Bioorthogonal-based antibody conjugates, methods of making and using the same

Abstract

The application discloses a bio-orthogonal-based antibody conjugate and a preparation method and application thereof, and the structural formula of the antibody conjugate is P-B-L-Y, wherein P is an ABDB6 fusion protein with a coupling short peptide, B is a cycloaddition product group, L is a linker, and Y is a functional group; the coupling short peptide is connected to the cycloaddition product group through a sulfide bond; and the cycloaddition product group is formed through bio-orthogonal reaction of a bio-orthogonal group connected to the coupling short peptide and a complementary reaction group connected to the linker. The application connects the coupling short peptide with a disulfide bond to the terminal of the ABDB6 fusion protein, so that the disulfide bond can be connected to the bio-orthogonal group at a fixed point after reduction, the coupling site consistency of different batches of products is ensured, the functional group can be quickly, efficiently and mildly connected to the ABDB6 fusion protein through bio-orthogonal reaction, the type and length of the linker can be flexibly selected, and the application scenarios of the antibody conjugate are enriched.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to antibody-drug conjugates based on bioorthogonality, their preparation methods, and applications. Background Technology

[0002] Antibody-drug conjugates (ADCs) have important applications in tumor diagnosis and treatment, such as antibody-drug conjugates (ADCs), antibody-nucleoside conjugates (ARCs), and antibody-fluorescent probe conjugates. Achieving efficient, site-specific, and controllable binding of antibodies to effector molecules such as drugs, nucleoside chelators, and fluorophores is a key technology in this field.

[0003] The inventors disclosed a bispecific nanobody ABDB6 in patent CN115925951A. This fusion protein is formed by bridging the CD70-targeting nanobody RCCB6 and the albumin-binding domain ABD035 via a GGGGS linker. It significantly prolongs the in vivo half-life by binding to albumin, and its smaller molecular weight enables faster blood clearance and better tumor penetration, making it particularly suitable as a molecular imaging probe platform. 18 F, 64 Cu、 68 Ga、 89 Zr and other nuclides are used in PET / CT diagnostic imaging, or as a linker 177 Targeted radionuclide therapy using therapeutic nuclides such as Lu enables integrated diagnosis and treatment of tumors.

[0004] Currently, antibody modification methods mainly utilize reduced cysteine ​​residues or lysine residues on the antibody surface for random coupling. Since the two disulfide bonds of ABDB6 are folded internally and are not easily reduced to thiol groups, patent CN115925951A uses lysine residues, i.e., primary amino groups, as coupling sites. The linker uses benzyl isothiocyanate (p-SCN-Bn) or dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS ester) to connect chelating groups such as NOA or deferoxamine, thereby complexing with radionuclides.

[0005] However, ABDB6 contains ten lysine residues, and the NHS or NCS groups of the linker may react with primary amino groups at different sites, resulting in a mixture of products with varying coupling ratios and coupling sites. This leads to significant batch-to-batch variability and can easily affect antigen-binding activity. Furthermore, the limited variety of linkers used, which only serve a linking function, makes it difficult to regulate and optimize the performance of antibody-drug conjugates, thus restricting their application scenarios to some extent. Summary of the Invention

[0006] One objective of this invention is to provide a bioorthogonal-based antibody-drug conjugate, which connects a short peptide containing a cysteine ​​(Cys) residue to the ABDB6 fusion protein to obtain the ABDB6-PT fusion protein. Then, the thiol group obtained after cysteine ​​reduction is used to site-specifically connect bioorthogonal groups, achieving consistency in the coupling sites across different batches of products. Simultaneously, one end of the linker is connected to a complementary reactive group, and the other end to a functional group. This not only enables rapid, efficient, and mild connection of the functional group to the ABDB6-PT fusion protein based on the bioorthogonal reaction between the bioorthogonal and complementary reactive groups, but also allows for flexible selection of the type and length of the linker to better regulate the performance of the antibody-drug conjugate and match different functional groups, thus enriching the application scenarios of antibody-drug conjugates.

[0007] This invention is achieved through the following technical solution: The antibody conjugate based on bioorthogonality has the structural formula PBLY, where P is an ABDB6 fusion protein with a conjugated short peptide, B is a cycloaddition product group, L is a linker, and Y is a functional group. The conjugated short peptide is linked to the cycloaddition product group via a thioether bond. The cycloaddition product group is formed by a bioorthogonal reaction between a bioorthogonal group attached to the coupled short peptide and a complementary reactive group attached to the linker.

[0008] The two disulfide bonds in the existing ABDB6 fusion protein are located inside the folded structure, making them difficult to reduce to thiol groups even with the addition of a reducing agent. In this technical solution, a short coupling peptide is linked to the existing ABDB6 fusion protein via a flexible linker. This short coupling peptide is attached to the end of the ABDB6 fusion protein, thus being located outside the folded structure. This allows the disulfide bonds on the short coupling peptide to be more easily reduced to two thiol groups. These thiol groups then react with a bioorthogonal precursor, and the bioorthogonal group is site-specifically linked via a thioether bond, ensuring the consistency of the coupling sites across different batches of antibody conjugates.

[0009] In this technical solution, effector functional groups, such as fluorescent groups, chelating groups, and drug molecules, can be linked to the ABDB6 fusion protein via linkers. Specifically, one end of the linker is connected to a functional group, and the other end is connected to a complementary reactive group capable of undergoing a bioorthogonal reaction with the bioorthogonal group. For example, the bioorthogonal group can be a tetrazine group, and its complementary group can be a dienophile. The two react via an inverse electron-demanding Diels-Alder reaction (iEDDA) to obtain a pyridazine cycloaddition product group. Another example is that the bioorthogonal group can be an azide group, and its complementary group can be a terminal alkyne compound. The two react via a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) to obtain a triazole cycloaddition product group. Therefore, through bioorthogonal reactions, functional groups can be rapidly, efficiently, and under mild conditions linked to the ABDB6 fusion protein. Furthermore, experiments have shown that the introduction of bioorthogonal groups does not affect the affinity of ABDB6 for CD70 protein, nor does the introduction of ABDB6 affect the rate of the bioorthogonal reaction.

[0010] This technical solution allows for more flexible selection of the type and length of linkers, enabling them to not only function as connectors but also to actively play a regulatory role. For example, in some embodiments, when PEG chains are used as linkers, the hydration of PEG chains can increase molecular hydrophilicity, reduce non-specific adsorption of plasma proteins, and reduce uptake by the reticuloendothelial system in organs such as the liver and spleen through a shielding effect, thereby improving background signals in normal organs throughout the body. Furthermore, by adjusting the length of the linker, the spatial matching degree between the target ligand and the receptor can be controlled, avoiding steric hindrance caused by excessively short linkers or clearance delay caused by excessively long linkers.

[0011] In this technical solution, the antibody conjugate has the following structural formula: or As can be seen, the cysteine ​​residues of the ABDB6-coupled short peptide are linked to the cycloaddition product group via a thioether bond. The cysteine ​​residues in the formula belong to the ABDB6-coupled short peptide and are shown to highlight the thioether bond. The cycloaddition product group is linked to the functional group via a linker. By selecting different functional molecules, various applications such as fluorescence imaging, PET imaging, radionuclide therapy, and drug delivery can be realized.

[0012] In a preferred embodiment of the present invention, the bioorthogonal group is a tetrazine group, and the complementary reactive group of the tetrazine group is a dienophile or an isonitrile group; or the bioorthogonal group is an azide group, and the complementary reactive group of the azide group is a terminal alkyne compound or a cyclic alkyne compound.

