Preparation method and application of high-homogeneity DAR1 oligonucleotide antibody conjugate

Abstract

The invention provides a preparation method and application of a high-homogeneity DAR1 oligonucleotide antibody conjugate, and the method comprises the following steps: incubating a reducing agent and transition metal ions with an antibody to be coupled in a buffer solution system, and selectively reducing an inter-chain disulfide bond in the antibody; adding an oxidizing agent to selectively oxidize the reduced sulfydryl; after the oxidation reaction, diPPBS is adopted for quenching oxidation; adding a metal chelating agent and a sulfydryl reactive load, and carrying out coupling reaction to generate an antibody linker conjugate with a DAR value of 1; and carrying out addition reaction on the antibody connexon conjugate and oligonucleotide containing an azide group to generate the oligonucleotide antibody conjugate with a DAR value of 1. According to the method, the average DAR value of the oligonucleotide antibody conjugate based on cysteine coupling between antibody chains is expanded to 1, and a product with good uniformity is obtained. The requirement of the current coupling field on the AOC with the homogeneous conjugate antibody ratio of 1 is further met, the applicability is wide, and the application potential is great.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to a preparation method and application of a DAR1 oligonucleotide-antibody conjugate with high homogeneity. Background Art

[0002] Antibody-drug conjugates refer to a group of therapeutic drugs that are connected to target-specific antibodies, directing the drug to the target and releasing it to produce a therapeutic effect. Among them, antibody-oligonucleotide drug conjugates (AOCs) play an important role in the fields of bioanalysis and targeted gene therapy as new synthetic bioconjugates, and the development of new AOCs as therapeutic drugs is of great significance. However, the traditional conjugation process randomly couples the drug moiety to different sites of the antibody, resulting in heterogeneity of the conjugated product. Depending on the reaction conditions, heterogeneous conjugates typically contain 0 to about 8 or more antibody distributions connected to the drug moiety. Each conjugate product in this mixture may have different pharmacokinetic, distribution, toxicity and effectiveness characteristics.

[0003] Homogeneous oligonucleotide antibody conjugates provide more accurate results in diagnostic, immunoassay, quantitative and imaging applications. Antibody drug conjugates with improved homogeneity benefit in treatment, such as higher therapeutic index, improved efficacy and reduced drug toxicity. Therefore, a new process for preparing highly homogeneous AOCs with simple process and controllable cost is very necessary and needs to be pursued in the long term.

[0004] At present, there are no AOC products approved for marketing worldwide, but at least more than 20 companies have more than 30 preclinical pipelines. Avidity Biosciences is a pioneer in the field of AOC drug research and development. Its AOC 1001 is the first AOC to enter clinical trials. It targets transferrin receptor I (TfFI) with siRNA payload for the treatment of myotonic dystrophy type I. In the current research and development of AOC drugs, its coupling technology is mainly divided into random coupling and fixed-site coupling. Random coupling is further divided into cysteine ​​and lysine coupling, which is prone to produce heterogeneous AOCs; fixed-site coupling mainly involves engineering antibody modification, such as adding non-natural amino acids to antibodies, changing antibody polysaccharide side chains, or adding short peptides to protein sequences, which may affect the performance of certain natural amino acids and have high production costs. Therefore, it is of great application value to provide a new process for preparing highly uniform AOCs with simple process and controllable cost. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for preparing a DAR1 oligonucleotide antibody conjugate with high uniformity and its application. The present invention provides a simple and efficient novel bioconjugation process for preparing a DAR1 oligonucleotide antibody conjugate (AOC) with improved uniformity without the need for protein engineering, linker payload modification and / or enzyme catalysis. Compared with the traditional coupling process involving a dithiol reaction, the uniformity of the DAR1 oligonucleotide antibody conjugate (AOC) product produced by the bioconjugation process of the present invention can be significantly improved. Specifically, in the AOC prepared by the process of the present invention, the content of D1 is usually more than 60wt%, preferably more than 75wt%, while in the ADC prepared by the conventional dithiol coupling process, the content of D1 is usually less than 50wt%.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a DAR1 oligonucleotide-antibody conjugate with high homogeneity, the method comprising:

[0008] (S1) incubating the antibody to be coupled with a reducing agent and a transition metal ion in a buffer system to selectively reduce the interchain disulfide bonds in the antibody;

[0009] (S2) adding an oxidant to selectively oxidize the reduced thiol groups; after the oxidation reaction, using diPPBS to quench the oxidation;

[0010] (S3) adding a metal chelator and a thiol-reactive load to generate an antibody-linker conjugate with a DAR value of 1 through a coupling reaction;

[0011] (S4) The antibody-linker conjugate and the oligonucleotide containing an azide group undergo an addition reaction to generate an oligonucleotide-antibody conjugate with a DAR value of 1.

