Bivalent bispecific antibody, and antibody-radionuclide conjugate and use thereof

Abstract

The present invention provides a bivalent bispecific antibody targeting an EGFR and a c-MET and a radioactive label thereof. The research shows that the bivalent bispecific antibody provided by the present invention has good targeting affinity, and an antibody-radionuclide conjugate (taking 177Lu radioactive label as an example) of the bivalent bispecific antibody has an excellent SPECT imaging result, can be continuously accumulated in tumors over time, is more quickly removed in organs such as the liver and normal tissues, and can exhibit lower toxic and side effects. This also indirectly shows that the bivalent bispecific antibody provided by the present invention can clinically exhibit higher safety potential.

Description

A bivalent bispecific antibody, antibody-coupled nuclide drug and its application Technical Field

[0001] The present invention relates to the fields of antibodies and nuclear medicine, and in particular to a bivalent bispecific antibody capable of binding to human epidermal growth factor receptor (EGFR) and MET proto-oncogene receptor tyrosine kinase (cMET), and a radionuclide label for the bivalent bispecific antibody. The present invention also relates to applications of the bivalent bispecific antibody and its radionuclide label. Background Art

[0002] Antibody Radionuclide Conjugates (ARC), also known as radiolabeled antibodies or radioimmunoconjugates, are conjugated drugs made by coupling radioisotopes to antibodies through chelators. They can utilize the selective targeting of antibodies to tumor-specific antigens to deliver diagnostic or therapeutic radioisotopes / nuclides, selectively image or kill tumor cells, and exert different functions such as in vivo diagnosis or treatment.

[0003] A key feature of antibody-conjugated radionuclide drugs is that the targeting moiety typically tends to be mouse or rat antibodies (Reference 1: Thurston, DE, & Pysz, I. (2021). Chemistry and Pharmacology of Anticancer Drugs (2nd ed.).). This is because, for other types of antibody therapies (such as antibody drugs and antibody-drug conjugates), the focus is on maintaining the drug's retention time in the body for as long as possible to maximize tumor exposure. However, for antibody-conjugated radionuclide drugs, the payload is a radionuclide. To ensure medical safety and protect the health rights of radiotherapy workers, patients, and the public, antibody-conjugated radionuclide drugs prefer short tumor exposure. Long-term tumor exposure often leads to hematological toxicity and a low tumor-to-background ratio. Therefore, mouse or rat antibodies with shorter half-lives are usually the conventional choice for antibody-conjugated radionuclide drugs. However, mouse antibodies or mouse-derived antibodies can be recognized by the human immune system, triggering human anti-mouse antibody reactions, which weaken the drug's efficacy and cause serious adverse reactions. Therefore, the selection of antibodies in antibody-conjugated radionuclide drugs often becomes the biggest problem limiting drug screening and clinical application.

[0004] The earliest representative drug of antibody-conjugated nuclide drug is Pemtumomab (Theragyn TM ), which is a 90A γ-conjugated mouse monoclonal antibody that specifically binds to the glycoform of the MUC1 mucin. This protein is overexpressed on the surface of epithelial tumor cells, including ovarian, gastric, breast, and lung cells. Although a phase II study of women with advanced ovarian cancer showed that 15 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 effect may be a problem that it has never been able to overcome (Reference 2: Angèle LMOei; Fred CGJSweep; Leon FAGMassuger; André J.Olthaar; Chris MG Thomas (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.), therefore, pemtumomab has not been further developed.

[0005] So far, the U.S. Food and Drug Administration has only approved two antibody-conjugated radionuclide drugs for marketing, namely ibritumomab tiuxetan (Zevalin TM ) and tositumomab (Bexxar) containing iodine-131 TM Bexxar is a radioactive isotope developed by GSK. 131 Zevalin is a radiolabeled CD20-targeting mouse IgG2a monoclonal antibody conjugate that was approved by the FDA in 2003 for the treatment of relapsed or refractory follicular or metastatic non-Hodgkin's lymphoma. However, in 2014, due to a significant decrease in demand in the US market, GSK withdrew the drug and believed that more beneficial methods were available for the treatment of these cancer types. Zevalin is a radioactive nuclide indium 111 ( 111 In) or Yttrium 90( 90Zevalin (Y) is a radiolabeled anti-CD20 murine IgG1k monoclonal antibody conjugate approved by the FDA in 2002 for the treatment of refractory relapsed B-cell non-Hodgkin's lymphoma. Although Zevalin has demonstrated favorable results in consolidation of first-remission advanced follicular lymphoma (extending progression-free survival by 2 years), a head-to-head comparison with a rituximab-based regimen did not reveal a difference in PFS. For various reasons, Zevalin faces numerous obstacles in its clinical application and commercialization.

[0006] With the application and development of nuclear medicine and the new generation of monoclonal antibody therapy targeting solid tumor antigens, antibody-conjugated radionuclide drugs, as an important product type of radionuclide drug conjugates (RDC), have gradually emerged as a number of candidate drugs in the clinical research stage, such as antibody-conjugated radionuclide drugs targeting tumor carcinoembryonic antigen (CEA). 225 Ac-DOTA-M5A (NCT05204147), targeting mesothelin, prostate-specific membrane antigen, or human epidermal growth factor receptor 2 (Her2) 227 Th-labeled RIT formulations (NCT03507452, NCT03724747, NCT04147819) targeting human kallikrein 2 225 Ac-DOTA-h11B6 (NCT04644770), and targeting insulin-like growth factor type I receptor 225 Ac-FPI-1434, etc. Despite this, there is still a huge unmet clinical need.

