Bifunctional fusion protein, radionuclide label, and use thereof

Abstract

Provided in the present invention is a bifunctional fusion protein radionuclide label, which has a high labeling yield and high radiochemical purity while maintaining high affinity and high functional activity, and has a suitable molecular weight, strong tissue penetration, a good imaging effect, a suitable circulation half-life, and strong molecular stability; the bifunctional fusion protein radionuclide label still has good affinity without being affected after radiolabeling a targeting carrier, has good biological metabolic activity and a good removal rate, and has reduced radiation toxicity, renal toxicity, hematological toxicity, and non-target organ toxicity; the bifunctional fusion protein radionuclide label has good enrichment in tumor cells, is conducive to implementing diagnosis and accurate staging of tumor lesions, and serves as a targeting carrier for implementing precision radiotherapy on primary lesions and metastatic lesions; and the bifunctional fusion protein radionuclide label has good safety and effectiveness potential, and has good application prospects in the field of solid tumor treatment.

Description

A bifunctional fusion protein, a radionuclide marker and its applications Technical Field

[0001] This invention relates to the fields of antibodies and nuclear medicine, specifically to a bifunctional fusion protein comprising an antigen-targeting binding portion and an albumin-targeting binding portion, and a radionuclide label of the bifunctional fusion protein. This invention also relates to the application of the above-mentioned bifunctional fusion protein and its radionuclide label. Background Technology

[0002] Cancer is the leading cause of death worldwide. Over the past few decades, cancer drugs (such as kinase inhibitors and monoclonal antibodies) have significantly improved patient prognosis. However, many patients still experience relapse, and advanced-stage patients may still experience disease progression even after treatment. In recent years, antibody-drug conjugates (ADCs) have seen rapid development in the field of anti-tumor therapy, reshaping treatment options for various cancers. However, some patients still experience disease progression after ADC treatment, and drug resistance to ADCs is receiving increasing attention. Resistance mechanisms exist at any stage of ADC drug entry into tumor cells, such as downregulation of tumor cell target protein expression or loss of antigen expression (preventing ADC drugs from recognizing and binding to target cells), defects in the endocytic flow pathway (preventing ADC drugs from entering cells), impaired lysosomal function or reduced lysosomal protein hydrolysis activity (preventing effective degradation of ADC drugs), and carrier resistance. To provide more clinical treatment options, novel carrier drugs such as immunostimulatory drug delivery, dual-drug delivery, and radionuclide labeling are receiving increasing attention.

[0003] Radionuclide drug conjugates (RDCs) are an emerging type of precision oncology therapy. Their structure consists of conjugates composed of antibodies or small molecules (ligands), linkers, chelators, and radioisotopes (radioactive isotopes) that mediate targeting. Under the precise targeting action of the antibody or small molecule ligand, the radionuclide reaches its target, exerting different functions such as imaging or therapy. Compared to ADCs, RDCs have significant competitive advantages. For example, RDCs do not need to enter cells or require linker breakage to release the drug; they can kill target cells using radiation generated during decay, thus improving the stability and safety of RDC drugs in vivo. RDCs kill cancer cells through radiation; within the radiation radius, even if tumor cells lack corresponding antigens, they can still act on tumor stromal cells, damaging or killing these cells and thus cutting off the stromal cells' supply of nutrients to the tumor cells, achieving a therapeutic effect. They also exhibit better resistance to drug resistance. With the approval and launch of Novartis' Lutathera and Pluvicto, RDC drugs have become wildly popular due to their significant efficacy, sparking a global RDC research boom and ushering in a new era of using radionuclide drugs for precision cancer treatment.

[0004] Since 2016, the FDA has approved 11 RDC drugs (see Table 1). Of these 11 drugs, 9 are for diagnosis and only 2 are for treatment. The indications and targets are concentrated in prostate cancer (PSMA) and neuroendocrine tumors (SSTRs), and the vast majority of the carriers are small molecule peptides. These small molecule peptides have small molecular weights and short blood half-lives, resulting in strong tumor penetration and low blood toxicity. However, these small molecule peptides are metabolized through the kidneys, exhibiting some nephrotoxicity, and it is difficult to screen for molecules with high affinity and high specificity. Furthermore, due to the small size of radioactive isotopes, using small molecule peptides as ligands often leads to low stability and uncontrollability of the radioactive isotopes. Therefore, it is desirable to conjugate and immobilize radioactive isotopes onto larger functional entities to achieve better control (e.g., antibodies).

[0005] Table 1. 11 RDC drugs approved by the FDA since 2016

[0006] Radionuclide-labeled antibodies can specifically recognize antigens on the surface of tumor cells, guiding the radionuclide to target the tumor. Due to their high accessibility, most RDCs primarily choose full-length monoclonal antibodies as carriers, such as... 90 Y-ibritumomab tiuxetan131 I-Omburtamab, 89 While common standard antibodies such as Zr-Girentixumab and Indium-111satumomab pendetide are structurally stable, their large molecular weight leads to poor tissue penetration, excessive retention in the bloodstream, and hematologic toxicity. Furthermore, the long half-life (up to several days) of antibodies results in a prolonged metabolic cycle after entering the body, causing the carried radionuclide to remain in the body for an extended period, leading to radiation toxicity. The long half-life also affects image quality and results in long detection intervals. Therefore, small-sized antibody-conjugated radioisotope probes have become a current research focus. However, developing RDCs based on small-sized antibodies also faces challenges. It is difficult to construct antibody fragments that possess high affinity, low toxicity, good pharmacokinetic characteristics, and structural stability. Additionally, the specific binding activity of some small-sized antibody fragments is easily affected by radiochemical activity, and some small-sized antibody fragments are more prone to aggregation. These factors limit the development and progress of RDC drugs. Therefore, there is an urgent need to provide more options for antibodies and RDCs to develop more effective targeted therapies. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a bifunctional fusion protein radionuclide label that not only has a suitable molecular weight, strong tissue penetration, and good imaging effect, but also a stable molecular structure. Even after conjugation with a radionuclide, the fusion protein molecule maintains good binding affinity and targeting specificity, has a suitable blood half-life, and strong tumor aggregation ability, making it suitable for widespread application in the diagnosis and treatment of solid tumor RDC.

[0008] Specifically, this invention provides a bifunctional fusion protein radionuclide marker, wherein the bifunctional fusion protein radionuclide marker comprises a radionuclide portion and a bifunctional fusion protein portion, wherein:

[0009] (1) The bifunctional fusion protein includes an albumin-binding portion and an antigen-targeting portion. The amino acid sequence of the albumin-binding portion is shown in SEQ ID NO: 1. The antigen-targeting portion is a Fab fragment. The antigen-targeting portion can target any selectable antigen binding site. The albumin-binding portion is directly connected to the C-terminus or N-terminus of the antigen-targeting portion or connected through any linking sequence.

[0010] (2) The radionuclide portion can be directly or through any chelating group conjugated to the bifunctional fusion protein portion, and the radionuclide can be any selectable nuclide.

[0011] Furthermore, the Fab (Antigen-binding fragment) is derived from human immunoglobulin G (IgG), such as human IgG1, IgG2, IgG3, and IgG4; preferably, the Fab fragment is derived from human immunoglobulin IgG1 and IgG4.

[0012] Furthermore, the Fab fragment comprises a heavy chain portion and a light chain portion. The heavy chain portion comprises a heavy chain variable domain (VH) and a heavy chain constant region 1 (CH1). The light chain portion comprises a light chain variable domain (VL) and a light chain constant region (CL).

[0013] Furthermore, the Fab fragment can specifically target Her2, PSMA, SSTR2, FAP-α, CA9, CD38, CD45, B7-H3, CEACAM5, IL2RA, Amyloid, CCKBR, CD22, CD33, CD37, CDH3, CEACAM1, FUT3, GD2, GRPR, IGF1R, KLK2, MSLN, NTSR1, PD-L1, MUC1, MUC16, 5T4, LIV-1, TROP2, NECTIN-4, CLDN18.2, CLDN6, GPRC5D, GPR56, CD205, BCMA, CD79b, FOLR1, etc.

[0014] In some preferred embodiments, the Fab fragment can specifically target Her2.

[0015] In some preferred embodiments, the Fab fragment is derived from the Fab fragment of pertuzumab (amino acid sequence see https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=11933), the Fab fragment of trastuzumab deruxtecan (amino acid sequence see https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=10516), or the Fab fragment of trastuzumab emtansine (trastuzumab). The Fab fragments of emtansine (amino acid sequence can be found at https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=9295), trastuzumab, disitamabvedotin (amino acid sequence can be found at https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=11048), and margetuximab (amino acid sequence can be found at https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=9799) are antibody or antigen-binding fragments.

