK-BR-3, and JIMT-1 in breast cancer cell lines, and very high HER2 expression was confirmed in the case of NCI-N87 (see Figures 8 to 11).

[0160]

[0161] Experimental Example 2. In vitro cytotoxicity evaluation of anti-HER2 antibodies

[0162] Herceptin, Perjeta, and Herzuma were treated at various concentrations (0, 1, 2, 5, 10, 20, and 50 ug / mL) in vitro, and cytotoxicity was evaluated after 5 days.

[0163] To confirm the antibody therapeutic effect of Herceptin, cytotoxicity tests were conducted in vitro. As a result, BT474 and SK-BR-3 cell lines were confirmed to be responsive to immunotherapy, while the therapeutic effect was confirmed to be minimal in KPL-4 and JIMT-1 cell lines known to be refractory to immunotherapy. In the case of MDA-MB-231, which does not express HER2, Herceptin was confirmed to have no therapeutic effect.

[0164] As a result of confirming the antibody therapeutic effect of Perjeta through a cytotoxicity test in a test tube, it was confirmed that there was no therapeutic effect in the Perjeta-only treatment group as previously known, and cytotoxicity was confirmed to be minimal in all cell lines.

[0165] The antibody therapeutic effect of Herzuma was confirmed through a cytotoxicity test in vitro. Similar to Herceptin, the BT474 and SK-BR-3 cell lines were confirmed to be responsive to immunotherapy, while the KPL-4 and JIMT-1 cell lines, known to be refractory to immunotherapy, were confirmed to have minimal therapeutic effect. In the case of MDA-MB-231, which does not express HER2, Herzuma was confirmed to have no therapeutic effect (see Figures 12 to 14).

[0166]

[0167] Experimental Example 3. Evaluation of the therapeutic effect of anti-HER2 antibodies in a tumor model.

[0168] BALB / c nude mice (Nara Biotech) were inoculated with 1x10 JIMT-1 breast cancer cell line and NCI-N87 human gastric cancer cell line on the right flank. 7 Anti-HER2 antibody-resistant breast and gastric cancer tumor models were prepared by subcutaneous injection of cells.

[0169] The above models were treated with Herceptin, Perjeta, Herzuma, or the control antibody, rituximab (Roche Korea), at a dose of 5 mg / kg twice a week for 4 weeks, and the tumor volume was measured for up to 30 days to evaluate the biological therapeutic effect.

[0170] The JIMT-1 breast cancer cell line has been previously reported to exhibit Herceptin resistance, and this example also confirmed the tumor model's resistance to Herceptin and Perjeta. That is, regardless of Herceptin or Perjeta administration, tumor growth increased at a rate similar to the control group, and no statistically significant difference was observed compared to the control group.

[0171] The NCI-N87 gastric cancer cell line has been previously reported to exhibit Herceptin resistance, and this example also confirmed the tumor model's resistance to Herceptin and Perjeta. Regardless of Herceptin and Perjeta administration, tumor growth increased at a rate similar to the control group, and no statistically significant difference was observed compared to the control group (see Figures 15 to 17).

[0172]

[0173] Experimental Example 4. 64 Cu / 177 Radiochemical Purity Analysis of Lu-PCTA-Anti-HER2 Antibody Conjugate

[0174] Manufactured 64 Cu / 177 The purpose of this study was to analyze the radiochemical purity of the Lu-PCTA-anti-HER2 antibody conjugate.

[0175] For this purpose, size exclusion HPLC analysis was first performed. The column was MabPac SEC-1 (5 μm, 4.0 × 300 mm) and the mobile phase was 0.3 M NaCl, 50 mM sodium PB pH 6.8, and was flowed at a flow rate of 0.2 mL / min. The absorbance at 280 nm was measured using a UV spectrophotometer and the radioactivity was measured using a Raytest measuring device. The manufactured 64 Cu / 177 The radiochemical purity of Lu-PCTA-anti-HER2 antibody conjugates was confirmed to be greater than 98% (see Figures 18 to 20).