[0013] In this technical solution, the bioorthogonal group can be a tetrazine group, and the corresponding complementary reactive group can be a dienophile. The tetrazine group and the dienophile form a cycloaddition product through a Diels-Alder reaction with reverse electron demand. For example, the dienophile can be bicyclic [6.1.0]nonyne (BCN), and the two react to obtain a pyridazine cycloaddition reactive group. In one or more embodiments, the dienophile can also be selected from other diens capable of undergoing bioorthogonal reactions with the tetrazine group, such as trans-cyclooctene (TCO), norbornene, cyclopropene, etc. When the bioorthogonal group is a tetrazine group, the corresponding complementary group can also be an isonitrile group. The isonitrile group and the tetrazine group form a pyrazole cycloaddition product through a [4+1] cycloaddition reaction. In some preferred embodiments, the structure of the linker connected to the isonitrile group can be: ; In the formula, the group R3 is selected from H or C1~C6 alkyl.

[0014] In this technical solution, the bioorthogonal group can also be an azide group indirectly linked to the coupled short peptide, and its corresponding complementary reactive group can be a terminal alkynyl compound. A CuAAC reaction between the azide group and the terminal alkynyl compound yields a triazole cycloaddition product. Alternatively, the complementary reactive group can also be a cyclic alkynyl compound, such as dibenzocyclooctyne (DBCO), where a strain-promoted azide-alkynyl cycloaddition reaction (SPAAC) between the azide group and the cyclic alkynyl compound yields a triazole cycloaddition product.

[0015] Furthermore, the coupled short peptide of the ABDB6 fusion protein includes one or two cysteine ​​residues, which are linked to the cycloaddition product group via a thioether bond after reduction.

[0016] Furthermore, the amino acid sequence of the ABDB6 fusion protein with coupled short peptides is shown in SEQ ID NO.1 or SEQ ID NO.3.

[0017] Furthermore, the linker is selected from one or more polyethylene glycol units, C1~C1. 12 At least one of alkylene, divalent glucose, C4-C6 aryl or heteroaryl, wherein the heteroatom of the heteroaryl is selected from N, O, or S.

[0018] In this technical solution, the physicochemical properties of the conjugate can be adjusted by selecting different linkers. In some embodiments, the linker can be one or more polyethylene glycol units, i.e., PEG chains. PEG chains are hydrophilic flexible chains that, by increasing the hydration radius and reducing plasma protein adsorption, adjust the drug from rapid renal clearance to moderately prolonged circulation without excessive accumulation, thereby increasing the cumulative tumor exposure dose. Simultaneously, the hydrophilic shielding layer of the PEG chain inhibits capture by the hepatic and splenic reticuloendothelial systems, the neutral charge avoids charge-mediated reabsorption in the renal tubules, and the metabolites maintain high water solubility for rapid excretion.

[0019] In one or more embodiments, the PEG chain In the n=1~12. In some preferred embodiments, n=4~8. By adjusting the appropriate PEG chain length, not only can the steric hindrance and decreased affinity caused by excessively short PEG chains be avoided, but the delay in normal tissue clearance caused by excessively long PEG chains can also be prevented.

[0020] In this technical solution, in addition to the PEG chain, the linker can also be selected as a hydrophobic alkylene chain. In one or more embodiments, the linker can be C1~C1. 12 Alkylene. In some embodiments, the linker may be methylene, ethylene, propylene, butylene, pentylene, or hexylene. Alternatively, the linker may be a hydrophilic rigid sugar ring, such as a divalent glucosyl group. In one or more embodiments, the linker may be glucose-1,6-diyl or glucose-1,4-diyl. In some embodiments, the linker may also be a C4-C6 aryl or heteroaryl group; in one or more embodiments, the linker may be phenyl, piperidinyl, piperazinyl, thiophene, pyridinyl, pyrroleyl, etc.

[0021] Further, the functional group is a fluorescent group, a chelating group complexed with a radionuclide, or a drug molecule; wherein, the fluorescent group is selected from cyanine fluorescent dyes or sulfonated cyanine fluorescent dyes; the chelating group is selected from any one of DOTA, NOTA, NODAGA, PCTA, Macropa, PYTA, TETA, DOTMA, DTPA, HBED-CC, Bispidine, HOPO, DFO, and TRAP; and the radionuclide is... 68 Ga、 89 Zr、 64 Cu、 124 I, 177 Lu、 90 Y, or 225 Ac; The drug molecule is selected from quinoline alkaloids, anthracycline antibiotics, or peptide drugs.

[0022] In some embodiments, the fluorescent group may be selected from cyanine fluorescent dyes, such as Cy series cyanine dyes such as Cy3, Cy5, and Cy7. In one or more embodiments, the fluorescent group may also be selected from sulfonated cyanine fluorescent dyes, such as at least one of AlexaFluor 647, DyLight 650, Atto 647N, or IRDye 680RD.

[0023] In some embodiments, the chelating group may be selected from macrocyclic chelating agents such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1,4,7-triacetic acid-glutaric acid), PCTA (1,4,7,10-tetraazacycloundecane-1,4,7-triacetic acid), Macropa, PYTA, TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), and DOTMA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tetramethyl derivative). In some embodiments, the chelating group may also be some non-macrocyclic chelating agents, such as linear aminocarboxylic acid complexing agent DTPA (diethylenetriaminepentaacetic acid), HBED-CC (N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid-cyclohexanecarboxylic acid), bicyclic [3.3.1]nonane chelating agent Bispidine, octate chelating agent HOPO, trihydroxyoxime acid iron chelating agent DFO, trihydroxyphosphonopropionic acid chelating agent TRAP, etc.

[0024] In one or more embodiments, the radionuclide may be used for diagnostic imaging. 68 Ga、 89 Zr、 64 Cu、 124 Class I radionuclides. For example, in one or more embodiments, the radionuclide may be used for antitumor therapy. 177 Lu、 90 Y、 225 Ac and other radionuclides. In some preferred embodiments, the radioactivity can be selected based on diagnostic or therapeutic purposes. 68 Ga / 177 Lu、 68 Ga / 225 Ac、 89 Zr / 177 Lu、 89 Zr / 225 Ac and other diagnostic and treatment options are available.

[0025] In some embodiments, the drug molecule may be selected from quinoline alkaloids, such as camptothecin, senna, and sinomenine; the drug molecule may also be selected from anthracycline antibiotics, such as doxorubicin, epirubicin, and pirarubicin; the drug molecule may also be selected from peptide drugs, such as MMAE, octreotide, and leuprorelin.

[0026] Another objective of this invention is to provide a method for preparing antibody-drug conjugates based on bioorthogonal synthesis. This method uses ABDB6-PT fusion protein with a short conjugated peptide as a raw material. The bioorthogonal precursor reacts with the reduced thiol group in the short conjugated peptide to form a thioether intermediate with one or two thioether bonds. The thioether intermediate is further subjected to a bioorthogonal reaction with a linker connected to complementary reactive and functional groups to obtain the antibody-drug conjugate. This method effectively overcomes the modification difficulties of ABDB6 in the prior art and allows for flexible connection of fluorescent probes and chelating groups containing radionuclides via linkers, thus possessing broad application value.

[0027] The preparation method of antibody conjugates based on bioorthogonality in this technical solution specifically includes the following steps: A reducing agent is added to an ABDB6 fusion protein containing a coupled short peptide to obtain a reduced ABDB6 fusion protein, wherein the coupled short peptide comprises one or two cysteine ​​residues. The reduced ABDB6 fusion protein reacts with a bioorthogonal precursor to obtain a thioether intermediate, the thioether intermediate having a bioorthogonal group, the bioorthogonal group being linked to the coupled short peptide via a thioether bond; The bioorthogonal group of the thioether bond intermediate undergoes a bioorthogonal reaction with the complementary reactive group at the first end of the linker to form a cycloaddition product group, and a functional group is attached to the second end of the linker.