[0007] EGFR (epidermal growth factor receptor, also known as ErbB-1 or HER1) is a member of the epidermal growth factor receptor family and plays an important regulatory role in cellular physiological processes. However, when EGFR expression or activity is uncontrolled, it can lead to cancer-related phenomena such as excessive cell proliferation, anti-apoptosis, angiogenesis, invasion, and metastasis. Studies have shown that EGFR is overexpressed or aberrantly expressed in many solid tumors (such as breast cancer, non-small cell lung cancer, colorectal cancer, and head and neck cancer). Therefore, the EGFR family is considered a key factor in the occurrence and progression of cancer and an important target for cancer treatment. However, due to the heterogeneity and plasticity of cancer cells and the complexity and redundancy of EGFR family signaling pathways, many patients develop primary or acquired drug resistance after using these drugs, leading to treatment failure. Therefore, overcoming resistance to EGFR family targeted drugs is a major challenge in the current field of cancer treatment. Several strategies have been proposed and explored, such as combining drugs of different types or targets and developing new or improved drugs.

[0008] C-Met (cellular-mesenchymal epithelial transition factor) is a member of the receptor tyrosine kinase family. The c-Met signaling pathway plays a key role in invasive growth during embryonic development and postnatal organ regeneration. While the c-Met signaling pathway is typically only fully activated in adults during wound healing and tissue regeneration, it can be frequently activated by cancer cells in tumors, promoting tumor formation, invasive growth, and metastasis. Studies have shown that the c-Met signaling pathway is abnormally expressed or mutated in various types of solid tumors, including lung, gastric, liver, breast, skin, and colorectal cancers, playing a crucial role in the development and progression of various tumors.

[0009] To date, a variety of bispecific EGFR x cMET antibodies have been described in the prior art, such as Castoldi R. et al. (Reference 3: Castoldi, R; Ecker, V; WiehIe, L; Majety, M; Busl-Schuller, R; Asmussen, M; Nopora, A; Jucknischke, U; Osl, F; Kobold, S; Scheuer, W; Venturi, M; Klein, C; Niederfellner, G; Sustmann, C (2013). A novel bispecific EGFR / Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity. Oncogene, 32 (50), 5593-5601.) described a bispecific EGFR x cMET antibody named MetHer1. cMET antibodies, which have the cMET binding site of antibody 5D5 (or MetMab) and the EGFR binding site of cetuximab. Patent publication number US20140378664A1 also describes cMET x EGFR bispecific antibodies with various structures.Moores.et al. (Literature 4: Moores, SL; Chiu, M.>; Bushey, BS; Chevalier, K.; Luistro, L.; Dorn, K.; Brezski, RJ; Haytko, P.; Kelly, T.; Wu, S.-J.; Martin, PL; Neijssen, J.; Parren, PW; Schuurman, J.; Attar, RM; Laquerre, S.; Lorenzi, MV; Anderson, GM (2016).A Novel Bispecific Antibody Targeting EGFR and cMet that is Effective Against EGFR Inhibitor-Resistant Lung Tumors.Cancer Research, 0008-5472.CAN-15-2833-.doi: 10.1158 / 0008-5472.can-15-2833) described a bispecific cMET x EGFR antibody, designated JNJ-61186372, generated by controlled Fab arm exchange (cFAE) with mutations at positions 405 and 409 according to EU numbering. In a Phase I clinical study (NCT02609776), 108 subjects with advanced NSCLC received JNJ-61186372 and demonstrated favorable safety and anti-tumor efficacy. However, due to the unique structure of antibody-conjugated radionuclides, there are currently no successful examples of radiolabeled conjugates of EGFR x cMET dual-targeting antibodies. Therefore, the development of antibody-conjugated radionuclides targeting the EGFR x cMET target combination is urgently needed to address unmet clinical needs. Summary of the Invention

[0010] In view of the above problems, the present invention provides a bivalent bispecific antibody with a novel structure and a radionuclide label for the bivalent bispecific antibody.

[0011] Specifically, the bivalent bispecific antibody or its antigenic structure fragment provided by the present invention can bind to human epidermal growth factor receptor (EGFR) and human MET proto-oncogene receptor tyrosine kinase (cMET), and structurally comprises a first targeting moiety, a second targeting moiety, and a first Fc domain and a second Fc domain;

[0012] The first targeting moiety can bind to human epidermal growth factor receptor EGFR, which comprises a first VH domain and a first VL domain, and the first VH domain is connected to the human epidermal growth factor receptor EGFR by (G x S) m The sequence is connected to the first VL domain;

[0013] The second targeting moiety can bind to human MET proto-oncogene receptor tyrosine kinase cMET, which comprises a second VH domain and a second VL domain, and the second VH domain is connected to the human MET proto-oncogene receptor tyrosine kinase cMET by (G y S) n The sequence is connected to the second VL domain;

[0014] in:

[0015] The amino acid sequence of the first VH domain is shown in SEQ ID NO: 1; The amino acid sequence of the first VH domain (SEQ ID NO: 1):

[0016] The amino acid sequence of the first VL domain is shown in SEQ ID NO: 2; The amino acid sequence of the first VL domain (SEQ ID NO: 2):

[0017] The amino acid sequence of the second VH domain is shown in SEQ ID NO: 3; The amino acid sequence of the second VH domain (SEQ ID NO: 3):

[0018] The amino acid sequence of the second VL domain is shown in SEQ ID NO: 4; The amino acid sequence of the second VL domain (SEQ ID NO: 4):

[0019] The x is an integer selected from 1, 2, 3, 4, 5, and 6;

[0020] The m is an integer selected from 1, 2, 3, 4, 5, and 6;

[0021] The y is an integer selected from 1, 2, 3, 4, 5, and 6;

[0022] The n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0023] In some preferred embodiments, the (G x S) m Can be selected from the following sequences:

[0024] In some preferred embodiments, the (Gy S) n Can be selected from the following sequences:

[0025] Preferably, the x is 4, the m is 3, and the (G x S) m is GGGGSGGGSGGGGS (i.e. (GGGGS)3) (SEQ ID NO: 13).

