Anti-her2 antibody, antibody-radionuclide conjugate, and use thereof

Abstract

Provided are a humanized / fully human HER2 antibody or an antigen-binding fragment thereof, and an antibody-radionuclide conjugate using same as a targeting group for radioactive labeling, showing a tumor-targeting enrichment ability.

Description

An anti-HER2 antibody, an antibody-nucleoside conjugate and its applications Technical Field

[0001] This invention relates to the fields of antibodies and nuclear medicine, specifically to an anti-HER2 antibody and its conjugated drug (especially antibody-nucleoside conjugates). This invention also relates to the application of the above-mentioned HER2 antibody and its conjugated drug (especially antibody-nucleoside conjugates). Background Technology

[0002] Antibody radionuclide conjugates (ARCs), also known as radiolabeled antibodies or radioimmunoconjugates, are an emerging type of precision oncology drug. They are conjugate drugs composed of antibodies (ligands), linkers, chelators, and radioisotopes that mediate targeting. The radionuclide reaches the target under the precise targeting action of the antibody and other ligands, exerting different functions such as imaging or therapy. Compared to antibody-drug conjugates (ADCs), antibody-nuclear conjugates (ARCs) have significant competitive advantages. For example, they do not require cell entry or linker breakage to release the drug; instead, they utilize the radiation generated during decay to kill target cells, thus improving the stability and safety of ARCs in vivo. ARCs kill cancer cells through radiation; within the radiation radius, even tumor cells without corresponding antigens can be targeted by stromal cells, damaging or killing them and disrupting the supply of nutrients from the stromal cells to the tumor cells, thereby achieving a therapeutic effect and exhibiting better resistance to drug resistance. With the approval of Novartis' Lutathera and Pluvicto, these drugs have become extremely popular due to their significant efficacy, sparking a global research boom and ushering in a new era of precision oncology using radionuclide drugs.

[0003] A key characteristic of antibody-nucleoside conjugates (ANCs) is that the target portion typically uses murine antibodies or murine-derived antibodies (Reference 1: Thurston, DE, & Pysz, I. (2021). Chemistry and Pharmacology of Anticancer Drugs (2nd ed.).). This is because for other types of antibody therapy (such as antibody drugs and antibody-drug conjugates), the focus is on maintaining the drug in the body for as long as possible to maximize tumor exposure. However, for ANCs, the effective payload is a radionuclide. To ensure medical safety and protect the health rights of radiation therapy staff, patients, and the public, ANCs tend to require shorter tumor exposure. Prolonged tumor exposure often leads to hematological toxicity and a lower tumor-to-background ratio. Therefore, murine antibodies or murine-derived antibodies with shorter half-lives are usually the standard choice for ANCs. However, mouse anti- or mouse-derived antibodies can be recognized by the human immune system, triggering a human anti-mouse antibody response, which weakens the efficacy of drugs and causes serious adverse reactions. Therefore, the selection of antibodies in antibody-nucleoside conjugates often becomes the biggest problem limiting drug screening and clinical application.

[0004] The earliest representative antibody-ribonucleoside conjugate was Pemtumomab (Theragyn). TM It is a kind of... 90Y-conjugated mouse monoclonal antibody specifically binds to the glycoform of MUC1 mucin. This protein is overexpressed on the surface of epithelial tumor cells, including ovarian, gastric, mammary, and lung cells. Although a phase II study in women with advanced ovarian cancer showed that 15 out of 21 women in remission responded well to pemtumomab treatment, and 14 patients survived for more than 8 years after treatment, the results of a subsequent phase III clinical trial were disappointing. The HAMA (human anti-murine antibody) effect may have been an insurmountable problem (Reference 2: Angèle LMOei; Fred CGJSweep; Leon FAGMassuger; André J. Olthaar; Chris MGThomas (2008). Transient human anti-mouse antibodies (HAMA) interference in CA 125 measurements during monitoring of ovarian cancer patients treated with murine monoclonal antibody.,109(2),199–202.), pemtumomab was not further developed.

[0005] To date, the U.S. Food and Drug Administration has approved only two antibody-nucleoside conjugates: tositumomab (Bexxar), which contains iodine-131. TM ) and ibritumomab tiuxetan (Zevalin) containing yttrium-90 TM Bexxar is a radioactive nuclide developed by GSK. 131 Zevalin is a radiolabeled mouse IgG2a monoclonal antibody conjugate targeting CD20, approved by the FDA in 2003 for the treatment of relapsed or refractory follicular or dysplastic non-Hodgkin's lymphoma. However, in 2014, due to a significant decrease in demand in the US market, GSK withdrew the drug, believing that more beneficial methods were available for treating these cancer types. 111 In) or yttrium 90 ( 90Y) A radiolabeled anti-CD20 mouse IgG1k monoclonal antibody conjugate, approved by the FDA in 2002 for the treatment of refractory relapsed B-cell non-Hodgkin lymphoma. Although Zevalin has shown favorable results in consolidation therapy for first-response advanced follicular lymphoma (prolonging progression-free survival by 2 years), no difference in PFS was observed between it and a rituximab-based regimen in a head-to-head comparison. For various reasons, Zevalin still faces many obstacles in its clinical application and commercialization.

[0006] With the application and development of nuclear medicine and next-generation monoclonal antibody therapies targeting solid tumor antigens, antibody-nucleoside conjugates (ANCs), as an important product type of radionuclide drug conjugates (RDCs), have gradually seen the emergence of many candidate drugs in clinical research stages, such as antibody-nucleoside conjugates targeting carcinoembryonic antigen (CEA). 225 Ac-DOTA-M5A (NCT05204147) targets mesothelin, prostate-specific membrane antigen, or human epidermal growth factor receptor 2 (HER2). 227 Th-labeled RIT formulations (NCT03507452, NCT03724747, NCT04147819) target human kallikrein 2. 225 Ac-DOTA-h11B6 (NCT04644770), and drugs targeting insulin-like growth factor type I receptors. 225 Despite the availability of products like Ac-FPI-1434, significant unmet clinical needs remain.

[0007] HER2 (human epidermal growth factor receptor 2), also known as ErbB-2 (Receptor tyrosine-protein kinase erbB-2), is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family, which includes EGFR (ErbB-1), HER2 / c-neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). The HER2 protein possesses extracellular ligand-binding domains (domains I-IV), a transmembrane domain, and an intracellular domain. HER2 forms a heterodimer with any of the other three receptors (ErbB-1, ErbB-3, and ErbB-4). Dimerization leads to autophosphorylation of tyrosine residues within the receptor's cytoplasmic domain, activating multiple signaling pathways, including MAPK (mitogen-activated protein kinase), PI3K / Akt (phosphoinositide 3-kinase), PKC (protein kinase C), and STAT (signal transducer and activator of transcription). HER2 gene amplification or overexpression plays a crucial role in the development and progression of some invasive breast cancers; 15%-30% of breast cancer patients are HER2-positive, making HER2 an important biomarker and therapeutic target for breast cancer. Furthermore, 7%-34% of gastric cancer patients and 30% of salivary duct carcinoma patients overexpress HER2. Therefore, HER2 expression detection is very important for the diagnosis and treatment of cancer, especially HER2-targeted molecular imaging, which has qualitative / quantitative and whole-body imaging features, and can simultaneously detect the dynamic changes in HER2 expression in primary lesions, single / multiple metastatic lesions, and during treatment.

[0008] Multiple studies have shown that HER2-targeted PET / CT molecular imaging can be used to visualize lesions in breast and gastric cancer, reveal the heterogeneity of HER2 expression in tumors, screen positive lesions in patients with negative primary HER2 expression, and accurately predict the efficacy of targeted therapy. 64 Cu or 89 Clinical studies of Zr-labeled intact antibodies (Trastuzumab, Pertuzumab) have emerged, demonstrating significant value in areas such as HER2-positive patient screening, prediction of HER2-targeted therapy efficacy, and monitoring of HER2 heterogeneity. For example, in 89In the Zr-Trastuzumab study, physicians completed questionnaires on diagnostic understanding and treatment decisions before, during, and ≥3 months after PET scans. A total of 20 patients were included. Eight patients had two types of primary cancer (HER2-positive and HER2-negative BC or BC and non-BC), seven patients were ineligible for biopsy, four had previous HER2-positive and HER2-negative metastases, and one patient had primary BC with uncertain HER2 status. Twelve patients had positive PET scans, seven had negative scans, and one had an indeterminate result. 89 Zr-Trastuzumab PET supported treatment decisions in 15 patients; imaging altered treatment in 8 patients; increased physician confidence without affecting patient treatment in 10 patients; and improved physician understanding of the disease in 18 patients. Circulating tumor cells (CTCs) were detected in 10 patients, with HER2 expression in 6. The HER2 status of CTCs was correlated with... 89 Zr-trastuzumab PET results are not related to treatment decisions. Studies have shown that when standard testing cannot determine HER2 status, 89 Zr-Trastuzumab PET can be used to support clinical decision-making (Reference 3: 89 Zr-Trastuzumab PET supports clinical decision making in breast cancer patients, when HER2 status cannot be determined by standard work up).

[0009] Therefore, HER2 antibody-nuclear conjugates can improve the diagnostic efficacy of HER2 status, providing a non-invasive and effective molecular imaging method for screening HER2-positive breast cancer and gastric cancer patients, monitoring HER2 expression, and predicting and evaluating the efficacy of targeted therapy. Radioactive molecular probes for the treatment of HER2-positive breast cancer and gastric cancer patients offer new treatment options after resistance to targeted therapy. This also provides new insights into the treatment of HER2-positive breast cancer and gastric cancer patients. However, while standard antibodies are structurally stable, their large molecular weight leads to poor tissue penetration, excessive retention in the blood, and hematologic toxicity. Furthermore, the long half-life of antibodies (up to several days) and their long metabolic cycle after entering the body result in prolonged retention of the carried nuclide, causing radiation toxicity. The long half-life also affects image quality and leads to long detection intervals. Therefore, there is an urgent need to provide more options for targeted HER2 antibodies and HER2 antibody-nuclear conjugates to address unmet clinical needs. Summary of the Invention

[0010] To address the aforementioned problems, this invention provides an antibody or antigen-binding fragment that specifically targets HER2, and an antibody-nucleoside conjugate with the HER2 antibody or antigen-binding fragment as the targeting group. This invention also provides applications of the antibody or antigen-binding fragment that specifically targets HER2 and its antibody-nucleoside conjugate.

