A modified affibody conjugate and methods of making and using the same

Abstract

The application provides a modified Affibody conjugate and a preparation method and application thereof, and belongs to the technical field of biological medicines.The application provides a modified Affibody conjugate and a preparation method and application thereof.The double modification strategy of site-specific S-glycosylation modification combined with small molecule albumin binding ligand effectively prolongs the in-vivo circulation half-life of the probe, enhances the specific accumulation and long-term retention of the probe at the tumor site, and significantly reduces the non-specific uptake of the kidney and the risk of radiation exposure on the basis of maintaining the small size advantage of the Affibody molecule and the original targeting affinity.After being labeled with a radionuclide, the conjugate can be applied to high-contrast molecular imaging and targeted radionuclide therapy of HER2 positive tumors, and provides a safe and effective radiopharmaceutical scheme for precise diagnosis and treatment of tumors, and has a good clinical transformation prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a modified Affibody conjugate, its preparation method, and its application. Background Technology

[0002] Human epidermal growth factor receptor 2 (HER2) is an important member of the ErbB receptor tyrosine kinase family, exhibiting gene amplification or protein overexpression in various malignant tumors, including breast cancer, gastric cancer, ovarian cancer, and lung cancer. Aberrant activation of HER2 can promote tumor cell proliferation, invasion, and metastasis, and is closely related to poor patient prognosis. Currently, targeted therapies against HER2 (such as trastuzumab, pertuzumab, and antibody-drug conjugates) are widely used clinically, significantly improving the survival prognosis of patients with HER2-positive tumors. However, a considerable proportion of patients still develop primary or secondary drug resistance, and there is significant heterogeneity in HER2 expression levels among different tumor types and metastatic lesions. Therefore, developing molecular imaging probes capable of accurately detecting HER2 expression levels and monitoring treatment response in real time, as well as targeted radiopharmaceuticals capable of delivering therapeutic radioactive doses, is of great significance for achieving personalized precision diagnosis and treatment of HER2-positive tumors.

[0003] Affibody molecules are small, artificial affinity proteins designed based on the Z-domain backbone of staphylococcal protein A, with a molecular weight of approximately 6.5 kDa. Compared to monoclonal antibodies, affibody molecules offer advantages such as small molecular size, strong tissue penetration, high target binding affinity, low immunogenicity, good structural stability, and the ability to be prepared through chemical synthesis or recombinant expression. They represent a novel targeting vector with significant application potential in nuclear medicine molecular imaging and targeted radiotherapy. Currently, several affibody radioactive probes targeting HER2, EGFR, and PD-L1 have entered preclinical or clinical research stages.

[0004] However, affibody's molecular weight is far below the glomerular filtration threshold, and after intravenous injection, it is primarily cleared rapidly by the kidneys. Some probes are efficiently reabsorbed in the proximal convoluted tubule epithelial cells of the kidneys, leading to nonspecific radioactive accumulation and long-term retention in the kidneys. This pharmacokinetic characteristic not only limits the imaging effectiveness of the probes in abdominal and pelvic lesions but also significantly increases the radiation dose burden on the kidneys, becoming a major technical bottleneck restricting the clinical translation and widespread application of affibody radiopharmaceuticals.

[0005] Therefore, there is an urgent need to develop a modification strategy that can precisely regulate the pharmacokinetic behavior of Affibody while maintaining its small molecule advantages. Summary of the Invention

[0006] The purpose of this invention is to provide a modified Affibody conjugate that solves the technical problems of short in vivo circulation time, high renal uptake and retention of existing Affibody radioactive probes, as well as the low modularity of existing modification strategies and the difficulty in balancing pharmacokinetic optimization and targeting activity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a modified Affibody conjugate, the Affibody conjugate comprising: Affibody protein; A glycosyl modification group covalently linked to the thiol group of the cysteine ​​residue in the Affibody protein via an S-glycosidic bond; and Small albumin-binding ligands covalently linked to the glycosyl-modified groups; The glycosyl modification group is N-acetylgalactosamine, N-acetylglucosamine, or N-acetylmannosamine.

[0008] Preferably, the small molecule albumin binding ligand is 4-iodophenylbutyric acid, 4-methylphenylbutyric acid, or palmitic acid.

[0009] Preferably, the Affibody conjugate further comprises a chelating agent group, which is covalently linked to the small molecule albumin binding ligand.

[0010] Preferably, the chelating agent group is DOTA.

[0011] Preferably, the Affibody protein is HER2-targeted Affibody.

[0012] Preferably, the small molecule albumin binding ligand is covalently linked to the glycosyl modification group via a linker arm, the linker arm comprising a triazole ring structure.

[0013] This invention provides a modified Affibody conjugate, which is any one of the following compounds 10a, 10b, and 10c: .

[0014] The present invention also provides a method for preparing the above-mentioned modified Affibody conjugate, comprising the following steps: (1) In the presence of an oxidant, the Affibody protein is reacted with a sodium glycosyl sulfinate compound containing an azide group to obtain a glycosylated Affibody intermediate containing an azide group; (2) The glycosylated Affibody intermediate with an azide group is coupled with the DBCO-small molecule albumin binding ligand conjugate through a strain-promoted azide-alkyne cycloaddition reaction to obtain the modified Affibody conjugate.

[0015] Preferably, the sodium glycosyl sulfinate compound with an azide group is sodium 2-acetamido-2-deoxyglycosyl sulfinate with an azide group; Step (1) is carried out in a buffer solution with a pH of 6.0 to 8.0, at a reaction temperature of 15 to 40°C, and for a reaction time of 15 to 60 minutes; The oxidant is tert-butyl hydroperoxide.

[0016] The present invention also provides a radioactive conjugate comprising the modified Affibody conjugate provided by the present invention and a radionuclide chelated therein.

[0017] Preferably, the radionuclide is selected from... 68 Ga、 18 F, 64 Cu、 44 Sc、 89 Zr、 99m Tc,¹ 77 Lu、 161 Tb, 90 Y、 225 Ac、 212 Pb or 188 Re.

[0018] The present invention also provides the use of modified Affibody conjugates or the above-mentioned radioactive conjugates in the preparation of medicaments for the diagnosis or treatment of HER2-positive tumors.

[0019] The beneficial effects of this invention are: This invention provides a modified affibody conjugate, its preparation method, and its applications. Through a dual modification strategy combining site-specific S-glycosylation modification with a small albumin-binding ligand, this invention effectively prolongs the probe's in vivo circulating half-life while maintaining the small size advantage and original targeting affinity of the affibody molecule. This enhances the specific accumulation and long-term retention at the tumor site, while significantly reducing non-specific renal uptake and radiation exposure risks. After radionuclide labeling, this conjugate can be applied to high-contrast molecular imaging and targeted radionuclide therapy for HER2-positive tumors, providing a safe and effective radiopharmaceutical approach for precision tumor diagnosis and treatment, and showing promising clinical translational potential. Attached Figure Description

[0020] Figure 1The LC-MS spectrum of compound 4c is shown below. Figure 2 The LC-MS spectrum of compound 4a is shown below. Figure 3 The LC-MS spectrum of compound 4b is shown below. Figure 4 This is a surface plasmon resonance (SPR) sensor map showing the binding interaction between the Affibody probe and HER2. Each map shows the superposition of all recorded sensor maps for the corresponding molecule. The colored curves are the experimentally measured sensor maps, and the black curves are the fitted curves generated based on the biosensor kinetics model. The concentration of the injected analyte is indicated on the right side of each sensor map. Figure 5 This is a surface plasmon resonance (SPR) sensor map of the binding interaction between the Affibody probe and mouse serum albumin (MSA). Each image shows the superposition of all recorded sensor maps for the corresponding molecule. The colored curves are the experimentally measured sensor maps, and the black curves are the fitted curves generated based on the biosensor kinetic model. The concentration of the injected analyte is indicated on the right side of each sensor map. Figure 6 The image shows the results of radioactive HPLC characterization and in vitro stability of the radiolabeled Affibody probe. (A) 68 Radio-HPLC chromatograms of Ga-labeled Affibody probes (5a-5c, 10a-10c); (B) 177 Radio-HPLC chromatograms of Lu-labeled Affibody probes (5c, 10a-10c); (C) [¹] 77 In vitro stability evaluation of Lu]Lu-10a in PBS (pH 7.4); (D) [¹ 77 In vitro stability evaluation of Lu]Lu-10a in fetal bovine serum (FBS); Figure 7 Image showing Micro-PET / CT imaging and in vivo distribution evaluation results of glycosylated Affibody probes. (A) SKOV3 tumor-bearing nude mice injected with glycosylated Affibody probes via tail vein. 68 Ga]Ga-5a, [ 68 Ga]Ga-5b and [ 68 Representative Micro-PET / CT images after Ga-5c, with dashed circles indicating tumor locations; (B) SKOV3 tumor-bearing nude mice injected via tail vein with GalNAc-modified albumin-binding ligand Affibody probe. 68 Ga]Ga-10a, [ 68 Ga]Ga-10b and [ 68Representative Micro-PET / CT images after Ga]Ga-10c, with dashed circles indicating tumor locations; (C) Quantitative analysis of radioactive uptake (%ID / g) of each probe in the tumor, kidney, and liver, as well as the tumor / kidney, tumor / liver, and tumor / muscle uptake ratios (mean ± standard deviation, n=3). Figure 8 Image showing the results of Micro-SPECT / CT imaging and in vivo distribution evaluation of glycosylated Affibody probes. (A) Glycosylated Affibody probes [¹] 77 Lu]Lu-5c and albumin-binding ligand-modified Affibody probe[¹ 77 Lu]Lu-10a、[¹ 77 Lu]Lu-10b and [¹ 77 Representative Micro-SPECT / CT images of Lu-10c in SKOV3 tumor-bearing nude mice. Dashed circles indicate tumor locations; (B) Quantitative analysis of radioactive uptake (%ID / g) of each probe in the tumor and kidney and the tumor / kidney ratio (mean ± standard deviation, n=3); Figure 9 Inject ICR mice¹ 77 The blood clearance curve results after Lu-labeled Affibody probe, where A is [ 177 The detection results of Lu]Lu-5c, B is [ 177 The detection results of Lu]Lu-10a, C is [ 177 Detection results of Lu]Lu-10b; Figure 10 for[ 177 The results of the Lu-10a radionuclide therapy efficacy evaluation are shown in the figure. (A) Schematic diagram of the treatment plan; (B) Tumor growth curves of each treatment group; (C) Weight changes of each treatment group; (D) Representative tumor photographs at the end of the experiment; (E) Tumor growth curves of mice in each group; (F) Micro-SPECT / CT monitoring results during treatment; (G) Quantitative analysis of tumor weight at the end of the experiment. *** p<0.001, **** p<0.0001; Figure 11 for[ 177 H&E staining results of tumor tissue and major organs (heart, lung, liver, spleen and kidney) of SKOV3 tumor-bearing mice after Lu]Lu-10a treatment (scale bar: 50 μm). Figure 12 for[ 177 Image of TUNEL cell apoptosis analysis and Ki67 immunohistochemical staining results in SKOV3 tumor tissue after Lu-10a treatment; Figure 13for[ 177 [Lu]Lu-10a treatment results are shown in the figure. The figures show (A) white blood cell (WBC, ×10) levels in the blood. 9 (L), (B) Red blood cells (RBC, ×10) 12 / L), (C) platelets (PLT, ×10 9 / L), (D)monocytes (MON, ×10 9 / L), (E) neutrophils (NEU, ×10 9 / L), (F) lymphocytes (LYM, ×10 9 The levels of (g / L), (G) hematocrit (HCT, %) and (H) hemoglobin (HGB, g / L); Figure 14 for[ 177 The following is a graph showing the results of blood biochemistry analysis after Lu-10a treatment. The graph shows the serum levels of (A) aspartate aminotransferase (AST, U / L), (B) alanine aminotransferase (ALT, U / L), (C) creatinine (CREA, μmol / L), (D) urea (UREA, mmol / L), and (E) uric acid (UA, μmol / L). Detailed Implementation