[0026] Preferably, the y is 4, the n is 3, and the (G y S) n is GGGGSGGGSGGGGS (i.e. (GGGGS)3) (SEQ ID NO: 13).

[0027] In some preferred embodiments, the C-terminus of the first VH domain is connected via (G x S) m Sequence is connected to the N-terminus of the first VL domain; and the C-terminus of the second VH domain is connected by (G y S) n The sequence is connected to the N-terminus of the second VL domain.

[0028] In other preferred embodiments, the N-terminus of the first VH domain is connected via (G x S) m Sequence is connected to the C-terminus of the first VL domain; and the N-terminus of the second VH domain is connected by (G y S) n The sequence is linked to the C-terminus of the second VL domain.

[0029] Preferably, the amino acid sequence of the first targeting moiety is shown in SEQ ID NO: 5. Amino acid sequence of the first targeting moiety (SEQ ID NO: 5):

[0030] Preferably, the amino acid sequence of the second targeting moiety is shown in SEQ ID NO: 6. The amino acid sequence of the second targeting moiety (SEQ ID NO: 6):

[0031] Furthermore, the first Fc domain is a human immunoglobulin Fc domain; preferably, the first Fc domain is a human IgG1, IgG2, IgG3 or IgG4 Fc domain; more preferably, the first Fc domain is a human IgG1 or IgG4 Fc domain.

[0032] Furthermore, the second Fc domain is a human immunoglobulin Fc domain; preferably, the second Fc domain is a human IgG1, IgG2, IgG3 or IgG4 Fc domain; more preferably, the second Fc domain is a human IgG1 or IgG4 Fc domain.

[0033] The first Fc domain and the second Fc domain of the present invention comprise a hinge region, a CH2 domain, and a CH3 domain, wherein the CH3 domain of the first Fc domain and the second Fc domain comprises a modified structure, and the modification is used to promote dimerization of the two; preferably, the modification is an amino acid substitution; more preferably, the modification is Knob-into-hole (KIH).

[0034] Knob-into-hole (KIH) is designed to form a "knob" or "hole" on the CH3 domain of the heavy chain to promote heterodimerization, which is used in the engineering modification of antibodies to obtain heterodimers. Among them, the "knob" is formed by replacing a smaller amino acid with a larger amino acid, while the "hole" is formed by replacing a larger amino acid with a smaller amino acid. Currently, there are many studies on knobs-into-holes, and many KIH structural patterns have been formed. The preferred knob-into-hole structures of the present invention are mainly the following three patterns: "1+1" pattern, "3+1" pattern and "4+2" pattern.

[0035] Preferably, the Knob-into-hole (KIH) structure pattern used in the present invention is the above-mentioned "3+1" pattern.

[0036] Furthermore, the amino acid sequence of the first Fc domain is as shown in SEQ ID NO: 7, or the amino acid sequence of the first Fc domain is a homologous sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) sequence identity to SEQ ID NO: 7. A preferred amino acid sequence of the first Fc domain (SEQ ID NO: 7) is:

[0037] Furthermore, the amino acid sequence of the second Fc domain is as shown in SEQ ID NO: 8, or the amino acid sequence of the second Fc domain is a homologous sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) sequence identity to SEQ ID NO: 8. A preferred amino acid sequence of the second Fc domain (SEQ ID NO: 8) is:

[0038] Preferably, the amino acid sequence of the first Fc domain is shown in SEQ ID NO: 7, and the amino acid sequence of the second Fc domain is shown in SEQ ID NO: 8.

[0039] In some preferred embodiments, the C-terminus of the first targeting moiety is linked to the N-terminus of the first Fc domain (its amino acid sequence is shown in SEQ ID NO: 9), and the C-terminus of the second targeting moiety is linked to the N-terminus of the second Fc domain (its amino acid sequence is shown in SEQ ID NO: 10). "First targeting moiety - first Fc domain" amino acid sequence (SEQ ID NO: 9): "Second targeting moiety - second Fc domain" amino acid sequence (SEQ ID NO: 10):

[0040] In other preferred embodiments, the C-terminus of the first targeting moiety is linked to the N-terminus of the second Fc domain (the amino acid sequence of which is shown in SEQ ID NO: 11), and the C-terminus of the second targeting moiety is linked to the N-terminus of the first Fc domain (the amino acid sequence of which is shown in SEQ ID NO: 12). "First targeting moiety-second Fc domain" amino acid sequence (SEQ ID NO: 11): "Second targeting moiety - first Fc domain" amino acid sequence (SEQ ID NO: 12):

[0041] The present invention also provides a bivalent bispecific antibody-conjugated radionuclide drug (i.e., antibody-conjugated radionuclide drug), which comprises a bivalent bispecific antibody or an antigenic structural fragment thereof conjugated to a radionuclide; wherein the bivalent bispecific antibody or the antigenic structural fragment thereof is the bivalent bispecific antibody described in any one of the foregoing items, and the radionuclide is a therapeutic radionuclide or a diagnostic radionuclide.

[0042] Furthermore, the radionuclide is used to label the bivalent bispecific antibody or its antigenic structure fragment via a chelating agent.

[0043] Furthermore, the therapeutic radionuclide is selected from 32 P. 33 P. 47 Sc, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 75 Se, 77 As、 89 Sr. 90 Y. 99 Mo, 105 Rh, 109 Pd, 125 I. 131 I. 111 Ag, 111 In, 111m In, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 169 Second, 177 Lu, 186 Re、 188 Re、 189 Re、 194 Ir, 198 Au, 199 Au, 199 Au, 211 At 211 pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra or 225 Ac.

[0044] Furthermore, the diagnostic radionuclide is selected from 18F. 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As、 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 169 Second, 175 Lu, 177 Lu, 186 Re、 188 Re、 189 Re、 194 Ir, 198 Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra or 225 Ac.