[0016] In some specific embodiments, the Fab fragment is derived from the Fab fragment of pertuzumab, and the amino acid sequences of its heavy chain portion and light chain portion are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively:

[0017] In some specific embodiments, the Fab fragment is derived from the Fab fragment of trastuzumab, and the amino acid sequences of its heavy chain portion and light chain portion are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively:

[0018] In some preferred embodiments, the albumin-binding portion is directly linked to the C-terminus or N-terminus of the heavy chain portion of the Fab fragment or linked through any linking sequence to form the heavy chain of the bifunctional fusion protein. In this case, the light chain portion of the Fab fragment is the light chain of the bifunctional fusion protein. More preferably, the albumin-binding portion is linked to the C-terminus or N-terminus of the heavy chain portion of the Fab fragment through any linking sequence.

[0019] In some other preferred embodiments, the albumin-binding portion is directly linked to the C-terminus or N-terminus of the light chain portion of the Fab fragment or linked through any linking sequence to form the light chain of the bifunctional fusion protein. In this case, the heavy chain portion of the Fab fragment is the heavy chain of the bifunctional fusion protein. More preferably, the albumin-binding portion is linked to the C-terminus or N-terminus of the light chain portion of the Fab fragment through any linking sequence.

[0020] In some preferred embodiments, the albumin-binding portion is directly linked to the C-terminus of the heavy chain portion of the Fab fragment or linked through any linking sequence to form the heavy chain of the bifunctional fusion protein. In this case, the light chain portion of the Fab fragment is the light chain of the bifunctional fusion protein. More preferably, the albumin-binding portion is linked to the C-terminus of the heavy chain portion of the Fab fragment through any linking sequence.

[0021] In some other preferred embodiments, the albumin-binding portion is directly linked to the C-terminus of the light chain portion of the Fab fragment or linked through any linking sequence to form the light chain of the bifunctional fusion protein. In this case, the heavy chain portion of the Fab fragment is the heavy chain of the bifunctional fusion protein. More preferably, the albumin-binding portion is linked to the C-terminus of the light chain portion of the Fab fragment through any linking sequence.

[0022] Furthermore, the connection sequence described in any of the above is preferably (G x S) m And x is an integer chosen from 1, 2, 3, 4, 5, 6, and m is an integer chosen from 1, 2, 3, 4, 5, 6.

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

[0024] In some preferred embodiments, x is 4, m is 3, and (G x S) mis GGGGSGGGSGGGGS (i.e. (GGGGS)3) (SEQ ID NO: 4).

[0025] In some specific embodiments, the albumin-binding moiety is linked to the C-terminus of the heavy chain portion of the Fab fragment via (GGGGS)3, and the bifunctional fusion protein comprises (from the N-terminus to the C-terminus):

[0026] In other specific embodiments, the albumin-binding portion is linked to the C-terminus of the light chain portion of the Fab fragment via (GGGGS)3, and the bifunctional fusion protein comprises (from the N-terminus to the C-terminus):

[0027] In other specific embodiments, the albumin-binding portion is linked to the C-terminus of the heavy chain portion of the Fab fragment via (GGGGS)3, and the bifunctional fusion protein of the XDC drug comprises (from N-terminus to C-terminus):

[0028] In other specific embodiments, the albumin-binding portion is linked to the C-terminus of the light chain portion of the Fab fragment via (GGGGS)3, and the bifunctional fusion protein comprises (from the N-terminus to the C-terminus):

[0029] Furthermore, the radionuclide may be a radionuclide with therapeutic function; or the radionuclide may be a radionuclide with diagnostic function; or the radionuclide may be a radionuclide with both diagnostic and therapeutic functions.

[0030] Preferably, the radionuclides include, but are not limited to, 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 Ge71 As、 72 As、 74 As、 73 If、 75 Br、 76 Br、 82 Rb、 82m Rb、 83 Sr、 86 THE, 89 Zr、 90 Nb、 94m Tc、 110m See, 118 Sb、 120 I、 122 I、 124 I、 152 Tb、 67 Though, 99m Tc、 111 See, 123 I、 125 I、 155 Tb、 201 Tl、 32 P、 47 Scr、 66 Cu、 67 Cu、 77 As、 77 Br、 89 Sr、 90 THE, 105 Rh、 103 Pd、 111 Ag、 117m Sn、 131 I、 133 It is、 149 Tb、 161 Tb、 149 Pm、 153 Sm、 166 Ho、 177 Lu、 186 Re, 188 Re, 195m Pt、 212 Wind、 213 Wind、 211 Available、 212 Pb、 223 Ra、 225 Ac、 230 U、 33 P、 59 Faith、 67 Cu、 67 Though, 75 If、 77 As、 99 For、109 Pd, 142 Pr, 143 Pr, 166 Dy、 169 Er、 189 Re、 194 Ir、 198 Au、 199 Au、 211 Pb, 212 Bi.

[0031] 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 bifunctional fusion protein provided by this invention without the use of a chelating agent, such as...). 77 As、 131 I, 211 At, etc., are all non-metallic radionuclides that can be covalently bound to the bifunctional fusion protein provided by this invention, and chelating agents with appropriate structures are selected according to the different properties of the radionuclides.

[0032] In some preferred embodiments, the radionuclide is directly conjugated to the bifunctional fusion protein.

[0033] In some other preferred embodiments, the radionuclide is labeled with the bifunctional fusion protein via a chelating agent.

[0034] 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.

[0035] 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 derivative of 2-AMPTA, 2-(N-(2-hydroxybenzyl)aminomethyl)piperidine (2-AMPTA-H), which has additional functional groups suitable for conjugation with peptide structures. B), 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, p-SCN-Bn-DFO, and wherein the chelating group optionally comprises a chelated radioactive or non-radioactive cation.

[0036] 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.

[0037] In some preferred embodiments, the chelating agent is p-SCN-Bn-DOTA or p-SCN-Bn-NOTA.

[0038] To achieve coupling, the chelating agent is preferably covalently linked to the bifunctional fusion protein provided by the present invention via cysteine ​​residues (i.e., thiol coupling as commonly referred to in the art), or it can be linked to the amino groups of the amino acid residues of the fusion protein (i.e., amino coupling as commonly referred to in the art). Of course, other coupling methods are also possible, such as site-directed coupling, including but not limited to the introduction of reactive cysteine ​​(Thiomab technology), disulfide re-bridging, non-natural amino acid technology, enzyme catalysis technology, glycosyl coupling technology, proximity-induced antibody coupling (pClick) and other technologies.

[0039] The present invention also provides a pharmaceutical composition comprising a bifunctional fusion protein radionuclide label as described in any of the preceding claims, and a pharmaceutically acceptable carrier.

[0040] The present invention also provides a kit comprising a radiolabeled bifunctional fusion protein as described in any of the preceding claims; or the kit comprising a pharmaceutical composition as described in any of the preceding claims.

[0041] The present invention also provides the use of the bifunctional fusion protein radionuclide labeling material described in any of the foregoing claims, or the pharmaceutical composition described in any of the foregoing claims, or the 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, etc.

[0042] Furthermore, the tumor or cancer mentioned is a cancer that expresses Her2.

[0043] Furthermore, the tumor or cancer mentioned is Her2 low-expression cancer, Her2 moderate-expression cancer, or Her2 high-expression cancer.

[0044] Furthermore, the tumors or cancers mentioned are 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.

[0045] The present invention also provides a bifunctional fusion protein comprising an albumin-binding moiety and an antigen-targeting moiety. The amino acid sequence of the albumin-binding moiety is shown in SEQ ID NO: 1. The antigen-targeting moiety is a Fab fragment. The antigen-targeting moiety can target any selectable antigen binding site. The albumin-binding moiety is directly linked to the C-terminus or N-terminus of the antigen-targeting moiety or linked through any linking sequence.

[0046] Furthermore, the Fab (Antigen-binding fragment) is derived from human immunoglobulin G (IgG), such as human IgG1, IgG2, IgG3, and IgG4; preferably, the Fab fragment is derived from human immunoglobulin IgG1 and IgG4.

[0047] Furthermore, the Fab fragment comprises a heavy chain portion and a light chain portion. The heavy chain portion comprises a heavy chain variable domain (VH) and a heavy chain constant region 1 (CH1). The light chain portion comprises a light chain variable domain (VL) and a light chain constant region (CL).

[0048] Furthermore, the Fab fragment can specifically target Her2, PSMA, SSTR2, FAP-α, CA9, CD38, CD45, B7-H3, CEACAM5, IL2RA, Amyloid, CCKBR, CD22, CD33, CD37, CDH3, CEACAM1, FUT3, GD2, GRPR, IGF1R, KLK2, MSLN, NTSR1, PD-L1, MUC1, MUC16, 5T4, LIV-1, TROP2, NECTIN-4, CLDN18.2, CLDN6, GPRC5D, GPR56, CD205, BCMA, CD79b, FOLR1, etc.