[0012] The bioconjugation process of the present invention is used to prepare DAR1 with better uniformity (oligonucleotide-antibody ratio is 1). In the preparation process, by controlling disulfide bond reduction and sulfhydryl oxidation, no protein engineering, enzyme catalysis or payload modification is required, so that the sulfhydryl group is selectively coupled to the linker to be reacted, one molecule of the linker is coupled to a pair of open disulfide bonds of the antibody, and then the oligonucleotide is coupled to the antibody linker. This process can produce DAR1 AOC with improved uniformity, and has the advantages of simple operation and low cost. Through this method, most natural human IgG1 antibodies can be coupled to DAR1ADC with various linkers (with disulfhydryl reactivity), and then the oligonucleotide and antibody linker are coupled to form DAR1 AOC.

[0013] In the present invention, the thiol-reactive load contains a reactive group coupled to the antibody and a reactive group connected to the target load.

[0014] In the present invention, the target load is, for example, a nucleic acid drug antisense nucleic acid, a phosphorothioate morpholino oligomer, a small interfering RNA, a microRNA, a nucleic acid aptamer or a CpG oligodeoxynucleotide, etc. The target load contains a reactive group connected to a thiol-reactive load.

[0015] The present invention extends the average DAR value of oligonucleotide antibody conjugates based on antibody interchain cysteine ​​coupling to 1, and obtains products with good uniformity. It further meets the current demand in the coupling field for AOCs with a uniform conjugate antibody ratio of 1, and has wide applicability and great application potential.

[0016] Preferably, in (S1), the buffer system is selected from any one of Hepes buffer, histidine buffer, PBS buffer or MES buffer.

[0017] Preferably, in (S1), the reducing agent is selected from: tri(2-carboxyethyl)phosphine (TCEP), THPP (tri(3-hydroxypropyl)phosphine), diPPB (2-diphenylphosphinobenzenesulfonic acid), DTT (dithiothreitol), DPAA (2-(diphenylphosphino)acetic acid), DTE (dithioerythritol), β-mercaptoethanol, LiAlH4, Na2S2O3, KBH4 or hydrazine, any one or a combination of at least two; preferably tri(2-carboxyethyl)phosphine (TCEP), THPP (tri(3-hydroxypropyl)phosphine), diPPB (2-diphenylphosphinobenzenesulfonic acid) or DPAA (2-(diphenylphosphino)acetic acid).

[0018] Preferably, in (S1), the transition metal ion is Zn 2+ .

[0019] In the present invention, transition metal ions selectively affect the reduction and oxidation of disulfide bonds. After adding an effective amount of a metal chelator (such as EDTA) and an excess of a linker that can react with a dithiol group (such as a bismaleimide-linked drug, etc.), a highly homogeneous DAR1 antibody-linker conjugate is obtained, which is then coupled with an excess of oligonucleotides to obtain a highly homogeneous DAR1AOC product.

[0020] Preferably, in (S1), the antibody is selected from any one of Trastuzumab, Cetuximab or Rituximab.

[0021] Preferably, in (S1), the molar ratio of transition metal ions to antibodies in the incubation system is (0.5-50):1, for example, it can be 0.5:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1, etc.; preferably, it is (1-16):1, for example, it can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 or 16:1, etc.; further preferably, it is (2-8):1.

[0022] Preferably, in (S1), the molar ratio of the reducing agent to the antibody in the incubation system is (1-20):1, for example, it can be 1:1, 5:1, 10:1, 15:1 or 20:1, etc.; preferably, it is (2-16):1, for example, it can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 or 16:1, etc.; further preferably, it is (3-8):1.