[0011] Specifically, the present invention provides an antibody or antigen-binding fragment that specifically targets HER2. The antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region has the same CDR sequence as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or has a substitution of 1-2 amino acids with the CDR sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. And / or the light chain variable region has the same CDR sequence as SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or has a substitution of 1-2 amino acids with the CDR sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0012] The CDRs described can be defined according to existing antibody numbering systems (e.g., Kabat, Chothia, IMGT, Gelfand, Aho, Martin, etc.). Different antibody numbering systems often result in differences in the CDR sequences defined for the same sequence. Therefore, CDR sequences defined based on SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 using different antibody numbering systems are often different. Therefore, it is understood that all CDR sequences defined based on SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 should be within the scope of protection and disclosure of this patent.

[0013] As a preferred approach, the CDR described in this patent is defined according to the Kabat antibody numbering system.

[0014] Furthermore, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0015] (1) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:9, the VH-CDR2 amino acid sequence described in SEQ ID NO:10, and the VH-CDR3 amino acid sequence described in SEQ ID NO:11; or

[0016] (2) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:12, the VH-CDR2 amino acid sequence described in SEQ ID NO:13, and the VH-CDR3 amino acid sequence described in SEQ ID NO:14; or

[0017] (3) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:15, the VH-CDR2 amino acid sequence described in SEQ ID NO:16, and the VH-CDR3 amino acid sequence described in SEQ ID NO:17; or

[0018] (4) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:18, the VH-CDR2 amino acid sequence described in SEQ ID NO:19, and the VH-CDR3 amino acid sequence described in SEQ ID NO:20; and / or

[0019] (5) The light chain variable region comprises the VL-CDR1 amino acid sequence described in SEQ ID NO:21, the VL-CDR2 amino acid sequence described in SEQ ID NO:22, and the VL-CDR3 amino acid sequence described in SEQ ID NO:23; or

[0020] (6) The light chain variable region comprises the VL-CDR1 amino acid sequence described in SEQ ID NO:24, the VL-CDR2 amino acid sequence described in SEQ ID NO:25, and the VL-CDR3 amino acid sequence described in SEQ ID NO:26; or

[0021] (7) The light chain variable region comprises the VL-CDR1 amino acid sequence described in SEQ ID NO:27, the VL-CDR2 amino acid sequence described in SEQ ID NO:28, and the VL-CDR3 amino acid sequence described in SEQ ID NO:29; or

[0022] (8) The light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO:30, the VL-CDR2 amino acid sequence described in SEQ ID NO:31, and the VL-CDR3 amino acid sequence described in SEQ ID NO:32.

[0023] Furthermore, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0024] (1) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; and / or

[0025] (2) The amino acid sequence of the light chain variable region is identical to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0026] In some preferred embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0027] (1) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:9, the VH-CDR2 amino acid sequence described in SEQ ID NO:10, and the VH-CDR3 amino acid sequence described in SEQ ID NO:11; and

[0028] (2) The light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO:21, the VL-CDR2 amino acid sequence described in SEQ ID NO:22, and the VL-CDR3 amino acid sequence described in SEQ ID NO:23.

[0029] In other preferred embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0030] (1) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:12, the VH-CDR2 amino acid sequence described in SEQ ID NO:13, and the VH-CDR3 amino acid sequence described in SEQ ID NO:14; and

[0031] (2) The light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO:24, the VL-CDR2 amino acid sequence described in SEQ ID NO:25, and the VL-CDR3 amino acid sequence described in SEQ ID NO:26.

[0032] In other preferred embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0033] (1) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:15, the VH-CDR2 amino acid sequence described in SEQ ID NO:16, and the VH-CDR3 amino acid sequence described in SEQ ID NO:17; and

[0034] (2) The light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO:27, the VL-CDR2 amino acid sequence described in SEQ ID NO:28, and the VL-CDR3 amino acid sequence described in SEQ ID NO:29.

[0035] In other preferred embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0036] (1) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:18, the VH-CDR2 amino acid sequence described in SEQ ID NO:19, and the VH-CDR3 amino acid sequence described in SEQ ID NO:20; and

[0037] (2) The light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO:30, the VL-CDR2 amino acid sequence described in SEQ ID NO:31, and the VL-CDR3 amino acid sequence described in SEQ ID NO:32.

[0038] In some further preferred embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0039] (1) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:1, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:1; and

[0040] (2) The amino acid sequence of the light chain variable region is consistent with the amino acid sequence shown in SEQ ID NO:5, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:5.

[0041] In some further preferred embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0042] (1) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:2, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:2; and

[0043] (2) The amino acid sequence of the light chain variable region is consistent with the amino acid sequence shown in SEQ ID NO:6, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:6.

[0044] In some further preferred embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0045] (1) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:3, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:3; and

[0046] (2) The amino acid sequence of the light chain variable region is consistent with the amino acid sequence shown in SEQ ID NO:7, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:7.

[0047] In some further preferred embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and:

[0048] (1) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:4, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:4; and

[0049] (2) The amino acid sequence of the light chain variable region is consistent with the amino acid sequence shown in SEQ ID NO:8, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0050] Furthermore, the antibody or antigen-binding fragment includes monoclonal antibodies, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, fully humanized antibodies, or fusion proteins containing an antigen-binding portion of the antibody; preferably, the antibody is a humanized antibody or a fully humanized antibody; more preferably, the antibody is a humanized monoclonal antibody or a fully human monoclonal antibody.

[0051] Furthermore, the antibody or antigen-binding fragment contains an Fc domain.

[0052] Furthermore, the Fc domain is the human immunoglobulin Fc domain; preferably, the Fc domain is a human IgG1 domain, a human IgG2 domain, a human IgG3 domain, or a human IgG4 Fc domain; more preferably, the Fc domain is a human IgG1 domain or a human IgG4 domain.

[0053] Furthermore, the Fc domain comprises a hinge region, a CH2 domain, and a CH3 domain. In some preferred embodiments, the amino acid sequence of the Fc domain is identical to the amino acid sequence shown in SEQ ID NO:33, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:33. In other preferred embodiments, the amino acid sequence of the Fc domain is identical to the amino acid sequence shown in SEQ ID NO:42, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:42.

[0054] In some specific embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain and a light chain, and:

[0055] (1) The amino acid sequence of the heavy chain is identical to the amino acid sequence shown in SEQ ID NO:34, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:34; and

[0056] (2) The amino acid sequence of the light chain is identical to the amino acid sequence shown in SEQ ID NO:38, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:38.

[0057] In other specific embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain and a light chain, and:

[0058] (1) The amino acid sequence of the heavy chain is identical to the amino acid sequence shown in SEQ ID NO:35, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:35; and

[0059] (2) The amino acid sequence of the light chain is identical to the amino acid sequence shown in SEQ ID NO:39, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0060] In other specific embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain and a light chain, and:

[0061] (1) The amino acid sequence of the heavy chain is identical to the amino acid sequence shown in SEQ ID NO:36, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:36; and

[0062] (2) The amino acid sequence of the light chain is identical to the amino acid sequence shown in SEQ ID NO:40, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:40.

[0063] In other specific embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain and a light chain, and:

[0064] (1) The amino acid sequence of the heavy chain is identical to the amino acid sequence shown in SEQ ID NO:37, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:37; and

[0065] (2) The amino acid sequence of the light chain is identical to the amino acid sequence shown in SEQ ID NO:41, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:41.

[0066] In other specific embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain and a light chain, and:

[0067] (1) The amino acid sequence of the heavy chain is identical to the amino acid sequence shown in SEQ ID NO:43, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:43; and

[0068] (2) The amino acid sequence of the light chain is identical to the amino acid sequence shown in SEQ ID NO:38, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:38.

[0069] In other specific embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain and a light chain, and:

[0070] (1) The amino acid sequence of the heavy chain is identical to the amino acid sequence shown in SEQ ID NO:44, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:44; and

[0071] (2) The amino acid sequence of the light chain is identical to the amino acid sequence shown in SEQ ID NO:39, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0072] In other specific embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain and a light chain, and:

[0073] (1) The amino acid sequence of the heavy chain is identical to the amino acid sequence shown in SEQ ID NO:45, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:45; and

[0074] (2) The amino acid sequence of the light chain is identical to the amino acid sequence shown in SEQ ID NO:40, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:40.

[0075] In other specific embodiments, the antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain and a light chain, and:

[0076] (1) The amino acid sequence of the heavy chain is identical to the amino acid sequence shown in SEQ ID NO:46, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:46; and

[0077] (2) The amino acid sequence of the light chain is identical to the amino acid sequence shown in SEQ ID NO:41, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:41.

[0078] Specifically, the heavy chain variable region of the antibody provided by this invention and its CDR sequence defined by the Kabat antibody numbering system, the light chain variable region and its CDR sequence defined by the Kabat antibody numbering system, and the heavy chain and light chain amino acid sequences of the antibody are shown below. As mentioned above, different antibody numbering systems often result in some differences in the CDR sequences defined for the same sequence. Based on the heavy chain variable region, light chain variable region, heavy chain and light chain provided by this invention, the CDR sequences defined by different antibody numbering systems (including but not limited to Kabat, Chothia, IMGT, Gelfand, Aho, Martin, etc.) are all within the scope of disclosure and protection of this invention.