[0176]

[0177] Experimental Example 5. 64 Cu / 177 In vitro stability analysis of Lu-PCTA-anti-HER2 antibody conjugates

[0178] Manufactured 64 Cu / 177 To evaluate the stability of Lu-PCTA-anti-HER2 antibody conjugate in human serum, equal amounts of 64 Cu / 177 Lu-PCTA-anti-HER2 antibody conjugate was mixed with human serum and reacted at 37°C for 24 hours. The stationary phase was ITLC-sg and the mobile phase was developed using sodium acetate buffer containing EDTA. The stability of the final product was confirmed using a thin layer chromatography scanner (TLC scanner AR-2000, Ecker & Ziegler). As a result, the stability of the final product was confirmed to be over 98% for 24 hours and 7 days (see Figures 21 to 23).

[0179]

[0180] Experimental Example 6. 64 Cu / 177 Cell binding assay of Lu-PCTA-anti-HER2 antibody conjugate

[0181] Manufactured 64 Cu / 177Cell binding assay was performed using Lu-PCTA-anti-HER2 antibody conjugate. 1 × 10 of each cell line 6 In the cell 64 Cu / 177 After treating with 100 ng of Lu-PCTA-anti-HER2 antibody conjugate and treating at 4°C for 1 hour, the cells were washed three times with 1% BSA-PBS, and the cell binding ability was measured and analyzed using a gamma counter (Gamma counter WIZARD 1480, Perkin-Elmer) using the added radioactive standard source.

[0182] In breast cancer cell lines, cell binding ability was confirmed in the order of BT474, SK-BR-3, KPL-4, and JIMT-1, and minimal binding was confirmed in MDA-MB-231. High cell binding ability was confirmed in the gastric cancer cell line NCI-N87, and minimal cell binding ability was confirmed in MKN-45 cells (see Figures 24 to 26).

[0183]

[0184] Experimental Example 7. 177 In vitro evaluation of the therapeutic efficacy of Lu-PCTA-anti-HER2 antibody conjugates

[0185] Manufactured 177 Evaluation of the in vitro radioimmunotherapeutic efficacy of Lu-PCTA-anti-HER2 antibody conjugates showed that BT474, SK-BR-3, and KPL-4 cell lines with high HER2 expression exhibited cell killing activity of approximately 50-60% at a radiation dose of 1.48 MBq, and JIMT-1 cell line with intermediate HER2 expression exhibited cell killing activity of approximately 40-50% at a radiation dose of 1.48 MBq.

[0186] Accordingly, it was confirmed that cytotoxicity was exhibited depending on the radiation dose delivered to tumor cells expressing HER2. In the case of the MDA-MB-231 cell line that does not express HER2, cytotoxicity was not exhibited.

[0187] In the gastric cancer cell line, the NCI-N87 cell line showed a high killing activity of more than 75% at a radiation dose of 1.48 MBq, and in the case of MKN-45, no statistically significant difference was observed compared to the control group (the group treated with the same dose of trastuzumab, pertuzumab, and herzuma alone) (see Figures 27 to 29).

[0188]

[0189] Experimental Example 8. 64 Biodistribution Evaluation of Cu-PCTA-anti-HER2 Antibody Conjugates in Antibody Therapy-Resistant Breast and Gastric Cancer Models

[0190] 1x10 of JIMT-1 breast cancer cell line and NCI-N87 gastric cancer cell line were inoculated into the right flank of BALB / c nude mice (Nara Biotech). 7 Breast cancer and gastric cancer tumor models were created by subcutaneous injection of cells. 64 After treatment with Cu-PCTA-anti-HER2 antibody conjugates, mice were sacrificed at 2, 24, and 48 hours, and each tissue of the mouse was excised and weighed.

[0191] Radioactivity was measured using a gamma counter (Gamma counter WIZARD 1480, Perkin-Elmer) and expressed as the percentage of injected radioactivity dose per gram of tissue (%ID / g, The percentage of injected radioactivity dose per gram of tissue) to evaluate biodistribution and confirm tumor targeting ability. The highest tumor uptake was observed after 48 hours, and a blocking experiment was performed by administering excessive amounts of Herceptin, Perjeta, and Herzuma without radioisotope labeling, confirming that uptake in the tumor decreased. 64 It was confirmed that the Cu-PCTA-anti-HER2 antibody conjugate specifically bound to HER2 and localized to the tumor (see Figures 30 to 32).