[0049] In some preferred embodiments, the Fab fragment specifically targets Her2; more preferably, the Fab fragment is derived from the Fab fragment of pertuzumab (amino acid sequence see https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=11933), the Fab fragment of trastuzumab deruxtecan (amino acid sequence see https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=10516), or the Fab fragment of trastuzumab emtansine (trastuzumab). The Fab fragments of emtansine (amino acid sequence can be found at https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=9295), trastuzumab, disitamabvedotin (amino acid sequence can be found at https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=11048), and margetuximab (amino acid sequence can be found at https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=9799) are antibody or antigen-binding fragments.

[0050] In some specific embodiments, the Fab fragment is derived from the Fab fragment of pertuzumab, and the amino acid sequences of its heavy chain portion and light chain portion are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively:

[0051] In some specific embodiments, the Fab fragment is derived from the Fab fragment of trastuzumab, and the amino acid sequences of its heavy chain portion and light chain portion are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively:

[0052] In some preferred embodiments, the albumin-binding portion is directly linked to the C-terminus or N-terminus of the heavy chain portion of the Fab fragment or linked through any linking sequence to form the heavy chain of the bifunctional fusion protein. In this case, the light chain portion of the Fab fragment is the light chain of the bifunctional fusion protein. More preferably, the albumin-binding portion is linked to the C-terminus or N-terminus of the heavy chain portion of the Fab fragment through any linking sequence.

[0053] In some other preferred embodiments, the albumin-binding portion is directly linked to the C-terminus or N-terminus of the light chain portion of the Fab fragment or linked through any linking sequence to form the light chain of the bifunctional fusion protein. In this case, the heavy chain portion of the Fab fragment is the heavy chain of the bifunctional fusion protein. More preferably, the albumin-binding portion is linked to the C-terminus or N-terminus of the light chain portion of the Fab fragment through any linking sequence.

[0054] In some preferred embodiments, the albumin-binding portion is directly linked to the C-terminus of the heavy chain portion of the Fab fragment or linked through any linking sequence to form the heavy chain of the bifunctional fusion protein. In this case, the light chain portion of the Fab fragment is the light chain of the bifunctional fusion protein. More preferably, the albumin-binding portion is linked to the C-terminus of the heavy chain portion of the Fab fragment through any linking sequence.

[0055] In some other preferred embodiments, the albumin-binding portion is directly linked to the C-terminus of the light chain portion of the Fab fragment or linked through any linking sequence to form the light chain of the bifunctional fusion protein. In this case, the heavy chain portion of the Fab fragment is the heavy chain of the bifunctional fusion protein. More preferably, the albumin-binding portion is linked to the C-terminus of the light chain portion of the Fab fragment through any linking sequence.

[0056] Furthermore, the connection sequence described in any of the above is preferably (G x S) m And x is an integer chosen from 1, 2, 3, 4, 5, 6, and m is an integer chosen from 1, 2, 3, 4, 5, 6.

[0057] In some preferred embodiments, the (G) x S) m The following sequences can be selected:

[0058] In some preferred embodiments, x is 4, m is 3, and (G x S) mis GGGGSGGGSGGGGS (i.e. (GGGGS)3) (SEQ ID NO: 4).

[0059] In some specific embodiments, the albumin-binding moiety is linked to the C-terminus of the heavy chain portion of the Fab fragment via (GGGGS)3, and the bifunctional fusion protein comprises (from the N-terminus to the C-terminus):

[0060] In other specific embodiments, the albumin-binding portion is linked to the C-terminus of the light chain portion of the Fab fragment via (GGGGS)3, and the bifunctional fusion protein comprises (from the N-terminus to the C-terminus):

[0061] In other specific embodiments, the albumin-binding portion is linked to the C-terminus of the heavy chain portion of the Fab fragment via (GGGGS)3, and the bifunctional fusion protein comprises (from the N-terminus to the C-terminus):

[0062] In other specific embodiments, the albumin-binding portion is linked to the C-terminus of the light chain portion of the Fab fragment via (GGGGS)3, and the bifunctional fusion protein comprises (from the N-terminus to the C-terminus):

[0063] The present invention also provides an isolated nucleic acid molecule that encodes the bifunctional fusion protein described in any of the preceding claims.

[0064] The present invention also provides an expression vector comprising the nucleic acid molecules described in any of the preceding claims.

[0065] 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.

[0066] The present invention also provides a method for preparing the bifunctional fusion protein described in any one of the preceding claims, the method comprising:

[0067] a) Culture the host cells described in any of the preceding claims under conditions sufficient to induce cell production of the bifunctional fusion protein, and

[0068] b) Collect the bifunctional fusion protein produced by the host cell.

[0069] The present invention also provides a pharmaceutical composition comprising the bifunctional fusion protein described in any of the preceding claims, and a pharmaceutically acceptable carrier.

[0070] The present invention also provides a kit comprising the bifunctional fusion protein described in any of the preceding claims; or the kit comprising the pharmaceutical composition described in any of the preceding claims.

[0071] The present invention also provides the use of the bifunctional fusion protein described in any of the foregoing claims, or the pharmaceutical composition described in any of the foregoing claims, or the 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, etc.

[0072] Furthermore, the tumor or cancer mentioned is a cancer that expresses Her2.

[0073] Furthermore, the tumor or cancer mentioned is Her2 low-expression cancer, Her2 moderate-expression cancer, or Her2 high-expression cancer.

[0074] Furthermore, the tumors or cancers mentioned are 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.

[0075] The present invention further provides the application of the bifunctional fusion protein described in any of the above claims in the preparation of XDC drugs.

[0076] The present invention also provides an XDC drug comprising the bifunctional fusion protein described in any of the preceding claims.

[0077] The XDC drugs mentioned in any of the foregoing claims of this invention are a general term for various drug-conjugated compounds, consisting of a target 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 drugs involved in this invention is the bifunctional fusion protein provided by this invention, and all drug-conjugated forms based on the bifunctional fusion protein provided by this invention are within the scope of the XDC drugs involved in this invention. In some preferred embodiments, the XDC drug refers to antibody-drug conjugates (ADCs); in other preferred embodiments, the XDC drug refers to radionuclide drug conjugates (RDCs); and in still other preferred embodiments, the XDC drug refers to fragment antibody-conjugated compounds (FDCs).

[0078] The present invention also provides a pharmaceutical composition comprising the XDC drug described in any of the preceding claims, and a pharmaceutically acceptable carrier.

[0079] The present invention also provides a kit comprising the XDC drug as described in any of the preceding claims; or the kit comprising a pharmaceutical composition as described in any of the preceding claims.

[0080] The present invention also provides the use of the XDC drug, the pharmaceutical composition, or the 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.

[0081] Furthermore, the tumor or cancer mentioned is a cancer that expresses Her2.

[0082] Furthermore, the tumor or cancer mentioned is Her2 low-expression cancer, Her2 moderate-expression cancer, or Her2 high-expression cancer.

[0083] Furthermore, the tumors or cancers mentioned are 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.

[0084] This invention provides a novel bifunctional fusion protein radionuclide label with a unique structural model. It exhibits high labeling efficiency and radiochemical purity while maintaining high affinity and functional activity. It has a suitable molecular weight, strong tissue penetration, good imaging effect, suitable circulating half-life, and strong molecular stability. Even after radiolabeling and targeting, it retains good affinity without being affected. It possesses good bio-metabolic activity and clearance rate, reduced radiotoxicity, nephrotoxicity, hematologic toxicity, and non-target organ toxicity. It accumulates well in tumor cells, facilitating accurate diagnosis and staging of tumor lesions and serving as a targeting carrier for precise radiotherapy of primary and metastatic lesions. It demonstrates good safety and efficacy potential. Furthermore, the bifunctional fusion protein radionuclide label provided by this invention has a suitable molecular size, good tumor infiltration, and facilitates the isotope's entry into solid tumors to kill tumor cells. It also has good blood circulation time, does not bind to immune cells expressing FcγR receptors, and has low background, ensuring good tumor treatment efficacy while minimizing toxic side effects. Therefore, the bifunctional fusion protein radionuclide markers provided by this invention have promising applications in the treatment of solid tumors. Attached Figure Description

[0085] Figure 1 shows the affinity assay results of the bifunctional fusion protein LNCX005-1H for HER2 protein.

[0086] Figure 2 shows the affinity of the bifunctional fusion protein LNCX005-1H to albumin.

[0087] Figure 3 shows the ELISA affinity assay results of LNCX005-2H and trastuzumab for HER2 protein.

[0088] Figure 4 shows the effect of HER2-positive cells on 68 Cellular uptake of Ga-LNCX005-1H.

[0089] Figure 5 shows the injection. 68 Ga-FabX005-1H and 68 Micro-PET image after Ga-LNCX005-1H.