[0079] The present invention also provides an isolated nucleic acid molecule that encodes an antibody or antigen-binding fragment thereof targeting HER2 as described in any of the preceding claims.

[0080] The present invention also provides an expression vector comprising the nucleic acid molecules described in any of the preceding claims.

[0081] The present invention also provides a host cell comprising any of the nucleic acid molecules described in any of the preceding claims or any of the expression vectors described in any of the preceding claims.

[0082] The present invention also provides a method for preparing an antibody or antigen-binding fragment thereof targeting HER2 as described in any of the preceding claims, the method comprising:

[0083] a) Culture the host cells described in any of the preceding claims under conditions sufficient to induce the cells to produce the antibody targeting HER2 or its antigen-binding fragment, and

[0084] b) Collect the antibody or antigen-binding fragment thereof targeting HER2 produced by the host cells.

[0085] The present invention also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof targeting HER2 as described in any of the preceding claims, and a pharmaceutically acceptable carrier.

[0086] The present invention also provides a kit comprising an antibody or antigen-binding fragment thereof targeting HER2 as described in any of the preceding claims; or the kit comprising a pharmaceutical composition as described in any of the preceding claims.

[0087] The present invention also provides the use of the antibody or antigen-binding fragment thereof targeting HER2 as described in any of the foregoing claims, or the pharmaceutical composition or kit as described in any of the foregoing claims, in the preparation of a medicament for treating tumors or cancer.

[0088] Preferably, the treatment includes, but is not limited to, improving or optimizing cancer cell killing, delaying the progression or recurrence of the tumor or cancer.

[0089] Furthermore, the tumor or cancer mentioned is a cancer that expresses HER2.

[0090] Furthermore, the tumor or cancer mentioned is a HER2-low expression cancer, a HER2-medium expression cancer, or a HER2-high expression cancer.

[0091] Furthermore, the tumor or cancer mentioned is not limited to being selected from: breast cancer, small cell lung cancer, ovarian cancer, endometrial cancer, bladder cancer, head and neck cancer, prostate cancer, gastric cancer, cervical cancer, uterine cancer, esophageal cancer, and colorectal cancer, etc.

[0092] The present invention further provides the use of the antibody or antigen-binding fragment thereof targeting HER2 as described in any of the above claims in the preparation of XDC conjugates.

[0093] The present invention also provides an XDC conjugate comprising an antibody or antigen-binding fragment thereof targeting HER2 as described in any of the preceding claims.

[0094] The XDC conjugates mentioned in any of the foregoing claims of this invention are a general term for various conjugated drugs, consisting of a targeting molecule, a linker, and a cytotoxic drug, where X is a carrier, D is the payload, and C is the conjugate. Specifically, the carrier of the XDC conjugates involved in this invention is the antibody targeting HER2 or its antigen-binding fragment provided by this invention. All conjugated drug forms based on the antibody targeting HER2 or its antigen-binding fragment provided by this invention are within the scope of the XDC conjugates involved in this invention. In some preferred embodiments, the XDC conjugate refers to antibody-drug conjugates (ADCs); in other preferred embodiments, the XDC conjugate refers to antibody-radionuclide conjugates (ARCs); and in still other preferred embodiments, the XDC conjugate refers to antibody fragment conjugates (FDCs).

[0095] The present invention also provides a pharmaceutical composition comprising the XDC conjugate described in any of the preceding claims, and a pharmaceutically acceptable carrier.

[0096] The present invention also provides a kit comprising the XDC conjugate described in any of the preceding claims; or the kit comprising the pharmaceutical composition described in any of the preceding claims.

[0097] The present invention also provides the use of the XDC conjugate, the XDC-containing pharmaceutical composition, or the XDC-containing kit described in any of the foregoing claims in the preparation of a medicament for treating tumors or cancer. Preferably, the treatment includes, but is not limited to, improving or optimizing cancer cell killing, delaying the progression or recurrence of the tumor or cancer.

[0098] Furthermore, the tumor or cancer mentioned is a cancer that expresses HER2.

[0099] Furthermore, the tumor or cancer mentioned is a HER2-low expression cancer, a HER2-medium expression cancer, or a HER2-high expression cancer.

[0100] Furthermore, the tumor or cancer mentioned is not limited to being selected from: breast cancer, small cell lung cancer, ovarian cancer, endometrial cancer, bladder cancer, head and neck cancer, prostate cancer, gastric cancer, cervical cancer, uterine cancer, esophageal cancer, and colorectal cancer, etc.

[0101] The present invention also provides an antibody-isotope conjugate comprising an antibody or antigenic fragment thereof targeting HER2 as described in any of the preceding claims.

[0102] Furthermore, the antibody-nucleoside conjugate comprises an antibody-targeting portion and a radionuclide portion, wherein the antibody-targeting portion is any of the aforementioned antibodies targeting HER2 or their antigenic structural fragments, and the radionuclide is any selectable nuclide, and the radionuclide can be conjugated to the antibody-targeting portion directly or through any chelating group.

[0103] Furthermore, the radionuclide mentioned is a diagnostic radionuclide or a therapeutic radionuclide.

[0104] Furthermore, the radionuclides mentioned are exemplary selected from the following: 11 C 13 N、 15 O、 18 F, 34m Cl、 38 K, 43 Sc、 44 Sc、 45 Ti、 51 Mn, 52 Mn, 52m Mn, 52 Fe、 55 Co、 60 Cu、 61 Cu、 62 Cu、 64 Cu、 63 Zn, 66 Ga、 68 Ga、 69 Ge 71 As、 72 As、 74 As、 73 Se、 75 Br、 76 Br、 82 Rb、 82m Rb、 83 Sr、 86 Y、 89 Zr、 90 Nb, 94m Tc, 110m In、 118 Sb, 120 I, 122 I, 124 I, 152 Tb, 67 Ga、 99m Tc, 111 In、 123 I, 125 I, 155 Tb, 201 Tl、 32 P,47 Sc、 66 Cu、 67 Cu、 77 As、 77 Br、 89 Sr、 90 Y、 105 Rh、 103 Pd, 111 Ag、 117m Sn、 131 I, 133 Xe, 149 Tb, 161 Tb, 149 Pm, 153 Sm、 166 Ho、 177 Lu、 186 Re、 188 Re、 195m Pt, 212 Bi、 213 Bi、 211 At、 212 Pb, 223 Ra、 225 Ac、 230 U、 33 P, 59 Fe、 67 Cu、 67 Ga、 75 Se、 77 As、 99 Mo、 109 Pd, 142 Pr、 143 Pr、 166 Dy、 169 Er、 189 Re、 194 Ir、 198 Au、 199 Au、 199 Au、 211 Pb, 212 Bi.

[0105] It is understandable that the choice of whether or not to use a chelating agent can be determined based on the properties of the selected radionuclide (for example, some radionuclides can also be directly linked to the antibody or its antigenic structural fragment targeting HER2 provided by this invention without a chelating agent, such as...). 77 As、 131 I, 211 At, etc., are all non-metallic radionuclides that can be covalently bound to the antibody or its antigenic structural fragment targeting HER2 provided by this invention, and chelating agents with appropriate structures are selected according to the different properties of the radionuclides.

[0106] In some preferred embodiments, the radionuclide is directly conjugated to the antibody targeting HER2 or its antigenic structural fragment.

[0107] In some other preferred embodiments, the radionuclide is labeled with the antibody or its antigenic structural fragment targeting HER2 via a chelating agent.

[0108] Generally, chelating agents can be selected from the group consisting of DOTA or NOTA and their derivatives, cross-linked macrocyclic chelating agents, and sterically confined acyclic chelating agents.

[0109] In some preferred embodiments, the chelating agent may be exemplary selected from diethylenetriaminepentamethylenephosphonic acid (EDTMP) and its derivatives, diethylenetriaminepentaacetic acid (DTPA) and its derivatives, bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradecyl-1-yl)-methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]-pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutyryl]-amino]pentyl]-N-hydroxybutyramide (DFO) and its derivatives. Compounds, 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), 1,4,7,10-tetraazacyclododecane-N,N',N”,N”'-tetraacetic acid (DOTA), 2-[1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-glutaric acid (DOTAGA or DOTA-GA), 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), N,N'-dipyridoxyethylenediamine-N,N'-diacetic acid ester-5,5'-bis(phosphate) (DPDP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethylene glycol -O,O-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-di(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetic acid ester (HP-DOA3), 6-hydrazino-N-methylpyridine-3-carboxamide (HYNIC), 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononane Triacetic acid (NOTA), 4,11-di(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), terpyridine-di(methyleneamine)tetraacetic acid (TMT), 1,4,7,10-tetraazacyclotridecane-N,N',N”,N”'-tetraacetic acid (TRITA), and triethylenetetraminehexaacetic acid (TTHA), N,N′-di[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (H2macropa), 4-amino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-Dihydropyridin-2-ylmethyl)-carbamoyl]-ethyl}heptanedioic acid di-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-2-ylmethyl)-amide](THP), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphonic acid (TRAP), 2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid (DO3AM), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra[methylene(2-carboxyethyl)phosphonic acid](DOTPI), S-2-(4-benzyl isothiocyanate)-1,4, 7,10-Tetraazacyclododecanetetraacetic acid, hydrazine (HYNIC), 6-amino-6-methylperhydro-1,4-diazacyclo-N,N,N',N'-tetraacetic acid (AAZTA) and its derivatives, such as (6-pentanoic acid)-6-(amino)methyl-1,4-diazacyclotriacetic acid ester (DATA), pentadecane-1,4,7,10,13-penta-aminopentaacetic acid (PEPA), hexadecane-1,4,7,10,13,16-hexamine-hexaacetic acid (HEHR), 4-{[bis(phosphonomethyl))carbamoyl]methyl}-7,10-di(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid (BPAMD), N-(4-{[ Bis(phosphonomethyl)carbamoyl]methyl}-7,10-di(carboxymethyl)-nonane-1,4,7-triaminetriacetic acid (BPAM), 1,2-[{6-(carboxylate)pyridin-2-yl}methylamine]ethane (DEDPA, H2DEDPA), deferoxamine (DFO) and its derivatives, deferoxone, (4-acetamido-4-yl){2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl}-pimelic acid di-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-amide](CP256) and its derivatives such as YM103; tetraazacyclododecane- Phosphocyanic acid (TEAP), 6-amino-6-methylperhydro-1,4-diazapheno-N,N,N',N'-tetraacetic acid (AAZTA); 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosano-1,8-diamine (SarAr), 6,6′-[{9-hydroxy-1,5-bis-(methoxycarbonyl)-2,4-bis(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl}bis(methylene)]bis(pyridin-2-carboxylic acid)(H2bispa2), 1,2-[{6-(carboxylate)pyridin-2-yl}methylamino]-ethane (H2dedpa), N,N′-Di(6-carboxy-2-pyridinylmethyl)-ethylenediamine-N,N′-diacetic acid (H4octapa), N,N′-di(2-hydroxy-5-sulfonylbenzyl)-N,N′-di-(2-methylpyridinyl)ethylenediamine (H6Sbbpen) and their derivatives, triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid (TTHA), 2-aminomethylpiperidinetriacetic acid (2-AMPTA) and their derivatives, such as the further functionalized derivatives of 2-AMPTA having additional functional groups suitable for conjugation with peptide structures, 2-(N-(2-hydroxybenzyl)aminomethyl)piperidine (2-A MPTA-HB), 4-nitro-2-hydroxybenzyl-2-{[(6)-trans-2-[benzyl(carboxymethyl)amino]cyclohexyl](carboxymethyl)amino}acetic acid (RESCA) and its derivatives, and 6-carboxyl-1,4,8,11-tetraazaundecane (N4) and its derivatives, p-SCN-Bn-NOTA, NOTA-NHS-ester, p-SCN-Bn-DOTA, DOTA-NHS-ester, p-NCS-Bz-DFO, p-SCN-Bn-DTPA, wherein the chelating group optionally comprises a chelated radioactive or non-radioactive cation.