[0192]

[0193] Experimental Example 9. 64 Immuno-PET imaging of Cu-PCTA-anti-HER2 antibody conjugates in antibody-resistant breast and gastric cancer models.

[0194] A subcutaneous tumor model was created in the same manner as in Experimental Example 8 using MDA-MB-231, a HER2-nonexpressing breast cancer cell line, JIMT-1 breast cancer cell line confirmed to be resistant to anti-HER2 antibody treatment, MKN-45, a HER2-nonexpressing gastric cancer cell line, and NCI-N87 gastric cancer cell line confirmed to be resistant to anti-HER2 antibody treatment. 64 Tumor targeting ability was evaluated by acquiring time-dependent PET images using Cu-PCTA-anti-HER2 antibody conjugates.

[0195] In non-expressing breast and gastric cancer models, it showed minimal tumor targeting ability, and in JIMT-1 and NCI-N87 tumors resistant to anti-HER2 antibody therapy, tumor uptake increased over time, with the highest tumor uptake observed after 48 hours. Blocking experiments performed by overdosing non-radiolabeled Herceptin, Perjeta, and Herzuma confirmed a decrease in tumor uptake. 64 We confirmed that the Cu-PCTA-anti-HER2 antibody conjugate specifically bound to HER2 and localized to JIMT-1 and NCI-N87 tumors that were resistant to anti-HER2 antibody treatment (see Figures 33 to 35).

[0196]

[0197] Experimental Example 10. 177 Biodistribution Evaluation of Lu-PCTA-anti-HER2 Antibody Conjugates in Antibody Therapy-Resistant Breast and Gastric Cancer Models

[0198] 1x10 of JIMT-1 breast cancer cell line and NCI-N87 gastric cancer cell line were injected into the right flank of 6-week-old BALB / c nude mice (Nara Biotech). 7 Breast cancer and gastric cancer tumor models were prepared by subcutaneous injection of cells.

[0199] After 3.7 MBq 177 Lu-PCTA-anti-HER2 antibody conjugate was intravenously administered into the tail of antibody-resistant breast cancer (JIMT-1) and gastric cancer (NCI-N87) model mice, respectively, and the mice were sacrificed at 2 h, 1, 3, 5, 7, and 14 days (n = 3 or 4 / group) after injection to evaluate biodistribution. In addition, excess unlabeled anti-HER2 antibodies (2 mg / head) were added for blocking experiments. 177 Lu-PCTA-anti-HER2 antibody conjugate was administered 30 minutes prior to administration. For the Lu-177 radiolabeled antibody in the blocking experiments, mice were sacrificed 5 days after administration, and biodistribution was measured.

[0200] Blood was collected via cardiac puncture, and each organ and tissue was excised and weighed. The radioactivity of each organ was measured using a gamma counter (Gamma counter WIZARD 1480, Perkin-Elmer), and the values ​​were calculated as the percentage of injected radioactivity per gram of tissue (%ID / g).

[0201] In the antibody-resistant breast cancer (JIMT-1) model, the highest tumor uptake was observed at 5 days, and a blocking experiment confirmed a significant decrease in tumor uptake, confirming specific tumor localization. In the antibody-resistant gastric cancer (NCI-N87) model, the highest tumor uptake was observed at 7 days, and a blocking experiment confirmed a significant decrease in tumor uptake. 177 It was confirmed that Lu-PCTA-anti-HER2 antibody conjugates were specifically localized to tumors (see Figures 36 to 38).

[0202]

[0203] Experimental Example 11. 177Single-photon emission computed tomography / computed tomography (SPECT / CT) imaging evaluation of Lu-PCTA-anti-HER2 antibody conjugates in antibody-resistant breast and gastric cancer models.

[0204] Small animal SPECT / CT yielded 12.95 MBq 177 Images were taken 10 days after administration of Lu-PCTA-anti-HER2 antibody conjugates to mice. In addition, excess unlabeled anti-HER2 antibodies (2 mg / head) were added for blocking experiments. 177 Lu-PCTA-anti-HER2 antibody conjugate 2.5 mg / kg (12.95 MBq) was pre-administered 30 minutes prior to administration.