[0023] In the present invention, the amount of the reducing agent transition metal ion used is more excessive than that of the antibody.

[0024] In a preferred embodiment, the transition metal ion is added at a 4-fold molar ratio relative to the antibody, ie, the molar ratio of transition metal ion / antibody is 4.

[0025] In a preferred embodiment, the reducing agent is added at an 8-fold molar ratio relative to the antibody, ie, the molar ratio of reducing agent / antibody is 8.

[0026] Preferably, in (S1), the incubation condition is: standing at 0-37°C for 0.5-72h.

[0027] Among them, 0-37℃ can be, for example, 0℃, 4℃, 8℃, 12℃, 16℃, 20℃, 24℃, 28℃, 32℃ or 37℃, etc.; 0.5-72h can be, for example, 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, 36h or 72h, etc.

[0028] Preferably, in (S2), the oxidant is selected from: hydrogen peroxide (H 2 O 2 ), nitrate compounds, potassium chloride (KClO 3 ), disulfuric acid peroxide (H 2 S 2 O 8 ), peroxymonosulfuric acid (H 2 SO 5 ), NaClO, sodium dichromate (Na2 Cr 2 O 7 ), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO 3 ), cerium sulfate, dehydroascorbic acid (DHAA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP) or 2-aminophenyl disulfide (DDD) or a combination of any one or at least two thereof.

[0029] Preferably, in (S2), the molar ratio of the oxidant to the antibody is (0.5-160):1, for example, it can be 0.5:1, 2:1, 3:1, 5:1, 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 120:1, 140:1 or 160:1, etc.

[0030] Preferably, in (S2), the oxidation conditions are: standing at 0-37°C (for example, 0°C, 4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C or 37°C, etc.) for 0.5-170h (for example, 0.5h, 1h, 2h, 4h, 24h, 47h or 170h).

[0031] Preferably, in (S2), the molar ratio of diPPBS to the oxidant is (0.3-4):1, for example, it can be 0.3:1, 0.5:1, 1:1, 2:1, 3:1, 3.5:1 or 4:1, etc.

[0032] Preferably, in (S2), the conditions for quenching oxidation are: 10-37°C (for example, 10°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C or 37°C, etc.) for 0.5-2h (for example, 0.5h, 1h, 1.5h or 2h, etc.).

[0033] Preferably, in (S3), the metal chelator is selected from any one of EDTA, DPTA or DOTA, or a combination of at least two of them.

[0034] Preferably, in (S2), the thiol-reactive load is a reactive group containing a sulfhydryl bridge group.

[0035] Preferably, the sulfide bridge group is selected from: any one of a dibromomaleimide linker, a 4,5-dibromo-1,2-dihydropyridazine-3,6-dione linker or a bismaleimide linker, or a combination of at least two thereof.

[0036] Preferably, the 4,5-dibromo-1,2-dihydropyridazine-3,6-dione type linker is DBDP-DBCO.

[0037] In the present invention, the above-mentioned thiol-reactive load can be used to obtain a highly homogeneous DAR1 antibody-linker conjugate, which is then coupled with an excess of oligonucleotide to obtain a DAR1 AOC product.

[0038] Preferably, in (S3), the molar ratio of the metal chelator to the antibody is (2-40):1, for example, it can be 2:1, 4:1, 8:1, 16:1, 32:1 or 40:1, etc., preferably (30-40):1.

[0039] Preferably, in (S3), the molar ratio of the thiol-reactive payload to the antibody is (1-20):1, for example, 1:1, 5:1, 10:1, 15:1 or 20:1, etc.

[0040] Preferably, in (S3), the conditions of the coupling reaction are: standing at 10-37°C (for example, it can be 10°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C or 37°C, etc.) for 0.5-24h (for example, it can be 0.5h, 1h, 2h, 4h, 8h, 12h or 24h, etc.).

[0041] Preferably, in (S3), the coupling reaction further comprises a purification step.

[0042] Preferably, the purification method comprises: any one of filtration, ion exchange, ultrafiltration or column chromatography, or a combination of at least two thereof.