[0045] It is understood that a suitable chelating agent can be selected according to the desired radionuclide, and the chelating agents provided in the present invention are only exemplary and should not be considered as limiting the present invention.

[0046] In some preferred embodiments, the chelating agent is selected from DFO (deferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), p-SCN-Bn-DOTA, DTPA (diethyltriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), TRITA (1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclotridecane), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), EDTA (ethylenediaminetetraacetic acid) and its derivatives.

[0047] In some specific embodiments, the radionuclide is preferably 177 Lu, the chelating agent is p-SCN-Bn-DOTA.

[0048] The present invention also provides an isolated nucleic acid molecule, which can encode any of the aforementioned bivalent bispecific antibodies or antigenic structural fragments thereof.

[0049] The present invention also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0050] The present invention also provides a host cell, which contains the aforementioned nucleic acid molecule or the aforementioned expression vector.

[0051] The present invention also provides a method for preparing a bivalent bispecific antibody or an antigenic structural fragment thereof, which method comprises at least the following steps: a) culturing the aforementioned host cells under conditions sufficient for the cells to produce the bivalent bispecific antibody or the antigenic structural fragment thereof, and b) collecting the bivalent bispecific antibody or the antigenic structural fragment thereof produced by the host cells.

[0052] The present invention also provides a pharmaceutical composition comprising any one of the aforementioned bivalent bispecific antibodies or antigenic structural fragments thereof, and a pharmaceutically acceptable carrier.

[0053] The present invention also provides another pharmaceutical composition, comprising any one of the aforementioned bivalent bispecific antibodies coupled to a nuclide drug, and a pharmaceutically acceptable carrier.

[0054] The present invention also provides a kit comprising any one of the aforementioned bivalent bispecific antibodies or antigenic structural fragments thereof, or any one of the aforementioned bivalent bispecific antibodies coupled with a nuclide drug, or any one of the aforementioned pharmaceutical compositions.

[0055] The present invention also provides a method for treating or diagnosing a disease characterized by EGFR and / or c-Met expression, the method comprising administering a therapeutically effective amount of any one of the aforementioned bivalent bispecific antibodies or antigenic fragments thereof, or any one of the aforementioned bivalent bispecific antibodies-conjugated nuclide drugs, or any one of the aforementioned pharmaceutical compositions, or any one of the aforementioned kits to a patient suffering from the disease characterized by EGFR and / or c-Met expression.

[0056] Furthermore, the patient is a mammal.

[0057] Furthermore, the patient is a human.

[0058] The present invention also provides the use of any of the aforementioned bivalent bispecific antibodies or antigenic structural fragments thereof, or any of the aforementioned bivalent bispecific antibodies coupled to a nuclide drug, or any of the aforementioned pharmaceutical compositions, or any of the aforementioned kits in the preparation of a drug for treating or diagnosing a disease characterized by EGFR and / or c-Met expression.

[0059] The present invention also provides a method for obtaining an image of a patient's part to be detected, the method comprising the following steps:

[0060] a) administering to a patient an effective amount of any one of the aforementioned bivalent, bispecific antibodies-conjugated nuclide drugs, any one of the aforementioned pharmaceutical compositions, or any one of the aforementioned kits; and

[0061] b) performing positron emission tomography (PET) or SPECT on the patient;

[0062] c) a detectable signal that identifies the radionuclide;

[0063] d) generating an image based on the detectable signal, thereby obtaining an image of the patient's part to be detected.

[0064] Furthermore, the patient is a mammal.

[0065] Furthermore, the patient is a human.

[0066] Furthermore, the patient suffers from or may suffer from a disease characterized by expression of EGFR and / or c-Met.

[0067] Furthermore, the disease characterized by EGFR and / or c-Met expression is cancer.

[0068] Furthermore, the cancer is an EGFR-positive tumor, a cMET-positive tumor, or an EGFR- and cMET-positive tumor.

[0069] Preferably, the cancer can be squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, liver cancer, kidney cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, salivary gland cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, leukemia, lymphoma, glioma, neuroblastoma, melanoma, sarcoma, endometrial cancer, testicular cancer or thyroid cancer or a subtype thereof; more preferably, lung cancer; more preferably, non-small cell lung cancer.

[0070] The bivalent bispecific antibody provided by the present invention has good targeting affinity, and its antibody-coupled nuclide drug (with 177 Lu radiolabeled as an example) has excellent SPECT imaging results, can accumulate in tumors over time, and is cleared more quickly in organs such as the liver and normal tissues, with lower toxic side effects. This indirectly demonstrates that the bivalent bispecific antibodies provided by the present invention may exhibit higher safety potential in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG1 is a schematic diagram of the structure of a bivalent bispecific antibody or an antigenic structure fragment thereof provided by the present invention.

[0072] Figure 2 shows the bivalent bispecific antibody provided by the present invention and 177 Schematic diagram of the antibody-conjugated nuclide drug structure formed by conjugation of Lu via the chelator p-SCN-Bn-DOTA.

[0073] FIG3 is a flow cytometry histogram of the bivalent bispecific antibody LNCX004 provided by the present invention.

[0074] Figure 4 shows 177 Radio-TLC radiochemical purity chromatogram of Lu-DOTA-LNCX004.

[0075] Figure 5 177 Schematic diagram of the changes in cell-specific binding of Lu-DOTA-LNCX004 over time.

[0076] Figure 6 177 Lu-DOTA-LNCX004 compared to the positive control 177 SPECT images of Lu-DOTA-Amivantamab (KIH) in a mouse model. DETAILED DESCRIPTION

[0077] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as they may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0078] Preferably, the terms used herein are as defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and KIbI, Hb eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0079] Unless the context requires otherwise, throughout the specification and the claims that follow, the word "comprise" and its variations such as "comprising" and "containing" will be understood to implicitly include the stated integers or steps, or groups of integers or steps, but not to 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 so defined can be combined with any other aspect or aspects unless there is a clear indication to the contrary. In particular, any feature that is optional, preferred or advantageous can be combined with any other feature or features that are optional, preferred or advantageous.