[0205] Imaging was performed using an Inveon SPECT / CT system equipped with two NaI detectors and a 1.0 mm mouse whole-body multi-penetration (5-penetration) collimator, and images were acquired using the Inveon Acquisition Workplace (IAW). A total of 30 projections were acquired in a 256 × 256 acquisition matrix with 0.5 rotations per projection for 180 s. Images were reconstructed using MAP 3D (16 iterations). CT images were presented overlaid with SPECT images using 180 projections, 200 ms / projection, 80 kVp, and 500 μA. Image fusion of SPECT and CT was performed using Inveon Research Workplace software, and digital whole-body autoradiography (DWBA) was performed after SPECT / CT imaging.

[0206] 177 Lu-PCTA-anti-HER2 antibody conjugates were selectively distributed in antibody-resistant breast cancer (JIMT-1) and gastric cancer (NCI-N87) tumors, and showed relatively low uptake in the liver. Furthermore, tumor uptake was significantly reduced in blocking experiments using excessive amounts of unlabeled anti-HER2 antibodies.177 It was confirmed that Lu-PCTA-anti-HER2 antibody conjugates were specifically distributed in HER2-expressing antibody-resistant breast cancer (JIMT-1) model and gastric cancer (NCI-N87) model tumors (see Figures 39 to 41).

[0207]

[0208] Experimental Example 12. 177 Evaluation of the therapeutic efficacy of Lu-PCTA-anti-HER2 antibody conjugates in antibody-resistant breast and gastric cancer models.

[0209] In antibody-resistant breast cancer (JIMT-1) and gastric cancer (NCI-N87) models, 177 To confirm the therapeutic effect of Lu-PCTA-anti-HER2 antibody conjugates, tumor volumes of JIMT-1 and NCI-N87 tumor-implanted mice were approximately 100 mm 3 When the tumor reached , the patients were divided into three groups (n=6 or 7 / group) and radioimmunotherapy was performed. For each group, saline, anti-HER2 antibody 2.5 mg / kg or 177 A single dose of 2.5 mg / kg (12.95 MBq) of Lu-PCTA-anti-HER2 antibody conjugate was administered intravenously, and tumor volume and body weight were measured three times a week. Tumor volume was calculated as (long-axis diameter × short-axis diameter2) / 2.

[0210] In antibody-resistant breast cancer (JIMT-1) and gastric cancer (NCI-N87) models, a single dose of saline and anti-HER2 antibody resulted in a sustained increase in tumor volume over time up to 28 days, reaching a volume greater than three times that before treatment.

[0211] the other side, 177In the treatment group administered a single dose of Lu-PCTA-anti-HER2 antibody conjugates, tumor volume increased compared to before treatment for 5 to 7 days after treatment, and then continued to decrease, showing a significant decrease compared to before treatment at 28 days. No significant decrease in body weight was observed compared to before treatment. 177 It was confirmed that the Lu-PCTA-anti-HER2 antibody conjugate treatment was well tolerated. From these results, in mice 177 A single dose of Lu-PCTA-anti-HER2 antibody conjugate was confirmed to exhibit therapeutic effects without observable toxicity (see Figures 42 to 44).

[0212]

[0213] Experimental Example 13. 177 Analysis of the therapeutic mechanism of Lu-PCTA-anti-HER2 antibody conjugate in antibody-resistant breast and gastric cancer models.

[0214] In antibody-resistant breast cancer (JIMT-1) and gastric cancer (NCI-N87) models, 177 To analyze the therapeutic mechanisms of Lu-PCTA-anti-HER2 antibody conjugates, changes in tumor glucose metabolism following treatment were evaluated using FDG PET imaging. Furthermore, TUNEL immunohistochemical staining, which assesses the degree of tumor apoptosis following treatment, and Ki-67 immunohistochemical staining, which indicates tumor growth potential, were performed.

[0215] In antibody-resistant breast cancer (JIMT-1) and gastric cancer (NCI-N87) models, FDG uptake was similarly high in the groups administered a single dose of saline and anti-HER2 antibody. 177In the treatment group treated with a single dose of Lu-PCTA-anti-HER2 antibody conjugates, FDG uptake decreased, confirming that glucose metabolism was reduced by tumor treatment. TUNEL staining showed similarly low staining in the saline and anti-HER2 antibody single-dose groups. 177 In the treatment group treated with a single dose of Lu-PCTA-anti-HER2 antibody conjugates, the TUNEL positivity rate increased, confirming that cell death was increased by beta radiation of the delivered therapeutic radioisotope.