[0028] Further, the bioorthogonal precursor is 3,6-divinyl-1,2,4,5-tetraazine or a monovinyltetraazine with a vinyl group attached at the 3 or 6 position, the bioorthogonal group of the thioether intermediate is a tetraazine group, and the complementary reactive group of the tetraazine group is a dienophile or an isonitrile group; or The bioorthogonal precursor is 1,1'-[5-(azidomethyl)-1,3-phenylene]bis(2-iodoethane-1-one), the bioorthogonal group of the thioether intermediate is an azide group, and the complementary reactive group of the azide group is a terminal alkyne compound or a cyclic alkyne compound.

[0029] Another object of the present invention is to provide the application of any of the aforementioned bioorthogonal-based antibody conjugates in the preparation of diagnostic or therapeutic molecules. For example, when the functional group is a fluorescent group, the antibody conjugate can be used to target tumor cells with ABDB6 and then be visualized, serving as an active substance in the preparation of tumor contrast agents; when the functional group is a drug molecule, the antibody conjugate can serve as an active substance in antitumor drugs; similarly, when the functional group is a chelating group complexed with a radionuclide, the antibody conjugate can be used for both tumor diagnosis and tumor treatment.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention connects a short peptide with a disulfide bond to the end of the ABDB6 fusion protein, so that the disulfide bond can be site-specifically linked to a bioorthogonal group after reduction, and then the bioorthogonal reaction is used to link functional groups, thus ensuring the consistency of the coupling sites of different batches of products. 2. This invention utilizes a bioorthogonal reaction to connect the bioorthogonal group on the ABDB6 fusion protein to the complementary reactive group at one end of the linker, enabling the functional group to be connected to the ABDB6 fusion protein rapidly, efficiently, and under mild conditions. 3. In this invention, the type and length of the linker can be flexibly selected to better adjust the performance of the antibody conjugate, match different functional groups, and enrich the application scenarios of the antibody conjugate. Attached Figure Description

[0031] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings: Figure 1 This is an electrophoresis image of the purified ABDB6-PT-1 fusion protein from Example 1 of this invention; Figure 2 This is a flowchart illustrating the preparation method of the antibody conjugate in a specific embodiment of the present invention; Figure 3 The HPLC chromatograms of ABDB6 and Tz-ABDB6-1 in Example 3 of this invention are shown below. Figure 4 This is the HPLC chromatogram of ABDB6-Cy5 in Example 3 of the present invention; Figure 5 The graph shows the stability of Tz-ABDB6-1 over time in a specific embodiment of the present invention; Figure 6 The bioorthogonal reaction rate of Tz-ABDB6-1 in a specific embodiment of the present invention is shown; Figure 7 illustrates the effect of Tz-ABDB6-1 and TCO-P4-Cy5 on CD70-positive SKOV3 cells in a specific embodiment of the present invention. Figure 7a ), positive cells 786-O ( Figure 7b ), and CD70-negative HUVECs ( Figure 7c Pre-targeted fluorescence imaging results were performed, with a scale bar of 20 μm; Figure 8 The imaging results of ABDB6-Cy5 accumulating at the tumor site over time are shown in a specific embodiment of the present invention; Figure 9 The HPLC chromatogram of DOTA-ABDB6-1 in a specific embodiment of the present invention is shown; Figure 10 A specific embodiment of the present invention is shown. 177 Micro SPECT / CT imaging results of Lu-ABDB6-1; Figure 11 The injection method is shown in a specific embodiment of the present invention. 177 Tumor volume after Lu-ABDB6-1. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0033] All raw materials used in this invention are not particularly limited in their source; they can be purchased commercially or prepared using conventional methods well-known to those skilled in the art. The purity of all raw materials used in this invention is not particularly limited; however, analytical grade or conventional purity requirements in the biopharmaceutical field are preferred. All raw materials used in this invention have brand names and abbreviations that are conventional in the field, and each brand name and abbreviation is clearly defined within its relevant application. Those skilled in the art can obtain these materials from commercial sources or prepare them using conventional methods based on the brand name, abbreviation, and corresponding application.

[0034] The present invention does not impose any particular restrictions on the expression of the substituents, and all expressions are well known to those skilled in the art. Based on common sense, those skilled in the art can correctly understand their meaning according to their expression.

[0035] In this invention, the specific operations for obtaining the ABDB6 fusion protein and constructing the vector can be performed by obtaining recombinant plasmids through commercial synthesis or conventional PCR cloning. The specific gene sequence design, codon optimization, whole-gene synthesis, and cloning into the target expression vector of the provided fusion protein recombinant plasmid are all routine operations that can be performed by those skilled in the art based on existing molecular biology techniques.

[0036] The ABDB6-PT fusion protein used in this invention is an ABDB6 fusion protein containing coupled short peptides. In some embodiments, for the sake of simplicity, it is also referred to as the ABDB6 fusion protein.

[0037] The terms "first," "second," etc. (e.g., first end, second end, etc.) used in this invention are merely for clarity of description and are not intended to limit any order or emphasize importance. Furthermore, the term "connection" used in this invention, unless otherwise specified, can refer to a direct connection or an indirect connection via other groups.

[0038] I. Preparation of ABDB6-PT fusion protein

Example 1

[0039] Plasmid extraction uses an alkaline lysis method, which includes the following steps: A1) Collect 100 mL of overnight cultured bacterial solution into a labeled 50 mL centrifuge tube. Collect about 45 mL of bacterial solution at a time. After centrifuging with an angle rotor centrifuge, discard the supernatant and retain the bacterial cells. Repeat the above operation once and mark the first centrifuge tube cap of each row on the acrylic rack as a sign that the bacterial collection is complete.

[0040] A2) Add 10 mL of Buffer P1 containing RNase A to the centrifuge tube and vortex until no obvious clumps of bacteria are visible. Then add 10 mL of Buffer P2 and mix well. At this point, the bacterial solution changes from turbid to viscous. Then add 10 mL of Buffer P3 and mix well. After a white flocculent precipitate appears, centrifuge. Finally, add 10 mL of ER Buffer to the centrifuged 50 mL centrifuge tube and mix well. A3) Add 25 mL of QBT reagent to the chromatography column, which is placed on a centrifuge rack and filled with packing material. Equilibrate the packing material. Filter the sample treated with ER Buffer to remove the white precipitate, and add the remaining liquid to a clean, labeled chromatography column. After the liquid has finished adding, add 60 mL of QC elution buffer to the column for gravity elution. After the QC elution is complete, transfer the chromatography column to a clean, labeled 50 mL centrifuge tube and verify that the tube numbers match. Add 10 mL of Elution Buffer and allow it to elute by gravity. A4) After elution, gently press the chromatography column against the centrifuge tube wall and then quickly remove it. Add 7 mL of isopropanol to the collected filtrate, mix well, centrifuge, and discard the supernatant. Place the centrifuge tubes on a rack in order and add 5 mL of 75% ethanol to each tube to thoroughly rinse the precipitate. After adding the sample, centrifuge again and discard the supernatant. Invert the centrifuge tubes onto a tissue in ascending order and add 50-500 μL of deionized water to the location of the plasmid. Swish the tubes several times to fully dissolve the plasmid in the water until there is no adsorption on the tube wall. Use a pipette to aspirate the dissolved solution and transfer it to the corresponding microporous filter column according to the centrifuge tube number. After all centrifugation is complete, transfer the plasmid to the corresponding EP tube according to the microporous filter column number.