[0080] Throughout this specification, some documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, operating instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to such disclosures as prior inventions. Certain documents cited herein are identified as "incorporated by reference." In the event that a definition or teaching in such an incorporated reference conflicts with a definition or teaching described in this specification, the text of this specification shall prevail.

[0081] The elements of the present invention are described below. These elements are listed together with specific embodiments. However, it should be understood that they can be combined in any manner and in any number to form other embodiments. The various described embodiments and preferred embodiments should not be interpreted as limiting the invention to only the embodiments explicitly described. This description should be understood to support and cover solutions that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. In addition, it should be considered that any permutation and combination of all elements described in this application is disclosed in the specification of this application unless the context indicates otherwise. Specific Examples

[0082] 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 used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0083] Example 1 Construction of bispecific antibody molecules

[0084] 1. Vector Construction

[0085] The antibody variable region sequence was obtained by gene synthesis, connected to the constant region by OverlapPCR, and loaded into the expression vector PcDNA3.4 using homologous recombination. The positive bacterial liquid with the correct clone was selected for sequencing, and the low-endotoxin plasmid was extracted after expansion culture and sequenced for verification.

[0086] 2. Plasmid Extraction

[0087] Materials: Packing column, microporous filter column, centrifuge tube, EP tube, filter screen

[0088] Reagents: Buffer P1 (P1 with RNase A added should be stored at 4°C), RNase A (stored at -20°C), Buffer P2 (SDS precipitation requires preheating in a 37-42°C water bath), Buffer P3, ER Buffer (stored at 4°C), Elution Buffer, QBT, QC, isopropanol, 75% ethanol, deionized water

[0089] Reagent preparation:

[0090] 75% ethanol: Prepare anhydrous ethanol and deionized water in a ratio of 3:1, mix well and store at room temperature.

[0091] Buffer P1 with RNase A added: Prepare RNase A and P1 solution at a ratio of 6:1000, mix well and store at 4°C.

[0092] Experimental steps:

[0093] (1) Collect 100 mL of overnight (12-16 h) cultured bacteria into a labeled 50 mL centrifuge tube, collecting about 45 mL of bacteria at a time (check the numbers for consistency). Centrifuge at 8000 rpm in a corner rotor for 4 min or at 4500 rpm in a horizontal rotor for 15 min. Pour off the supernatant and retain the bacteria. Repeat the above operation once and mark the first centrifuge tube cap in each row of the 36-well acrylic rack to indicate that the collection is complete.

[0094] (2) Add 10 mL of Buffer P1 suspension containing RNase A to the centrifuge tube and vortex for 6 min until no obvious bacterial clumps are visible.

[0095] (3) Add 10 mL of Buffer P2 to the centrifuge tube (if SDS precipitates, preheat in a 37-42°C water bath until no precipitation occurs). Immediately rotate gently by hand 3-9 times or place on a mixer at 45,000 rpm for 60 seconds to mix thoroughly. The bacterial solution will then turn from turbid to viscous.

[0096] (4) Add 10 mL of Buffer P3 to the tube and immediately mix by inverting it 5-15 times or by placing it on a mixer at 70,000 rpm for 90 seconds. Once a white flocculent precipitate appears, centrifuge it at 11,000 rpm for 4 minutes in a corner rotor or at 4,500 rpm for 4 minutes in a swing-out rotor.

[0097] (5) Add 10 mL of ER Buffer to the centrifuged 50 mL centrifuge tube and mix thoroughly by inverting the tube 3-5 times or by placing the tube on a mixer at 45,000-70,000 rpm for 10 seconds.

[0098] (6) Before the filtration and loading step, add 25 mL of QBT reagent to the chromatography column filled with filler placed in a centrifuge tube rack to equilibrate the filler.

[0099] (7) Filter the sample treated with ER Buffer to remove the white precipitate, and add the remaining liquid to a clean, labeled chromatography column.

[0100] (8) After the liquid has dripped off, add 60 ml of QC to the chromatography column and let it pass through the column by gravity.

[0101] (9) After the QC solution is dripped, place the column into a clean, labeled 50 mL centrifuge tube and check that the numbers are consistent. Add 10 mL of Elution Buffer and allow it to elute by gravity.

[0102] (10) After elution, gently place the column against the wall of the centrifuge tube and then quickly remove it. Add 7 mL of isopropanol to the collected filtrate, mix it by inverting it upside down 3-5 times, and centrifuge it in an angle rotor centrifuge at 11,000 rpm for 15 minutes or in a horizontal centrifuge at 4,500 rpm for 15 minutes.

[0103] (11) Add 75% ethanol: After centrifugation, gently pour off the supernatant, place the centrifuge tubes in order on the rack and add 5 mL of 75% ethanol to each tube to thoroughly rinse the precipitate. After adding the sample, place the tubes in an angle rotor centrifuge and centrifuge at 11,000 rpm for 10 minutes or in a horizontal centrifuge at 3,700 rpm for 10 minutes.

[0104] (12) After centrifugation, gently pour off the supernatant and turn the centrifuge tubes upside down on tissue paper in ascending order. Add 50-500 μl of deionized water to the location of the plasmid and pipette 5-25 times to fully dissolve the plasmid in the water. When the liquid is no longer adsorbed on the tube wall, the plasmid is completely eluted. Use a pipette to draw up the dissolved liquid and transfer it to the microporous filter column with the corresponding serial number according to the serial number of the centrifuge tube. Place it in an angle rotor centrifuge and centrifuge at 14000 rpm for 15-30 minutes. After all the centrifugation is completed, transfer the plasmid to the corresponding EP tube according to the serial number of the microporous filter column.