[0110] More preferably, the chelating agent may be further non-limitingly selected from DFO (deferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, DTPA (diethyltriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), TRITA (1,4,7,10-tetra(carboxymethyl)-1,4,7,10-tetraazacyclotridecane), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8, 11-Tetraacetic acid), EDTA (ethylenediaminetetraacetic acid), NODASA (1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid), NODAGA (1-(1-carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7-triazacyclononane), 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), DOTAGA, HP-DOA3, HYNIC, NCS-MP-NODA, NH2-MPAA-NODA, NODA and its derivatives, etc.

[0111] In some non-limiting examples, the chelating agent is preferably p-SCN-Bn-NOTA or p-SCN-Bn-DOTA.

[0112] It is understood that the purpose of this invention is to provide an antibody or antigen-binding fragment targeting HER2, and an antibody-nucleoside conjugate using the antibody or antigen-binding fragment targeting HER2 as the targeting unit. Based on the antibody or antigen-binding fragment targeting HER2 provided by this invention, it can be conjugated with any radionuclide in the prior art and necessary and selectable chelating agents according to clinical needs to form a target antibody-nucleoside conjugate. Therefore, the type and structure of the radionuclide and chelating agent, and the conjugation method should not be regarded as a limitation of this invention.

[0113] The present invention also provides a pharmaceutical composition comprising the antibody-nucleoside conjugate described in any of the preceding claims and a pharmaceutically acceptable carrier.

[0114] The present invention also provides a kit comprising the antibody-nucleoside conjugate described in any of the preceding claims; or the kit comprising a pharmaceutical composition described in any of the preceding claims.

[0115] In addition, the present invention provides the use of the antibody-nucleoside conjugates described in any of the foregoing claims, or the pharmaceutical compositions containing antibody-nucleoside conjugates described in any of the foregoing claims, or the reagent kits containing antibody-nucleoside conjugates described in any of the foregoing claims, in the preparation of medicaments for treating tumors or cancer.

[0116] In some preferred embodiments, the tumor or cancer is a cancer that expresses HER2.

[0117] More specifically, the tumor or cancer mentioned is a HER2-low expression cancer, a HER2-medium expression cancer, or a HER2-high expression cancer.

[0118] Furthermore, the tumor or cancer mentioned is not limited to being selected from: breast cancer, small cell lung cancer, ovarian cancer, endometrial cancer, bladder cancer, head and neck cancer, prostate cancer, gastric cancer, cervical cancer, uterine cancer, esophageal cancer, and colorectal cancer, etc.

[0119] The present invention also provides a method for treating or diagnosing a disease characterized by HER2 expression, the method comprising administering to a patient suffering from the disease characterized by HER2 expression a therapeutically effective amount of any of the preceding HER2 antibodies or antigen-binding fragments, or any of the preceding HER2 antibody-nucleoside conjugates, or any of the preceding pharmaceutical compositions, or any of the preceding kits.

[0120] Furthermore, the patient in question is a mammal.

[0121] Furthermore, the patient in question is a person.

[0122] The present invention also provides a method for acquiring images of a patient's target area, the method comprising the following steps:

[0123] a) administering to a patient an effective amount of any of the preceding antibody-isotope conjugates, or any of the preceding pharmaceutical compositions comprising the antibody-isotope conjugates, or any of the preceding kits comprising the antibody-isotope conjugates; and

[0124] b) Perform positron emission tomography (PET) or SPECT on the patient;

[0125] c) Detectable signals that identify radionuclides;

[0126] d) Generate an image based on the detectable signal to obtain an image of the area to be detected in the patient.

[0127] Furthermore, the patient in question is a mammal.

[0128] Furthermore, the patient in question is a person.

[0129] Furthermore, the patient has or may have a disease characterized by HER2 expression.

[0130] Furthermore, the disease characterized by HER2 expression is cancer.

[0131] Furthermore, the cancer mentioned refers to cancer with low HER2 expression, moderate HER2 expression, or high HER2 expression.

[0132] Preferably, the tumor or cancer is selected, without limitation, from: breast cancer, small cell lung cancer, ovarian cancer, endometrial cancer, bladder cancer, head and neck cancer, prostate cancer, gastric cancer, cervical cancer, uterine cancer, esophageal cancer, and colorectal cancer, etc.

[0133] The HER2-targeting antibody or its antigen-binding fragment provided by this invention is preferably a humanized / fully human antibody or its antigen-binding fragment. While possessing excellent targeting affinity and binding activity, it effectively avoids immune reactions, ensuring efficacy while reducing the risk of immune rejection and increasing drug safety. The antibody-nucleoside conjugate, radiolabeled with the HER2-targeting antibody or its antigen-binding fragment provided by this invention as the targeting group, exhibits high radiolabeling efficiency, structural stability, strong tissue penetration, low hematologic toxicity, high tumor uptake, low background, and continuous accumulation in tumors over time. It demonstrates excellent tumor uptake imaging at the tumor site, producing excellent imaging results with high image quality. Furthermore, it exhibits rapid clearance in non-target organs such as the liver and normal tissues. This indicates that the antibody provided by this invention not only has superior tumor-targeting enrichment ability but also a suitable blood circulation half-life and metabolic cycle, allowing the carried nuclide to remain in the body for an appropriate time, thereby ensuring tumor uptake while reducing radiation toxicity side effects. In summary, the HER2-targeting antibodies or their antigen-binding fragments and antibody-nucleoside conjugates provided by this invention can not only achieve specific uptake and accumulation in the target organ during diagnosis / treatment, but also exhibit lower toxicity and side effects, showing excellent clinical application potential. Attached Figure Description

[0134] Figure 1 shows the binding activity assay (flow cytometry) of the antibodies LNCX006-002, LNCX006-095, LNCX006-072, LNCX006-190 and the positive control pertuzumab provided in this invention in the breast cancer cell line SK-BR-3.

[0135] Figure 2 shows the binding activity assay (flow cytometry) of the antibodies LNCX006-002, LNCX006-095, LNCX006-072, LNCX006-190 and the positive control pertuzumab provided in this invention in the breast cancer cell line SKOV-3.

[0136] Figure 3 shows the affinity test results (ELISA method) of the antibodies LNCX006-002, LNCX006-095, LNCX006-072, LNCX006-190, the positive control pertuzumab, and trastuzumab provided in this invention for HER2.

[0137] Figure 4 is 177 Antibody labeling rate of Lu-DOTA-LNCX006-002.

[0138] Figure 5 is 177 Radiochemical purity of Lu-DOTA-LNCX006-002.

[0139] Figure 6 is 177 Antibody labeling rate of Lu-DOTA-LNCX006-095.

[0140] Figure 7 is 177 Radiochemical purity of Lu-DOTA-LNCX006-095.

[0141] Figure 8 is 177 Antibody labeling rate of Lu-DOTA-LNCX006-072.

[0142] Figure 9 is 177 Radiochemical purity of Lu-DOTA-LNCX006-072.

[0143] Figure 10 is 177 Antibody labeling rate of Lu-DOTA-LNCX006-190.

[0144] Figure 11 is 177 Radiochemical purity of Lu-DOTA-LNCX006-190.

[0145] Figure 12 shows antibody-nucleoside conjugates. 177 Lu-DOTA-LNCX006-072 and 177 Specific binding of Lu-DOTA-LNCX006-190 in the CT26-HER2 cell line.

[0146] Figure 13 is 177 SPECT image of Lu-DOTA-LNCX006-002 in a mouse model.

[0147] Figure 14 is 177 SPECT image of Lu-DOTA-LNCX006-190 in a mouse model.

[0148] Figure 15 is 177 SPECT images of Lu-DOTA-Pertuzumab in a mouse model.

[0149] Figure 16 is 177 SPECT images of Lu-DOTA-Trastuzumab in a mouse model.

[0150] Figure 17 is 177 SPECT image of Lu-DOTA-LNCX006-095 in a mouse model.