[0041] Place 1.5 mL EP tubes on a 96-well aliquot plate for plasmid aliquoting. Use the sample to be tested for various assays, measure and record the concentration using an ELISA reader, and record the corresponding concentration on the side wall of the EP tube.

[0042] B) Expression and purification. Mammalian cells (CHO cells) were used. The parameters for CHO cell resuscitation and three passages are shown in Table 1. Table 1: Once the cell density reaches the requirements for electroporation, centrifuge the cells to be transfected, remove the supernatant, add the electroporation buffer, mix well by pipetting, add the plasmid to obtain a suspension, then add the suspension to the electroporation tube for electroporation. After electroporation, add the suspension to the preheated culture medium for incubation. After incubation, place the shake flask in a 37°C, 120 rpm, 8% CO2 incubator. After 24 hours, add feed and continue culturing for 4-6 days.

[0043] After expression, the protein was purified using a Ni affinity chromatography column. Following equilibration, washing, elution, and dialysis, the purified ABDB6-PT fusion protein was obtained. The electrophoresis results are shown below. Figure 1 As shown in the figure, R represents the sample with the reducing agent added before electrophoresis, and NR represents the sample without the reducing agent added before electrophoresis. As shown in the figure, the main band positions of lanes R and NR are completely consistent, indicating that the ABDB6-PT backbone was not destroyed after reduction.

[0044] C) Purity detection. SEC-HPLC was performed using an LC-20AT high-performance liquid chromatograph and a gel chromatography column. The results are shown in Table 2. The retention times (Ret. Time) of the control sample (Detector A Channel 1) and the purified sample (Detector A Channel 2) were basically the same, and the concentration (Conc.) of the ABDB6-PT fusion protein reached 82.72%.

[0045] Table 2: In some preferred embodiments, the number of amino acids in the coupled short peptide can be 5 to 15. In a more preferred embodiment, the number of amino acids separating the two cysteine ​​residues in the coupled short peptide is 2 to 4, in order to better react with the bioorthogonal precursor to form a thioether bond.

[0046] In some embodiments, the flexible linker peptide connecting ABDB6 to the conjugated short peptide has at least one GGGGS linker. In one or more embodiments, the flexible linker peptide has 1 to 10 GGGGS linkers.

[0047] Those skilled in the art will understand that the ABDB6-PT-1 conjugated short peptide is formed by the reaction of the thiol group generated from cysteine ​​reduction with the linker bioorthogonal precursor to form a thioether bond intermediate. Therefore, without substantially affecting the performance of the fusion protein, any conjugated short peptide containing cysteine ​​residues should be considered to fall within the scope of protection claimed in this application.

[0048]

Example 2

Example 3

[0049] A) Reaction of ABDB6-PT fusion protein with dienetetrazine The ABDB6 stock solution was diluted to 50 μM with buffer (20 mM, pH=8.0) to obtain a diluted solution. Then, 5 equivalents (eq) of the reducing agent tris(2-carboxyethyl)phosphine (TCEP) were added to the diluted solution, and the reaction was carried out at 37°C for 2 hours to obtain a reduced solution. The reduced solution was placed in a 10 kDa ultrafiltration tube and centrifuged at 4°C and 10000g for 15 minutes to remove excess TCEP. The filtrate was collected to obtain reduced Re-ABDB6.

[0050] The Re-ABDB6 solution was then diluted to 20 μM with PBS buffer (100 mM, pH = 8.0) to obtain a diluted Re-ABDB6 solution. Subsequently, 5 equivalents of dienetetrazine (Tz) stock solution were added, and the reaction was carried out at 37°C for 2 hours to obtain thioether intermediate 1, namely Tz-ABDB6-1.

[0051] The labeled yield and molecular weight of the product were analyzed by HPLC-MS. The HPLC conditions were as follows: Poroshell 120 EC-C18 column (3.0 × 150 mm, 2.7 μm); column temperature: 35 °C; elution gradient: 10% in phase B at 0 min, increasing from 10% to 100% in phase B over 0–20 min; flow rate: 0.5 mL / min.

[0052] The calculated value of reactant ABDB6 was 22348 Da, and the observed value was 22341 Da. The calculated value of product Tz-ABDB6-1 was 22482 Da, and the observed value was 22475 Da. The HPLC data for ABDB6 and Tz-ABDB6 are as follows: Figure 3 As shown.

[0053] In some embodiments, the ABDB6 stock solution is diluted with buffer to a concentration of 10-200 μM.

[0054] In some embodiments, the amount of TCEP added is 1 to 10 equivalents.

[0055] In some embodiments, the ultrafiltration tube has a molecular weight cutoff of 3 to 30 kDa.

[0056] In some embodiments, the Re-ABDB6 solution was diluted to 5–50 μM.

[0057] B) Preparation of TCO-P4-Cy5 (4E)-TCO-PEG4-amine (20 mg, 0.05 mmol) and Cyanine 5-NHS ester (34 mg, 0.055 mmol) were dissolved in 5 mL of anhydrous acetonitrile, and triethylamine (28 µL, 0.2 mmol) was added. The mixture was stirred at room temperature, and the reaction was monitored by TLC. After the reaction was complete, water was added, and the mixture was extracted with dichloromethane. The organic phases were combined, washed successively with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by reversed-phase column chromatography to give the target product TCO-P4-Cy5 (32 mg, 76% yield).

[0058] C) The antibody conjugate ABDB6-Cy5 is prepared by a bioorthogonal reaction between thioether intermediate 1 and TCO-P4-Cy5. The synthetic route is as follows: Thioether intermediate 1 and TCO-P4-Cy5 were dissolved in PBS buffer at an equivalent ratio of 1:1.1 and reacted completely at room temperature via a bioorthogonal reaction. After the reaction, excess TCO-P4-Cy5 was removed by ultrafiltration (molecular weight cutoff 10 kDa) to obtain the antibody conjugate ABDB6-Cy5. Its purity and molecular weight were determined by HPLC-MS. The calculated value of the product ABDB6-Cy5 was 23307 Da, and the observed value was 23300 Da. The HPLC data of ABDB6-Cy5 are as follows: Figure 4 As shown.

[0059]

Example 4

[0060]

Example 5

[0061] Specifically, the testing method was surface plasmon resonance (SPR) technology, and the CD70 protein was recombinant human CD70 protein (CDLH52Da, ACRO Biosystems). The experimental method included the following steps: CD70 was immobilized on a CM5 sensor chip (Cytiva) using a standard amine coupling method. First, a 1:1 mixture of EDC / NHS solution (Cytiva, BR-1006-33) was injected at a flow rate of 10 μL / min to activate the chip surface for 7 minutes. Subsequently, the CD70 protein was diluted to 5 μg / mL with 10 mM sodium acetate buffer (pH 5.5, Cytiva BR100352) and injected into the chip to achieve an immobilization level of 320.5 RU. Finally, 1 M ethanolamine (pH 8.5, Cytiva BR-1006-33) was injected to block any remaining active ester groups.

[0062] The ABDB6 or Tz-ABDB6 samples to be tested were serially diluted with HEPES buffer (pH 7.4) and flowed sequentially through the chip surface. The binding time was set to 120 seconds and the dissociation time to 1200 seconds; all measurements were performed at 25°C. Chip regeneration was achieved by rinsing the chip surface with glycine-hydrochloric acid buffer (pH 2.5) for 20 seconds after each binding-dissociation cycle. Instrument control and data acquisition were performed using Biacore Control software. The obtained sensor data were kinetically fitted using Biacore Evaluation software, and the binding rate constant (k) was calculated as shown in Table 4. a ), dissociation rate constant (k d ) and equilibrium rate constant (K D ).