[0105] 3. Transfection

[0106] Transfection and expression: expression in 400ml

[0107] (1) Take 2900M cells, centrifuge and remove the supernatant.

[0108] (2) Add about 4.5 ml of electroporation solution to the cells, mix well, and then add an appropriate amount of plasmid (concentration above 500 ng / ul).

[0109] (3) After the above cell plasmid suspension is fully mixed, 10 ml is taken and added to a 10 ml electric shock tube, and the electric shock tube is placed in an electroporator for electric shock.

[0110] (4) After the electroporation is completed, the cells in the electroporated tube are divided into a shake flask containing 130 ml of culture medium prepared in advance and incubated statically for 40 minutes.

[0111] (5) After the incubation, the shake flask was placed in 37°C, 120 rpm, 8% CO2 for culture. After 24 hours, feed / sodium butyrate / double antibody were added and culture was continued for 4 days.

[0112] 4. Antibody Purification

[0113] Experimental method: Protein A prepacked column affinity chromatography purification

[0114] (1) Equilibration column: 1xPBS, flow rate 1ml / min, 20ml

[0115] (2) Sample loading: flow rate 1 ml / min

[0116] (3) Washing: 1xPBS, flow rate 1ml / min, 20ml

[0117] (4) Elution: Sodium acetate buffer (pH 3.4), 1 ml / min, collect in separate tubes, approximately 500 μl per tube. Collect 10 tubes in total and read the absorbance at 280 nm using a NanoDrop instrument.

[0118] (5) Dialysis: Pipette the high-concentration protein into a dialysis bag and place it in a beaker containing PBS, pH 7.2-7.4 for dialysis.

[0119] Vector construction, plasmid extraction, transfection expression, and antibody purification were performed according to the above methods to obtain the target bivalent bispecific antibody of the present invention, named LNCX004, the amino acid sequence of the first targeting moiety of which is shown in SEQ ID NO: 5, the amino acid sequence of the second targeting moiety is shown in SEQ ID NO: 6, the amino acid sequence of the first Fc domain is shown in SEQ ID NO: 7, and the amino acid sequence of the second Fc domain is shown in SEQ ID NO: 8, wherein the first targeting moiety is connected to the first Fc domain (the amino acid sequence of which is shown in SEQ ID NO: 9), and the second targeting moiety is connected to the second Fc domain (the amino acid sequence of which is shown in SEQ ID NO: 10). The amino acid sequence of the first targeting moiety (SEQ ID NO: 5) is: Amino acid sequence of the second targeting moiety (SEQ ID NO: 6): Amino acid sequence of the first Fc domain (SEQ ID NO: 7): Amino acid sequence of the second Fc domain (SEQ ID NO: 8): First targeting moiety - first Fc domain amino acid sequence (SEQ ID NO: 9): Second targeting moiety - second Fc domain amino acid sequence (SEQ ID NO: 10):

[0120] Example 2 Affinity Determination

[0121] The affinity of LNCX004 was determined by flow cytometry. The positive control group was Amivantamab (KIH) (Amivantamab is an EGFR-cMET bispecific antibody. Its amino acid sequence can be found at https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=11030. In this example, the Fc domain of Amivantamab was sequence-modified in a "3+1" pattern Knob-into-hole. The obtained bispecific antibody was designated as Amivantamab (KIH)). The method was as follows: 5×10 5 The HCC827 cells were divided into EP tubes, washed with 1X PBS, centrifuged and discarded the supernatant. If necessary, the washing can be repeated once. The primary antibody was diluted to a concentration of 10μg / ml to a volume of 100μl, and the cells were resuspended and incubated on ice for 1 hour. Centrifuged and washed with incubation buffer. The supernatant was discarded. The cells were resuspended in 100μl of diluted fluorescent dye-labeled secondary antibody. Incubated on ice for 1 hour. Centrifuged and washed with incubation buffer. The supernatant was discarded. Repeat once and tested on the machine. Referring to Figure 3, it can be seen from the flow cytometry histogram that the bivalent bispecific antibody LNCX004 provided by the present invention has good affinity on HCC827 cells, and its affinity is comparable to that of Amivantamab (KIH).

[0122] Example 3 Radiolabeling of Bispecific Antibodies

[0123] The bispecific antibody LNCX004 obtained in Example 1 above was mixed with the chelating agent p-SCN-Bn-DOTA at a molar ratio of 1:20 in a 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 30 kDa molecular cutoff, and the solvent was replaced with 0.01 M PBS buffer (pH 7.4) to obtain a conjugate of the target antibody and the chelating agent, which was named DOTA-LNCX004.

[0124] To 300uL of 0.1M sodium acetate solution (pH 4.5-5) add 177 LuCl3 solution and DOTA-LNCX004, wherein DOTA-LNCX004 and 177 The ratio of LuCl3 was 1 μg: 10 μCi, and the reaction was carried out in a constant temperature shaker at 37°C and 400 rpm for 1 hour. After the reaction, the product was purified using a PD-10 pre-packed gel column. 177 The Lu-labeled bispecific antibody conjugate was named 177Lu-DOTA-LNCX004, the labeling rate is greater than 75%, and the radiochemical purity is greater than 99% (see Figure 4).

[0125] Amivantamab (KIH) was tested using the above method. 177 Lu-labeled bispecific antibody conjugates are named 177 Lu-DOTA-Amivantamab (KIH), spare.