[0021] In the preparation method of the modified Affibody conjugate provided by the present invention, the sodium glycosyl sulfinate compound with an azide group in step (1) is a glycosyl donor, and its structure contains a sodium sulfinate group (-SO2Na). This group can undergo a nucleophilic substitution reaction with the thiol group of the cysteine ​​residue of the Affibody protein in the presence of an oxidant to form an S-glycosidic bond. The sodium glycosyl sulfinate compounds with an azide group suitable for the present invention include, but are not limited to, sodium N-acetylgalactosamine sulfinate, sodium N-acetylglucosamine sulfinate, or sodium N-acetymannosamine sulfinate with an azide group. The role of the oxidant is to activate the sulfinate group in the sodium glycosyl sulfinate compound into a reactive intermediate, thereby promoting the nucleophilic attack of the thiol group. The reaction in step (1) is carried out in an aqueous medium, without the need for organic solvents, which has the advantages of being environmentally friendly and having mild reaction conditions.

[0022] In the above preparation method, the DBCO-small molecule albumin-binding ligand conjugate mentioned in step (2) refers to a bifunctional molecule formed by covalently linking a DBCO group with a small molecule albumin-binding ligand, containing both a DBCO group and a small molecule albumin-binding ligand in its structure. The DBCO group contains a cyclooctyne structure and can undergo a strain-promoted azido-acetyl cycloaddition reaction with an azide group under copper-free catalytic conditions. The strain-promoted azido-acetyl cycloaddition reaction (SPAAC) is a type of click chemistry reaction with advantages such as fast reaction rate, high selectivity, no catalyst required, and good biocompatibility, and is especially suitable for the mild modification of biomacromolecules such as proteins. Through the SPAAC reaction, the DBCO group in the DBCO-small molecule albumin-binding ligand conjugate undergoes a cycloaddition reaction with the azide group in the glycosylated Affibody intermediate containing an azide group, forming a linker arm containing a triazole ring structure, thereby covalently linking the small molecule albumin-binding ligand to the glycosylated Affibody intermediate.

[0023] In one embodiment of the present invention, the sodium glycosyl sulfinate compound with an azide group is sodium 2-acetamido-2-deoxyglycosyl sulfinate with an azide group. In the structure of sodium 2-acetamido-2-deoxyglycosyl sulfinate, an acetamido group (-NHAc) is attached to the carbon atom at position 2, and a sodium sulfinate group (-SO2Na) is attached to the carbon atom at position 1 (anomeric position), while the sugar ring also contains an azide group. More preferably, the sodium 2-acetamido-2-deoxyglycosyl sulfinate with an azide group is selected from sodium N-acetylgalactosamine sulfinate with an azide group, sodium N-acetylglucosamine sulfinate with an azide group, or sodium N-acetylmannosamine sulfinate with an azide group. More preferably, the sodium glycosyl sulfinate compound with an azide group is sodium N-acetylgalactosamine sulfinate with an azide group.

[0024] In one embodiment of the present invention, step (1) is carried out in a buffer solution. The buffer solution is an aqueous solution capable of maintaining the pH value of the reaction system within a specific range, selected from phosphate buffer, borate buffer, or Tris-hydrochloric acid buffer. In step (1), the pH of the buffer solution is 6.0–8.0. More preferably, the pH of the buffer solution is 6.8–7.4. More preferably, the pH of the buffer solution is 6.9–7.1. Most preferably, the pH of the buffer solution is 7.0. The reaction temperature is 15–40°C, more preferably, the reaction temperature is 20–30°C, more preferably, the reaction temperature is 22–28°C, and most preferably, the reaction temperature is 25°C. The reaction time is 15–60 minutes, more preferably, the reaction time is 20–40 minutes, more preferably, the reaction time is 25–35 minutes, and most preferably, the reaction time is 30 minutes.

[0025] In one embodiment of the present invention, the oxidant is tert-butyl hydroperoxide. t BuOOH is an organic peroxide capable of oxidizing sulfinate ions to reactive intermediates under mild conditions. Besides tert-butyl hydroperoxide, other suitable oxidants for this invention can be selected from hydrogen peroxide, peracetic acid, or m-chloroperoxybenzoic acid, but tert-butyl hydroperoxide exhibits the best performance in terms of reaction selectivity and side reaction control. In step (1), the amount of the oxidant is 3.0 to 10.0 equivalents of the Affibody protein, preferably 4.0 to 6.0 equivalents, and more preferably 5.0 equivalents. The amount of the glycosyl sulfinate compound with an azido group is 3.0 to 10.0 equivalents of the Affibody protein, preferably 4.0 to 6.0 equivalents, and more preferably 5.0 equivalents.

[0026] In one embodiment of the present invention, the preparation method further includes a purification step of the reaction product after step (2). The purification method is selected from preparative high-performance liquid chromatography (HPLC). Preparative HPLC uses a C18 reversed-phase column with gradient elution of acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, and collects the corresponding fractions according to the retention time of the target product. The collected fractions are freeze-dried to remove the solvent, yielding the purified modified Affibody conjugate. The freeze-drying temperature is -80℃ to -50℃, the vacuum degree is 0.1 to 10 Pa, and the drying time is 12 to 48 hours.

[0027] In one embodiment of the present invention, in the DBCO-small molecule albumin-binding ligand conjugate, the DBCO group is covalently linked to the small molecule albumin-binding ligand via an amide bond. The preparation method of the DBCO-small molecule albumin-binding ligand conjugate includes: reacting a carboxyl-containing small molecule albumin-binding ligand with a DBCO-amine compound in the presence of a condensing agent to form an amide bond. The condensing agent is selected from HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), or DCC (dicyclohexylcarbodiimide). In a specific embodiment of the present invention, the DBCO-small molecule albumin-binding ligand conjugate is a DBCO-4-iodophenylbutyrate conjugate (8a), a DBCO-4-methylphenylbutyrate conjugate (8b), or a DBCO-palmitic acid conjugate (8c). The aforementioned DBCO-small molecule albumin-binding ligand conjugates can be further linked with chelating agents to form a DBCO-small molecule albumin-binding ligand-chelating agent three-functional module, such as DBCO-4-iodophenylbutyric acid-DOTA (9a), DBCO-4-methylphenylbutyric acid-DOTA (9b), or DBCO-palmitic acid-DOTA (9c), to achieve one-step coupling to simultaneously introduce albumin-binding ligands and chelating agents.

[0028] This invention also provides a radioconjugate comprising a modified Affibody conjugate provided by this invention and a radionuclide chelated therein. The radionuclide is chelated in the chelating agent group of the modified Affibody conjugate. When the modified Affibody conjugate contains a DOTA chelating agent group, the four nitrogen atoms and four carboxyl oxygen atoms in the DOTA provide eight coordination sites, forming a stable eight-coordinate complex with the radionuclide. When the modified Affibody conjugate contains a NOTA chelating agent group, the three nitrogen atoms and three carboxyl oxygen atoms in the NOTA provide six coordination sites, forming a stable six-coordinate complex with the radionuclide. The preparation method of the radioconjugate includes: dissolving the modified Affibody conjugate in a buffer solution, adding a radionuclide solution, incubating at a certain temperature for a certain time to allow the radionuclide to fully coordinate with the chelating agent group, and then removing uncoordinated free radionuclides through a purification step.

