DAR1 antibody drug conjugate as well as preparation method and application thereof

Abstract

The invention provides a DAR1 antibody drug conjugate as well as a preparation method and application thereof, a drug is connected to a sulfydryl reactive load, and the sulfydryl reactive load is coupled with a Fab of an antibody or a sulfydryl of a hinge structural domain; the sulfydryl reactive load is a reactive group coupled with double sulfydryl; the medicine is methyl auristatin E; the sulfydryl reactive load is a reactive group containing a sulfur bridge group. The product prepared by the preparation process of the DAR1 antibody drug conjugate is high in uniformity, simple to operate and low in cost, and has important application value in production of the antibody drug conjugate.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a DAR1 antibody-drug conjugate and a preparation method and application thereof. Background Art

[0002] Antibody-drug conjugates are a group of therapeutic drugs that are linked to target-specific antibodies, directing the drug to the target and releasing it to produce a therapeutic effect. The development of new ADCs 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 linked to the drug moiety. Each conjugated product in this mixture may have different pharmacokinetic, distribution, toxicity, and effectiveness characteristics.

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

[0004] In recent years, with the breakthrough of coupling technology, the introduction of new linkers and payloads, the new generation of ADCs have shown better specificity and cytotoxicity. Nevertheless, there are still many challenges in the development of ADC drugs, including how to further optimize the antibody-to-load ratio, improve the "utilization rate" of the load, and then reduce the drug safety risks caused by the narrow therapeutic window of the special load while increasing the drug efficacy. This is the top priority in the current ADC drug development process.

[0005] Currently, there are 14 ADC drugs approved worldwide, and more than 100 ADC drugs are in different stages of clinical research. In the current development of ADC drugs, an important ADC parameter is the drug-antibody ratio (DAR), which usually varies between 2 and 8, depending on the cytotoxicity of the payload; the least potent payloads, such as topoisomerase 1 inhibitors (SN-38, DXd) are usually applied with high drug loading (DAR7-8), while ADCs with high-efficiency payloads (microtubule inhibitors, such as MMAE and maytansine) are usually used to produce an average DAR4, while super-potent molecules (such as calicheamicin, PBD dimer, PNU-159682 and amanitin) are mainly coupled with low stoichiometry (average DAR2). Given that most tumor-associated antigens (TAAs) are endogenously expressed on healthy tissues in addition to tumor cells, such low ADC doses may not achieve systemic targeting saturation. Therefore, these lower doses may not be conducive to optimal pharmacokinetic and biodistribution characteristics, thereby reducing efficacy and tolerability. It can therefore be hypothesized that, in the case of ultra-potent payloads, ADCs with DAR < 2 may be highly beneficial, as lower DARs may tolerate higher ADC doses (at the same payload dose), thereby improving tissue penetration.

[0006] Dean, AQ et al reported a disulfide bridging strategy to prepare a homogeneous ADC with a DAR of 1 using a bismaleimide pyrrole benzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab). They compared it with a site-specific ADC with a DAR of 2 prepared using a monomaleimide PBD dimer (SG3249), and the ADC was twice as well tolerated in rats at its dose. In addition to the above-mentioned technologies, there is also a selenocysteine-based coupling technology that has the potential to prepare DAR1 products. In order to obtain DAR1 ADCs more efficiently and without genetic modification, Laureen de Bever et al proposed to remodel the sugar-coupled enzyme glycan and then couple a molecule of payload to a molecule of antibody based on a bimetallic free click reaction. They found that at the same payload dose, DAR1 ADC was observed to have a significant improvement in tumor spheroid penetration compared to DAR2 ADC, emphasizing the potential of ADCs with lower DARs to carry super-strong payloads.