[0126] Example 4 Specific Binding of Antibody-Conjugated Nuclide Drugs

[0127] HCC827 human non-small cell lung cancer cell line (Xiamen Yimo Biotechnology Co., Ltd.) was used in a 24-well plate with approximately 2 × 10 cells per well. 5 500 μL serum-free 1640 medium was added to each well. The experiment set up a blocking group and an experimental group. The blocking group was incubated with 2 μg of non-radioactive LNCX004 antibody 2 h in advance. After that, 74 kBq (2 μCi) of LNCX004 was added to each well of the blocking group and the experimental group. 177 Lu-DOTA-LNCX004 was incubated at 37°C for 1 h, 2 h, 4 h, 12 h, and 24 h. The culture medium was discarded and the cells were washed twice with PBS. 200 μL of 0.1 M NaOH solution was added to each well to lyse the cells, and the lysate was collected. 2 μCi was detected using an automated γ counter. 177 The radioactivity of Lu-DOTA-scFv-Fc and the lysate in each well was counted, and the cellular uptake was expressed as %.

[0128] The analysis results are shown in Figure 5. The results show that the antibody-coupled nuclide drug provided by the present invention 177 Lu-DOTA-LNCX004 can specifically bind to HCC827 cells, which indicates that antibody-conjugated nuclide drugs 177 Lu-DOTA-LNCX004 has good cellular uptake ability.

[0129] Example 5 177 In vivo SPECT imaging study of Lu-DOTA-LNCX004

[0130] Humanized lung cancer cell line HCC827 was cultured at a rate of approximately 1×10 7 The number of cells was inoculated into the dorsal side of the right lower abdomen of BALB / c nude mice (4 weeks, female), and the tumor size was monitored every other day. When the long diameter was about 1 cm, in vivo imaging was performed. The experiment was set up in an experimental group and a positive control group, with 3-5 mice in each group. The experimental group used the antibody-coupled nuclide drug provided by the present invention. 177 Lu-DOTA-LNCX004, the positive control group used 177Lu-DOTA-Amivantamab (KIH). Mice in the experimental group and the positive control group were injected with about 150 μCi (about 200 μL) of 177 Lu-DOTA-LNCX004 or 177 Lu-DOTA-Amivantamab (KIH), in vivo SPECT imaging was performed at 4h, 16h, 24h, 48h, 72h, 96h, 120h, 144h and 168h after injection.

[0131] The imaging results are shown in FIG6 , which show that the antibody-coupled nuclide drug provided by the present invention 177 Lu-DOTA-LNCX004 compared to the positive control 177 Lu-DOTA-Amivantamab (KIH) has better SPECT imaging results. In addition, it can be seen that 177 Lu-DOTA-LNCX004 can accumulate in tumors over time and be cleared more quickly from organs such as the liver and normal tissues, suggesting that 177 Lu-DOTA-LNCX004 compared 177 Lu-DOTA-Amivantamab (KIH) has lower toxic side effects, which indirectly indicates that the bivalent bispecific antibody provided by the present invention may exhibit higher safety potential in clinical practice.

[0132] The above description is merely a preferred embodiment, which is intended to be illustrative and non-limiting of the combinations of features necessary to implement the present invention. The titles provided are not intended to limit the various embodiments of the present invention. Terms such as "comprising," "containing," and "including" are not intended to be limiting. In addition, unless otherwise indicated, the absence of a numeral modifier includes plural forms, and "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.

[0133] All disclosures and patents mentioned in this application are incorporated herein by reference. Without departing from the scope and spirit of the present invention, multiple modifications and variants of the described method and composition of the present invention will be apparent to those skilled in the art. Although the present invention has been described by specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, those multiple variants of the described pattern that are apparent to those skilled in the relevant art are intended to be included in the scope of the appended claims.

Claims

1. A bivalent bispecific antibody or an antigenic structure fragment thereof, characterized in that: The bispecific antibody or its antigenic structure fragment comprises: The first targeting moiety comprises a first VH domain and a first VL domain, and the first VH domain is x S) m The sequence is connected to the first VL domain; The second targeting moiety comprises a second VH domain and a second VL domain, and the second VH domain is y S) n The sequence is connected to the second VL domain; and a first Fc domain and a second Fc domain; in: The amino acid sequence of the first VH domain is shown in SEQ ID NO: 1; The amino acid sequence of the first VL domain is shown in SEQ ID NO: 2; The amino acid sequence of the second VH domain is shown in SEQ ID NO: 3; The amino acid sequence of the second VL domain is shown in SEQ ID NO: 4; The x is an integer selected from 1, 2, 3, 4, 5, and 6; The m is an integer selected from 1, 2, 3, 4, 5, and 6; The y is an integer selected from 1, 2, 3, 4, 5, and 6; The n is an integer selected from 1, 2, 3, 4, 5, and 6.

2. The bivalent bispecific antibody or antigenic structure fragment thereof according to claim 1, characterized in that: The amino acid sequence of the first targeting moiety is shown in SEQ ID NO:

5.

3. The bivalent bispecific antibody or antigenic structure fragment thereof according to any one of claims 1 or 2, characterized in that: The amino acid sequence of the second targeting moiety is shown in SEQ ID NO:

6.

4. The bivalent bispecific antibody or antigenic structure fragment thereof according to any one of claims 1 to 3, characterized in that: The first Fc domain and the second Fc domain are human immunoglobulin Fc domains; preferably, the first Fc domain and the second Fc domain are human IgG1 domains, human IgG2 domains, human IgG3 domains or human IgG4 Fc domains; more preferably, the first Fc domain and the second Fc domain are human IgG1 domains or human IgG4 Fc domains.

5. The bivalent bispecific antibody or antigenic structure fragment thereof according to any one of claims 1 to 4, characterized in that: The CH3 domains of the first Fc domain and the second Fc domain comprise modified structures, and the modification is used to promote dimerization of the two; preferably, the modification is an amino acid replacement; more preferably, the modification is Knob-into-hole (KIH).

6. The bivalent bispecific antibody or antigenic structure fragment thereof according to claim 1, characterized in that: The amino acid sequence of the first Fc domain is shown in SEQ ID NO: 7, or the amino acid sequence of the first Fc domain is a homologous sequence having at least 80% sequence identity with SEQ ID NO:

7.