[0029] In one embodiment of the present invention, the radionuclide is selected from diagnostic radionuclides that can be used in positron emission tomography (PET), diagnostic radionuclides that can be used in single-photon emission computed tomography (SPECT), or therapeutic radionuclides that can be used for targeted radionuclide therapy. The radionuclide includes, but is not limited to, those used in positron emission tomography (PET) and single-photon emission computed tomography (SPECT).68 Ga、 18 F, 64 Cu、 44 Sc、 89 Zr、 99m Tc,¹ 77 Lu、 161 Tb, 90 Y、 225 Ac、 212 Pb or 188 Re. Among them. 68 Ga、 18 F, 64 Cu、 44 Sc and 89 Zr is a positron-emitting nuclide that can be used in PET imaging. 99m Tc is a gamma-ray emitting nuclide that can be used for SPECT imaging;¹ 77 Lu、 161 Tb, 90 Y、 225 Ac、 212 Pb and 188 Re is a β- or α-ray emitting nuclide and can be used for targeted radionuclide therapy.¹ 77 Lu emits low-energy beta particles (maximum energy 0.5 MeV) and gamma rays (energy 113 keV and 208 keV), and has both therapeutic and imaging functions, making it an ideal radionuclide for integrated diagnosis and treatment. 90 Y emits high-energy beta particles (maximum energy 2.3 MeV), suitable for the treatment of large tumors. 225 Ac emits high-energy-density alpha particles, suitable for the treatment of small lesions and disseminated tumors. 68 Ga has a half-life of 68 minutes, making it suitable for rapid imaging; 177 Lu has a half-life of 6.65 days, making it suitable for long-term in vivo therapy and imaging monitoring.

[0030] This invention also provides the use of the above-described modified Affibody conjugate or the above-described radioactive conjugate in the preparation of medicaments for the diagnosis or treatment of HER2-positive tumors. The diagnosis includes positron emission tomography (PET) and single-photon emission computed tomography (SPECT). When the modified Affibody conjugate chelates diagnostic radionuclides (such as...) 68 After Ga), PET imaging can be used for high-sensitivity, high-resolution molecular imaging diagnosis of HER2-positive tumors, for early detection, staging, efficacy evaluation, and recurrence monitoring of tumors. When the modified Affibody conjugate chelates therapeutic radionuclides (such as ¹), 77Following Lu), HER2-positive tumors can be treated with targeted radionuclide therapy via internal irradiation. The β- or α particles emitted by the radionuclide produce ionizing radiation effects within the tumor tissue, inducing DNA damage and apoptosis in tumor cells, thereby inhibiting tumor growth. The diagnosis and treatment can be performed individually or in combination, i.e., using the same precursor molecule to label different radionuclides for integrated diagnosis and treatment. HER2-positive tumors include, but are not limited to, HER2-positive breast cancer, HER2-positive gastric cancer, HER2-positive ovarian cancer, HER2-positive lung cancer, HER2-positive colorectal cancer, and other malignant tumors associated with HER2 overexpression.

[0031] In one embodiment of the present invention, the drug is a sterile lyophilized powder for injection, which is reconstituted with sterile physiological saline or water for injection before use and administered intravenously. The drug may also contain pharmaceutically acceptable carriers, excipients, or stabilizers, wherein the carriers include, but are not limited to, physiological saline, phosphate buffer, glucose solution, or human serum albumin. The content of the modified Affibody conjugate or radioconjugate in the drug is a therapeutically effective dose or a diagnostically effective dose, and the specific dosage is determined based on factors such as the patient's weight, condition, tumor burden, and the type and activity of the radionuclide used.

[0032] In one embodiment of the invention, when the modified Affibody conjugate or radioconjugate is used for diagnosis or treatment, it can be administered intravenously, with the injection dose determined according to the specific radionuclide and indication. For example, when used for PET imaging, 68 The injection activity of Ga-labeled modified Affibody conjugates can be 37–185 MBq; when used for radionuclide therapy,¹ 77 The injectable activity of Lu-labeled modified Affibody conjugates can range from 1 to 7.4 GBq, and can be administered as a single dose or in multiple doses. In the multiple-dose regimen, the interval between two consecutive doses can be 2 to 8 weeks, preferably 4 to 7 weeks. After administration, the tumor lesion can be imaged using nuclear medicine imaging equipment (such as PET / CT, SPECT / CT), or the absorbed dose to major organs (including tumor, kidney, liver, bone marrow, etc.) can be calculated using dosimetric assessment methods to guide the implementation of individualized treatment plans.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] The HPLC method in this example: HPLC analysis was performed using a gradient system consisting of solvent A (acetonitrile) and solvent B (0.1% trifluoroacetic acid aqueous solution) at a flow rate of 1.0 mL / min. The gradient program was set as follows: 0-5 min, 10% A / 90% B; 5-12 min, 50% A / 50% B; 12-18 min, 90% A / 10% B.

[0035] Example 1: Synthesis of sodium glycosyl sulfinate donors (compounds 3a, 3b, 3c)

[0036] This embodiment describes a method for synthesizing sodium glycosyl sulfinate donors for site-specific S-glycosylation modification of Affibody. Using acetyl-protected sugars as raw materials, N-acetylmmannosamine sulfinate (3a), N-acetylglucosamine sulfinate (3b), and N-acetylglucosamine sulfinate (3c) were prepared via a three-step reaction involving thioetherification, oxidation, and deprotection.

[0037] Step 1: Synthesis of thioether S1-2. Acetyl-protected starting material S1-1 (1 equiv.) was dissolved in anhydrous CH2Cl2, and methyl 3-mercaptopropionate (1.5 equiv.) was added. BF3·Et2O (2.0 equiv.) was slowly added dropwise, and the mixture was stirred at room temperature (25°C) for several hours (the reaction progress was monitored by TLC). After the reaction was complete, it was quenched with saturated NaHCO3 solution. The aqueous phase was extracted three times with CH2Cl2, and the combined organic phases were dried over anhydrous Na2SO4. The concentrate yielded thioether S1-2 (no further purification required, directly used in the next step).

[0038] Step 2: Synthesis of S1-3. Thioether S1-2 (1 equiv.) was dissolved in anhydrous CH2Cl2. After cooling to 0°C, m-CPBA (3.5 equiv.) was slowly added. The mixture was then brought back to room temperature (25°C) and stirred for several hours (monitored by TLC). After the reaction was complete, the solid was removed by filtration. The filtrate was washed successively with saturated NaHCO3, Na2SO3, and NaCl aqueous solutions. The organic phase was dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography to obtain S1-3.

[0039] Step 3: Deprotection to obtain the final product. Dissolve S1-3 in methanol (MeOH), cool to 0°C, and slowly add MeONa (1.0 equiv.) while stirring. Return to room temperature and stir for several hours until TLC shows complete consumption of the sulfone feedstock. Concentrate the reaction solution, and purify the residue under N2 protection using anhydrous CH2Cl2 and anhydrous EtOAc to obtain the final product with purity sufficient for subsequent reactions.

[0040] The synthesis route is as follows: Specifically, based on the above synthetic route, 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose was used as the starting glycosyl donor. Methyl 3-mercaptopropionate was added to anhydrous CH2Cl2, and a thioglycosylation reaction was carried out at room temperature under the action of Lewis acid catalyst BF3·Et2O to obtain the corresponding thioether intermediate. Subsequently, the thioether was oxidized with m-chloroperoxybenzoic acid (m-CPBA) to generate the corresponding sulfone intermediate. Finally, sodium methoxide (MeONa) was added to the methanol (MeOH) system for deacetylation protection to obtain the target product 3a.

[0041] 3a White solid:

[0042] 1 H NMR (400 MHz, D2O) δ 4.05 (dt, J = 9.8, 4.8 Hz, 2H), 3.94 - 3.78(m, 4H), 3.68 (t, J = 9.1 Hz, 1H), 3.52 - 3.44 (m, 1H), 3.38 - 3.30 (m, 2H). 13 C NMR (101 MHz, D2O) 13 C NMR (101 MHz, D2O) δ 170.50, 99.43, 77.30, 70.03, 66.31, 60.44, 49.61, 48.85. Specifically, based on the above synthetic route, 2-[(azidoacetyl)amino]-2-deoxy-D-glucopyranose 1,3,4,6-tetraacetic acid ester was used as the glycosyl donor, and the target product 3b was obtained by the same method as product 3a, through thioglycosylation reaction promoted by methyl 3-mercaptopropionate / BF3·Et2O, m-CPBA oxidation and MeONa deprotection reaction.

[0043] 3b White solid:

[0044] 1 H NMR (400 MHz, D2O) δ 4.06 - 3.94 (m, 3H), 3.88 - 3.71 (m, 2H), 3.72- 3.57 (m, 2H), 3.49 - 3.45 (m, 2H). 13C NMR (101 MHz, D2O) δ 169.21, 91.31,78.82, 73.69, 68.34, 59.71, 50.66, 50.26. Specifically, based on the above synthetic route, 1,3,4,6-tetra-O-acetyl-2-[(2-azidoacetyl)amino]-2-deoxy-D-galactose was used as the glycosyl donor, and the target product 3c was obtained by the same method as product 3a, through thioglycosylation reaction promoted by methyl 3-mercaptopropionate / BF3·Et2O, m-CPBA oxidation and MeONa deprotection reaction.