[0007] Although the technologies of the DAR1 ADCs mentioned above still need more thorough research, they all prove that DAR1 products may have advantages in pharmacokinetics and efficacy compared to DAR2 ADCs in the field of highly active toxin-conjugated drugs. Therefore, it is necessary to prepare high-quality DAR1 ADCs through simple processes. The currently known DAR1 platform involves protein engineering, linker payload modification and / or enzyme catalysis, and has disadvantages such as low antibody expression levels, complex purification and high costs. Therefore, it is of great application value to provide a simple and efficient new bioconjugation process for preparing DAR1 antibody-drug conjugates (ADCs) with improved uniformity without the need for protein engineering, linker payload modification and / or enzyme catalysis. Summary of the invention

[0008] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a DAR1 antibody-drug conjugate and a preparation method and application thereof. The present invention prepares a DAR1 antibody-drug conjugate with improved homogeneity, wherein the drug is coupled to the antibody through a dithiol-coupled reactive group; the homogeneity of the DAR1 antibody-drug conjugate (ADC) product produced by the bioconjugation process of the present invention can be significantly improved. In the ADC prepared by the process of the present invention, the content of D1 is usually more than 65wt%, preferably more than 75wt%, which is much better than the conventional process.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a DAR1 antibody-drug conjugate, wherein the drug is connected to a thiol-reactive payload, and the thiol-reactive payload is coupled to a thiol group of a Fab or hinge domain of an antibody; the thiol-reactive payload is a reactive group coupled to a dithiol group;

[0011] The drug is monomethyl auristatin E (MMAE); the thiol-reactive payload is a reactive group containing a sulfhydryl bridge group.

[0012] The advantage of the present invention is that the dithiol reactive linker can obtain a product with a DAR of 1 under the preparation method of the present invention. It is assumed that in the case of ultra-potent payloads, ADCs with DAR<2 may be very beneficial because lower DAR may tolerate higher ADC doses (at the same payload dose), thereby improving tissue penetration.

[0013] Preferably, the antibody is selected from any one of Trastuzumab, Daratumumab or Basiliximab.

[0014] 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.

[0015] Preferably, the 4,5-dibromo-1,2-dihydropyridazine-3,6-dione type linker is STB.

[0016] Preferably, the bismaleimide linker is Bismaleimide.

[0017] Preferably, the dibromomaleimide linker is DBM.

[0018] In a second aspect, the present invention provides a method for preparing the DAR1 antibody-drug conjugate according to the first aspect, the preparation method comprising:

[0019] (S1) incubating the antibody with a reducing agent and a transition metal ion in a buffer system to selectively reduce interchain disulfide bonds in the antibody;

[0020] (S2) selectively oxidizing the reduced thiol groups with an oxidant, and quenching the oxidation with diPPBS after the oxidation reaction;

[0021] (S3) mixing the metal chelator, the thiol-reactive load and the antibody to perform a coupling reaction to generate a conjugate with a DAR value of 1;

[0022] (S4) obtaining a DAR1 antibody-drug conjugate by purifying small molecule impurities in the system.

[0023] The preparation process of the present invention bypasses protein engineering, enzymatic reaction or additional payload modification. Based on the natural interchain disulfide bond, only trace transition metal ions need to be introduced into the reaction to control redox, thereby obtaining a highly uniform DAR1 product. Therefore, compared with the currently known process for preparing DAR1 ADC, the process of the present invention is simple, and the uniformity of the obtained antibody drug conjugate is significantly improved compared with the conventional method.

[0024] Preferably, in (S1), the buffer system is selected from: any one of Hepes buffer, histidine buffer, PBS buffer or MES buffer; the pH value of the buffer system is 5-8, for example, it can be 5, 5.5, 6, 6.5, 7, 7.5 or 8.

[0025] 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).

[0026] Preferably, in (S1), the transition metal ion is selected from Zn 2+ , Mn 2+ 、Ni 2+ , Fe 2+ , Fe 3+ , Ca 2+ Cu 2+ ; preferably Zn 2+ .

[0027] In the present invention, the transition metal ions are added to the buffer system in the form of transition metal salts, and the transition metal salts are selected from: ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, Zn(HCOO)2 or Zn(BF4)2, etc.

[0028] In the present invention, transition metal ions selectively affect disulfide bond reduction and oxidation, and after adding an effective amount of a metal chelator (such as EDTA) and an excess of a load that can react with dithiol groups (such as bismaleimide-linked drugs, etc.), a highly homogeneous DAR1 ADC product is obtained.

[0029] Compared with the conventional coupling process involving thiol reaction, the antibody drug conjugate produced by the process of the present invention has higher uniformity than that produced by the conventional coupling process.

[0030] 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.

[0031] 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.