[0151] Figure 18 is 177 SPECT image of Lu-DOTA-LNCX006-072 in a mouse model.

[0152] Figure 19 is 177 Tumor growth curve of Lu-DOTA-LNCX006-072 mice.

[0153] Figure 20 is 77 Curve of weight change in Lu-DOTA-LNCX006-072 mice.

[0154] Figure 21 is 89 PET imaging of Zr-DFO-LNCX006-002 in the human body.

[0155] Figure 22 is 89 PET images of Zr-DFO-LNCX006-002 at different time points in the human body Detailed Implementation

[0156] Before describing the invention in detail, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as they can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims.

[0157] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are as defined in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)” (Leuenberger, HGW, Nagel, B. and Klbl, Hb, eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). Numerous documents are referenced throughout this specification. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturer’s instructions, operating guidelines, etc.) is incorporated herein by reference in its entirety, both above and below.

[0158] Unless the context otherwise requires, throughout this specification and the following claims, the word "comprising" and its variations such as "including" or "containing" will be understood to implicitly include the stated integers or steps, or groups of integers or steps, but not exclude any other integers or steps, or groups of integers or steps. In the following paragraphs, the same aspects of the invention will be defined in more detail. Each aspect thus defined may be combined with any other one or more aspects unless expressly indicated to the contrary. In particular, any of the optional, preferred, or advantageous features may be combined with any other optional, preferred, or advantageous feature.

[0159] In this document, antibody refers to a naturally occurring or partially or completely synthetically produced immunoglobulin. Antibodies can be isolated from natural sources such as naturally occurring plasma or serum, from the supernatant of a culture of antibody-producing hybridoma cells, or can be synthesized partially or completely using techniques such as genetic recombination. Preferred examples of antibodies include immunoglobulin isotypes and subtypes of those isotypes. Nine classes (isotypes) of human immunoglobulins are known: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. The antibodies of the present invention may include IgG1, IgG2, IgG3, and IgG4 of these isotypes. Various allotype sequences of human IgG1, human IgG2, human IgG3, and human IgG4 constant regions due to genetic polymorphism are described in Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, but in this invention, any of them may be used. Specifically, as a sequence of human IgG1, the amino acid sequence at positions 356-358, indicated by the EU number, can be DEL or EEM. Furthermore, for the human Igκ (Kappa) constant region and the human Igλ (Lambda) constant region, multiple allotropic sequences due to genetic polymorphism are described in the protein sequences of immunological interest of this invention, NIH Publication No. 91-3242, and any one of them can be used.

[0160] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0161] Example 1: Construction of HER2 antibody molecules

[0162] 1. Carrier Construction

[0163] The variable region sequence of the antibody was obtained through gene synthesis, ligated to the constant region by overlap PCR, and loaded into the expression vector PcDNA3.4 using homologous recombination. Positive bacterial cultures with correctly sequenced clones were selected, expanded cultured, and low endotoxin plasmids were extracted and sequenced for verification.

[0164] 2. Plasmid extraction

[0165] Materials: Packed column, microporous filter column, centrifuge tube, EP tube, filter screen

[0166] Reagents: Buffer P1 (P1 with added RNase A, store at 4℃), RNase A (store at -20℃), Buffer P2 (if SDS precipitation occurs, preheat in a 37-42℃ water bath), Buffer P3, ER Buffer (store at 4℃), Elution Buffer, QBT, QC, isopropanol, 75% ethanol, deionized water

[0167] Reagent preparation:

[0168] 75% ethanol: Prepare anhydrous ethanol and deionized water in a 3:1 ratio, mix well and store at room temperature.

[0169] Buffer P1 with added Rnase A: Rnase A and P1 solutions were prepared at a ratio of 6:1000, mixed well and stored at 4°C.

[0170] Experimental steps:

[0171] (1) Collect 100 mL of overnight (12-16 h) cultured bacterial solution into a labeled 50 mL centrifuge tube. Collect about 45 mL of bacterial solution at a time (check that the numbers are consistent). Centrifuge at 8000 rpm for 4 min using an angle rotor centrifuge or at 4500 rpm for 15 min using a horizontal rotor centrifuge. After centrifuging, discard the supernatant and retain the bacterial cells. Repeat the above operation once more and mark the first centrifuge tube cap in each row on a 36-well acrylic rack with a checkmark to indicate that the bacterial collection is complete.

[0172] (2) Add 10 mL of Buffer P1 containing RNase A to the centrifuge tube and vortex for 6 min until no obvious clumps of bacteria are visible.

[0173] (3) Add 10 mL of Buffer P2 to the centrifuge tube (if SDS precipitates, preheat in a 37-42℃ water bath until no reagent precipitates), then immediately and gently rotate manually 3-9 times or place on a mixer at 45000 rpm for 60 seconds to mix. At this point, the bacterial solution will change from turbid to viscous liquid.

[0174] (4) After adding 10 mL of Buffer P3 to the centrifuge tube, immediately invert it manually 5-15 times or place it on a mixer at 70,000 rpm for 90 seconds to mix. When a white flocculent precipitate appears, centrifuge it at 11,000 rpm for 4 minutes in an angle rotor centrifuge or at 4,500 rpm for 4 minutes in a horizontal rotor centrifuge.

[0175] (5) Add 10 mL of ER Buffer to the 50 mL centrifuge tube after centrifugation, manually invert the tube 3-5 times to mix thoroughly, or place it on a mixer at 45000-70000 rpm for 10 seconds to mix.

[0176] (6) Before the filtration and loading step, add 25 mL of QBT reagent to the chromatography column that is placed on the centrifuge tube rack and filled with packing material to equilibrate the packing material.

[0177] (7) Filter the sample treated with ER Buffer to remove the white precipitate, and add the remaining liquid to a clean, labeled chromatography column.

[0178] (8) After the liquid has finished dripping, add 60 ml of QC gravity column chromatography solution to the chromatography column.

[0179] (9) After the QC titration is complete, place the chromatography column into a clean, labeled 50mL centrifuge tube and check that the numbers match. Add 10mL of Elution Buffer and allow it to elute by gravity.

[0180] (10) After elution, gently press the chromatography column against the centrifuge tube wall and then quickly remove it. Add 7 mL of isopropanol to the collected filtrate, invert and mix 3-5 times, and centrifuge at 11000 rpm for 15 min in an angle rotor centrifuge or at 4500 rpm for 15 min in a horizontal centrifuge.

[0181] (11) Add 75% ethanol: After centrifugation, gently pour off the supernatant, place the centrifuge tubes on the rack in order, and add 5 mL of 75% ethanol to each tube to rinse the precipitate thoroughly. After adding the sample, centrifuge at 11000 rpm for 10 min in an angle rotor centrifuge or at 3700 rpm for 10 min in a horizontal centrifuge.

[0182] (12) After centrifugation, gently pour off the supernatant and invert the centrifuge tubes onto a tissue in ascending order. Add 50-500 μl of deionized water to the location of the plasmid and blow it 5-25 times to fully dissolve the plasmid in the water. When there is no adsorption of liquid on the tube wall, the plasmid has been completely eluted. Use a pipette to aspirate the dissolved liquid and transfer it to the corresponding microporous filter column according to the serial number of the centrifuge tube. Centrifuge in an angle rotor centrifuge at 14000 rpm for 15-30 min. After centrifugation, transfer the plasmid to the corresponding EP tube according to the serial number of the microporous filter column.

[0183] 3. Transfection

[0184] Transfection and expression: expression in 400ml

[0185] (1) Take 2900M cells and centrifuge to remove the supernatant.

[0186] (2) Add about 4.5 ml of electroporation buffer to the cells, mix well, and then add an appropriate amount of plasmid (concentration above 500 ng / ul).

[0187] (3) After thoroughly mixing the above cell plasmid suspension, take 10 ml and add it to a 10 ml electroporation tube. Place the electroporation tube into the electroporation instrument for electroporation.

[0188] (4) After the electroporation is completed, the cells in the electroporation tube are dispensed into a shake flask containing 130 ml of culture medium and incubated for 40 min.

[0189] (5) After incubation, place the shake flask at 37°C, 120 rpm, and 8% CO2 for 24 hours. Add feed / sodium butyrate / double antibiotics and continue culturing for 4 days.

[0190] 4. Antibody purification

[0191] Experimental method: Protein A pre-packed column affinity chromatography purification

[0192] (1) Equilibration chromatography column: 1xPBS, flow rate 1ml / min, 20ml

[0193] (2) Sample loading: flow rate 1 ml / min

[0194] (3) Washing: 1xPBS, flow rate 1ml / min, 20ml

[0195] (4) Elution: Sodium acetate buffer (pH 3.4), 1 ml / min, collected in aliquots, approximately 500 μl per tube. A total of 10 tubes were collected, and the absorbance at 280 nm was read using a NanoDrop instrument.

[0196] (5) Dialysis: Aspirate high-concentration protein into a dialysis bag and place it in a beaker containing PBS and pH 7.2-7.4 for dialysis.

[0197] The vector was constructed, plasmids were extracted, transfected and expressed, and antibodies were purified according to the above method to obtain the specific HER2 antibodies of the present invention, which were named LNCX006-002, LNCX006-072, LNCX006-095, and LNCX006-190.

[0198] The CDR sequence (defined by Kabat), variable region sequence, and heavy and light chain amino acid sequences of LNCX006-002 are shown below.

[0199] The CDR sequence (defined by Kabat), variable region sequence, and heavy and light chain amino acid sequences of LNCX006-072 are shown below.

[0200] The CDR sequence (defined by Kabat), variable region sequence, and heavy and light chain amino acid sequences of LNCX006-095 are shown below.

[0201] The CDR sequence (defined by Kabat), variable region sequence, and heavy and light chain amino acid sequences of LNCX006-190 are shown below.