[0045] 3c White solid:

[0046] 1 H NMR (400 MHz, CD3OD) δ 4.17 (t, J = 10.4 Hz, 1H), 3.99 - 3.88 (m,3H), 3.83 - 3.76 (m, 1H), 3.67 (dd, J = 10.3, 3.3 Hz, 1H), 3.64 - 3.55 (m,2H), 3.52 (d, J = 10.5 Hz, 1H). 13 C NMR (101 MHz, DMSO-D6) δ 173.90, 167.81,93.11, 79.38, 72.45, 67.99, 60.96, 51.00, 49.03.

[0047] Example 2: Synthesis of glycosylated Affibody intermediates (compounds 4a, 4b, 4c)

[0048] In this embodiment, the sodium glycosyl sulfinate donor prepared in Example 1 is used to undergo a site-specific S-glycosylation reaction with Affibody 1 (HER2-targeted Affibody with a cysteine ​​residue at the C-terminus) in the presence of an oxidant to obtain a glycosylated Affibody intermediate with an azide group, which is used for subsequent bioorthogonal coupling.

[0049] Under nitrogen protection, Affibody 1 (8.0 mg, 1.18 μmol, 1.0 equiv.) was dissolved in H2O (1 mL), followed by the addition of isothiazolo[5,4-b]pyridin-3(2H)-one (0.54 mg, 3.5 μmol, 3.0 equiv.). The reaction mixture was stirred at room temperature for 15 minutes. Then, 3c (2.0 mg, 5.9 μmol, 5.0 equiv.) and... t BuOOH (8.2 μL, 5.9 μmol, 7% aqueous solution, 5.0 equiv.). The reaction mixture was stirred for 30 minutes, and the reaction progress was monitored by LC-MS. After the intermediate was completely consumed, the reaction mixture was separated by preparative HPLC. The elution program was set as follows: initial elution at 5% MeCN concentration for 15 minutes, followed by a linear gradient from 5% to 26% MeCN over 15 minutes, and then a linear gradient from 26% to 30% MeCN over 30 minutes. The target product was typically eluted at a constant concentration of 28% MeCN. After lyophilization to remove the solvent, a white solid product 4c (TFA salt, 3.2 mg, yield 38%) was obtained. The purity of the product was confirmed by LC-MS analysis. HRMS (ESI⁺): [M+7H] 7+ (C) 307 H 493 N 91 O 96 S2 7+ The calculated value is 1175.7650; the measured value is 1175.7848. The corresponding LC-MS spectrum is shown below. Figure 1 The LC-MS retention time was 2.69 minutes.

[0050] The synthesis methods for 4a and 4b are similar to those for 4c. Refer to the method for 4c, but replace the raw materials with 3a and 3b: HRMS (ESI⁺): [M+6H] 6+ Calculated for C 307 H 493 N 91 O 96 S2 6+ ,1175.7650; found, 1175.7848. The corresponding LC-MS spectrum is shown below. Figure 2 The LC-MS retention time was 2.77 minutes.

[0051] HRMS (ESI⁺): [M+6H] 6+ Calculated for C 307 H493 N 91 O 96 S2 6 + , 1175.7650; found, 1175.7859. The corresponding LC-MS spectrum is shown below. Figure 3 The LC-MS retention time is 3.02 minutes.

[0052] Example 3: Synthesis of small molecule albumin-binding ligand-DBCO-NH2 modules (compounds 8a, 8b, 8c) In this embodiment, different small molecule albumin binding ligands (4-iodophenylbutyric acid, 4-methylphenylbutyric acid, palmitic acid) are coupled to the DBCO linker via an amide condensation reaction, and then deprotected to obtain a modular compound with a free amino group, which is used for the subsequent introduction of DOTA chelating agent.

[0053] Step 1: The carboxylic acid derivative of the albumin ligand (1.5 equiv.) was dissolved in anhydrous CH2Cl2, followed by the addition of HATU (1.5 equiv.) and DIPEA (3.0 equiv.). The reaction mixture was stirred at room temperature for 0.5 hours, and then N-((9H-fluorene-9-yl)methoxycarbonyl)-L-lysine methyl ester hydrochloride (1.0 equiv.) was added. After the reaction was complete, the solvent was removed under reduced pressure to obtain the crude product, which was purified by column chromatography to obtain compound PRE-6.

[0054] Step 2: Under nitrogen protection, compound PRE-6 and LiI (20 equiv.) were dissolved in EA. The mixture was reacted at 80 °C for 8 hours. After the reaction was complete, the mixture was diluted with ethyl acetate and washed with 1 M hydrochloric acid aqueous solution. The organic layer was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by column chromatography to obtain compound 6.

[0055]

[0056] Compound PRE-6a was prepared from 4-(P-iodophenyl)butyric acid according to general procedure 1. The reaction system was monitored by LC / MS. A yellow solid, PRE-6a, was finally obtained.

[0057] 1 H NMR (400 MHz, CDCl3) δ = 7.77 (d, J =7.6, 2H), 7.60 (dd, J =7.7, 4.1,2H), 7.40 (t, J =7.5, 2H), 7.31 (t, J=7.4, 2H), 7.04 (q, J =7.8, 4H), 5.50 (t, J =5.6, 2H), 5.47 (d, J =8.2, 1H), 4.41 (dd, J =10.6, 7.1, 1H), 4.35 (dq, J =10.8, 7.1, 6.3, 2H), 4.21 (t, J =7.0, 1H), 3.74 (s, 3H), 3.24 (q, J =6.6, 2H), 2.58 (t, J =7.5, 2H), 2.29 (s, 3H), 2.14 (t, J =7.6, 2H), 1.93 (p, J =7.5, 2H), 1.86 (qd, J =10.8, 7.0, 5.4, 1H), 1.76 – 1.66 (m, 1H), 1.52 (p, J =6.5, 2H),1.44 – 1.32 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 173.00, 172.70, 156.21, 144.01, 143.82,141.44, 141.26, 137.52, 130.71, 127.88, 127.21, 125.19, 120.15, 91.11, 67.16,53.68, 52.59, 47.29, 39.04, 35.80, 34.78, 32.32, 29.09, 26.95, 22.51.

[0058] Compound PRE-6b was prepared from 4-p-tolylbutyric acid using general procedure 1. The reaction system was monitored by LC / MS. A yellow solid, PRE-6b, was finally obtained.

[0059] 1 H NMR (400 MHz, CDCl3) δ = 7.77 (d, J =7.6, 2H), 7.60 (t, J =6.9, 2H), 7.55 (d, J=7.9, 2H), 7.40 (t, J =7.5, 2H), 7.34 – 7.28 (m, 2H), 6.88 (d, J =7.9, 2H), 5.52 (t, J =5.8, 1H), 5.46 (d, J =8.2, 1H), 4.40 (dd, J =10.7, 7.1, 1H), 4.35 (td, J =10.5, 9.1, 5.3, 2H), 4.21 (t, J =7.0, 1H), 3.75 (s, 3H), 3.23(q, J =6.6, 2H), 2.54 (t, J =7.6, 2H), 2.11 (t, J =7.5, 2H), 1.91 (p, J =7.6,2H), 1.85 (dt, J =10.6, 5.8, 1H), 1.71 (d, J =8.6, 1H), 1.52 (dh, J =13.8, 6.8,2H), 1.38 (tq, J =20.7, 8.5, 6.6, 2H). 13 C NMR (151 MHz, CDCl3) δ 173.02, 156.20, 144.02, 143.86, 141.44,138.49, 135.50, 129.17, 128.46, 127.86, 127.20, 125.22, 120.12, 67.15, 53.72,52.56, 47.29, 39.00, 36.06, 34.87, 32.27, 29.17, 27.35, 22.51, 21.09.

[0060] Compound PRE-6c was prepared from palmitic acid using general procedure 1. The reaction system was monitored by LC / MS. A yellow solid, PRE-6c, was finally obtained.

[0061] 1 H NMR (400 MHz, CDCl3) δ 7.76 (d, J= 7.6 Hz, 2H), 7.60 (dd, J = 7.8, 3.6 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2fH), 5.68 (s,0.5H), 5.55 (d, J = 8.3 Hz, 0.5H), 4.45 – 4.31 (m, 2H), 4.22 (t, J = 7.3 Hz,1H), 3.75 (s, 3H), 3.28 – 3.17 (m, 1H), 2.68 (s, 1H), 2.13 (t, J = 7.8 Hz, 2H), 1.85 (dq, J = 14.2, 6.6 Hz, 1H), 1.69 (dt, J = 14.5, 7.3 Hz, 1H), 1.62 –1.48 (m, 2H), 1.44 – 1.33 (m, 2H), 1.23 (d, J = 7.9 Hz, 24H), 0.87 (t, J =6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 173.92, 173.11, 172.27, 156.29, 143.95,143.78, 141.39, 127.86, 127.20, 125.21, 120.13, 67.15, 53.63, 52.65, 47.20,39.07, 36.95, 32.27, 32.04, 29.82, 29.78, 29.75, 29.63, 29.49, 29.44, 28.98,25.94, 25.50, 22.82, 22.48, 14.27.

[0062] Compound 6a was prepared according to general procedure 2. The reaction system was monitored using LC / MS. A yellow oily substance, 6a, was finally obtained.