[0090] Figure 6 shows the injection. 177 Lu-FabX005-1H and 177 SPECT image after Lu-LNCX005-1H.

[0091] Figure 7 shows the injection. 177 SPECT images were observed up to 168 hours after Lu-LNCX005-1H.

[0092] Figure 8 shows the injection. 177 Lu-Trastuzumab, 177 Lu-FabX005-2 and 177 SPECT image after Lu-LNCX005-2H.

[0093] Figure 9 is 177 Plot of tumor growth inhibition over time for Lu-X005-2H and DS8201.

[0094] Figure 10 shows a single intravenous administration in CT26-HER2 tumor-bearing mice. 89 PET images after Zr-X005-2H (100μCi).

[0095] Figure 11 shows a single intravenous administration in NCI-N87 tumor-bearing mice. 89 PET images after Zr-X005-2H (100μCi). Detailed Implementation

definition

[0096] Before describing the invention in detail below, 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. 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.

[0097] Preferably, the terms used herein are as defined in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, edited by Leuenberger, HGW, Nagel, B. and Klbl, Hb (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0098] 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.

[0099] Several documents are referenced throughout this specification. Every document referenced herein (including all patents, patent applications, scientific publications, manufacturers' specifications, operating instructions, etc.) is incorporated herein by reference in its entirety, both above and below. Nothing herein shall be construed as an admission that the invention does not preclude any prior disclosure of such invention. Some documents referenced herein are marked "incorporated by reference." In the event of any conflict between definitions or teachings in such incorporated references and those set forth in this specification, the text of this specification shall prevail.

[0100] The elements of the invention will now be described. 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 construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and cover schemes that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, it should be considered that any permutation and combination of all the elements described in this application is disclosed in the specification of this application unless the context otherwise requires. [Specific Embodiments]

[0101] 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 for illustrative purposes only and should not be considered as limiting the scope of the present invention. Example 1: Construction of a bifunctional fusion protein molecule

[0102] The sequences SEQ ID NO:5 and SEQ ID NO:3 were obtained through gene synthesis. The sequences were then loaded into the expression vector PcDNA3.4 using homologous recombination. Positive bacterial cultures with correctly sequenced clones were selected, expanded, and low-endotoxin plasmids were extracted and sequenced for verification.

[0103] CHO-S cells were co-transfected using antibody heavy chain recombinant plasmid and antibody light chain plasmid via electroporation. Cells were electroporated at 160V for 15ms in a 0.4cm electroporation cuvette. The electroporated cells were then transferred to a shake flask containing 100mL of culture medium and incubated statically for 40min. After incubation, the shake flask was placed in a 37℃, 120rpm, 8% CO2 environment for 24h. Feed / sodium butyrate / antibiotics were added, and the cells were cultured for another 4 days.

[0104] Take the supernatant of the above cell culture medium and boil it at 5000 r·min -1 Centrifuge at 4℃ for 30 min, then filter through a 0.22 μm membrane. Refer to CaptureSelect. TM The CH1-XL affinity packing material instructions state that the concentration should be 0.5 mL / min. -1 Load the sample at a flow rate of [value missing], then remove non-specific adsorption with 10 column volumes of equilibration buffer, followed by elution with elution buffer. Collect the eluent and use 1 mol·L [concentration missing] solution. -1 Neutralization with Tris solution and replacement with PBS yielded the bifunctional fusion protein molecule involved in this embodiment (named LNCX005-1H), whose amino acid sequences are shown in SEQ ID NO:5 and SEQ ID NO:3, respectively.

[0105] The Fab fragment (denoted as FabX005-1) was constructed using the same method, and its amino acid sequences are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0106] Other bifunctional fusion proteins provided by this invention can be constructed according to the above method, such as: the bifunctional fusion protein "LNCX005-1L", whose heavy chain amino acid sequence is shown in SEQ ID NO:2 and whose light chain amino acid sequence is shown in SEQ ID NO:6; the bifunctional fusion protein "LNCX005-2H", whose heavy chain amino acid sequence is shown in SEQ ID NO:9 and whose light chain amino acid sequence is shown in SEQ ID NO:8; the bifunctional fusion protein "LNCX005-2L", whose heavy chain amino acid sequence is shown in SEQ ID NO:7 and whose light chain amino acid sequence is shown in SEQ ID NO:10; and the Fab fragment "FabX005-2", whose heavy chain amino acid sequence is shown in SEQ ID NO:7 and whose light chain amino acid sequence is shown in SEQ ID NO:8. Example 2: Radiolabeling of bifunctional fusion proteins

[0107] In this embodiment, a universal labeling method was used to radiolabel the bifunctional fusion protein LNCX005-1H antibody. The specific labeling method is as follows:

[0108] (1) 68 Ga labeling was performed using p-SCN-Bn-NOTA as the chelating agent.

[0109] Take 1 mg of the bifunctional fusion protein LNCX005-1H and add an equal volume of carbonate-bicarbonate buffer (pH = 9.2) to it. Simultaneously, dissolve 0.15 mg of p-SCN-Bn-NOTA in 10 μL of DMSO. Add the p-SCN-Bn-NOTA DMSO solution to LNCX005 and adjust the pH to 8.9-9.0. Incubate at room temperature for 2 hours. Meanwhile, prepare a PD-10 desalting column and wash it four times with PBS. Purify the above reaction solution using the washed PD-10 desalting column to obtain the purified chelating agent antibody conjugate, denoted as NOTA-p-NCS-Bn-LNCX005-1H.

[0110] Take 0 μg of NOTA-p-NCS-Bn-LNCX005-1H2O and rinse with 4 mL of 0.05 mol / L HCl. 68 Add 0.8 mL of sodium acetate solution (0.25 mol / L) to adjust the pH to 4.5, then take 1 mL of... 68 Ga liquid was added to the Nota-p-NCS-Bn-LNCX005-1H bifunctional fusion protein. The total volume should not be too large. The mixture was immediately vortexed and shaken at room temperature for 30 minutes. The protein was then purified using a PD-10 column to obtain the desired product. 68Ga radiolabeling yielded the bifunctional fusion protein LNCX005-1H (denoted as Ga). 68 (Ga-LNCX005-1H). Detected using Radio-TLC method. 68 The radiolabeling rate of Ga-LNCX005-1H is greater than 95%, and after purification by a PD-10 pre-packed gel column, it is further enhanced. 68 The radiochemical purity of Ga-LNCX005-1H is greater than 99%.

[0111] (2) 177 Lu labeling, using p-SCN-Bn-DOTA as the chelating agent.

[0112] Take 1 mg of the bifunctional fusion protein LNCX005-1H and add an equal volume of carbonate-bicarbonate buffer (pH = 9.2) to it. Simultaneously, dissolve 0.23 mg of p-SCN-Bn-DOTA in 10 μL of DMSO. Add the p-SCN-Bn-DOTA DMSO solution to the bifunctional fusion protein LNCX005-1H and adjust the pH to 8.9-9.0. Incubate at room temperature for 2 hours. Meanwhile, prepare a PD-10 desalting column for purification, washing the PD-10 column four times with PBS. Purify the above reaction solution using the washed PD-10 desalting column to obtain the purified chelating agent antibody conjugate, denoted as DOTA-p-NCS-Bn-LNCX005-1H.

[0113] Prepare 300 μL of sodium acetate solution (0.1 mol / L). 177 Lu was added to the sodium acetate solution, and the pH was adjusted to 4.5–5.5. Then, DOTA-p-NCS-Bn-LNCX005-1H was added to the solution. The reaction was carried out at 37°C (300–500 rpm) for 30 min using a constant temperature shaker. The solution was then purified using a PD-10 column to obtain… 177 Lu radiolabeling yielded the bifunctional fusion protein LNCX005-1H (denoted as...). 177 Lu-LNCX005-1H), detected by Radio-TLC method. 177 The radiolabeling rate of Lu-LNCX005-1H is greater than 95%, and after purification by a PD-10 pre-packed gel column, it is further enhanced. 177 The radiochemical purity of Lu-LNCX005-1H is greater than 99%.

[0114] (3) 89 Zr labeling was performed using p-SCN-Bn-DFO as the chelating agent.

[0115] Take 3 mg of LNCX005-2H antibody solution and add 0.2 M sodium carbonate-sodium bicarbonate solution (pH 9.0) to the LNCX005-2H antibody solution to make the solution volume 2100 μL. Dissolve 1 mg of p-SCN-Bn-DFO in 100 μL of DMSO. Take 84 μL of the p-SCN-Bn-DFO DMSO solution and add it to the LNCX005-2H antibody solution. Incubate in a constant temperature mixer (37℃) at 400 rpm for 2 h. After the reaction is complete, purify the reaction solution by ultrafiltration centrifugation to obtain the purified chelating agent antibody conjugate, denoted as DFO-p-NCS-Bn-LNCX005-2H.