[0063] Table 4: As shown in Table 4, compared with ABDB6, the interaction between Tz-ABDB6 and CD70 protein was not significantly affected by the modification of the tetrazine group.

[0064]

Example 6

[0065] Add 100 μL of Tz-ABDB6-1 stock solution (400 μM, dissolved in PBS) to 900 μL of fetal bovine serum (FBS), mix well, and incubate at 37°C. At 0, 2, 4, 8, 12, and 24 hours after incubation, aspirate 100 μL of the incubation mixture into new centrifuge tubes. Add 50 μL of TCO-P4-Cy5 solution (100 μM, dissolved in PBS containing 10% DMSO) to each aspirated incubation mixture, mix thoroughly to ensure complete reaction of Tz-ABDB6-1 with TCO. After the reaction is complete, use an ultrafiltration centrifuge tube (Amicon-Ultra-0.5, Millipore, molecular weight cutoff 10 kDa) to remove excess and unreacted TCO-P4-Cy5. The filtrate was collected, and its fluorescence intensity was detected using a microplate reader with an excitation wavelength of 633 nm and an emission wavelength of 678 nm.

[0066] 100 μL of fetal bovine serum was collected, and its fluorescence value was measured following the same procedure as for the samples, serving as the background value. The stability retention rate at each time point was calculated using the formula to characterize the binding ability of Tz-ABDB6-1 to TCO in serum. All samples were measured in equal volumes, and the fluorescence intensity at 0 h was normalized to 100% as a reference for stability calculation. Independent measurements were performed three times, and results are expressed as mean ± standard deviation. The binding stability of Tz-ABDB6 to TCO in serum is expressed as the percentage of binding activity retained relative to the initial value.

[0067] The test results are as follows Figure 5 As shown, Tz-ABDB6-1 exhibits good stability.

[0068]

Example 7

[0069] Specifically, Tz-ABDB6-1 (20 μM) was mixed with an equal volume of TCO-Cy5 (2 μM), and samples were taken at different time points after the reaction (Tz-trastuzumab: 0~900 s; Tz-ABDB6-1: 0~600 s). Excess TCO (200 μM) was added immediately to quench the reaction, and the fluorescence intensity was measured after centrifugation and washing twice with a 10 kDa ultrafiltration tube.

[0070] The apparent first-order rate constant k_obs was obtained by fitting the fluorescence decay curve, and the second-order rate constant k2 was calculated according to k_obs / c0 (c0 = 20 μM). Each experiment was repeated three times. Figure 6As shown, the second-order rate constant of the bioorthogonal reaction is k2 = 1169.84 ± 27.21 M. -1 s -1 Therefore, it can be seen that the bioorthogonal reaction rate of Tz-ABDB6-1 is not affected after the fusion protein ABDB6 is linked.

[0071]

Example 8

[0072] Specifically, in CD70-positive SKOV3 and 786-O cells and CD70-negative HUVEC cells, a pre-positional imaging strategy was used to verify the targeting binding affinity of Tz-ABDB6 at the live cell level and the bioorthogonal ability of the tetrazine in the structure. First, cells were counted using a hemocytometer and then seeded into 35 mm glass-bottomed culture dishes. Experiments began when the cell count reached 80%-90%.

[0073] Cells were incubated with the nuclear dye Hoechst 33342 (250 nM) for 5 minutes, followed by washing three times with PBS. Cells were then incubated with 1 μM Tz-ABDB6 for 1 hour to allow for sufficient receptor binding. In the blocking assay, cells were pre-incubated with 10 µM MABDB6 for 1 hour before the addition of Tz-ABDB6. Subsequently, 1 μM TCO-P4-Cy5 was added, and incubation was performed for 20 minutes to allow for a fully orthogonal reaction between TCO-P4-Cy5 and the Tz-ABDB6 bound intracellularly (on the cell surface). Cells were then washed with PBS and imaged using a confocal microscope at an excitation wavelength of λ. ex =633 nm, λ em =640~750 nm.

[0074] Theoretically, the pre-targeting strategy requires Tz-ABDB6 to bind well to cells, while the tetrazine in its structure retains its bioorthogonal function to achieve fluorescence activation. CD70-negative HUVEC cells served as a control experiment to demonstrate the binding of Tz-ABDB6 to its specific receptor. As shown in Figure 7, for CD70-positive SKOV3 and 786-O cells, fluorescence activation proved that the tetrazine activity in Tz-ABDB6 was retained and that bioorthogonality could be achieved at the live cell level, consistent with the previous in vitro bioorthogonal rate measurement results. However, the negative control group showed no fluorescence activation in HUVEC cells, indicating that Tz-ABDB6 did not specifically bind to cells with weak or no CD70.

[0075]

Example 9

[0076] Specifically, in vivo imaging was performed on tumor-bearing mice. Female Balb / c-nu mice (3-5 weeks old) were subcutaneously inoculated with 1×10 7 SKOV3 cells. 3-4 weeks post-inoculation, the tumor volume was approximately 200-400 mm. 3 At that time, imaging studies were conducted.

[0077] In the fluorescence imaging study, 4 nmol of ABDB6-Cy5 was administered intravenously, and images were acquired at multiple time points from 1 to 72 h post-injection. In the biodistribution study, tissues and organs were collected at 24, 48, and 72 hours post-injection, and favorable tumor-to-organ ratios were observed. Collected organs were weighed, and fluorescence intensity was quantified. Tissue uptake is expressed as mean ± SD for each group (n=3 per group), and results are shown as a percentage of the injected dose per gram of tissue (%ID / g).

[0078] The test results are as follows Figure 8 As shown, ABDB6-Cy5 rapidly accumulated in the tumor after 4 hours, and a good tumor / background image was obtained after 24 hours, indicating that the antibody-drug conjugate construction strategy was successful and that ABDB6-Cy5 has good tumor specificity. Table 5 shows the accumulation of ABDB6-Cy5 in tumors and various organs at 24, 48, and 72 hours. As can be seen from Table 5, the biodistribution results are consistent with the imaging results. After 24 hours, ABDB6-Cy5 had low accumulation in non-target organs and a good tumor / non-target organ ratio, further demonstrating the excellent in vivo distribution characteristics of this antibody-drug conjugate.

[0079] Table 5: III. Preparation and Performance Testing of Antibody Conjugates with Radionuclide Chelating Groups

Example 10

[0080] A) Preparation of DOTA-PEG5-BCN DOTA was dissolved in anhydrous DMF. Under an inert gas atmosphere, EDC·HCl (1.0 eq), N-hydroxysuccinimide (NHS, 1.0 eq), and N,N-diisopropylethylamine (DIPEA, 1.5 eq) were added sequentially, and the mixture was stirred at room temperature to activate the carboxyl groups. The activated solution was then slowly added dropwise to an anhydrous N,N-dimethylformamide solution (DMF, 0.2 eq) of NH2-PEG5-NH2 while cooling in an ice-water bath. After the addition was complete, the ice bath was removed, and the mixture was allowed to rise naturally to room temperature before stirring continued. After the reaction was complete, the mixture was concentrated under reduced pressure below 40°C. The residue was poured into cold diethyl ether to precipitate the solid, which was then collected and purified by preparative high-performance liquid chromatography (HPLC). Finally, the solid was freeze-dried to obtain DOTA-PEG5-NH2, with a yield of 88%. In one or more embodiments, the amount of anhydrous DMF used is 0.05~0.1 M.

[0081] In one or more embodiments, the reaction was stirred at room temperature for 2 to 4 hours to complete the activation of the carboxyl group.