[0216] In Ki-67 staining, the groups that received a single dose of saline and anti-HER2 antibody showed similar, consistent staining intensities. 177 In the treatment group treated with a single dose of Lu-PCTA-anti-HER2 antibody conjugates, Ki-67 staining intensity was significantly reduced, confirming that tumor growth was reduced by beta radiation from the delivered therapeutic radioisotope. Quantitative analysis also showed similar results to the staining images (see Figures 45 to 50).

Claims

1. Radioisotope-labeled anti-HER2 antibody-chelator conjugate.

2. An anti-HER2 antibody-chelator conjugate according to claim 1, wherein the chelator is p-SCN-Bn-PCTA.

3. An anti-HER2 antibody-chelator conjugate according to claim 1, wherein the anti-HER2 antibody is selected from Trastuzumab and Pertuzumab.

4. In claim 1, the radioactive isotope is 64 Cu and 177 An anti-HER2 antibody-chelator conjugate selected from Lu.

5. An anti-HER2 antibody-chelator conjugate according to claim 1, wherein the binding ratio of the chelator per molecule of the anti-HER2 antibody is 1.0 to 5.

0.

6. A diagnostic kit for HER2-expressing cancer, comprising an anti-HER2 antibody-chelator conjugate according to any one of claims 1 to 5.

7. A pharmaceutical composition for diagnosing or treating HER2-expressing cancer, comprising an anti-HER2 antibody-chelator conjugate according to any one of claims 1 to 5.

8. A pharmaceutical composition according to claim 7, wherein the HER2-expressing cancer is a cancer resistant to anti-HER2 antibody treatment.

9. A pharmaceutical composition according to claim 7, wherein the HER2-expressing cancer is a cancer resistant to combination therapy with an anti-HER2 antibody and a chemical drug. 10.(a) a step of producing a conjugate by reacting anti-HER2 antibody and p-SCN-Bn-PCTA at a molar ratio of 1:10 to 1:20; and (b) comprising a step of labeling the conjugate with a radioisotope by reacting the conjugate with the radioisotope, The above anti-HER2 antibody is trastuzumab, Method for preparing a radioisotope-labeled anti-HER2 antibody-chelator conjugate. 11.(a) a step of producing a conjugate by reacting an anti-HER2 antibody and p-SCN-Bn-PCTA at a molar ratio of 1:5 to 1:15; and (b) comprising a step of labeling the conjugate with a radioisotope by reacting the conjugate with the radioisotope, The above anti-HER2 antibody is pertuzumab, Method for preparing a radioisotope-labeled anti-HER2 antibody-chelator conjugate.

12. In claim 10 or claim 11, the radioisotope of step (b) is 64 Cu and 177 A method for preparing an anti-HER2 antibody-chelator conjugate selected from Lu.

13. A method for producing an anti-HER2 antibody-chelator conjugate according to claim 10 or 11, wherein step (b) is performed at a temperature of 20 to 40°C for 50 to 70 minutes in a sodium acetate buffer solution having a concentration of 500 to 600 mM at pH 6.

5.

14. In claim 13, the radioactive isotope 64 When labeling Cu, it is performed at a temperature of 23 to 27°C, and the radioisotope 177 A method for preparing an anti-HER2 antibody-chelator conjugate, wherein the process is performed at 35 to 40°C when labeling Lu.

15. A radioisotope-labeled anti-HER2 antibody-chelator conjugate manufactured by the manufacturing method of claim 10 or 11.

16. A method for diagnosing or treating HER2-expressing cancer, comprising administering to a subject an anti-HER2 antibody-chelator conjugate of any one of claims 1 to 5.

17. Use of an anti-HER2 antibody-chelator conjugate of any one of claims 1 to 5 for diagnosing or treating HER2 expressing cancer.

18. Use of an anti-HER2 antibody-chelator conjugate of any one of claims 1 to 5 for preparing a medicament for diagnosing or treating HER2-expressing cancer.

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