[0116] Take 500 μL of HEPES solution (0.5 M, pH 7.2), and add 5 mCi 89 Zr was added to the HEPES solution, and the pH was adjusted to 7.0-7.5 with 2M sodium carbonate solution. 1.6 mg of DFO-p-NCS-Bn-LNCX005-2H solution was added to the above solution system, and the mixture was reacted in a constant temperature mixer (25℃) at 400 rpm for 1 h to obtain the desired product. 89 Zr radiolabeled LNCX005-2H (denoted as...) 89 The radiolabeling rate of Zr-LNCX005-2H was greater than 90% as detected by Radio-TLC (iTLC, developing solvent: 0.5M citric acid / sodium citrate, pH 5.5).

[0117] Other bifunctional fusion proteins (such as LNCX005-1L, LNCX005-2H, LNCX005-2L) and Fab fragments (such as FabX005-1, FabX005-2) provided by this invention can all be labeled according to the above method to obtain... 68 Ga-LNCX005-1L, 68 Ga-LNCX005-2H, 68 Ga-LNCX005-2L, 68 Ga-FabX005-1 68 Ga-FabX005-2; 177 Lu-LNCX005-1L, 177 Lu-LNCX005-2H, 177 Lu-LNCX005-2L, 177 Lu-FabX005-1 177 Lu-FabX005-2 and 89 Zr-LNCX005-1L, 89 Zr-LNCX005-2H, 89Zr-LNCX005-2L 89 Zr-FabX005-1 89 Zr-FabX005-2.

[0118] It is understood that the above method is merely an exemplary method, and other feasible labeling methods can also be used to radiolabel the above bifunctional fusion protein and Fab fragment. Alternatively, a commonly used radiolabeling method in the prior art can be selected to label the above fragments according to the radionuclide required. Of course, any suitable chelating agent can be used in this process. Example 3 Affinity Evaluation

[0119] (1) Affinity of LNCX005-1H

[0120] The affinity of LNCX005-1H at the protein level was determined using SPR (surface plasmon resonance) technology. The results showed that the bifunctional fusion protein LNCX005-1H has good affinity for both HER2 protein (see Figure 1) and albumin (see Figure 2).

[0121] (2) Affinity of LNCX005-2H

[0122] The affinity of LNCX005-2H was detected using an ELISA method. A solution containing human ERBB2 / HER2 / CD340-His protein (2 μg / mL, in 1% PBS) was added to each well of an ELISA plate, 30 μL per well, and incubated overnight at 4°C. The plate was washed five times with PBST, and then incubated with 5% PBSM blocking buffer at room temperature for 2 hours. The plate was washed five times with PBST, and then 100 μL of diluted antibody (in 1% PBSM) was added to each well, and incubated at room temperature for 60 minutes. The plate was washed five times with PBST, and then 100 μL of Goat-Anti-Human-Fab-HRP secondary antibody, diluted 1:5000 (in 1% PBSM), was added to each well, and incubated at room temperature for 60 minutes. The plate was washed five times with PBST, and then TMB substrate was added. The reaction was stopped using 2M stop buffer, and OD450 was measured.

[0123] The test results are shown in Figure 3. The results indicate that both the bifunctional fusion protein LNCX005-2H and the full-length trastuzumab antibody can specifically bind to the HER2 protein, meaning that the bispecific antibody fusion protein effectively retains the antibody's binding function to the HER2 terminus. Example 4 68 Specific binding of Ga-LNCX005-1H

[0124] HER2-positive cells (SKOV3 cells) were seeded into 24-well plates, 1.5 x 10 cells per well.5 Cells were cultured overnight until they adhered to the culture medium. The original medium was discarded, and 0.4 mL of serum-free medium was added. Experimental and blocking groups were set up. Pertuzumab was added to the blocking group half an hour beforehand. Then, 5 uCi of [unclear text - possibly a specific drug or treatment] was added to both the experimental and blocking groups. 68 Ga-LNCX005-1H. After incubation for 0.5h, 1h, 1.5h and 2h, the supernatant was discarded, and the cells were washed with PBS. 500ul of NaOH was added to each well to vigorously flush the bottom of the plate until cells detached. The cells and washing solution were collected, and the cell viability of each well was measured using a γ-counter.

[0125] The results are shown in Figure 4. HER2-positive cells showed... 68 The uptake of Ga-LNCX005-1H increased significantly over time, while the results of the blocking group showed that pre-incubation with the positive antibody significantly reduced the uptake of the probe by tumor cells. This demonstrates... 68 Ga-LNCX005-1H specifically binds to the HER2 receptor in cells, and its specific binding activity is not affected by radionuclide labeling. Example 5: In vivo imaging and targeted therapy study of the bifunctional fusion protein LNCX005-1H with radionuclide labeling.

[0126] (1) 68 Ga radiolabeled in vitro imaging study

[0127] The CT26 colorectal cancer cell line expressing the humanized HER2 receptor was implanted subcutaneously into the hind leg of Balb / C mice. After the tumors grew to a suitable size, the mice were randomly divided into experimental group 1 and control group 1. In control group 1, 200 uCi was injected via the tail vein. 68 Ga-FabX005-1 (the radiolabeling method in Example 2 was used to label FabX005-1) 68 Ga radiolabeled), experimental group 1 received 200 uCi via tail vein injection. 68 Ga-LNCX005-1H was used to anesthetize each group under isoflurane (2% isoflurane-30% oxygen / air) at 1h, 2h, 3h, 4h and 6h after injection, and imaging studies were performed on small animals using Micro-PET.

[0128] The results of the Micro-PET imaging are shown in Figure 5. 68 The Ga-FabX005-1 group (i.e., control group 1) showed weaker tumor enrichment at the tumor site, while 68 In the Ga-FabX005-1 group (i.e., control group 1), tumor-bearing mice showed higher renal and bladder signaling levels within 6 hours post-injection. In contrast, 68The Ga-LNCX005-1H group (i.e., experimental group 1) showed a significant signal at the tumor site, and the uptake at the tumor site increased over time, continuing until 6 hours. This indicates that the bifunctional fusion protein radionuclide label provided by this invention has a suitable blood circulation time, which, compared with the single Fab fragment radionuclide label, can significantly increase the probe's circulation time in vivo, delay probe excretion, and thus improve probe enrichment at the tumor site.

[0129] (2) 177 Lu radiolabeled in vitro imaging study

[0130] CT26 mouse colorectal cancer cells expressing the human HER2 receptor were implanted subcutaneously into the hind legs of Balb / C mice. After the tumors grew to a suitable size, the mice were randomly divided into experimental group 2 and control group 2. Control group 2 mice received a tail vein injection of 200 uCi of [a specific drug / method / treatment]. 177 Lu-FabX005-1 (the radiolabeling method in Example 2 was used to label FabX005-1) 177 Lu (radiolabeled); Experimental group 2 received 200 uCi via tail vein injection. 177 Lu-LNCX005-1H. At 12h, 24h, 48h, 72h, 96h and 120h after injection, the groups were anesthetized with isoflurane (2% isoflurane-30% oxygen / air) and imaging studies were performed on small animal SPECT.

[0131] The SPECT imaging results are shown in Figure 6. Within 120 hours after injection, 177 Lu-FabX005-1H did not show significant tumor enrichment at any tumor site. Meanwhile, at all time points post-injection, the liver, kidneys, and bladder of tumor-bearing mice exhibited elevated signal intensity. In contrast, at 12 hours post-injection, 177 Lu-LNCX005-1H initially showed a significant signal at the tumor site, and the signal contrast at the tumor site increased progressively over time, particularly within 48 hours post-injection. Simultaneously, uptake was observed in the heart, liver, and kidneys at various post-injection time points, but uptake at non-target sites gradually decreased over time until it almost disappeared. This further demonstrates that the bifunctional fusion protein radionuclide label provided by this invention significantly improves the probe's blood circulation time, delays probe excretion, and thus ensures effective enrichment of the probe at the tumor site.

[0132] (3) 177 Further research on Lu radiolabeled in vitro imaging

[0133] To further explore 177 Lu radiolabeled 177The imaging effect of Lu-LNCX005-1H was also studied in this embodiment, with imaging results at multiple observation points including 1h, 4h, 24h, 48h, 72h, 96h, and 168h. Specifically, mouse colorectal cancer CT26 cell line stably expressing high levels of human HER2 protein was implanted subcutaneously into the axilla of Balb / C mice. After the tumor grew to a suitable size, 0.8-1 mCi of [a specific drug / treatment] was injected via the tail vein. 177 Lu-LNCX005-1H. At 1 h, 4 h, 24 h, 48 h, 72 h, 96 h and 168 h after injection, the groups were anesthetized with isoflurane (2% isoflurane-30% oxygen / air) and imaging studies were performed on small animal SPECT.