[0063] 1 H NMR (400 MHz, CD3OD) δ 7.78 (dd, J= 7.4, 2.1 Hz, 2H), 7.65 (t, J =6.9 Hz, 2H), 7.58 – 7.51 (m, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.4Hz, 2H), 7.00 – 6.90 (m, 2H), 4.39 – 4.24 (m, 2H), 4.23 – 4.05 (m, 2H), 3.15(t, J = 6.8 Hz, 2H), 2.52 (t, J = 7.8 Hz, 2H), 2.14 (t, J = 7.5 Hz, 2H), 1.86(q, J = 7.6 Hz, 3H), 1.70 (s, 1H), 1.51 (p, J = 6.8 Hz, 2H), 1.46 – 1.37 (m,2H). 13 C NMR (101 MHz, CD3OD) δ 175.64, 158.50, 145.35, 145.15, 142.76,142.55, 138.51, 131.76, 128.78, 128.18, 126.31, 126.27, 120.93, 91.54, 67.84,40.10, 36.36, 35.66, 32.86, 30.78, 29.94, 28.51, 24.27.

[0064] Compound 6b was prepared according to general procedure 2. The reaction system was monitored using LC / MS. A yellow oily substance, 6b, was finally obtained.

[0065] 1 H NMR (400 MHz, CD3OD) δ 7.85 – 7.75 (m, 2H), 7.69 – 7.59 (m, 2H),7.39 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.4 Hz, 2H), 7.02 (s, 3H), 4.30 (d, J = 7.1 Hz, 1H), 4.18 (t, J= 6.8 Hz, 1H), 4.07 (s, 1H), 3.16 (t, J = 6.8 Hz, 2H), 2.53 (t, J = 7.7 Hz, 2H), 2.24 (s, 3H), 2.15 (t, J = 7.5 Hz, 2H), 1.86(p, J = 7.6 Hz, 3H), 1.77 – 1.64 (m, 1H), 1.52 (h, J = 6.5 Hz, 2H), 1.46 –1.39 (m, 2H), 1.36 – 1.24 (m, 4H). 13 C NMR (101 MHz, Chloroform- d ) δ 190.04, 185.15, 178.57, 148.04,147.83, 145.20, 142.41, 138.97, 132.60, 131.99, 131.41, 130.80, 128.92,123.55, 70.44, 42.76, 39.21, 38.50, 35.75, 32.64, 31.61, 26.88, 23.68.

[0066] Compound 6c was prepared according to general procedure 2. The reaction system was monitored using LC / MS. A yellow oily substance, 6c, was finally obtained.

[0067] 1 H NMR (400 MHz, CD3OD) δ 7.79 (d, J = 7.5 Hz, 1H), 7.68 (t, J = 7.4Hz, 1H), 7.39 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 7.4 Hz, 1H), 4.33 (d, J = 7.1Hz, 1H), 4.22 (t, J = 7.0 Hz, 1H), 4.09 (dd, J = 8.8, 4.5 Hz, 0H), 3.17 (t, J = 6.8 Hz, 1H), 2.14 (t,J = 7.6 Hz, 1H), 1.85 (ddt, J = 13.3, 8.2, 4.6 Hz,0H), 1.73 – 1.67 (m, 0H), 1.55 (dt, J = 14.8, 7.7 Hz, 2H), 1.46 – 1.38 (m,0H), 1.27 (dd, J = 9.0, 4.3 Hz, 14H), 0.89 (t, J = 6.8 Hz, 1H). 13 C NMR (101 MHz, CD3OD) δ 176.30, 158.51, 145.40, 145.20, 142.59,128.79, 128.18, 126.31, 120.92, 67.89, 56.15, 40.13, 37.17, 33.10, 32.95,30.82, 30.79, 30.75, 30.66, 30.52, 30.46, 30.33, 29.98, 24.27, 23.77, 14.49. Compound 6a (64 mg, 0.10 mmol, 1.0 equiv.) was dissolved in CH2Cl2 (1 mL), followed by the sequential addition of HATU (114 mg, 0.30 mmol, 3.0 equiv.) and DIPEA (27 μL, 0.15 mmol, 1.5 equiv.). The mixture was stirred at room temperature for 0.5 h, followed by the addition of compound 7 (86 mg, 0.15 mmol). The reaction mixture was stirred for another 30 min and monitored by TLC and LC-MS. After complete consumption of 6b, the reaction mixture was diluted with EA and then washed with 1 M HCl aqueous solution. The crude product was obtained by drying with anhydrous Na2SO4. Without separating the DBCO amide intermediate, diethylamine (1 mL) and CH2Cl2 (1 mL) were added directly to the above reaction mixture. The resulting solution was stirred at room temperature for 30 min, and the reaction progress was monitored by TLC and LC-MS. After completion, insoluble matter was removed by filtration, and the solvent was evaporated under reduced pressure to obtain the crude product. Purified by column chromatography (DCM / MeOH = 90 / 10, v / v Compound 8a was obtained, which was a yellow oily liquid (3 mg, 53%). 1H NMR (400 MHz, CD3OD): δ 7.66 (d, J = 7.4 Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.51 - 7.41 (m,4H), 7.35 (dt, J = 16.3, 7.5 Hz, 2H), 7.25 (d, J = 7.4 Hz, 1H), 6.98 (d, J =8.0 Hz, 2H), 5.13 (d, J = 14.0 Hz, 1H), 3.70 (d, J = 14.0 Hz, 1H), 3.26 (dt,J = 13.1, 6.4 Hz, 1H), 3.20-3.08 (m, 4H), 2.57 (t, J = 7.7 Hz, 2H), 2.54 -2.46 (m, 1H), 2.16 (t, J = 7.5 Hz, 2H), 2.03 (dt, J = 15.3, 7.0 Hz, 1H), 1.89(q, J = 7.7 Hz, 2H), 1.56 - 1.39 (m, 4H), 1.30 - 1.26 (m, 2H). 13 C NMR (101MHz, CD3OD): δ 176.76, 176.57, 175.63, 173.06, 173.02, 152.49, 152.45,149.51, 149.47, 142.81, 138.57, 133.48, 131.81, 130.46, 130.03, 129.73,129.27, 128.96, 128.18, 126.49, 124.30, 124.26, 123.60, 115.62, 115.58,108.89, 108.87, 91.56, 56.62, 56.59, 55.64, 55.52, 40.10, 36.46, 36.39,35.71, 35.60, 35.55, 35.26, 30.11, 28.55, 23.88, 23.80. HRMS (ESI⁺): [M+H]⁺(C 34 H 38 IN4O3⁺), calculated value 677.1983; measured value 677.1981.

[0068]

[0069] Compounds 8b and 8c were prepared using the same synthetic method as compound 8a. Briefly, the corresponding substrates 6b or 6c were dissolved in CH₂Cl₂, activated sequentially with HATU and DIPEA, followed by amide coupling with compound 7. After the reaction, without separating the DBCO amide intermediate, deprotection was performed directly with diethylamine / CH₂Cl₂. Following post-treatment and purification by silica gel column chromatography (DCM / MeOH = 90 / 10, v / v), compounds 8b and 8c were obtained, respectively.

[0070] 8b Yellow oily substance

[0071] 1 H NMR (400 MHz, CD3OD): δ 7.66 (d, J = 7.4 Hz, 1H), 7.52-7.42 (m,4H), 7.40-7.30 (m, 2H), 7.26 (d, J = 7.4 Hz, 1H), 7.09 -7.01 (d, J = 1.3 Hz, 4H), 5.12 (d, J = 14.0 Hz, 1H), 3.67 (d, J = 13.9 Hz, 1H), 3.25 (dt, J =13.0, 6.4 Hz, 1H), 3.19 - 3.07 (m, 4H), 2.57 (td, J = 7.8, 2.2 Hz, 2H), 2.54- 2.45 (m, 1H), 2.27 (s, 3H), 2.16 (t, J = 7.6 Hz, 2H), 2.03 (dt, J = 15.8, 7.3 Hz, 1H), 1.88 (qd, J = 8.1, 2.0 Hz, 2H), 1.57 - 1.39 (m, 4H), 1.33 - 1.25 (m, 2H). 13¹³C NMR (101 MHz, CD₃OD): δ 176.96, 176.80, 175.86, 173.05, 173.01, 152.49, 152.45, 149.50, 149.47, 139.80, 136.39, 133.49, 133.47, 130.46, 130.00, 129.70, 129.38, 129.26, 128.95, 128.16, 115.62, 115.58, 108.90, 108.86, 56.58, 56.56, 55.68, 55.56, 40.11, 36.59, 36.44, 36.37, 35.89, 35.59, 35.54, 35.37, 30.14, 28.97, 23.92, 23.84, 21.08. HRMS (ESI⁺): [M+H]⁺ calculated for C 35 H 41 N₄O₃⁺, 565.3173; found, 565.3156 8c Yellow oily substance

[0072] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.68 (dd, J J = 7.1, 2.7 Hz, 1H), 7.42 - 7.27 (m, 7H), 7.24 (dd, J J = 7.5, 1.5 Hz, 1H), 5.88 (dt, J J = 12.6, 5.8 Hz, 1H), 5.14 (dd, J J = 13.9, 2.4 Hz, 1H), 3.67 (d, J J = 13.9 Hz, 1H), 3.39 - 3.12 (m, 5H), 2.50 - 2.37 (m, 1H), 2.12 (td, J J = 7.7, 3.1 Hz, 1H), 1.99 (tdd, J J = 16.2, 7.5, 4.6 Hz, 1H), 1.74 - 1.39 (m, 8H), 1.35 - 1.17 (m, 24H), 0.89 - 0.84 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 174.28, 174.16, 173.67, 173.61, 171.97,151.11, 148.13, 132.39, 129.15, 128.81, 128.55, 128.46, 127.94, 127.35,125.57, 123.09, 122.43, 122.39, 114.79, 114.73, 108.07, 108.03, 55.55, 55.52,54.66, 39.07, 38.99, 36.95, 35.10, 35.01, 34.02, 33.86, 32.04, 29.82, 29.78, 29.66, 29.53, 29.49, 29.23, 25.98, 22.82, 22.70, 14.28. HRMS (ESI⁺): [M+H]⁺ calculated for C 40 H 59 N4O3⁺, 643.4582; found, 643.4581.