[0082] In one or more embodiments, the reaction was allowed to rise naturally to room temperature and then stirred for 12 to 24 hours.

[0083] Raw material I (1.0 eq) and DOTA-PEG5-NH2 (1.5 eq) were dissolved in 2 mL of acetonitrile / dimethyl sulfoxide (0.05 M, 1:1, v / v) mixed solvent, and triethylamine (about 4.0 eq) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, water was added to quench the reaction, and the mixture was purified by preparative high performance liquid chromatography to obtain a white solid with a yield of 80%.

[0084] B) The thioether intermediate 1 undergoes a bioorthogonal reaction with DOTA-PEG5-BCN, and its synthetic pathway is as follows: DOTA-PEG5-BCN was freshly dissolved in PBS buffer containing 10% DMSO and added to 400 μM Tz-ABDB6-1 solution at a molar ratio of 1.1:1. The reaction was carried out at room temperature for 30 minutes, followed by purification by ultrafiltration (10 kDa cutoff) to obtain the precursor compound DOTA-ABDB6-1. The calculated value of the product DOTA-ABDB6-1 was 23296 Da, and the observed value was 23290 Da. The HPLC data of DOTA-ABDB6-1 are as follows. Figure 9 As shown.

[0085] C) Preparation of antibody conjugates 177 Lu-ABDB6-1 Take 30 μL of DOTA-ABDB6-1 (400 μM) and 1 mL of freshly eluted... 177 Lu (200-250 MBq) was mixed, and the pH was adjusted to 5.0-5.5 with 1 M sodium acetate buffer. The mixture was reacted at 50°C for 30 minutes. The resulting antibody conjugate was purified and concentrated by sequentially passing it through a PD-10 desalting column (pre-equilibrated with PBS) and ultrafiltration (10 kDa). 177 The maximum specific activity of Lu-ABDB6 is approximately 40 MBq / nmol; its radiochemical purity was determined by rapid thin-layer chromatography (iTLC) using 0.1 M sodium citrate as the developing solvent. The radiochemical purity was greater than 95%, indicating an antibody-drug conjugate. 177 The structural formula of Lu-ABDB6-1 is:

Example 11

[0086] A) ABDB6-PT fusion protein and monoalkenyltetrazine After reducing the ABDB6-PT-2 fusion protein using TCEP, the Re-ABDB6 solution was diluted to 20 μM with PBS buffer (100 mM, pH = 8.0) to obtain a diluted Re-ABDB6 solution. Subsequently, 5 equivalents of monoalkenyltetraazine stock solution were added, and the reaction was carried out at 37°C for 2 hours to obtain thioether intermediate 2, namely Tz-ABDB6-2.

[0087] The labeled yield and molecular weight of the product were analyzed by HPLC-MS. The HPLC conditions were as follows: Poroshell 120 EC-C18 column (3.0 × 150 mm, 2.7 μm); column temperature: 35 °C; elution gradient: 10% in phase B at 0 min, increasing from 10% to 100% in phase B over 0–20 min; flow rate: 0.5 mL / min.

[0088] The calculated value of product Tz-ABDB6-2 is 22316 Da, and the observed value is 22301 Da.

[0089] B) The thioether intermediate 2 undergoes a bioorthogonal reaction with DOTA-PEG5-BCN, and its synthetic pathway is as follows: DOTA-PEG5-BCN was freshly dissolved in PBS buffer containing 10% DMSO and added to 400 μM Tz-ABDB6-2 solution at a molar ratio of 1.1:1. The mixture was reacted at room temperature for 30 minutes and then purified by ultrafiltration (10 kDa cutoff) to obtain the precursor compound DOTA-ABDB6-2, which had a calculated value of 22500 Da and an observed value of 22488 Da.

[0090] C) Preparation of antibody conjugates 177 Lu-ABDB6-2 Take 30 μL of this DOTA-ABDB6-2 (400 μM) and 1 mL of freshly eluted... 177 Lu (200-250 MBq) was mixed, and the pH was adjusted to 5.0-5.5 with 1 M sodium acetate buffer. The mixture was reacted at 50°C for 30 minutes. The resulting antibody conjugate was purified and concentrated by sequentially passing it through a PD-10 desalting column (pre-equilibrated with PBS) and ultrafiltration (10 kDa). 177 The maximum specific activity of Lu-ABDB6 is approximately 40 MBq / nmol; its radiochemical purity was determined by rapid thin-layer chromatography (iTLC) using 0.1 M sodium citrate as the developing solvent. The radiochemical purity was greater than 95%, indicating an antibody-drug conjugate. 177 The structural formula for Lu-ABDB6-2 is:

Example 12

[0091] A) Reaction of reduced Re-ABDB6 with 1,1'-[5-(azidomethyl)-1,3-phenylene]bis(2-iodoethane-1-one) Re-ABDB6 solution was added to 1M 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) (pH=8.0) buffer, followed by a DMSO solution of 1,1'-[5-(azidomethyl)-1,3-phenylene]bis(2-iodoethane-1-one), and the reaction was carried out at room temperature for 60 min. The precursor compound was purified by ultrafiltration (10 kDa cutoff) and the calculated value of the precursor compound was 22561 Da, and the observed value was 22554 Da.

[0092] B) Preparation of alkyne-PEG6-DOTA DOTA was dissolved in anhydrous DMF, and EDC·HCl (1.0 eq), NHS (1.0 eq), and DIPEA (1.5 eq) were added sequentially under an inert gas atmosphere. The reaction was stirred at room temperature to activate the carboxyl group. Subsequently, the activated solution was slowly added dropwise to an anhydrous DMF solution of acetylene-PEG6-NH2 (0.2 eq) under an ice-water bath. After the addition was complete, the ice bath was removed, and the mixture was allowed to rise naturally to room temperature before stirring was continued. After the reaction was completed, the mixture was concentrated under reduced pressure below 40°C. The residue was poured into cold diethyl ether to precipitate the solid. The solid was collected and purified by preparative high-performance liquid chromatography. Finally, it was freeze-dried to obtain acetylene-PEG6-DOTA with a yield of 75%.

[0093] In one or more embodiments, the amount of anhydrous DMF used is 0.05~0.1 M.

[0094] In one or more embodiments, the reaction was stirred at room temperature for 2 to 4 hours to complete the activation of the carboxyl group.

[0095] In one or more embodiments, the reaction was allowed to rise naturally to room temperature and then stirred for 12 to 24 hours.

[0096] C) The thioether intermediate 3 undergoes a bioorthogonal reaction with alkyne-PEG6-DOTA, and its synthetic pathway is as follows: Under nitrogen protection, azide-modified ABDB6 and alkyne-PEG6-DOTA were dissolved in PBS buffer (pH 7.4) that had been degassed by bubbling with nitrogen or argon for 15–20 minutes. Then, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), CuSO4 solution, and sodium ascorbate were added sequentially. After thorough mixing, the reaction system was incubated at 25 °C in the dark for 2–4 hours. After the reaction, EDTA was added to chelate residual Cu(II) ions to terminate the catalytic reaction. The product was purified by ultrafiltration (10 kDa cutoff), with a calculated value of 23266 Da and an observed value of 23260 Da.

[0097] D) Preparation of antibody conjugates 177 Lu-ABDB6-3 Take 30 μL of ABDB6-1 (400 μM) and 1 mL of freshly eluted... 177 Lu (200-250 MBq) was mixed, and the pH was adjusted to 5.0-5.5 with 1 M sodium acetate buffer. The mixture was reacted at 50°C for 30 minutes, and then purified and concentrated sequentially by PD-10 desalting column (pre-equilibration with PBS) and ultrafiltration (10 kDa). The radiochemical purity was determined by rapid thin-layer chromatography (iTLC) using 0.1 M sodium citrate as the developing solvent. The radiochemical purity was greater than 95%.