[0134] As shown in Figure 7, 4 hours after injection, 177 Lu-LNCX005-1H initially showed a significant signal at the tumor site, and over time, the signal contrast at the tumor site increased within 24 hours post-injection, maintaining significant uptake at the tumor site until 168 hours, while uptake at non-target sites gradually decreased until it almost disappeared. This indicates that the bifunctional fusion protein radionuclide label (using...) provided by this invention... 177 Taking Lu-LNCX005-1H as an example, it significantly improves the probe's blood circulation time and delays its excretion from the body, thereby ensuring that the probe can be effectively enriched at the tumor site. This further illustrates that the bifunctional fusion protein radionuclide label provided by this invention (taking Lu-LNCX005-1H as an example) significantly improves the probe's blood circulation time and delays its excretion from the body, thus ensuring that the probe can be effectively enriched at the tumor site. 177 Taking Lu-LNCX005-1H as an example, it has good prospects for clinical treatment applications. Example 6 177 In vivo imaging and targeted therapy study of Lu-LNCX005-2H

[0135] (1) 177 Lu-LNCX005-2H and full-length antibody radionuclide marker 177 Lu-Trastuzumab and Fab fragment radionuclide markers 177 Comparative Study of Lu-FabX005-2

[0136] The mouse colorectal cancer CT26 cell line, stably transfected with high expression of human HER2 protein, was implanted subcutaneously into the axillary region of Balb / C mice. Once the tumors reached a suitable size, 0.8-1 mCi of [a specific drug / method] was injected via the tail vein. 177 Lu-Trastuzumab (using full-length Trastuzumab antibody, labeled according to the radionuclide labeling method provided in Example 2 of this invention) 177 Lu radiolabeling), 177 Lu-FabX005-2 and 177Lu-LNCX005-2H. At 1 h, 4 h, 24 h, 48 h, 72 h and 96 h after injection, the groups were anesthetized with isoflurane (2% isoflurane-30% oxygen / air) and imaging studies were performed on small animal SPECT.

[0137] As shown in Figure 8, the results are as follows: 177 Lu-LNCX005-2H showed good imaging performance in the CT26 tumor model. 24 hours after injection, 177 Lu-LNCX005-2H initially showed a significant signal at the tumor site, and the signal contrast at the tumor site increased over time. Even after 96 hours of experimentation, significant uptake was maintained at the tumor site, while uptake at non-target sites gradually decreased until it almost disappeared. This indicates that the bifunctional fusion protein radionuclide label (using...) provided by this invention... 177 Taking Lu-LNCX005-2H as an example, it significantly improved the probe's blood circulation time, delayed probe excretion, and thus ensured effective enrichment of the probe at the tumor site; full-length antibody 177 Lu-Trastuzumab also showed some tumor uptake signals, but the method provided by this invention... 177 Compared to Lu-LNCX005-2H 177 Lu-Trastuzumab significantly demonstrated better tumor infiltration ability, resulting in better imaging and a lower background. Furthermore, as can be seen from Figure 8, 177 Lu-Trastuzumab also exhibits some non-targeted uptake in the salivary glands of mice, which increases the risk of toxic side effects; radiolabeling using the Fab fragment... 177 Lu-FabX005-2 has a short half-life and is easily metabolized by the kidneys, showing a very short tumor uptake time and high renal uptake.

[0138] In summary, the present invention provides 177 Compared to full-length antibody radionuclide labeling, Lu-LNCX005-2H... 177 Lu-Trastuzumab and Fab fragment radionuclide markers 177 Lu-FabX005-2 both exhibited good tumor-targeted uptake and imaging effects, which further demonstrates the effectiveness of the bifunctional fusion protein radionuclide marker (using...) provided by this invention. 177Taking Lu-LNCX005-2H as an example, compared with full-length antibodies and Fab fragment radionuclide markers, it has better tumor-targeting uptake effect and longer blood circulation time. It can not only delay the excretion of probes from the body, thus ensuring that probes can be effectively enriched at the tumor site, but also effectively achieve the advantages of high tumor-targeting specificity, fast infiltration, and uniformity. It has strong safety and good clinical treatment and application prospects.

[0139] (2) 177 Comparative Study of Lu-LNCX005-2H and DS8201

[0140] For further research 177 Regarding the application prospects of Lu-LNCX005-2H, this embodiment also compares it with DS8201 (Trastuzumab deruxtecan). Specifically, N87 tumor cells were divided into groups of approximately 5 × 10⁻⁶ cells per tumor cell. 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, 177 The Lu-LNCX005-2H treatment group and the DS8201 treatment group each contained 8 mice. 177 Mice in the Lu-LNCX005-2H treatment group received a tail vein injection of approximately 18.5 MBq (approximately 200 μL) of radiopharmaceutical. Mice in the DS8201 treatment group received a tail vein injection of 5.4 mg / kg of the ADC drug DS8201. Mice in the saline group received 200 μg of saline per mouse. Tumor size, body weight, and survival status were monitored every other day after injection. Mice with tumors exceeding 1500 mm² were considered to have tumors. 3 As the monitoring endpoint.

[0141] As shown in Figure 9, the results are as follows: 177 Tumor growth in mice treated with Lu-LNCX005-2H was significantly inhibited, with no significant increase in tumor volume observed even after 40 days. In contrast, tumors in the saline group grew almost uncontrollably, with some tumors exceeding 1500 mm² by 40 days. 3 The average tumor volume also exceeded 1000 mm. 3 In the DS8201 treatment group, tumor growth was significantly inhibited in the early stages of drug injection, but the tumor growth rate accelerated after 10 days. Although it also showed a significant delay compared to the saline group, it was still faster than... 177 The tumor volume increased significantly in the Lu-LNCX005-2H treatment group, indicating that a single dose... 177Lu-LNCX005-2H showed more significant efficacy in treating tumors, while the ADC drug DS8201, although inhibiting tumor growth to some extent after a single dose, was less effective than [the other drug]. 177 Lu-LNCX005-2H. Example 8 89 In vivo imaging and targeted therapy study of Zr-LNCX005-2H

[0142] (1) 89 Efficacy of Zr-LNCX005-2H in CT26-HER2 Tumor-Bearing Mouse Model

[0143] CT26-HER2 tumor-bearing mice (3 males) were injected via tail vein. 89 Zr-LNCX005-2H injection solution (approximately 100 μCi / animal). The administration time and injection dose were recorded. PET imaging was performed at 1 h, 4 h, 6 h, 24 h, 48 h, and 144 h after administration, and the imaging time was recorded. The percentage of radioactivity per unit mass of the administered dose in the region of interest (ROI) was determined by the instrument's built-in image processing software (%ID / g).

[0144] As shown in Figure 10, the results indicate that a single intravenous administration of approximately 100 μCi to CT26-HER2 tumor-bearing mice... 89 Following Zr-LNCX005-2H administration, the radioactive material was initially distributed primarily in the heart and blood pool. Over time, the amount of radiopharmaceutical in the blood gradually decreased, while the radioactive uptake by the tumor gradually increased. From 1 to 48 hours post-administration, radioactive uptake in tumor tissue showed an upward trend, while radioactivity in other tissues showed a continuous downward trend. By 48 hours post-administration, radioactive uptake in the tumor reached its peak, and the uptake signal in other organs gradually decreased to background levels. This indicates... 89 After Zr-LNCX005-2H was administered via the tail vein into CT26-HER2 tumor-bearing mice, it rapidly distributed to target organs via the bloodstream and was quickly cleared from non-target tissues. Tumor uptake gradually increased, and good imaging contrast was observed at time points 24 hours and beyond after injection.

[0145] (2) 89 Efficacy study of Zr-LNCX005-2H in NCI-N87 tumor-bearing mouse model

[0146] NCI-N87 tumor-bearing mice (3 males) were injected via tail vein. 89Zr-LNCX005-2H injection (100 μCi / animal) was administered at the following times and doses. SPECT imaging was performed at 1 h, 4 h, 6 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h post-administration. The imaging time was recorded. The percentage of radioactivity per unit mass of the administered dose (%ID / g) in the region of interest (ROI) was determined using the instrument's built-in image processing software.

[0147] As shown in Figure 11, the results indicate that a single tail vein injection of 100 μCi into NCI-N87 tumor-bearing mice... 89 Following Zr-LNCX005-2H administration, the radioactive material was primarily distributed in the blood and heart. From 1 to 48 hours post-administration, radioactive uptake in tumor tissue continuously increased, while uptake in other organs gradually decreased. The tumor's radioactive uptake peaked at 48 hours post-administration, while signals in other organs gradually decreased to background levels. Good imaging contrast was observed at the tumor site at 48, 72, 96, and 120 hours post-injection. This indicates... 89 After being administered intravenously to NCI-N87 tumor-bearing mice, Zr-LNCX005-2H rapidly distributed to the tumor tissue via the bloodstream and was quickly cleared from non-target tissues, exhibiting good imaging contrast at all imaging time points.