[0202] Other antibodies can also be constructed using the above construction method, such as LNCX006-0021 (heavy chain amino acid sequence as shown in SEQ ID NO:43, light chain amino acid sequence as shown in SEQ ID NO:38), LNCX006-0721 (heavy chain amino acid sequence as shown in SEQ ID NO:44, light chain amino acid sequence as shown in SEQ ID NO:39), LNCX006-0951 (heavy chain amino acid sequence as shown in SEQ ID NO:45, light chain amino acid sequence as shown in SEQ ID NO:40), and LNCX006-1901 (heavy chain amino acid sequence as shown in SEQ ID NO:46, light chain amino acid sequence as shown in SEQ ID NO:41).

[0203] Example 2: Combining Affinity Measurement

[0204] 1. Flow cytometry

[0205] The HER2-targeting binding activity of antibodies LNCX006-002, LNCX006-095, LNCX006-072, and LNCX006-190 was measured by flow cytometry. (5 × 10⁻⁶ cells were used.) 5 SK-BR-3 and SKOV3 cells were aliquoted into EP tubes, washed with 1×PBS, and centrifuged, discarding the supernatant. Primary antibody (10 μg / ml) was diluted to 100 μl, and the cells were resuspended and incubated on ice for 1 hour. Cells were separated by centrifugation and washed with incubation buffer. The supernatant was discarded. Cells were resuspended in 100 μl of a diluted fluorescently labeled secondary antibody. Cells were incubated on ice for 1 hour. Cells were separated by centrifugation and washed with incubation buffer. The supernatant was discarded, and the cells were ready for analysis.

[0206] The experiment included a blank control group, a negative control group, and a positive control group. Pertuzumab was used as the positive control group. The blank control group consisted of SK-BR-3 cells and SKOV3 cells. The negative control group consisted of fluorescent secondary antibody (FITC). The pertuzumab used in the positive control group was commercially available.

[0207] The results showed (see Figures 1 and 2) that the HER2 antibodies LNCX006-002, LNCX006-095, LNCX006-072, and LNCX006-190 provided by this invention exhibited good binding activity on the breast cancer cell line SK-BR-3 and the ovarian cancer cell line SKOV3, and their flow cytometry fluorescence intensity was comparable to that of the positive control group (pertuzumab). This indicates that the antibodies provided by this invention exhibited good targeted binding activity at the cellular level.

[0208] 2. ELISA testing

[0209] The affinity of antibodies LNCX006-002, LNCX006-095, LNCX006-072, and LNCX006-190 was determined. Human HER2-antigen was coated onto ELISA plates at a concentration of 2 μg / mL in 1×PBS, 30 μL per well, and incubated overnight at 4°C. The plates were then washed three times with PBST and blocked with 5% PBSM for 2 hours at room temperature. After washing three times with PBST, 30 μL of diluted antibody in 1% PBSM was added to each well and incubated for 60 minutes at room temperature. The plates were then washed three times with PBST, and 30 μL of Goat-Anti-Human-IgG-Fc-HRP (abcam) was added at a 1:8000 dilution in 1% PBSM, incubated for 50 minutes at room temperature. Finally, the plates were washed six times with PBST, TMB was added, and the reaction was stopped with 2M stop solution. The OD value was read at 450.

[0210] The experiment included pertuzumab and trastuzumab as positive control groups, with trastuzumab being a commercially available drug.

[0211] The results (see Figure 3) show that the ELISA binding EC50 values ​​of the HER2 antibodies LNCX006-002, LNCX006-095, LNCX006-072, and LNCX006-190 provided by this invention are comparable to those of the positive control groups (pertuzumab and trastuzumab). This indicates that the HER2 antibodies LNCX006-002, LNCX006-095, LNCX006-072, and LNCX006-190 provided by this invention are comparable to those of the positive control groups (pertuzumab and trastuzumab). X006-190 all exhibited good binding affinity to the HER2 protein. Moreover, the EC50 values ​​of LNCX006-002, LNCX006-072, and LNCX006-190 were lower than those of the positive control group (pertuzumab and trastuzumab), indicating that LNCX006-002, LNCX006-072, and LNCX006-190 showed even better affinity than pertuzumab and trastuzumab.

[0212] Example 3: Preparation of Antibody-Nucleoside Conjugates

[0213] The HER2 antibody provided by this invention is radiolabeled using a common antibody radiolabeling method. In this embodiment, the radionuclide selected is exemplarily... 177 Lu, the chelating agent selected is p-SCN-Bn-DOTA.

[0214] The antibody LNCX006-002 obtained in Example 1 was mixed with the chelating agent p-SCN-Bn-DOTA at a molar ratio of 1:20 in carbonate-bicarbonate buffer (pH 9.2) at room temperature for 2 hours. After the reaction, the mixture was purified using an ultrafiltration tube with a molecular cutoff of 30 kDa. The solvent was replaced with 0.01 M pH 7.4 PBS buffer to obtain the conjugate of the target product antibody and the chelating agent, which was named DOTA-LNCX006-002.

[0215] Add the following sequentially to 300 μL of 0.1 M sodium acetate solution (pH 4.5-5) 177 LuCl3 solution and DOTA-LNCX006-002, wherein DOTA-LNCX006-002 and 177 The LuCl3 ratio was 1 μg:10 μCi, and the reaction was carried out in a constant temperature shaker at 37 °C and 400 rpm for 1 h. After the reaction, the solution was purified using a PD-10 pre-packed gel column to obtain... 177 The Lu-labeled antibody-nuclein conjugate was named... 177Lu-DOTA-LNCX006-002, with an antibody labeling rate of 90.66% (see Figure 4) and a radiochemical purity greater than 99% (see Figure 5). Antibodies LNCX006-095, LNCX006-072, and LNCX006-190 were radiolabeled using the above method to obtain antibody-nucleoside conjugates. 177 Lu-DOTA-LNCX006-095 177 Lu-DOTA-LNCX006-072 177 Lu-DOTA-LNCX006-190, where: 177 The antibody labeling rate of Lu-DOTA-LNCX006-095 was 87.47% (see Figure 6), and the radiochemical purity was greater than 98% (see Figure 7). 177 The antibody labeling rate of Lu-DOTA-LNCX006-072 was 85.65% (see Figure 8), and the radiochemical purity was greater than 98% (see Figure 9). 177 The antibody labeling rate of Lu-DOTA-LNCX006-190 was 72.12% (see Figure 10), and the radiochemical purity was greater than 97% (see Figure 11). This indicates that the antibody provided by the present invention can be well labeled with radionuclides and is suitable for preparation into antibody-nuclide conjugates.

[0216] Example 4: Specific binding of antibody-isotope conjugates

[0217] CT26-HER2 mouse colorectal cancer cells transfected with human HER2 were seeded in 24-well plates, with approximately 1 × 10⁶ cells per well. 5 500 μL of serum-free 1640 medium was added to each well. The experiment included a closed group and an experimental group. The closed group was incubated with 10 μg of unlabeled pertuzumab or trastuzumab 0.5 h in advance. Then, 37 kBq (1 μCi) of serum-free 1640 medium was added to each well in both the closed and experimental groups. 177 Lu-DOTA-LNCX006-190 or 177 After incubating Lu-DOTA-LNCX006-072 at 37℃ for 0.5h, 1h, 2h, 4h, 6h, and 24h, the culture medium was discarded, and the cells were washed twice with PBS. 500μL of pH=4 dissociation buffer was added to each well, and the reaction was allowed to proceed for 10min. The dissociation buffer was collected, followed by washing once with 500μL of PBS per well. The PBS wash was collected and combined with the dissociation buffer, and the two were used as dissociation products for radioactivity counting to obtain cell membrane binding counts. Subsequently, 500μL of 0.1M NaOH solution was added to each well to lyse the cells, and the lysis products were collected for radioactivity counting to obtain cell internalization counts. An automated gamma counter was used to detect 1μCi... 177 Lu-DOTA-LNCX006-190 or177 Lu-DOTA-LNCX006-072, radioactivity counts of membrane dissociation products and lysis products per well, expressed as a percentage of cellular uptake. Cellular uptake % = (membrane-bound count + internalized count) / 1 μCi 177 Lu-DOTA-LNCX006-190 or 177 Lu-DOTA-LNCX006-072 count value × 100.

[0218] The results show (see Figure 12) that the antibody-nucleoside conjugates of HER2 antibodies LNCX006-072 and LNCX006-190 provided by this invention are effective. 177 Lu-DOTA-LNCX006-072 and 177 Lu-DOTA-LNCX006-190 exhibited good uptake activity in the CT26-HER2 cell line. The same method was used to analyze the antibody-isotope conjugates of LNCX006-002 and LNCX006-095. 177 Lu-DOTA-LNCX006-002 and 177 Lu-DOTA-LNCX006-095 was verified, and the results all indicated that... 177 Lu-DOTA-LNCX006-002 and 177 Lu-DOTA-LNCX006-095 exhibited good uptake activity in the CT26-HER2 cell line.

[0219] Example 5: In vivo SPECT imaging study in the CT26-HER2 model

[0220] The CT26-HER2 cell line, which has been stably converted to human HER2, was divided into groups of approximately 5 × 10⁻⁵ cells per cell. 5 A certain number of cells were inoculated into the axilla of BALB / c mice, and the tumor size was monitored every other day. When the long diameter reached about 1 cm, in vivo imaging was performed. The experiment was set up with an experimental group and a positive control group, with 3-5 mice in each group. The experimental group used the antibody-nucleoside conjugate provided in this invention. 177 Lu-DOTA-LNCX006-002 177 Lu-DOTA-LNCX006-072, the positive control group used was 177 Lu-DOTA-Pertuzumab and 177 Lu-DOTA-Trastuzumab (using the method provided in Example 3 of this invention to radiolabel pertuzumab and trastuzumab to obtain antibody-nucleoside conjugates) 177 Lu-DOTA-Pertuzumab and 177Lu-DOTA-Trastuzumab). Mice in the experimental group and the positive control group were injected with approximately 500 μCi (approximately 200 μL) via the tail vein, and in vivo SPECT imaging was performed at 1 h, 4 h, 24 h, 48 h, 72 h, 96 h, and 168 h after injection.