[0073] Example 4: Synthesis of small molecule albumin-binding ligand-DBCO-DOTA modules (compounds 9a, 9b, 9c) In this embodiment, a DOTA chelating agent is introduced through the amidation reaction of NHS-DOTA with the amino modules (8a, 8b, 8c) prepared in Example 3, thereby constructing a functional module that can be used for radioactive metal nuclide labeling.

[0074] Compound 8a (6.0 mg, 8.9 μmol, 1.0 equiv.) was dissolved in DMSO (0.2 mL), followed by the addition of NHS-DOTA (13.3 mg, 26.6 μmol, 3.0 equiv.) and DIPEA (15.7 μL, 0.09 mmol, 10.0 equiv.). The reaction mixture was stirred at room temperature for 2 hours, and the reaction progress was monitored by LC-MS. After the reaction was complete, the solvent was removed under reduced pressure to obtain the crude product. After 8a was completely consumed, the reaction mixture was directly injected into preparative HPLC for separation. The elution program was maintained at a constant 5% MeCN for 20 min, then a linear gradient from 5% to 40% MeCN over 10 min, and then further increased from 40% to 60% MeCN over 30 min. The target product was typically eluted with a constant concentration of 50% MeCN. After solvent removal by lyophilization, 9a (8.4 mg, 89%) was obtained as a white solid. The purity of the product was confirmed by HPLC. HPLC analysis of product 9a (C18 column, mL / min) showed a purity of 96.9% and a retention time (tR) of 7.6 min. HRMS (ESI⁺): [M+H]⁺ (C 50 H 64 IN8O 10 ⁺), calculated value 1063.3785; measured value 1063.3728.

[0075]

[0076] Compounds 9b and 9c were synthesized using the same method as compound 9a. Briefly, the corresponding compound 8b or 8c was dissolved in DMSO, and NHS-DOTA (3.0 equiv.) and DIPEA (10.0 equiv.) were added. The reaction mixture was stirred at room temperature for 2 hours, and the reaction progress was monitored by LC-MS. After the starting material was completely consumed, the reaction mixture was directly purified by preparative reversed-phase HPLC. Separation was performed using gradient elution conditions similar to those used for 9a. The target product fractions were collected, and after lyophilization to remove the solvent, white solid compounds 9b and 9c were obtained, respectively.

[0077] 9b:

[0078] HRMS (ESI⁺): [M+H]⁺ calculated for C 51 H 67 N8O 10 ⁺, 951.4975; found, 951.4959. HPLC purity: 96.94%.

[0079] 9c:

[0080] HRMS (ESI⁺): [M+H]⁺ calculated for C 56 H 85 N8O 10 ⁺, 1029.6383; found, 1029.6400. HPLC purity 98.55%.

[0081] Example 5: Synthesis of site-specific S-glycosylation and small albumin-binding ligand-modified Affibody conjugates (compounds 10a, 10b, 10c) In this embodiment, the glycosylated Affibody intermediate (4c) prepared in Example 2 was coupled with the DOTA-modified modules (9a, 9b, 9c) prepared in Example 4 through strain-promoted azido-alkyne cycloaddition reaction (SPAAC) to obtain a dual-modified Affibody conjugate that simultaneously possesses glycosylation modification, small molecule albumin-binding ligand modification, and DOTA chelating agent.

[0082] Synthesis of compound 10a: Compound 4c (2.4 mg, 0.34 μmol, 1.0 equiv.) was dissolved in H2O (0.2 mL), followed by the addition of 9a (1.8 mg, 1.7 μmol, 5.0 equiv.) dissolved in H2O (0.2 mL). The reaction mixture was stirred at room temperature for 1 hour, and the reaction progress was monitored by LC-MS. After 4c was completely consumed, the reaction mixture was directly injected into preparative HPLC for separation. The elution program was set as follows: initial elution at 5% MeCN concentration for 20 min, followed by a linear gradient from 5% MeCN to 30% MeCN over 10 min, and then from 30% MeCN to 50% MeCN over 30 min. The target product was typically eluted at a constant concentration of 40% MeCN. After lyophilization to remove the solvent, 10a (TFA salt, 1.0 mg, yield 36%) was obtained as a white solid. The purity of the product was confirmed by HPLC. HPLC analysis of product 10a (C18 column, flow rate 1 mL / min) showed a purity of 97.4% and a retention time (tR) of 6.9 min. HRMS (ESI⁺): [M+6H] 6+ (C) 357 H 556 IN 99 O 106 S 26 + ), calculated value 1352.8269; measured value 1353.1687.

[0083]

[0084] Compounds 10b and 10c were synthesized using the same method as compound 10a. Briefly, compound 4c was dissolved in water, and the corresponding compounds 9b or 9c (5.0 equiv.) were added, respectively. The reaction system was stirred at room temperature for 1 hour, and the reaction progress was monitored by LC-MS. After compound 4c was completely consumed, the reaction mixture was directly purified by preparative reversed-phase HPLC. The target product fraction was collected, and after lyophilization to remove the solvent, white solid compounds 10b and 10c (trifluoroacetate) were obtained, respectively. The purity of the products was confirmed by HPLC.

[0085] 10b:

[0086] HRMS (ESI⁺): [M+6H] 6+ Calculated for C 358 H 559 N 99 O 106 S2 6+ , 1334.1801;found, 1334.5143. HPLC purity data: 95.36%.

[0087] 10c:

[0088] HRMS (ESI⁺): [M+6H] 6+ Calculated for C 363 H 577 N 99 O 106 S2 6+ , 1347. 2035;found, 1347.3754. HPLC purity data: 98.77%.

[0089] Example 6: Evaluation of in vitro binding affinity of Affibody conjugates The binding affinity of the constructed affibody probe to HER2 protein and mouse serum albumin (MSA) was evaluated using surface plasmon resonance (SPR) technology. The effects of site-specific S-glycosylation and albumin-binding ligand modification on affibody targeting activity were evaluated by measuring the binding kinetics of the probe to HER2. Simultaneously, the binding affinity of the probe to MSA was measured to evaluate the regulatory effects of different albumin-binding ligands on serum albumin binding affinity and long-term circulating characteristics.

[0090] Table 1. Affinity determination of each compound with HER2 and MSA

[0091] The results (Table 1) show that glycosylation did not affect the targeting ability of Affibody to HER2; the glycosylated probes (5a-5c) all maintained picomolar HER2 binding affinity (KD = 3.08 × 10⁻¹). 0 ~4.03×10⁻¹ 0 M). Further introduction of a small albumin-binding ligand (ABM) maintained nanomolar HER2 binding activity in the probes, with the IPBA-modified probe 10a exhibiting the highest HER2 affinity (KD = 7.58 × 10⁻¹). 0 M).

[0092] Figure 4 This is a surface plasmon resonance (SPR) sensor map showing the binding interaction between the Affibody probe (3.125–200 nM) and HER2. Each map shows the overlay of all recorded sensor maps for the corresponding molecule. The colored curves are the experimentally measured sensor maps, and the black curves are the fitted curves generated based on the biosensor kinetics model. The concentration of the injected analyte is indicated on the right side of each sensor map.

[0093] Figure 5 This is a surface plasmon resonance (SPR) sensor map of the binding interaction between Affibody probes (0.15625-50 μM) and mouse serum albumin (MSA). Each figure shows the superposition of all recorded sensor maps for the corresponding molecule. The colored curves are the experimentally measured sensor maps, and the black curves are the fitted curves generated based on the biosensor kinetic model. The concentration of the injected analyte is indicated on the right side of each sensor map.

[0094] The results of the MSA binding assay showed that Affibody and glycosylated probes without ABM exhibited only weak nonspecific binding; after introducing ABM, the binding affinity of the probes to albumin increased by approximately 100–1000 times. Among them, the IPBA-modified probe 10a showed the highest albumin binding affinity (KD = 4.58 × 10⁻⁻⁴). 6 M), which is superior to MBA and Palm modified probes.

[0095] The above results demonstrate that the site specificity established in this invention... S - The glycosylation strategy can significantly enhance the albumin binding capacity of Affibody by introducing small molecule albumin-binding ligands in a modular manner while maintaining its excellent HER2-targeting activity, laying the foundation for subsequent optimization of in vivo pharmacokinetics and improvement of radiotherapy efficacy.

[0096] Example 7: 68 Ga and¹ 77 Lu radiolabeling and in vitro stability evaluation Using diagnostic radionuclides 68 Ga and therapeutic nuclides 177 Lu radiolabeled the Affibody precursor. Radiometal coordination was completed under optimized labeling conditions (heating at 70℃ for 30 min, pH 3-4), and radiochemical purity and labeling efficiency were detected by radio-high performance liquid chromatography (radio-HPLC) to obtain a radioactive probe that meets the requirements for subsequent in vivo evaluation.