[0148] The results from the two mouse models described above demonstrate that the radionuclide markers provided by this invention... 89 Zr-LNCX005-2H both exhibited good tumor-targeted uptake and imaging effects, which further demonstrates the effectiveness of the bifunctional fusion protein radionuclide marker (using...) provided by this invention. 89 Taking Zr-LNCX005-2H as an example, it can effectively achieve the advantages of high tumor targeting specificity, rapid infiltration, and uniformity, with strong safety and good clinical treatment and application prospects.

[0149] In summary, this invention provides a novel structural mode of bifunctional fusion protein radionuclide labeling, which has the following technical advantages:

[0150] (1) The bifunctional fusion protein provided by the present invention was radiolabeled with a high labeling rate and radiochemical purity, which indicates that the bifunctional fusion protein provided by the present invention is suitable for radiolabeling in the field of RDC drugs.

[0151] (2) The bifunctional fusion protein provided by the present invention still maintains good specific binding activity after radionuclide labeling, which indicates that the bifunctional fusion protein provided by the present invention has good structural stability and its antibody activity after conjugation with radionuclides is not affected by radionuclide radioactivity.

[0152] (3) The contrast of the bifunctional fusion protein radionuclide marker provided by this invention increases over time at the tumor site after injection. This indicates that the bifunctional fusion protein in the bifunctional fusion protein radionuclide marker provided by this invention has an appropriate molecular weight and strong tissue penetration.

[0153] (4) Although uptake by the heart, liver, and kidneys is observed at the initial time point after injection of the bifunctional fusion protein radionuclide provided by this invention, uptake at non-target sites gradually decreases over time until it almost disappears. This indicates that the bifunctional fusion protein radionuclide provided by this invention has a suitable circulating half-life, good bio-metabolic activity and clearance rate, and good blood toxicity. It can effectively achieve the therapeutic purpose while significantly reducing the patient's own radiation toxicity, demonstrating strong safety and excellent clinical application prospects.

[0154] (5) Compared to 177 Lu-FabX005-1, the present invention provides a bifunctional fusion protein radionuclide marker (using... 177 Taking Lu-LNCX005-1H as an example, a significant signal began to appear at the tumor site 12 hours after injection, and the signal contrast at the tumor site increased over time, with the signal contrast increasing within 48 hours after injection. This indicates that the bifunctional fusion protein radionuclide marker provided by this invention has good tumor uptake and can achieve effective enrichment at the tumor site. This also shows that the bifunctional fusion protein radionuclide marker provided by this invention has good clinical therapeutic efficacy and application prospects.

[0155] (6) Compared to 177 Lu-FabX005-1 (high signal intensity in the liver, kidneys, and bladder of tumor-bearing mice at all time points after injection), the bifunctional fusion protein radionuclide marker provided by this invention (with... 177 (Taking Lu-LNCX005-1H as an example) At various time points after injection, significant uptake was observed in the heart, liver, and kidneys. However, as time progressed, uptake at non-target sites gradually decreased until it almost disappeared. This indicates that the bifunctional fusion protein radionuclide marker provided by this invention can significantly reduce hematologic and renal toxicity, as well as non-target organ toxicity, demonstrating excellent safety potential.

[0156] (7) 89 The results of the two mouse models described above demonstrate that the radionuclide marker provided by this invention, Zr-LNCX005-2H, is... 89 Zr-LNCX005-2H both exhibited good tumor-targeted uptake and imaging effects, which further demonstrates the effectiveness of the bifunctional fusion protein radionuclide marker (using...) provided by this invention.89 Taking Zr-LNCX005-2H as an example, it can effectively achieve the advantages of high tumor targeting specificity, rapid infiltration, and uniformity, with strong safety and good clinical treatment and application prospects.

[0157] In summary, the bifunctional fusion protein radionuclide markers provided by this invention exhibit high labeling rates and radiochemical purity, while maintaining high affinity and functional activity. Further preclinical animal studies have confirmed their excellent imaging effects and good enrichment in tumor cells, facilitating accurate diagnosis and staging of tumor lesions, as well as their use as targeted carriers for precise radiotherapy of primary and metastatic lesions. These bifunctional fusion protein radionuclide markers increase the absorbed tumor dose and reduce the dose to non-target tissues, which is particularly crucial for the development of RDCs in solid tumors. Furthermore, the bifunctional fusion protein radionuclide markers provided by this invention have a suitable molecular size and good tumor invasiveness, facilitating the isotope's entry into the solid tumor to kill tumor cells. They also have good blood circulation time, do not bind to immune cells expressing FcγR receptors, and have low background, ensuring good tumor therapeutic effects while minimizing toxic side effects. Therefore, the bifunctional fusion protein radionuclide markers provided by this invention have promising applications in the treatment of solid tumors.

[0158] 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.

[0159] 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. A bifunctional fusion protein radionuclide label, characterized in that, The bifunctional fusion protein radionuclide label comprises a radionuclide moiety and a bifunctional fusion protein moiety, wherein: the bifunctional fusion protein moiety comprises an albumin binding moiety and an antigen targeting moiety, the albumin binding moiety has an amino acid sequence as shown in SEQ ID NO: 1, the antigen targeting moiety is a Fab fragment, the antigen targeting moiety can target any selectable antigen binding site, and the albumin binding moiety is directly connected to the C-terminal end / N-terminal end of the antigen targeting moiety or connected through any connecting sequence; the radionuclide moiety can be conjugated to the bifunctional fusion protein moiety directly or through any chelating group, and the radionuclide is any selectable nuclide.

2. The bifunctional fusion protein radionuclide label of claim 1, wherein, The Fab fragment is derived from human immunoglobulin G; preferably, the Fab fragment is derived from human immunoglobulin IgG1, IgG2, IgG3, IgG4; more preferably, the Fab fragment is derived from human immunoglobulin IgG1, IgG4.

3. The bifunctional fusion protein radionuclide label of claim 1, wherein, The Fab fragment specifically targets Her2, PSMA, SSTR2, FAP-alpha, CA9, CD38, CD45, B7-H3, CEACAM5, IL2RA, Amyloid, CCKBR, CD22, CD33, CD37, CDH3, CEACAM1, FUT3, GD2, GRPR, IGF1R, KLK2, MSLN, NTSR1, PD-L1, MUC1, MUC16, 5T4, LIV-1, TROP2, NECTIN-4, CLDN18.2, CLDN6, GPRC5D, GPR56, CD205, BCMA, CD79b, FOLR1.

4. The bifunctional fusion protein radionuclide label of claim 3, wherein, The Fab fragment specifically targets Her2.

5. The bifunctional fusion protein radionuclide label of claim 4, wherein, The Fab fragment comprises a heavy chain portion and a light chain portion, the amino acid sequence of the heavy chain portion of the Fab is as shown in SEQ ID NO: 2, and the amino acid sequence of the light chain portion of the Fab is as shown in SEQ ID NO: 3; or the amino acid sequence of the heavy chain portion of the Fab is as shown in SEQ ID NO: 7, and the amino acid sequence of the light chain portion of the Fab is as shown in SEQ ID NO:

8.

6. The bifunctional fusion protein radionuclide label of claim 5, wherein, The albumin binding moiety is directly connected to the C-terminal end / N-terminal end of the heavy chain portion of the Fab fragment or connected through any connecting sequence, or the albumin binding moiety is directly connected to the C-terminal end / N-terminal end of the light chain portion of the Fab fragment or connected through any connecting sequence; preferably, the albumin binding moiety is directly connected to the C-terminal end of the heavy chain portion of the Fab fragment or connected through any connecting sequence, or the albumin binding moiety is directly connected to the C-terminal end of the light chain portion of the Fab fragment or connected through any connecting sequence.

7. The bifunctional fusion protein radionuclide label of claim 6, wherein, The connecting sequence is (G x S) m , and x is an integer optionally selected from 1, 2, 3, 4, 5, 6, and m is an integer optionally selected from 1, 2, 3, 4, 5, 6.

8. The bifunctional fusion protein radionuclide label according to claim 7, characterized in that, The connecting sequence (G x S) m x is 4 and m is 3, the connecting sequence is GGGGSGGGGSGGGGS (i.e., (GGGGS)3) (SEQ ID NO: 4).