[0098] Antibody conjugates 177 The structural formula for Lu-ABDB6-3 is:

Example 13

[0099] A) Preparation of DBCO-PEG5-DOTA Raw material III (1.0 eq) and DOTA-PEG5-NH2 (1.5 eq) were dissolved in an acetonitrile / dimethyl sulfoxide (0.05 M, 1:1, v / v) mixed solvent, and triethylamine (approximately 4.0 eq) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, water was added to quench the reaction, and the mixture was purified by preparative high performance liquid chromatography to obtain a white solid with a yield of 73%.

[0100] B) The thioether intermediate 3 undergoes a bioorthogonal reaction with DBCO-PEG5-DOTA, and its synthetic pathway is as follows: Azide-labeled ABDB6 was dissolved in degassed PBS buffer (pH 7.4), and then DBCO-PEG5-DOTA was added. The reaction was carried out at room temperature in the dark. After the reaction, the product was purified by ultrafiltration (10 kDa cutoff), with a calculated value of 23514 Da and an observed value of 23506 Da.

[0101] C) Preparation of antibody conjugates 177 Lu-ABDB6-4 Take 30 μL of ABDB6-2 (400 μM) and 1 mL of freshly eluted... 177 Lu (200-250 MBq) was mixed, and the pH was adjusted to 5.0-5.5 with 1 M sodium acetate buffer. The mixture was reacted at 50°C for 30 minutes, and then purified and concentrated sequentially by PD-10 desalting column (pre-equilibration with PBS) and ultrafiltration (10 kDa). The radiochemical purity was determined by rapid thin-layer chromatography (iTLC) using 0.1 M sodium citrate as the developing solvent. The radiochemical purity was greater than 95%.

[0102] Antibody conjugates 177 The structural formula for Lu-ABDB6-4 is:

Example 14

[0103] A) Preparation of linkers Methyl bromoformate (1.2 eq) was slowly added dropwise to a THF solution of 4-(piperazin-1-ylmethyl)piperidin-1-carboxylate (1.0 eq) and triethylamine (1.5 eq) under ice bath conditions, and the reaction was slowly brought to room temperature. After the reaction was complete, water was added, and the mixture was extracted with ethyl acetate. The organic phase was concentrated and purified by silica gel column chromatography. The purified product was dissolved in a 50% aqueous solution of tetrahydrofuran, and 1M NaOH aqueous solution (5 eq) was added. The mixture was reacted at room temperature for 1 h, extracted with DCM, and dried to obtain intermediate-1.

[0104] Intermediate-1 (1.0 eq) was dissolved in anhydrous DMF. Under inert gas protection, EDC·HCl (1.5 eq), HOBt (1.5 eq), DOTA-PEG5-NH2 (1.2 eq), and N,N-diisopropylethylamine (DIPEA, 2.0 eq) were added sequentially. The mixture was allowed to rise naturally to room temperature, and the reaction was continued with stirring. After the reaction was complete, the mixture was concentrated under reduced pressure below 40°C. The residue was poured into cold diethyl ether to precipitate the solid, which was then collected and purified by preparative high-performance liquid chromatography (HPLC). Finally, it was freeze-dried to obtain the intermediate. The intermediate was dissolved in a 30% TFA solution in DCM to remove the Boc protecting group, and then dried to obtain intermediate-2, with a yield of 56%.

[0105] Starting material I (1.0 eq) and intermediate-2 (1.5 eq) were dissolved in 2 mL of acetonitrile / dimethyl sulfoxide (0.05 M, 1:1, v / v) mixed solvent, and triethylamine (approximately 4.0 eq) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, water was added to quench the reaction, and the mixture was purified by preparative high performance liquid chromatography to obtain linker A with BCN attached to one end and DOTA attached to the other end, with a yield of 67%.

[0106] B) The thioether bond intermediate 1 undergoes a bioorthogonal reaction with linker A. Linker A was dissolved in PBS buffer containing 10% DMSO and added to 400 μM Tz-ABDB6-1 solution at a molar ratio of 1.1:1. The reaction was carried out at room temperature for 30 minutes, followed by purification by ultrafiltration (10 kDa cutoff) to obtain compound DOTA-ABDB6-3. The calculated value of the product was 23519 Da, and the observed value was 23510 Da.

[0107] C) Preparation of antibody conjugates 177 Lu-ABDB6-5 Take 30 μL of DOTA-ABDB6-3 (400 μM) and 1 mL of freshly eluted... 177 Lu (200-250 MBq) was mixed, and the pH was adjusted to 5.0-5.5 with 1M sodium acetate buffer. The mixture was reacted at 50°C for 30 minutes, followed by purification and concentration via PD-10 desalting column (pre-equilibration with PBS) and ultrafiltration (10 kDa). Radiochemical purity was determined by rapid thin-layer chromatography (iTLC) using 0.1 M sodium citrate as the developing solvent. The radiochemical purity was greater than 95%. Antibody conjugate. 177 The structural formula for Lu-ABDB6-5 is: In some embodiments, the linker may also be other aryl or heteroaryl groups, such as phenyl, pyridyl, pyrrolithyl, thiophenyl, etc. In one or more embodiments, the linker may also be C1~C1. 12 Alkylene or divalent glucose.

[0108]

Example 15

[0109] Specifically, micro-SPECT / CT imaging was performed in SKOV3 tumor-bearing mice. The mice were injected via the tail vein. 177Lu-ABDB6 (100 μL, approximately 7.4 MBq) was administered to all animals in a prone position, and imaging was performed from 1 h to 240 h post-injection. During imaging, mice were anesthetized with isoflurane and maintained in anesthesia. Image acquisition, reconstruction, and analysis were all performed using the instrument's dedicated image acquisition and analysis software package.

[0110] The test results are as follows Figure 10 As shown, antibody conjugates 177 Lu-ABDB6-1 can accumulate in tumors, exhibiting good tumor targeting.

[0111]

Example 16

[0112] Specifically, evaluation in the SKOV3 xenograft tumor model. 177 The antitumor activity of Lu-ABDB6-1. When the tumor volume reaches approximately 100-200 mm... 3 Mice were randomly divided into four groups (n=6 per group). The treatment group received a single intravenous injection via the tail vein. 177 Lu-ABDB6-1 was administered at a dose of ~18.5 MBq or ~37 MBq, or a repeated dosing regimen of ~18.5 MBq every two weeks (days 0 and 14); the control group received intravenous saline. Tumor volume was measured every 2–3 days and calculated using the formula: V = 0.5 × (small axis). 2 × (major axis).

[0113] The test results are as follows Figure 11 As shown, during the injection of antibody-drug conjugates 177 Following Lu-ABDB6-1, tumor volume was effectively reduced, demonstrating the excellent anti-tumor effect of the antibody-drug conjugate.

[0114] IV. Preparation of Antibody-Drug Conjugates Containing Drug Molecules In Examples 17 to 19, a bioorthogonal reaction was performed on the thioether bond intermediate 1 with a linker having complementary reactive groups BCN and a drug molecule attached to both ends, respectively, to link the drug molecule to the ABDB6-PT fusion protein.