[0221] The results showed that antibody-nucleoside conjugates 177 Lu-DOTA-LNCX006-002 (see Figure 13), 177 Lu-DOTA-LNCX006-190 (see Figure 14) both showed good tumor uptake imaging at the tumor site, and compared with the positive control, 177 Lu-DOTA-LNCX006-002 177 Lu-DOTA-LNCX006-190 showed high tumor uptake, low background, and superior imaging performance compared to the positive control group. 177 Lu-DOTA-Pertuzumab (see Figure 15) and 177 Lu-DOTA-Trastuzumab (see Figure 16). Furthermore, it can be observed that... 177 Lu-DOTA-LNCX006-002 and 177 Compared to Lu-DOTA-LNCX006-190 177 Lu-DOTA-Pertuzumab and 177 The significantly reduced uptake of Lu-DOTA-Trastuzumab in the blood and some glands such as the salivary glands further enhances its safety.

[0222] Using the above method to 177 Lu-DOTA-LNCX006-095 and 177 The imaging performance of Lu-DOTA-LNCX006-072 was studied, and the results all indicate that... 177 Lu-DOTA-LNCX006-095 (see Figure 17) and 177 Lu-DOTA-LNCX006-072 (see Figure 18) showed good tumor uptake imaging at the tumor site, and its uptake in blood and some glands such as salivary glands was significantly better than that in other tumor sites. 177 Lu-DOTA-Pertuzumab and 177 Lu-DOTA-Trastuzumab.

[0223] Example 6: Treatment Study in the NCI-N87 Model

[0224] For further research 177In this embodiment, Lu-DOTA-LNCX006-002 was used in a therapeutic study within the NCI-N87 tumor model. Specifically, NCI-N87 tumor cells were divided into groups of approximately 5 × 10⁻⁶ cells per tumor cell line. 6 A certain number of cells were inoculated into the right upper limb of BALB / c nude mice, and the tumor size was monitored the next day. When the tumor volume reached approximately 100 mm², the tumor was counted. 3 A treatment experiment was conducted, with the experiment set up as a saline group and a 0.25mCi group. 177 Lu-DOTA-LNCX006-002 group, dual-dose 0.1mCi 177 The Lu-DOTA-LNCX006-002 group, with 7-8 mice per group, was given different doses of... 177 Lu-DOTA-LNCX006-002 was administered via tail vein injection (approximately 200 μL). Mice in the saline group received 200 μg of saline per injection. Tumor size, body weight, and survival status were monitored every other day after injection. Tumors exceeding 1500 mm² were considered normal. 3 Or a weight loss of more than 20% can be used as the monitoring endpoint.

[0225] As shown in Figures 19-20, the results are as follows: 177 Lu-DOTA-LNCX006-002 showed good inhibitory effects on NCI-N87 tumors. After 46 days of monitoring, the tumor volume in the saline group mice had exceeded 1500 mmHg. 3 In contrast 177 The Lu-DOTA-LNCX006-002 treatment group showed no significant increase in tumor size, and the tumor growth rate was significantly slowed. Both the 0.25 mCi group and the 0.1 mCi dual-dose treatment group produced significant tumor inhibition, and the mouse body weight did not change significantly, demonstrating its good safety profile. This indicates that the invention provides… 177 Lu-DOTA-LNCX006-002 produced good tumor suppression in a HER2-positive NCI-N87 tumor model, with both 0.25 mCi and 0.1 mCi dual-dose regimens showing positive results. 177 The tumor suppression effects of the Lu-DOTA-LNCX006-002 treatment group were similar, both significantly better than the saline control group, while maintaining a high level of safety.

[0226] Example 7 89 In vivo imaging study of Zr-DFO-LNCX006-002

[0227] Subject preparation: Fasting and water restriction are not required before the imaging; subjects can undergo the examination under normal conditions. Basic vital signs should be monitored to ensure the patient's general condition is stable.

[0228] Radiopharm injection: 89 Zr-DFO-LNCX006-002 was prepared under GMP conditions, with a radiochemical purity >95% and a specific activity meeting clinical requirements. Subjects received intravenous injection. 89 Zr-DFO-LNCX006-002, dosage approximately 74–111 MBq (approximately 2–3 mCi), corresponding to an antibody dose of 2–3 mg. After intravenous injection, subjects should rest in a quiet environment and avoid strenuous activity.

[0229] Imaging acquisition: Whole-body PET / CT imaging was performed at different time points (4h, 24h, 48h, 96h) after injection. The PET acquisition range was from the head to the thigh, and the acquisition time for the head and body was 4.0 minutes per bed.

[0230] Image Analysis: Image Reconstruction and Analysis: The OSEM (ordered-subset expectation maximization) algorithm was used for image reconstruction, followed by CT attenuation correction. Images were reviewed to observe lesion morphology, size, and CT values, and visual and semi-quantitative analyses (SUVmax) were performed.

[0231] As shown in Figures 21-22, the results indicate that the patient received an intravenous injection. 89 Following Zr-DFO-LNCX006-002, PET / CT imaging was performed at different time points: 4h, 24h, 48h, and 96h. Results showed that tumor lesions in all patients were clearly visualized, and the imaging contrast gradually increased over time; SUV max The range is approximately 2.76–12.44, significantly higher than the surrounding normal tissue; high-quality imaging can still be obtained after 96 hours, demonstrating a long in vivo retention time and good imaging stability. This indicates that the invention provides… 89 Zr-DFO-LNCX006-002 imaging agent enables highly specific HER2 imaging of tumors at multiple time points. No fasting or glucose control is required before imaging, simplifying the procedure; it exhibits high HER2 specificity for tumor tissue; and its long imaging duration meets the needs of long-term dynamic observation and multi-time-point assessment, demonstrating significant advantages in molecular imaging of HER2-positive tumors.

[0232] In summary, the HER2-targeting antibody or its antigen-binding fragment provided by this invention is preferably a humanized / fully human antibody or its antigen-binding fragment. While possessing excellent targeting affinity and binding activity, it effectively avoids immune reactions, ensuring efficacy while reducing the risk of immune rejection and increasing drug safety. The antibody-nucleoside conjugates radiolabeled with the HER2-targeting antibody or its antigen-binding fragment provided by this invention as the targeting group exhibit high radiolabeling efficiency, structural stability, strong tissue penetration, low hematologic toxicity, high tumor uptake, low background, and can continuously accumulate in tumors over time, demonstrating excellent tumor uptake imaging effects at the tumor site, with excellent imaging results and high image quality. Furthermore, it exhibits rapid clearance rates in non-target organs such as the liver and normal tissues. This indicates that the antibody provided by this invention not only has superior tumor-targeting enrichment ability but also a suitable blood circulation half-life and metabolic cycle, allowing the carried nuclide to have an appropriate residence time in the body, thereby ensuring tumor uptake while reducing the toxic side effects of radiation on the human body. In summary, the HER2-targeting antibodies or their antigen-binding fragments and antibody-nucleoside conjugates provided by this invention can not only achieve specific uptake and accumulation in the target organ during diagnosis / treatment, but also exhibit lower toxicity and side effects, showing excellent clinical application potential.

[0233] The above description represents only preferred embodiments and is provided as an example only, not as a limitation on the combination of features necessary for carrying out the invention. The provided headings are not intended to limit the various embodiments of the invention. Terms such as “comprising,” “including,” and “including” are not intended to be limiting. Furthermore, unless otherwise stated, the plural form is included when not modified by a numeral, and “or” or “or” means “and / or.” Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0234] All disclosures and patents mentioned in this application are incorporated herein by reference. Various modifications and variations of the methods and compositions described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described through specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various variations of the described modes of carrying out the invention that will be apparent to those skilled in the art are intended to be included within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment specifically targeting HER2, characterized in that, The antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region has the same CDR sequence as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or has a substitution of 1-2 amino acids with the CDR sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4. And / or the light chain variable region has the same CDR sequence as SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or has a substitution of 1-2 amino acids with the CDR sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:

8.

2. The antibody or antigen-binding fragment specifically targeting HER2 according to claim 1, characterized in that, The antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and: (1) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:9, the VH-CDR2 amino acid sequence described in SEQ ID NO:10, and the VH-CDR3 amino acid sequence described in SEQ ID NO:11; or (2) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:12, the VH-CDR2 amino acid sequence described in SEQ ID NO:13, and the VH-CDR3 amino acid sequence described in SEQ ID NO:14; or (3) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:15, the VH-CDR2 amino acid sequence described in SEQ ID NO:16, and the VH-CDR3 amino acid sequence described in SEQ ID NO:17; or (4) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:18, the VH-CDR2 amino acid sequence described in SEQ ID NO:19, and the VH-CDR3 amino acid sequence described in SEQ ID NO:20; and / or (5) The light chain variable region comprises the VL-CDR1 amino acid sequence described in SEQ ID NO:21, the VL-CDR2 amino acid sequence described in SEQ ID NO:22, and the VL-CDR3 amino acid sequence described in SEQ ID NO:23; or (6) The light chain variable region comprises the VL-CDR1 amino acid sequence described in SEQ ID NO:24, the VL-CDR2 amino acid sequence described in SEQ ID NO:25, and the VL-CDR3 amino acid sequence described in SEQ ID NO:26; or (7) The light chain variable region comprises the VL-CDR1 amino acid sequence described in SEQ ID NO:27, the VL-CDR2 amino acid sequence described in SEQ ID NO:28, and the VL-CDR3 amino acid sequence described in SEQ ID NO:29; or (8) The light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO:30, the VL-CDR2 amino acid sequence described in SEQ ID NO:31, and the VL-CDR3 amino acid sequence described in SEQ ID NO:

32.