[0043] Preferably, in (S4), the oligonucleotide is selected from small interfering RNA and / or antisense oligonucleotide.

[0044] Preferably, in (S4), an azide group is attached to the oligonucleotide.

[0045] Preferably, in (S4), the molar ratio of the oligonucleotide to the antibody linker conjugate is (1-20):1, for example, it can be 1:1, 5:1, 10:1, 15:1 or 20:1, etc.

[0046] Preferably, in (S4), the conditions of the addition reaction are: standing at 10-37°C (for example, it can be 10°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C or 37°C, etc.) for 0.5-24h (for example, it can be 0.5h, 1h, 2h, 4h, 8h, 12h or 24h, etc.).

[0047] In a second aspect, the present invention provides a DAR1 antibody-nucleic acid conjugate, wherein the antibody-nucleic acid conjugate is prepared by the method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate according to the first aspect.

[0048] In a third aspect, the present invention provides the use of the method for preparing the highly homogeneous DAR1 oligonucleotide-antibody conjugate described in the first aspect in the preparation of AOC drugs.

[0049] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) The present invention adopts metal-assisted coupling technology to control disulfide bond reduction and sulfhydryl oxidation through chemical reactions, without the need for protein engineering, enzyme engineering introduction, and payload modification. After reacting with a disulfhydryl-reactive payload, DAR1 AOC with improved uniformity can be produced, and the operation is simple and the cost is low.

[0052] (2) In the present invention, transition metal ions are selective in disulfide bond reduction and oxidation. After adding an effective amount of a metal chelator (such as EDTA) and an excess of a linker that can react with a dithiol group (such as a bismaleimide-linked drug, etc.), a highly homogeneous DAR1 antibody-linker conjugate is obtained, which is then coupled with an excess of oligonucleotides to obtain a DAR1 AOC product. The high content of D1 (the portion of the AOC with DAR1 of 1) in the obtained AOC improves the homogeneity of the AOC.

[0053] (3) The antibody nucleic acid conjugates produced by the process of the present invention have a higher uniformity than those produced by conventional conjugation processes. Specifically, the content of D1 in the AOC prepared by the process of the present invention is usually more than 60wt%, preferably more than 75wt%, while the content of D1 in the AOC prepared by the conventional bisthiol conjugation process is usually less than 50wt%. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Trastuzumab-DBDP-DBCO-N prepared by the method of the present invention 3 -IEX profile of RNA conjugates.

[0055] Figure 2 Trastuzumab-DBDP-DBCO-N prepared by the method of the present invention 3 -RNA conjugates.

[0056] Figure 3 Trastuzumab-DBDP-DBCO-N prepared by different methods of the present invention 3 -IEX profile of RNA conjugates.

[0057] Figure 4 Trastuzumab-DBDP-DBCO-N prepared by different methods of the present invention 3 -RNA conjugates.

[0058] Figure 5 Cetuximab-DBDP-DBCO-N prepared by the method of the present invention 3 -IEX profile of RNA conjugates.

[0059] Figure 6 Cetuximab-DBDP-DBCO-N prepared by the method of the present invention 3 -RNA conjugates.

[0060] Figure 7 Cetuximab-DBDP-DBCO-N prepared by different methods of the present invention 3 -IEX profile of RNA conjugates.

[0061] Figure 8 Cetuximab-DBDP-DBCO-N prepared by different methods of the present invention 3 -IEX profile of RNA conjugates.

[0062] Fig. 9 Rituximab-DBDP-DBCO-N prepared by the method of the present invention 3 -IEX profile of RNA conjugates.

[0063] Fig.10 Rituximab-DBDP-DBCO-N prepared by the method of the present invention 3 -RNA conjugates.

[0064] Fig.11 Rituximab-DBDP-DBCO-N prepared by different methods of the present invention 3 -RNA conjugates.

[0065] Fig.12 DBDP-PEG 3 -DBCO chemical structure diagram.

[0066] Fig.13 is the siRNA sequence.