[0097] Will 68 Ga / 177 Lu-labeled probes were placed in PBS buffer and fetal bovine serum (FBS) and incubated at 37°C. The radiochemical purity was detected by HPLC at different time points to evaluate the stability of the probes in a simulated physiological environment, providing experimental basis for subsequent in vivo imaging and radiotherapy studies.

[0098] The results showed that ( Figure 6 All probes exhibit good radiolabeling performance, among which 68 The radiochemical yields of Ga-labeled probes ranged from 66.73% to 72.53%, with radiochemical purities all exceeding 94%;¹ 77 The radiochemical yields of the Lu-labeled probes ranged from 93.29% to 98.80%, and the radiochemical purity was all above 92%, indicating that the Affibody probes constructed in this invention have excellent radioactive metal coordination ability and can meet the requirements for subsequent in vivo evaluation.

[0099] In vitro stability experiments showed that ( Figure 6 ), 68 The Ga-labeled probe maintained a radiochemical purity of over 91% after incubation in PBS and fetal bovine serum for 4 hours. 177 The Lu-labeled probes maintained a radiochemical purity of over 93% after incubation at 37°C for 120 h. Among them, the IPBA-modified probes [¹] 77 Lu]Lu-10a exhibits the best stability, with a radiochemical purity of over 98% in PBS and over 93% in fetal bovine serum.

[0100] The above results demonstrate that the site-specific S-glycosylated Affibody probe constructed in this invention possesses excellent radiolabeling efficiency and in vitro stability, meeting the application requirements of nuclear medicine molecular imaging and radionuclide therapy, and providing a reliable foundation for subsequent in vivo pharmacokinetic and radiotherapy research.

[0101] Example 8:68 Micro-PET / CT Imaging Study of Ga-labeled Affibody Probes In the SKOV3 tumor-bearing nude mouse model, the effects of different glycosyl modifications and small molecule albumin-binding ligand modifications on the early in vivo distribution, tumor targeting ability, and pharmacokinetic behavior of the Affibody probe were evaluated using Micro-PET / CT dynamic imaging. SKOV3 tumor-bearing nude mice were injected with approximately 3.7 MBq via the tail vein. 68 Ga-labeled affibody probes were used for micro-PET / CT imaging at 0.25, 0.5, 1, 2, 3, and 4 h post-injection. The blockade group simultaneously received a 20-fold molar dose of unlabeled affibody via tail vein injection to verify HER2 receptor-mediated specific uptake. The ROIs of tumors and major organs were delineated using image analysis software, and tissue radioactive uptake values ​​(%ID / g) and tumor / kidney and tumor / liver ratios were calculated to evaluate the effects of different glycosylation modifications on the early in vivo distribution and tumor targeting ability of affibody.

[0102] result( Figure 7 This indicates that all glycosylated Affibody probes can rapidly target HER2-positive tumors and achieve high tumor uptake within 4 hours post-injection. Among them, the GalNAc-modified probe […]. 68 Ga]Ga-5c exhibited the best tumor targeting performance, with a tumor uptake value of 18.16 ± 0.44 %ID / g, significantly higher than that of ManNAc and GlcNAc modified probes. At the same time, it maintained a high tumor / kidney and tumor / liver ratio, indicating that different glycosyl structures can significantly affect the in vivo pharmacokinetic behavior of Affibody. Among them, GalNAc is the best glycosyl module for optimizing the pharmacokinetics of Affibody.

[0103] Based on the above results, different small-molecule albumin-binding ligands (IPBA, MBA, and Palm) were further introduced into the GalNAc-modified probe to construct a series of long-circulating Affibody probes. Micro-PET / CT results showed that all three ABM-modified probes exhibited significantly prolonged blood circulation time and sustained blood pool activity, indicating that albumin-binding ligands can effectively prolong the in vivo circulation of the probes. Among them, the IPBA-modified probe […]. 68 Ga]Ga-10a exhibits the highest blood retention capacity and significantly reduces renal uptake; in contrast, while Palm-modified probes have strong albumin binding capacity, they show significant liver accumulation, which is detrimental to subsequent applications.

[0104] In summary, site-specific S-glycosylation can optimize the early in vivo distribution of Affibody by regulating glycosylation structure, while modular introduction of small albumin-binding ligands can further prolong blood circulation, improve pharmacokinetics, and enhance tumor delivery. Among these, GalNAc-IPBA (10a) exhibited the best overall performance and was therefore selected as the subsequent target. 177 Candidate probes for Lu-labeled and radionuclide therapy research.

[0105] Example 9:¹ 77 Study of Lu-labeled Affibody probes in micro-SPECT / CT longitudinal dynamic imaging To further evaluate the long-term in vivo behavior of the albumin-binding ligand-modified Affibody probe, the probe was modified with GalNAc [¹]. 77 Lu]Lu-5c was used as a control. A series of probes (10a-10c) were used for 192 hours of longitudinal dynamic micro-SPECT / CT imaging in SKOV3 tumor-bearing nude mice. Approximately 18.5 MBq of [a specific substance / method] was injected into the tail vein of the SKOV3 tumor-bearing nude mice. 177 Lu-labeled Affibody probes were used for longitudinal dynamic micro-SPECT / CT imaging at 4, 24, 48, 72, 120, and 192 h post-injection. Image analysis software was used to quantitatively analyze tumors and major organs, calculating tissue uptake values ​​(%ID / g). The focus was on evaluating the blood circulation time, tumor retention capacity, renal clearance, and long-term pharmacokinetic characteristics of the probe modified with albumin-binding ligands.

[0106] The results showed that ( Figure 8 ), control probe [¹ 77 Lu-5c is rapidly cleared in vivo, with tumor uptake peaking 4 hours post-injection and then gradually declining, accompanied by persistently high renal uptake. Palm-modified probe [¹] 77 Although Lu]Lu-10c has a relatively long in vivo circulation time, it exhibits low tumor uptake and significant liver accumulation, indicating that excessive hydrophobicity is not conducive to the in vivo distribution of the probe.

[0107] In contrast, IPBA-modified probes[¹] 77Lu-10a exhibited the best overall pharmacokinetic performance. This probe showed significant blood pool activity in the early post-injection period, indicating that albumin binding significantly prolonged circulation time. With prolonged circulation time, tumor uptake gradually increased, peaking at 24 h post-injection (10.79 ± 0.42 %ID / g), and subsequently maintained high tumor retention, demonstrating its ability to continuously deliver radionuclide to the tumor and prolong tumor irradiation time. Simultaneously, 10a showed lower renal uptake and a higher tumor / kidney (T / K) ratio in the early stages, resulting in superior overall pharmacokinetic performance compared to the MBA-modified probe 10b.

[0108] In summary, the Micro-SPECT / CT results further confirm that site-specific S-glycosylation binding to small albumin-binding ligands can effectively prolong the in vivo circulation time of Affibody probes, improve tumor retention, and reduce renal burden. Among them, the GalNAc-IPBA modified probe (10a) exhibits the best long-term in vivo distribution characteristics and is a preferred candidate probe for subsequent radionuclide therapy.

[0109] Example 10:¹ 77 Blood clearance kinetics study of Lu-labeled Affibody probes Evaluation using normal ICR mice¹ 77 Hemodynamics of Lu-labeled Affibody probes. Following tail vein injection of the radioactive probe, tail vein blood samples were collected at multiple time points. Blood radioactivity was measured using a gamma counter, and blood radioactive uptake (%ID / g) was calculated. Blood clearance curves were plotted, and the circulating half-life and pharmacokinetic parameters of the probe were further analyzed to evaluate the effect of albumin-bound ligands on improving the in vivo circulation time of Affibody.

[0110] Blood pharmacokinetic analysis further confirmed the imaging results (see Figure 9 ). with glycosylated Affibody probes [¹ 77 Compared to Lu-5c, albumin-binding ligand modification significantly prolonged the probe's blood circulation time. Among them, IPBA-modified probes [¹] 77 Lu]Lu-10a exhibited the longest slow-phase half-life (484.70 min), approximately 18 times that of the control probe, significantly outperforming the MBA-modified probe 10b, further demonstrating that IPBA possesses the best long-cycle capability.

[0111] Table 2¹ 77 Lu-labeled Affibody probe blood clearance kinetics <![CDATA[[ 177 Lu]Lu-5c]]> 0.32 28.84 2047 <![CDATA[[ 177 Lu]Lu-10a]]> 6.49 484.70 29304 <![CDATA[[ 177 Lu]Lu-10b]]> 1.43 51.52 9720

[0112] Example 11:¹ 77In vivo biodistribution of Lu-labeled probes and human dosimetric extrapolation In vivo biodistribution experiments were conducted in SKOV3 tumor-bearing nude mice to systematically evaluate tissue radioactive uptake at different time points. OLIDA / EXM software was used to perform human dosimetric extrapolation to assess the clinical application potential and radioactive safety of the probe.

[0113] (1) Distribution of organisms in the body SKOV3 tumor-bearing nude mice were randomly divided into groups and injected via tail vein¹ 77 Lu-labeled Affibody probes were used to euthanize animals at different time points, and tumors and major tissues and organs, including blood, heart, liver, spleen, lungs, kidneys, stomach, intestines, muscles, bones, and brain, were collected. The radioactivity of each tissue was measured using a gamma counter after weighing, and the tissue radioactive uptake value (%ID / g) was calculated after decay correction. The in vivo distribution characteristics, tumor targeting ability, and renal clearance effects of different probes were systematically evaluated.