3. The antibody or antigen-binding fragment specifically targeting HER2 according to any one of claims 1-2, characterized in that, The antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and: (1) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; and / or (2) The amino acid sequence of the light chain variable region is identical to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

4. The antibody or antigen-binding fragment specifically targeting HER2 according to any one of claims 1-3, characterized in that, The antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and: (1) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:9, the VH-CDR2 amino acid sequence described in SEQ ID NO:10, and the VH-CDR3 amino acid sequence described in SEQ ID NO:11; and (2) The light chain variable region comprises the VL-CDR1 amino acid sequence described in SEQ ID NO:21, the VL-CDR2 amino acid sequence described in SEQ ID NO:22, and the VL-CDR3 amino acid sequence described in SEQ ID NO:23, or (3) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:12, the VH-CDR2 amino acid sequence described in SEQ ID NO:13, and the VH-CDR3 amino acid sequence described in SEQ ID NO:14; and (4) The light chain variable region comprises the VL-CDR1 amino acid sequence described in SEQ ID NO:24, the VL-CDR2 amino acid sequence described in SEQ ID NO:25, and the VL-CDR3 amino acid sequence described in SEQ ID NO:26, or (5) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:15, the VH-CDR2 amino acid sequence described in SEQ ID NO:16, and the VH-CDR3 amino acid sequence described in SEQ ID NO:17; and (6) The light chain variable region comprises the VL-CDR1 amino acid sequence described in SEQ ID NO:27, the VL-CDR2 amino acid sequence described in SEQ ID NO:28, and the VL-CDR3 amino acid sequence described in SEQ ID NO:29, or (7) The heavy chain variable region comprises the VH-CDR1 amino acid sequence described in SEQ ID NO:18, the VH-CDR2 amino acid sequence described in SEQ ID NO:19, and the VH-CDR3 amino acid sequence described in SEQ ID NO:20; and (8) The light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO:30, the VL-CDR2 amino acid sequence described in SEQ ID NO:31, and the VL-CDR3 amino acid sequence described in SEQ ID NO:

32.

5. The antibody or antigen-binding fragment specifically targeting HER2 according to any one of claims 1-4, characterized in that, The antibody targeting HER2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and: (1) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:1, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:1; and (2) The amino acid sequence of the light chain variable region is identical to the amino acid sequence shown in SEQ ID NO:5, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:5, or (3) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:2, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:2; and (4) The amino acid sequence of the light chain variable region is identical to the amino acid sequence shown in SEQ ID NO:6, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:6, or (5) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:3, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:3; and (6) The amino acid sequence of the light chain variable region is identical to the amino acid sequence shown in SEQ ID NO:7, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:7, or (7) The amino acid sequence of the heavy chain variable region is identical to the amino acid sequence shown in SEQ ID NO:4, or has at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:4; and (8) The amino acid sequence of the light chain variable region is consistent with the amino acid sequence shown in SEQ ID NO:8, or has at least 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

6. The antibody or antigen-binding fragment specifically targeting HER2 according to any one of claims 1-5, characterized in that, The antibody or antigen-binding fragments include monoclonal antibodies, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or fusion proteins containing an antigen-binding portion of an antibody.

7. The antibody or antigen-binding fragment specifically targeting HER2 according to any one of claims 1-6, characterized in that, The antibody or antigen-binding fragment that specifically targets HER2 contains an Fc domain.

8. The antibody or antigen-binding fragment specifically targeting HER2 according to claim 7, characterized in that, The Fc domain is a human immunoglobulin Fc domain; preferably, the Fc domain is a human IgG1 domain, a human IgG2 domain, a human IgG3 domain, or a human IgG4 Fc domain; more preferably, the Fc domain is a human IgG1 domain or a human IgG4 domain.

9. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof targeting HER2 as described in any one of claims 1-8.

10. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 9.

11. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 9 or the expression vector of claim 10.

12. A method for preparing the antibody targeting HER2 or its antigen-binding fragment according to any one of claims 1-8, wherein the method comprises: a) Culture the host cells of claim 11 under conditions sufficient to induce the cells to produce the antibody targeting HER2 or its antigen-binding fragment, and b) Collect the antibody or antigen-binding fragment thereof targeting HER2 produced by the host cells.

13. The use of the antibody targeting HER2 or its antigen-binding fragment as described in any one of claims 1-8 in the preparation of the XDC drug.

14. An XDC coupling, characterized in that, The XDC conjugate comprises the antibody or antigen-binding fragment thereof targeting HER2 as described in any one of claims 1-8.

15. The use of the antibody targeting HER2 or its antigen-binding fragment as described in any one of claims 1-8 in the preparation of antibody-isotope conjugate drugs.

16. An antibody-nucleoside conjugate, characterized in that, The antibody-nucleoside conjugate comprises the antibody targeting HER2 or an antigenic fragment thereof as described in any one of claims 1-8.

17. The antibody-nucleoside conjugate according to claim 16, characterized in that, The antibody-nucleoside conjugate comprises an antibody-targeting portion and a radionuclide portion, wherein the antibody-targeting portion is the antibody or antigenic structural fragment of the HER2-targeting conjugate as described in any one of claims 1-8, and the radionuclide is any selectable nuclide, and the radionuclide can be conjugated to the antibody-targeting portion directly or through any chelating group.

18. The antibody-nucleoside conjugate according to claim 17, characterized in that, The radionuclide mentioned is a diagnostic radionuclide or a therapeutic radionuclide.

19. The antibody-nucleoside conjugate according to claim 18, characterized in that, The radionuclides mentioned are selected from 11 C 13 N、 15 O、 18 F, 34m Cl、 38 K, 43 Sc、 44 Sc、 45 Ti、 51 Mn, 52 Mn, 52m Mn, 52 Fe、 55 Co、 60 Cu、 61 Cu、 62 Cu、 64 Cu、 63 Zn, 66 Ga、 68 Ga、 69 Ge 71 As、 72 As、 74 As、 73 Se、 75 Br、 76 Br、 82 Rb、 82m Rb、 83 Sr、 86 Y、 89 Zr、 90 Nb, 94m Tc, 110m In、 118 Sb、 120 I, 122 I, 124 I, 152 Tb, 67 Ga、 99m Tc, 111 In、 123 I, 125 I, 155 Tb, 201 Tl、 32 P, 47 Sc、 66 Cu、 67 Cu、 77 As、 77 Br、 89 Sr、 90 Y、 105 Rh、 103 Pd, 111 Ag、 117m Sn、 131 I, 133 Xe, 149 Tb, 161 Tb, 149 Pm, 153 Sm、 166 Ho、 177 Lu、 186 Re、 188 Re、 195m Pt, 212 Bi、 213 Bi、 211 At、 212 Pb, 223 Ra、 225 Ac、 230 U、 33 P, 59 Fe、 67 Cu、 67 Ga、 75 Se、 77 As、 99 Mo、 109 Pd, 142 Pr, 143 Pr, 166 Dy、 169 Er、 189 Re、 194 Ir、 198 Au、 199 Au、 199 Au、 211 Pb, 212 Any one of Bi.

20. The antibody-nucleoside conjugate according to claim 19, characterized in that, The radionuclide is directly conjugated to the antibody or its antigenic structural fragment that targets HER2.

21. The antibody-nucleoside conjugate according to claim 19, characterized in that, The radionuclide is used to label the antibody or its antigenic structural fragment targeting HER2 via a chelating agent.

22. A pharmaceutical composition comprising an antibody targeting HER2 according to any one of claims 1-8 or an antigen-binding fragment thereof, or comprising an XDC conjugate according to claim 14, or comprising an antibody-nucleoside conjugate according to any one of claims 16-21, and a pharmaceutically acceptable carrier.

23. A kit comprising an antibody targeting HER2 according to any one of claims 1-8 or an antigen-binding fragment thereof, or comprising the XDC conjugate according to claim 14, or comprising the antibody-nucleoside conjugate according to any one of claims 16-21, or comprising the pharmaceutical composition according to claim 22.

24. The use of the antibody targeting HER2 according to any one of claims 1-8 or its antigen-binding fragment, or the XDC conjugate according to claim 14, or the antibody-nucleoside conjugate according to any one of claims 16-21, or the pharmaceutical composition according to claim 22, or the kit according to claim 23, in the preparation of a medicament for treating tumors or cancer.

25. The application according to claim 24, characterized in that, The treatments include, but are not limited to, improving or optimizing the killing of cancer cells, delaying the progression or recurrence of the tumor or cancer.

26. The application according to any one of claims 24-25, characterized in that, The tumor or cancer mentioned is a cancer that expresses HER2.

27. The application according to any one of claims 24-26, characterized in that, The tumor or cancer mentioned is a HER2-low expression cancer, a HER2-medium expression cancer, or a HER2-high expression cancer.

28. The application according to any one of claims 23-27, characterized in that, The tumors or cancers mentioned are not limited to those selected from: breast cancer, small cell lung cancer, ovarian cancer, endometrial cancer, bladder cancer, head and neck cancer, prostate cancer, stomach cancer, cervical cancer, uterine cancer, esophageal cancer, and colorectal cancer, etc.

29. A method for treating or diagnosing a disease characterized by HER2 expression, the method comprising administering to a patient suffering from the disease characterized by HER2 expression a therapeutically effective amount of an antibody targeting HER2 as described in any one of claims 1-8 or an antigen-binding fragment thereof, or an XDC conjugate as described in claim 14, or an antibody-nucleoside conjugate as described in any one of claims 16-21, or a pharmaceutical composition as described in claim 22, or a kit as described in claim 23.

30. A method for acquiring an image of a patient's area to be detected, the method comprising the following steps: a) administering to a patient an effective amount of the antibody-nucleoside conjugate according to any one of claims 16-21; and b) Perform positron emission tomography (PET) or SPECT on the patient; c) Detectable signals that identify radionuclides; d) Generate an image based on the detectable signal to obtain an image of the area to be detected in the patient.

31. The method according to claim 29 or claim 30, characterized in that, The patient is a mammal; preferably, the patient is a human.

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