[0067] Fig.14 It is a reaction schematic diagram of the preparation process of oligonucleotide antibody conjugates. DETAILED DESCRIPTION

[0068] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0069] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0070] Example 1

[0071] Preparation of Trastuzumab-DBDP-DBCO-N 3 - siRNA conjugates and homogeneity testing of said conjugates

[0072] Trastuzumab-DBDP-DBCO-N 3 The specific preparation steps of -siRNA conjugates are as follows:

[0073] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl 2 A solution of Trastuzumab (prepared by WuXi Biologics according to the published corresponding protein sequence by standard methods for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) (STBK6550, Sigma-Aldrich) (0.15 mM) was added and the reaction mixture was allowed to stand at 4°C overnight.

[0074] (2) DTNB (5,5'-dithiobis(2-nitrobenzoic acid), D8130-500MG, Sigma-Aldrich) (0.25 mM) was introduced and the reaction was continued at 12°C for 0.5 hour. Then, 0.05 mM diPPBS (2-diphenylphosphonobenzenesulfonic acid, prepared by WuXi Biologics) was added to quench the oxidation reaction and the reaction was continued at 12°C for 0.5 hour.

[0075] (3) Add EDTA (5 mM) to capture Zn 2+ , while introducing DBDP-PEG in DMA (dimethylacetamide, commercially available from AldrichSigma) 3 -DBCO (prepared from XDC, 0.15 mM), and the reaction was carried out at 22°C overnight.

[0076] (4) The product buffer was replaced with phosphate buffer (50 mM phosphate buffer, pH 7.4) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0077] (5) N 3 -siRNA solution (prepared by WuXi Biologics, 0.06 mM in phosphate buffer, pH 7, 40 mM) was added to Trastuzumab-DBDP-DBCO solution (0.03 mM) and the reaction mixture was allowed to stand at 22°C overnight.

[0078] The protein sequence of trastuzumab is available in Trastuzumab: Uses, Interactions, Mechanism of Action | DrugBank Online.

[0079] The unpurified AOC was characterized and the results are shown in Table 1.

[0080] Table 1

[0081]

[0082] As can be seen from Table 1, compared with the known solution, DAR1 is less than 50%. The present invention can increase the DAR1 ratio on this antibody, which can be demonstrated by different detection methods.

[0083] Example 2

[0084] Preparation of Trastuzumab-DBDP-DBCO-N by Different Processes 3 - siRNA conjugates and homogeneity testing of said conjugates

[0085] Trastuzumab-DBDP-DBCO-N 3 The specific preparation steps of -siRNA conjugates are as follows:

[0086] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl 2 A solution of Trastuzumab (prepared by WuXi Biologics according to the published corresponding protein sequence by standard methods for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) (STBK6550, Sigma-Aldrich) (0.15 mM) was added and the reaction mixture was allowed to stand at 4°C overnight.

[0087] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.25 mM) was introduced and the reaction was continued at 12° C. for 0.5 hour. Then 0.05 mM diPPBS (manufactured by WuXi Biologics) was added to quench the oxidation reaction, and the reaction was continued at 12° C. for 0.5 hour.

[0088] (3) Add EDTA (5 mM) to capture Zn 2+ , while introducing DBDP-PEG in DMA (dimethylacetamide, commercially available from AldrichSigma) 3 -DBCO (prepared from XDC, 0.15 mM), and the reaction was carried out at 22°C overnight.

[0089] (4) The product buffer was replaced with phosphate buffer (50 mM phosphate buffer, pH 7.4) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0090] (5) N 3 -siRNA solution (prepared by WuXi Biologics, 0.20 mM in phosphate buffer, pH 7, 40 mM) was added to Trastuzumab-DBDP-DBCO solution (0.03 mM) and the reaction mixture was allowed to stand at 22°C overnight.

[0091] The unpurified AOC was characterized and the results are shown in Table 2.

[0092] Table 2

[0093]

[0094] As can be seen from Table 2, compared with the known solution, DAR1 is less than 50%. The present invention can increase the DAR1 ratio on this antibody, which can be demonstrated by different detection methods.

[0095] Example 3

[0096] Preparation of Cetuximab-DBDP-DBCO-N 3 - siRNA conjugates and homogeneity testing of said conjugates

[0097] Cetuximab-DBDP-DBCO-N 3 The specific preparation steps of -siRNA conjugates are as follows:

[0098] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl 2A solution of Cetuximab (prepared by WuXi Biologics according to the published corresponding protein sequence by standard methods for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) (STBK6550, Sigma-Aldrich) (0.15 mM) was added and the reaction mixture was allowed to stand at 4°C overnight.