[0115]

Example 17

[0116] Intermediate 1 was dissolved in anhydrous DMF, and under ice bath and inert gas protection, EDC·HCl (1.2 eq), HOBt (1.2 eq), aminoethyl-disulfide-ethanol (1.1 eq), and N,N-diisopropylethylamine (DIPEA, 1.5 eq) were added sequentially. The reaction was carried out under ice bath conditions. After the reaction was confirmed to be complete by TLC, an aqueous solution was added, followed by DCM extraction, concentration, and column chromatography to obtain intermediate 2 in 53% yield. The structural formula of intermediate 2 is as follows: Bis(4-nitrophenyl) carbonate (1.2 eq) and DIEA (3 eq) were dissolved in dichloromethane (DCM). Then, a dichloromethane solution of intermediate-2 (1 eq) was added dropwise to the solution. The mixture was stirred at room temperature for 6 hours under argon protection. The reaction was monitored by TLC, and the reaction was stopped after 6 hours. The reaction solution was diluted with dichloromethane, washed successively with water and saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and filtered to obtain intermediate-3. Intermediate 3 (1.0 eq), camptothecin (1.5 eq), DMAP (1.5 eq), and DIEA (2.0 eq) were dissolved in dichloromethane and stirred overnight at 0 °C. The reaction mixture was washed successively with water and saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and then purified by HPLC to give the linker BCN-PEG5-camptothecin in 61% yield.

[0117] B) The thioether intermediate 1 undergoes a bioorthogonal reaction with BCN-PEG5-camptothecin to prepare ABDB6-camptothecin. BCN-PEG5-camptothecin was dissolved in PBS buffer containing 10% DMSO and added to a 400 μM solution of thioether intermediate 1 at a molar ratio of 1.1:1. The reaction was carried out at room temperature for 30 minutes, and the compound ABDB6-camptothecin was obtained by ultrafiltration (10 kDa cutoff). The calculated value of the product is 23671 Da, and the observed value is 23658 Da.

[0118]

Example 18

[0119] B) The thioether intermediate 1 undergoes a bioorthogonal reaction with BCN-PEG5-doxorubicin to prepare ABDB6-doxorubicin. BCN-PEG5-doxorubicin was dissolved in PBS buffer containing 10% DMSO and added to a 400 μM solution of thioether intermediate 1 at a molar ratio of 1.1:1. The reaction was carried out at room temperature for 30 minutes, and the compound ABDB6-doxorubicin was obtained by ultrafiltration (10 kDa cutoff). The calculated value of the product is 23822 Da, and the observed value is 23812 Da.

[0120]

Example 19

[0121] B) The thioether intermediate 1 undergoes a bioorthogonal reaction with BCN-PEG5-MMAE to prepare ABDB6-MMAE. BCN-PEG5-MMAE was dissolved in PBS buffer containing 10% DMSO and added to a 400 μM solution of thioether intermediate 1 at a molar ratio of 1.1:1. The reaction was carried out at room temperature for 30 minutes, followed by purification by ultrafiltration (10 kDa cutoff) to obtain compound ABDB6-MMAE. The calculated value of the product is 23996 Da, and the observed value is 23985 Da.

[0122] In this embodiment, the mechanism of drug molecule release in tumor cells is as follows: after the drug molecule targets the tumor cells with ABDB6, GSH (glutathione) in the tumor cells acts as a reducing agent to cleave the SS bond in the linker, resulting in a thiol group. The thiol group further attacks the carbonate bond or carbamate bond in the molecule, forming a stable five-membered ring and releasing the drug molecule at the same time.

[0123] Those skilled in the art should understand that although only three drug molecules—camptothecin, doxorubicin, and monomethylolpropionate E—are listed in the embodiments, other drug molecules that can be linked to the linker can also be released after targeting tumor cells based on the same release mechanism, and these drug molecules should also be considered to fall within the scope of protection claimed in this application.

[0124] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody-drug conjugate based on bioorthogonality, characterized in that, Its structural formula is PBLY, where P is the ABDB6 fusion protein with a coupled short peptide, B is the cycloaddition product group, L is the linker, and Y is the functional group. The coupled short peptide is linked to the cycloaddition product group through a thioether bond. The cycloaddition product group is formed by a bioorthogonal reaction between a bioorthogonal group attached to the coupled short peptide and a complementary reactive group attached to the linker.

2. The antibody conjugate based on bioorthogonality according to claim 1, characterized in that, The bioorthogonal group is a tetrazine group, and the complementary reactive group of the tetrazine group is a dienophile or an isonitrile group; or The bioorthogonal group is an azide group, and the complementary reactive group of the azide group is a terminal alkyne compound or a cyclic alkyne compound.

3. The antibody conjugate based on bioorthogonality according to claim 1, characterized in that, The ABDB6 fusion protein includes one or two cysteine ​​residues, which, after reduction, are linked to the cycloaddition product group via a thioether bond.

4. The antibody conjugate based on bioorthogonality according to claim 3, characterized in that, The amino acid sequence of the ABDB6 fusion protein with coupled short peptides is shown in SEQ ID NO.1 or SEQ ID NO.

3.

5. The antibody conjugate based on bioorthogonality according to claim 1, characterized in that, The linker is selected from one or more polyethylene glycol units, C1~C1. 12 At least one of alkylene, divalent glucose, C4-C6 aryl or heteroaryl, wherein the heteroatom of the heteroaryl is selected from N, O, or S.

6. The antibody conjugate based on bioorthogonality according to any one of claims 1 to 5, characterized in that, The functional group is a fluorescent group, a chelating group containing a radioactive nuclide, or a drug molecule; Wherein, the fluorescent group is selected from cyanine fluorescent dyes or sulfonated cyanine fluorescent dyes; the chelating group is selected from any one of DOTA, NOTA, NODAGA, PCTA, Macropa, PYTA, TETA, DOTMA, DTPA, HBED-CC, Bispidine, HOPO, DFO, and TRAP; and the radionuclide is... 68 Ga、 89 Zr、 64 Cu、 124 I, 177 Lu、 90 Y, or 225 Ac; The drug molecule is selected from quinoline alkaloids, anthracycline antibiotics, or peptide drugs.

7. A method for preparing antibody-conjugates based on bioorthogonality, characterized in that, Includes the following steps: A reducing agent is added to an ABDB6 fusion protein containing a coupled short peptide to obtain a reduced ABDB6 fusion protein, wherein the coupled short peptide comprises one or two cysteine ​​residues. The reduced ABDB6 fusion protein reacts with a bioorthogonal precursor to obtain a thioether intermediate, the thioether intermediate having a bioorthogonal group, the bioorthogonal group being linked to the coupled short peptide via a thioether bond; The bioorthogonal group of the thioether bond intermediate undergoes a bioorthogonal reaction with the complementary reactive group at the first end of the linker to form a cycloaddition product group, and a functional group is attached to the second end of the linker.

8. The method for preparing antibody conjugates based on bioorthogonality according to claim 7, characterized in that, The bioorthogonal precursor is 3,6-divinyl-1,2,4,5-tetraazine or a monovinyltetraazine with a vinyl group attached at the 3 or 6 position; the bioorthogonal group of the thioether intermediate is a tetraazine group; and the complementary reactive group of the tetraazine group is a dienophile or an isonitrile group; or The bioorthogonal precursor is 1,1'-[5-(azidomethyl)-1,3-phenylene]bis(2-iodoethane-1-one), the bioorthogonal group of the thioether intermediate is an azide group, and the complementary reactive group of the azide group is a terminal alkyne compound or a cyclic alkyne compound.

9. The method for preparing antibody conjugates based on bioorthogonality according to claim 7 or 8, characterized in that, The amino acid sequence of the ABDB6 fusion protein with coupled short peptides is shown in SEQ ID NO.1 or SEQ ID NO.

3.

10. The use of the bioorthogonal antibody conjugate as described in any one of claims 1 to 6 in the preparation of reagents for diagnosing tumors or medicaments for treating tumors.

Images