[0114] The results (Table 3) show that the glycosylated Affibody probe [¹] 77 Lu-5c reached peak tumor uptake 4 hours after injection, and then gradually decreased; while IPBA-modified glycosylated probes [¹] 77 Lu]Lu-10a exhibited sustained tumor accumulation, with tumor uptake peaking at 24 h (22.84 ± 1.96 %ID / g) and remaining at a high level (13.62 ± 1.77 %ID / g) at 120 h. Meanwhile, [¹ 77 The renal uptake of Lu]Lu-10a was significantly lower than that of the control probe. At 24 h, the renal uptake decreased from 108.20 ± 10.04 %ID / g to 44.53 ± 1.61 %ID / g, and the tumor / kidney (T / K) ratio increased from 0.07 to 0.51, indicating that it has better in vivo distribution characteristics.

[0115] Table 3 177 Biodistribution data of Lu-labeled probes

[0116] (2) Human dosimetry extrapolation Based on mouse biodistribution data, human dosimetric extrapolation was performed using OLIDA / EXM software to calculate the absorbed dose (AD) and effective dose (ED) for each organ, evaluating the clinical application potential and radioactivity safety of the Affibody probe. The results showed (Table 4), [¹] 77 The renal absorption dose of Lu]Lu-10a is determined by [¹ 77The radiation dose of Lu]Lu-5c decreased from 3.13 mSv / MBq to 1.01 mSv / MBq, while the effective dose was further reduced, indicating that it can effectively reduce the radiation burden on the kidneys and improve the safety of radiotherapy.

[0117] Table 4 Extrapolation data of human doses

[0118] In summary, site-specific S-glycosylation and synergistic modification with small albumin-binding ligands significantly prolong the in vivo circulation time of Affibody probes, enhance tumor accumulation, and reduce renal uptake and radiation dose. Among them, the IPBA-modified probe (10a) exhibits the best overall performance, providing a promising candidate probe for HER2-targeted radiotherapy.

[0119] Example 12:¹ 77 Evaluation of the efficacy of radionuclide therapy using Lu-labeled preferred probes In the SKOV3 tumor-bearing nude mouse model, the preferred probes were evaluated [¹]. 77 The antitumor effects of Lu]Lu-10a under different dosing regimens were verified by tumor volume monitoring, Micro-SPECT / CT dynamic imaging, and tumor weight analysis as the experimental endpoint.

[0120] When the tumor volume in SKOV3 tumor-bearing nude mice reached 50-100 mm³, the animals were randomly divided into a saline control group, a single high-dose treatment group (18.5 MBq), a single low-dose treatment group (11.1 MBq), and a fractionated low-dose treatment group (11.1 MBq × 2, administered 7 days apart), with corresponding radioactive probes injected via the tail vein. Tumor volume and body weight were measured regularly during treatment, and the retention of probes within the tumor was dynamically monitored using Micro-SPECT / CT imaging to evaluate the antitumor effects of different treatment regimens. After treatment, tumors and major organs were collected for H&E staining and histopathological analysis. Simultaneously, complete blood counts and blood biochemical indicators were measured to comprehensively evaluate the safety and major organ toxicity of radiotherapy.

[0121] The results showed that ( Figure 10 Compared with the saline control group, all treatment groups were able to significantly inhibit tumor growth. Among them, the single high-dose group and the fractionated low-dose group showed the best treatment effect, with tumor growth inhibition rate (TGI) reaching 83.68% and 86.25%, respectively, which were better than the single low-dose group (72.11%).

[0122] Continuous Micro-SPECT / CT imaging monitoring was performed during treatment, and the results showed [¹] 77Lu-10a can remain in the tumor for a long time. Continuous tumor radioactivity signals can be observed in both the single high-dose group and the fractionated low-dose group, indicating that site-specific S-glycosylation binding albumin-binding ligands can prolong the retention time of the probe in the tumor, continuously release the therapeutic dose, and thus improve the efficacy of radionuclide therapy.

[0123] After treatment, tumor tissue and major organs were collected for H&E staining to observe histopathological changes. Tumor tissue was also collected for TUNEL cell apoptosis detection and Ki67 immunohistochemical staining to assess tumor cell apoptosis and proliferation. Blood samples were collected for complete blood count and blood biochemical markers to assess potential hematological toxicity and liver and kidney function after treatment. Figure 11-14 ).

[0124] The safety evaluation results show that ( Figure 11-14 Animals in all treatment groups maintained stable body weight, and no significant tissue damage was observed in H&E staining of major organs. Blood biochemical indicators were all within the normal range. Only mild and reversible bone marrow suppression was observed, and no significant systemic toxicity was found. The fractionated low-dose administration regimen achieved superior antitumor efficacy compared to a single high-dose administration while maintaining good safety, suggesting that dose fractionation can further improve the therapeutic index.

[0125] In summary, the GalNAc-IPBA modified Affibody probe constructed in this invention [¹] 77 Lu-Lu-10a exhibits excellent radionuclide therapeutic efficacy and good biocompatibility. Site-specific S-glycosylation and synergistic modification with small albumin-binding ligands can significantly prolong probe circulation and tumor retention, increase tumor radiation dose, and reduce radiation burden on normal tissues, thereby effectively enhancing the performance of HER2-targeted radiotherapy.

[0126] As shown in the above embodiments, this invention provides a modified Affibody conjugate. A series of dual-modified probes were successfully constructed by introducing S-glycosyl modification at specific sites of the Affibody protein and coupling it with a small molecule albumin-binding ligand. In vitro characterization results showed that this series of probes maintained good HER2 targeting affinity and possessed reversible binding ability to mouse serum albumin. Radiolabeling experiments demonstrated that this series of probes could efficiently chelate diagnostic and therapeutic radionuclides, and the labeled products exhibited excellent radiochemical purity and in vitro stability. In vivo molecular imaging studies and biodistribution experiments further confirmed that the Affibody probes modified by this invention exhibited significantly prolonged blood circulation time, enhanced tumor-specific uptake and long-term retention capacity, and significantly reduced renal non-specific uptake in tumor-bearing mouse models. Radionuclide therapy experiments showed that the probes described in this invention could effectively inhibit the growth of HER2-positive tumors, and tumor-bearing mice showed good tolerance to the therapeutic dose, with no obvious systemic toxicity or major organ damage observed. In summary, this invention has successfully established a chemical modification strategy that can simultaneously optimize the pharmacokinetic behavior and radiotherapeutic properties of Affibody, providing a novel radiopharmaceutical with clinical translational potential for the precise diagnosis and targeted radionuclide therapy of HER2-positive tumors.

[0127] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A modified Affibody conjugate, characterized in that, The Affibody conjugate comprises: Affibody protein; A glycosyl modification group covalently linked to the thiol group of the cysteine ​​residues of the Affibody protein via an S-glycosidic bond; as well as Small albumin-binding ligands covalently linked to the glycosyl-modified groups; The glycosyl modification group is N-acetylgalactosamine, N-acetylglucosamine, or N-acetylmannosamine.

2. The modified Affibody conjugate according to claim 1, characterized in that, The small molecule albumin binding ligand is 4-iodophenylbutyric acid, 4-methylphenylbutyric acid, or palmitic acid.

3. The modified Affibody conjugate according to claim 1, characterized in that, The Affibody conjugate further comprises a chelating group, which is covalently linked to the small molecule albumin binding ligand.

4. The modified Affibody conjugate according to claim 3, characterized in that, The chelating agent group is DOTA.

5. The modified Affibody conjugate according to claim 1, characterized in that, The Affibody protein is HER2-targeted Affibody.

6. The modified Affibody conjugate according to any one of claims 1 to 5, characterized in that, The small molecule albumin binding ligand is covalently linked to the glycosyl modification group via a linker arm, the linker arm comprising a triazole ring structure.

7. A modified Affibody conjugate, characterized in that, It is any one of the following compounds 10a, 10b, and 10c: .

8. The method for preparing the modified Affibody conjugate according to any one of claims 1 to 7, characterized in that, Includes the following steps: (1) In the presence of an oxidant, the Affibody protein is reacted with a sodium glycosyl sulfinate compound containing an azide group to obtain a glycosylated Affibody intermediate containing an azide group; (2) The glycosylated Affibody intermediate with an azide group is coupled with the DBCO-small molecule albumin binding ligand conjugate through a strain-promoted azide-alkyne cycloaddition reaction to obtain the modified Affibody conjugate.

9. The preparation method according to claim 8, characterized in that, The sodium glycosyl sulfinate compound with an azide group is sodium 2-acetamido-2-deoxyglycosyl sulfinate with an azide group; Step (1) is carried out in a buffer solution with a pH of 6.0 to 8.0, at a reaction temperature of 15 to 40°C, and for a reaction time of 15 to 60 minutes; The oxidant is tert-butyl hydroperoxide.

10. A radioactive conjugate, characterized in that, It comprises the modified Affibody conjugate according to any one of claims 1 to 7 and the radionuclide chelated therein.

11. The radioactive conjugate according to claim 10, characterized in that, The radionuclides are selected from 68 Ga、 18 F, 64 Cu、 44 Sc、 89 Zr、 99m Tc,¹ 77 Lu、 161 Tb, 90 Y、 225 Ac、 212 Pb or 188 Re.

12. Use of the modified Affibody conjugate of any one of claims 1 to 7 or the radioactive conjugate of any one of claims 10 to 11 in the preparation of a medicament for the diagnosis or treatment of HER2-positive tumors.

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