[0099] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.25 mM) was introduced and the reaction was continued at 12° C. for 0.5 hour. Then 0.05 mM diPPBS (manufactured by WuXi Biologics) was added to quench the oxidation reaction, and the reaction was continued at 12° C. for 0.5 hour.

[0100] (3) Add EDTA (5 mM) to capture Zn 2+ , while introducing DBDP-PEG in DMA (dimethylacetamide, commercially available from AldrichSigma) 3 -DBCO (prepared from XDC, 0.15 mM), and the reaction was carried out at 22°C overnight.

[0101] (4) The product buffer was replaced with phosphate buffer (50 mM phosphate buffer, pH 7.4) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0102] (5) N 3 -siRNA solution (prepared by WuXi Biologics, 0.06 mM in phosphate buffer, pH 7, 40 mM) was added to Cetuximab-DBDP-DBCO solution (0.03 mM) and the reaction mixture was allowed to stand at 22°C overnight.

[0103] The unpurified AOC was characterized and the results are shown in Table 3.

[0104] Table 3

[0105]

[0106] As can be seen from Table 3, compared with the known solution, DAR1 is less than 50%. The present invention can increase the DAR1 ratio on this antibody, which can be demonstrated by different detection methods.

[0107] Example 4

[0108] Preparation of Cetuximab-DBDP-DBCO-N by Different Processes 3 - siRNA conjugates and homogeneity testing of said conjugates

[0109] Cetuximab-DBDP-DBCO-N 3 The specific preparation steps of -siRNA conjugates are as follows:

[0110] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl 2 A solution of Cetuximab (prepared by WuXi Biologics according to the published corresponding protein sequence by standard methods for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) (STBK6550, Sigma-Aldrich) (0.15 mM) was added and the reaction mixture was allowed to stand at 4°C overnight.

[0111] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.25 mM) was introduced and the reaction was continued at 12° C. for 0.5 hour. Then 0.05 mM diPPBS (manufactured by WuXi Biologics) was added to quench the oxidation reaction, and the reaction was continued at 12° C. for 0.5 hour.

[0112] (3) Add EDTA (5 mM) to capture Zn 2+ , while introducing DBDP-PEG in DMA (dimethylacetamide, commercially available from AldrichSigma) 3 -DBCO (prepared from XDC, 0.15 mM), and the reaction was carried out at 22°C overnight.

[0113] (4) The product buffer was replaced with phosphate buffer (50 mM phosphate buffer, pH 7.4) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0114] (5) N 3 -siRNA solution (prepared by WuXi Biologics, 0.20 mM in phosphate buffer, pH 7, 40 mM) was added to Cetuximab-DBDP-DBCO solution (0.03 mM) and the reaction mixture was allowed to stand at 22°C overnight.

[0115] The unpurified AOC was characterized and the results are shown in Table 4.

[0116] Table 4

[0117]

[0118] As can be seen from Table 4, compared with the known solution, DAR1 is less than 50%. The present invention can increase the DAR1 ratio on this antibody, which can be demonstrated by different detection methods.

[0119] Example 5

[0120] Preparation of Rituximab-DBDP-DBCO-N 3 - siRNA conjugates and homogeneity testing of said conjugates

[0121] Rituximab-DBDP-DBCO-N 3 The specific preparation steps of -siRNA conjugates are as follows:

[0122] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl 2 A solution of Rituximab (prepared by WuXi Biologics according to the published corresponding protein sequence by standard methods for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) (STBK6550, Sigma-Aldrich) (0.15 mM) was added and the reaction mixture was allowed to stand at 4°C overnight.

[0123] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.25 mM) was introduced and the reaction was continued at 12° C. for 0.5 hour. Then 0.05 mM diPPBS (manufactured by WuXi Biologics) was added to quench the oxidation reaction, and the reaction was continued at 12° C. for 0.5 hour.