9. The bifunctional fusion protein radionuclide label of claim 8, wherein, The bifunctional fusion protein comprises a heavy chain and a light chain, the heavy chain amino acid sequence of the bifunctional fusion protein is shown as SEQ ID NO: 5 and the light chain amino acid sequence of the bifunctional fusion protein is shown as SEQ ID NO: 3, or the heavy chain amino acid sequence of the bifunctional fusion protein is shown as SEQ ID NO: 2 and the light chain amino acid sequence of the bifunctional fusion protein is shown as SEQ ID NO: 6, or the heavy chain amino acid sequence of the bifunctional fusion protein is shown as SEQ ID NO: 9 and the light chain amino acid sequence of the bifunctional fusion protein is shown as SEQ ID NO: 8, or the heavy chain amino acid sequence of the bifunctional fusion protein is shown as SEQ ID NO: 7 and the light chain amino acid sequence of the bifunctional fusion protein is shown as SEQ ID NO:

10.

10. The bifunctional fusion protein radionuclide label of any one of claims 1-9, wherein, The radionuclide is selected from the group consisting of 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, 211 Pb, 212 Bi.

11. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the bifunctional fusion protein radionuclide marker of any one of claims 1-10 and a pharmaceutically acceptable carrier.

12. A kit characterized in that, The kit comprises the bifunctional fusion protein radionuclide marker of any one of claims 1-10 or the pharmaceutical composition of claim 11.

13. Use of the bifunctional fusion protein radionuclide marker of any one of claims 1-10, or the pharmaceutical composition of claim 11, or the kit of claim 12 in the preparation of a medicament for treating a tumor or cancer.

14. Use according to claim 13, characterized in that, The tumor or cancer is a Her2-expressing cancer.

15. Use according to claim 14, characterized in that, The tumor or cancer is a Her2-low-expressing cancer, a Her2-medium-expressing cancer or a Her2-high-expressing cancer.

16. The use according to claim 15, characterized in that, The tumor or cancer is non-limitingly 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.

17. A bifunctional fusion protein, characterized in that, The bifunctional fusion protein comprises an albumin binding moiety and an antigen targeting moiety, the albumin binding moiety has an amino acid sequence shown as SEQ ID NO: 1, the antigen targeting moiety is a Fab fragment, the antigen targeting moiety can target any selectable antigen binding site, and the albumin binding moiety is directly connected to the C-terminus of the antigen targeting moiety or connected through any connecting sequence.

18. The bifunctional fusion protein of claim 17, wherein, The Fab fragment is derived from human immunoglobulin G; preferably, the Fab fragment is derived from human immunoglobulin IgG1, IgG2, IgG3, IgG4; more preferably, the Fab fragment is derived from human immunoglobulin IgG1, IgG4.

19. The bifunctional fusion protein of claim 18, wherein, the Fab fragment specifically targets Her2, PSMA, SSTR2, FAP-alpha, CA9, CD38, CD45, B7-H3, CEACAM5, IL2RA, Amyloid, CCKBR, CD22, CD33, CD37, CDH3, CEACAM1, FUT3, GD2, GRPR, IGF1R, KLK2, MSLN, NTSR1, PD-L1, MUC1, MUC16, 5T4, LIV-1, TROP2, NECTIN-4, CLDN18.2, CLDN6, GPRC5D, GPR56, CD205, BCMA, CD79b, FOLR1.

20. The bifunctional fusion protein of claim 19, wherein, the Fab fragment specifically targets Her2.

21. The bifunctional fusion protein of claim 20, wherein, the Fab fragment comprises a heavy chain portion and a light chain portion, and the amino acid sequence of the heavy chain portion of the Fab is as shown in SEQ ID NO: 2 and the amino acid sequence of the light chain portion of the Fab is as shown in SEQ ID NO: 3, or the amino acid sequence of the heavy chain portion of the Fab is as shown in SEQ ID NO: 7 and the amino acid sequence of the light chain portion of the Fab is as shown in SEQ ID NO:

8.

22. The bifunctional fusion protein of claim 21, wherein, the albumin binding moiety is directly connected to the C-terminus of the heavy chain portion of the Fab fragment or is connected through any connecting sequence, or the albumin binding moiety is directly connected to the C-terminus of the light chain portion of the Fab fragment or is connected through any connecting sequence.

23. The bifunctional fusion protein of claim 22, wherein, The connection sequence is (G) x S) m And x is an integer chosen from 1, 2, 3, 4, 5, 6, and m is an integer chosen from 1, 2, 3, 4, 5, 6.

24. The bifunctional fusion protein of claim 23, wherein, The connecting sequence (G x S) m x is 4 and m is 3, the connecting sequence is GGGGSGGGGSGGGGS (i.e., (GGGGS)3) (SEQ ID NO: 4).

25. The bifunctional fusion protein of claim 24, wherein, the bifunctional fusion protein comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain of the bifunctional fusion protein is as shown in SEQ ID NO: 5 and the amino acid sequence of the light chain of the bifunctional fusion protein is as shown in SEQ ID NO: 3, or the amino acid sequence of the heavy chain of the bifunctional fusion protein is as shown in SEQ ID NO: 2 and the amino acid sequence of the light chain of the bifunctional fusion protein is as shown in SEQ ID NO: 6, or the amino acid sequence of the heavy chain of the bifunctional fusion protein is as shown in SEQ ID NO: 9 and the amino acid sequence of the light chain of the bifunctional fusion protein is as shown in SEQ ID NO: 8, or the amino acid sequence of the heavy chain of the bifunctional fusion protein is as shown in SEQ ID NO: 7 and the amino acid sequence of the light chain of the bifunctional fusion protein is as shown in SEQ ID NO:

10.

26. Use of the bifunctional fusion protein of any one of claims 17-25 in the preparation of an XDC medicament.

27. An XDC medicament, characterized in that, the XDC medicament comprises the bifunctional fusion protein of any one of claims 17-25.

28. An isolated nucleic acid molecule encoding the bifunctional fusion protein of any one of claims 17-25.

29. An expression vector comprising the nucleic acid molecule of claim 28.

30. A host cell comprising the nucleic acid molecule of claim 28 or the expression vector of claim 29.

31. A method of producing the bifunctional fusion protein of any one of claims 17-25, comprising, the method comprises: the Fab fragment specifically targets Her2, PSMA, SSTR2, FAP-alpha, CA9, CD38, CD45, B7-H3, CEACAM5, IL2RA, Amyloid, CCKBR, CD22, CD33, CD37, CDH3, CEACAM1, FUT3, GD2, GRPR, IGF1R, KLK2, MSLN, NTSR1, PD-L1, MUC1, MUC16, 5T4, LIV-1, TROP2, NECTIN-4, CLDN18.2, CLDN6, GPRC5D, GPR56, CD205, BCMA, CD79b, FOLR1. the Fab fragment specifically targets Her2. the Fab fragment comprises a heavy chain portion and a light chain portion, and the amino acid sequence of the heavy chain portion of the Fab is as shown in SEQ ID NO: 2 and the amino acid sequence of the light chain portion of the Fab is as shown in SEQ ID NO: 3, or the amino acid sequence of the heavy chain portion of the Fab is as shown in SEQ ID NO: 7 and the amino acid sequence of the light chain portion of the Fab is as shown in SEQ ID NO:

8. the albumin binding moiety is directly connected to the C-terminus of the heavy chain portion of the Fab fragment or is connected through any connecting sequence, or the albumin binding moiety is directly connected to the C-terminus of the light chain portion of the Fab fragment or is connected through any connecting sequence. the bifunctional fusion protein comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain of the bifunctional fusion protein is as shown in SEQ ID NO: 5 and the amino acid sequence of the light chain of the bifunctional fusion protein is as shown in SEQ ID NO: 3, or the amino acid sequence of the heavy chain of the bifunctional fusion protein is as shown in SEQ ID NO: 2 and the amino acid sequence of the light chain of the bifunctional fusion protein is as shown in SEQ ID NO: 6, or the amino acid sequence of the heavy chain of the bifunctional fusion protein is as shown in SEQ ID NO: 9 and the amino acid sequence of the light chain of the bifunctional fusion protein is as shown in SEQ ID NO: 8, or the amino acid sequence of the heavy chain of the bifunctional fusion protein is as shown in SEQ ID NO: 7 and the amino acid sequence of the light chain of the bifunctional fusion protein is as shown in SEQ ID NO:

10.

26. Use of the bifunctional fusion protein of any one of claims 17-25 in the preparation of an XDC medicament. the XDC medicament comprises the bifunctional fusion protein of any one of claims 17-25.

28. An isolated nucleic acid molecule encoding the bifunctional fusion protein of any one of claims 17-25.

29. An expression vector comprising the nucleic acid molecule of claim 28.

30. A host cell comprising the nucleic acid molecule of claim 28 or the expression vector of claim 29. the method comprises: a) culturing the host cell of claim 30 under conditions sufficient for the cell to produce the bifunctional fusion protein, and b) collecting the bifunctional fusion protein produced by the host cell.

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