7. The bivalent bispecific antibody or antigenic structure fragment thereof according to claim 1, characterized in that: The amino acid sequence of the second Fc domain is shown in SEQ ID NO: 8, or the amino acid sequence of the second Fc domain is a homologous sequence having at least 80% sequence identity with SEQ ID NO:

8.

8. The bivalent bispecific antibody or antigenic structure fragment thereof according to any one of claims 1 to 7, characterized in that: The C-terminus of the first targeting moiety is connected to the N-terminus of the first Fc domain, and the C-terminus of the second targeting moiety is connected to the N-terminus of the second Fc domain.

9. The bivalent bispecific antibody or antigenic structure fragment thereof according to any one of claims 1 to 7, characterized in that: The C-terminus of the first targeting moiety is connected to the N-terminus of the second Fc domain, and the C-terminus of the second targeting moiety is connected to the N-terminus of the first Fc domain.

10. A bivalent bispecific antibody-coupled nuclide drug, characterized in that: The bivalent bispecific antibody-conjugated radionuclide drug comprises a bivalent bispecific antibody or an antigenic structural fragment thereof conjugated to a radionuclide, wherein the bivalent bispecific antibody or the antigenic structural fragment thereof is the bivalent bispecific antibody according to any one of claims 1 to 9, and the radionuclide is a therapeutic radionuclide or a diagnostic radionuclide.

11. The bivalent bispecific antibody-coupled nuclide drug according to claim 10, characterized in that: The therapeutic radionuclide is selected from 32 P. 33 P. 47 Sc, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 75 Se, 77 As、 89 Sr. 90 Y. 99 Mo, 105 Rh, 109 Pd, 125 I. 131 I. 111 Ag, 111 In, 111m In, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 169 Second, 177 Lu, 186 Re、 188 Re、 189 Re、 194 Ir, 198 Au, 199 Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra or 225 Ac.

12. The bivalent bispecific antibody-coupled nuclide drug according to claim 10, characterized in that: The diagnostic radionuclide is selected from 18 F. 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As、 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 169 Second, 175 Lu, 177 Lu, 186 Re、 188 Re、 189 Re、 194 Ir, 198 Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 pb, 213 Bi, 223 Ra or 225 Ac.

13. The bivalent bispecific antibody-conjugated nuclide drug according to claim 10, characterized in that: The radionuclide is used to label the bivalent bispecific antibody or its antigenic structure fragment via a chelating agent.

14. The bivalent bispecific antibody-conjugated nuclide drug according to claim 13, characterized in that: The chelating agent is selected from DFO (deferoxamine), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), p-SCN-Bn-DOTA, DTPA (diethyltriaminepentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), TRITA (1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclotridecane), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), EDTA (ethylenediaminetetraacetic acid) and its derivatives.

15. An isolated nucleic acid molecule encoding the bivalent, bispecific antibody or antigenic structural fragment thereof according to any one of claims 1 to 9. An expression vector comprising the nucleic acid molecule of claim 15 .

17. A host cell comprising the nucleic acid molecule of claim 15 or the expression vector of claim 16.

18. A method for preparing a bivalent bispecific antibody or an antigenic structural fragment thereof, characterized in that: The method includes: a) culturing the host cell of claim 17 under conditions sufficient for the cell to produce the bivalent, bispecific antibody or antigenic fragment thereof, and b) collecting the bivalent bispecific antibody or antigenic structural fragment thereof produced by the host cell.

19. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the bivalent bispecific antibody or antigenic structure fragment thereof according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier; or the pharmaceutical composition comprises the bivalent bispecific antibody-coupled nuclide drug according to any one of claims 10 to 14, and a pharmaceutically acceptable carrier.

20. A kit, characterized in that The kit comprises the bivalent bispecific antibody or antigenic structure fragment thereof according to any one of claims 1 to 9, or the bivalent bispecific antibody-coupled nuclide drug according to any one of claims 10 to 14, or the pharmaceutical composition according to claim 19.

21. A method for treating or diagnosing a disease characterized by expression of EGFR and / or c-Met, the method comprising administering to a patient suffering from the disease characterized by expression of EGFR and / or c-Met a therapeutically effective amount of the bivalent bispecific antibody or antigenic fragment thereof according to any one of claims 1 to 9, or the bivalent bispecific antibody-conjugated nuclide drug according to any one of claims 10 to 14, or the pharmaceutical composition according to claim 19, or the kit according to claim 20.

22. The method according to claim 21, characterized in that The patient is a mammal; preferably, the patient is a human.

23. Use of the bivalent bispecific antibody or antigenic fragment thereof according to any one of claims 1 to 9, or the bivalent bispecific antibody-conjugated nuclide drug according to any one of claims 10 to 14, or the pharmaceutical composition according to any one of claim 19, or the kit according to claim 20 in the preparation of a medicament for treating or diagnosing a disease characterized by EGFR and / or c-Met expression.

24. A method for obtaining an image of a patient's part to be detected, characterized in that: The method comprises the following steps: a) administering to a patient an effective amount of the bivalent, bispecific antibody-conjugated nuclide drug according to any one of claims 10 to 14, or the pharmaceutical composition according to claim 19, or the kit according to claim 20; and b) performing positron emission tomography (PET) or SPECT on the patient; c) a detectable signal that identifies the radionuclide; d) generating an image based on the detectable signal, thereby obtaining an image of the patient's part to be detected.

25. The method according to claim 24, characterized in that The patient is a mammal; preferably, the patient is a human.

26. The method according to claim 24, characterized in that The patient suffers from or may suffer from a disease characterized by expression of EGFR and / or c-Met.

27. The method according to claim 21, or the use according to claim 23, or the method according to claim 24, characterized in that: The disease characterized by EGFR and / or c-Met expression is cancer; further, the cancer is an EGFR-positive tumor, a cMET-positive tumor, or an EGFR- and cMET-positive tumor.

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