[0124] (3) Add EDTA (5 mM) to capture Zn 2+ , while introducing DBDP-PEG in DMA (dimethylacetamide, commercially available from AldrichSigma) 3 -DBCO (prepared from XDC, 0.15 mM), and the reaction was carried out at 22°C overnight.

[0125] (4) The product buffer was replaced with phosphate buffer (50 mM phosphate buffer, pH 7.4) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0126] (5) N 3-siRNA solution (prepared by WuXi Biologics, 0.06 mM in phosphate buffer, pH 7, 40 mM) was added to Rituximab-DBDP-DBCO solution (0.03 mM) and the reaction mixture was allowed to stand at 22°C overnight.

[0127] The unpurified AOC was characterized and the results are shown in Table 5.

[0128] Table 5

[0129]

[0130]

[0131] As can be seen from Table 5, compared with the known solution, DAR1 is less than 50%. The present invention can increase the DAR1 ratio on this antibody, which can be demonstrated by different detection methods.

[0132] Example 6

[0133] Preparation of Rituximab-DBDP-DBCO-N by different processes 3 - siRNA conjugates and homogeneity testing of said conjugates

[0134] Rituximab-DBDP-DBCO-N 3 The specific preparation steps of -siRNA conjugates are as follows:

[0135] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl 2 A solution of Rituximab (prepared by WuXi Biologics according to the published corresponding protein sequence by standard methods for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) (STBK6550, Sigma-Aldrich) (0.15 mM) was added and the reaction mixture was allowed to stand at 4°C overnight.

[0136] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.25 mM) was introduced and the reaction was continued at 12° C. for 0.5 hour. Then 0.05 mM diPPBS (manufactured by WuXi Biologics) was added to quench the oxidation reaction, and the reaction was continued at 12° C. for 0.5 hour.

[0137] (3) Add EDTA (5 mM) to capture Zn 2+ , while introducing DBDP-PEG in DMA (dimethylacetamide, commercially available from AldrichSigma)3 -DBCO (prepared from XDC, 0.15 mM), and the reaction was carried out at 22°C overnight.

[0138] (4) The product buffer was replaced with phosphate buffer (50 mM phosphate buffer, pH 7.4) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0139] (5) N 3 -siRNA solution (prepared by WuXi Biologics, 0.20 mM in phosphate buffer, pH 7, 40 mM) was added to Rituximab-DBDP-DBCO solution (0.03 mM) and the reaction mixture was allowed to stand at 22°C overnight.

[0140] The unpurified AOC was characterized and the results are shown in Table 6.

[0141] Table 6

[0142]

[0143] As can be seen from Table 6, compared with the known solution, DAR1 is less than 50%, and the present invention can increase the DAR1 ratio on this antibody.

[0144] Fig.12 DBDP-PEG 3 -DBCO chemical structure diagram. Fig.13 It is the siRNA sequence, SEQ ID NO: 1: CCUCGGUAUUUAUUGUCUA. Fig.14 It is a reaction schematic diagram of the preparation process of oligonucleotide antibody conjugates.

[0145] The present invention provides a bioconjugation process for preparing DAR1 (oligonucleotide-antibody ratio is 1) AOC with better uniformity. In the preparation process of the present invention, by controlling disulfide bond reduction and sulfhydryl oxidation, protein engineering, enzyme catalysis or payload modification are not required, so that the sulfhydryl group is selectively coupled with the linker to be reacted, a molecule of the linker is coupled to a pair of open disulfide bonds of the antibody, and then the oligonucleotide is coupled to the antibody linker. The process can produce DAR1 AOC with improved uniformity, and has the advantages of simple operation and low cost. The method has wide applicability and great application potential.

[0146] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate, characterized in that: The method comprises: (S1) incubating the antibody to be coupled with a reducing agent and a transition metal ion in a buffer system to selectively reduce the interchain disulfide bonds in the antibody; (S2) adding an oxidant to selectively oxidize the reduced thiol groups; after the oxidation reaction, using diPPBS to quench the oxidation; (S3) adding a metal chelator and a thiol-reactive load to generate an antibody-linker conjugate with a DAR value of 1 through a coupling reaction; (S4) The antibody-linker conjugate and the oligonucleotide containing an azide group undergo an addition reaction to generate an oligonucleotide-antibody conjugate with a DAR value of 1.

2. The method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate according to claim 1, characterized in that: (S1), the buffer system is selected from any one of Hepes buffer, histidine buffer, PBS buffer or MES buffer; Preferably, in (S1), the reducing agent is selected from: any one or a combination of at least two of tris(2-carboxyethyl)phosphine, THPP, diPPB, DTT, DPAA, DTE, β-mercaptoethanol, LiAlH4, Na2S2O3, KBH4 or hydrazine; preferably tris(2-carboxyethyl)phosphine, THPP, diPPB or DPAA; Preferably, in (S1), the transition metal ion is Zn 2+ ; Preferably, in (S1), the antibody is selected from any one of Trastuzumab, Cetuximab or Rituximab.

3. The method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate according to claim 1 or 2, characterized in that: In (S1), the molar ratio of transition metal ions to antibodies in the incubation system is (0.5-50):1, preferably (1-16):1, and more preferably (2-8):1; Preferably, in (S1), the molar ratio of the reducing agent to the antibody in the incubation system is (1-20):1, preferably (2-16):1, and more preferably (3-8):1; Preferably, in (S1), the incubation condition is: standing at 0-37°C for 0.5-72h.

4. The method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate according to any one of claims 1 to 3, characterized in that: In (S2), the oxidant is selected from any one or a combination of at least two of hydrogen peroxide, nitrate compounds, potassium chloride, peroxydisulfuric acid, peroxymonosulfuric acid, NaClO, sodium dichromate, permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate, cerium sulfate, dehydroascorbic acid, 5,5'-dithiobis(2-nitrobenzoic acid), nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate or 2-aminophenyl disulfide; Preferably, in (S2), the molar ratio of the oxidant to the antibody is (0.5-160):1; Preferably, in (S2), the oxidation conditions are: standing at 0-37° C. for 0.5-170 h; Preferably, in (S2), the molar ratio of diPPBS to the oxidant is (0.3-4):1; Preferably, in (S2), the conditions for quenching oxidation are: 10-37° C. for 0.5-2 h.

5. The method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate according to any one of claims 1 to 4, characterized in that: (S3), the metal chelator is selected from: any one of EDTA, DPTA or DOTA or a combination of at least two thereof; Preferably, in (S3), the thiol-reactive payload is a reactive group containing a sulfhydryl bridge group; Preferably, the sulfide bridge group is selected from: any one or a combination of at least two of a dibromomaleimide linker, a 4,5-dibromo-1,2-dihydropyridazine-3,6-dione linker or a bismaleimide linker; Preferably, the 4,5-dibromo-1,2-dihydropyridazine-3,6-dione type linker is DBDP-DBCO.

6. The method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate according to any one of claims 1 to 5, characterized in that: (S3), the molar ratio of the metal chelator to the antibody is (2-40):1, preferably (30-40):1; Preferably, in (S3), the molar ratio of the thiol-reactive load to the antibody is (1-20):1; Preferably, in (S3), the coupling reaction is carried out at 10-37° C. for 0.5-24 h.

7. The method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate according to any one of claims 1 to 6, characterized in that: (S3), the coupling reaction further comprises a purification step; Preferably, the purification method comprises: any one of filtration, ion exchange, ultrafiltration or column chromatography, or a combination of at least two thereof.

8. The method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate according to any one of claims 1 to 7, characterized in that: (S4), wherein the oligonucleotide is selected from small interfering RNA and / or antisense oligonucleotide; Preferably, in (S4), the oligonucleotide is linked to an azide group; Preferably, in (S4), the molar ratio of the oligonucleotide to the antibody linker conjugate is (1-20):1; Preferably, in (S4), the addition reaction is carried out under the conditions of: standing at 10-37° C. for 0.5-24 h.

9. A DAR1 antibody-nucleic acid conjugate, characterized in that: The antibody nucleic acid conjugate is prepared by the method for preparing a highly homogeneous DAR1 oligonucleotide antibody conjugate according to any one of claims 1-8.

10. Use of the method for preparing a highly homogeneous DAR1 oligonucleotide-antibody conjugate according to any one of claims 1 to 8 in preparing an AOC drug.

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