[0032] In the present invention, the amount of the reducing agent transition metal ion used is more excessive than that of the antibody.

[0033] 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.

[0034] 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.

[0035] Preferably, the antibody incubation conditions are: standing at 0-37°C for 0.5-72h.

[0036] 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.

[0037] Preferably, in (S2), the oxidant is selected from the group consisting of: hydrogen peroxide (H2O2), nitrate compounds, potassium chloride (KClO3), peroxydisulfuric acid (H2S2O8), peroxymonosulfuric acid (H2SO5), NaClO, sodium dichromate (Na2Cr2O7), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO3), 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), any one or a combination of at least two thereof; preferably DHAA or DTNB.

[0038] 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, 5:1, 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 120:1, 140:1 or 160:1, etc.

[0039] 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, etc.).

[0040] 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.

[0041] Preferably, in (S2), the conditions for quenching oxidation are: 10-37°C (for example, it can be 10°C, 12°C, 14°C, 16°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C or 37°C, etc.) for 0.5-2 hours (for example, it can be 0.5, 1, 1.5 or 2, etc.).

[0042] In the present invention, the oxidant used is 5,5-dithiobis(2-nitrobenzoic acid), which has a fast oxidation rate and few side reactions, and can avoid the next step of purification to a certain extent.

[0043] In the present invention, the oxidation time needs to be reasonably controlled. If the oxidation time is too slow, the cost will increase and side reactions will occur, which will affect the subsequent coupling.

[0044] In the present invention, the diPPBS used for quenching oxidation can terminate the oxidation reaction and control the oxidation time to avoid affecting the subsequent experimental steps. In the preparation of existing antibody-drug conjugates, no quencher has been introduced. In the present invention, diPPBS is first used for quenching oxidation, which cooperates with the aforementioned oxidant to increase the reaction efficiency and facilitate the conjugation reaction.

[0045] In the present invention, the optimized quenching oxidation conditions can make the reaction more stable and can also avoid side reactions caused by too long a reaction time.

[0046] 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.

[0047] Preferably, in (S3), the molar ratio of the metal chelator to the antibody is (30-40):1, for example, it can be 30:1, 32:1, 34:1, 36:1, 38:1 or 40:1, etc.

[0048] 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.

[0049] Preferably, in (S3), the conditions of the coupling reaction are: standing at 10-37°C (for example, it can be 10°C, 12°C, 14°C, 16°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C or 37°C, etc.) for 0.5-24h (for example, it can be 0.5h, 1h, 2h, 4h, 8h, 12h or 24h, etc.).

[0050] Preferably, in (S4), the purification method comprises any one of filtration, ion exchange, ultrafiltration or column chromatography, or a combination of at least two thereof.

[0051] In an embodiment of the present invention, the method for preparing a DAR1 antibody-drug conjugate comprises:

[0052] (S1) Incubating the antibody to be coupled with a reducing agent and a transition metal ion in a buffer system, wherein the molar ratio of the transition metal ion to the antibody in the incubation system is (0.5-50):1, and the molar ratio of the reducing agent to the antibody is (1-20):1; selectively reducing the interchain disulfide bonds in the antibody; and the antibody incubation conditions are: standing at 0-37°C for 0.5-72h.

[0053] (S2) selectively oxidizing the reduced thiol group in (S1) with an oxidant, wherein the molar ratio of the oxidant to the antibody is (0.5-160):1; the oxidation conditions are: standing at 0-37°C for 0.5-170h; after the oxidation reaction is completed, the oxidation reaction is quenched with diPPBS; the molar ratio of diPPBS to the oxidant is (0.3-4):1; the quenching oxidation conditions are: 10-37°C for 0.5-2h;

[0054] (S3) mixing a metal chelator, a thiol-reactive payload and the oxidized antibody in (S2) to carry out a coupling reaction, wherein the molar ratio of the metal chelator to the antibody is (30-40):1, and the molar ratio of the thiol-reactive payload to the antibody is (1-20):1; the coupling reaction is carried out under the conditions of standing at 10-37° C. for 0.5-24 h; and the reaction generates a conjugate with a DAR value of 1;

[0055] (S4) purifying small molecule impurities in the system to obtain DAR1 antibody-drug conjugate.

[0056] In the present invention, the antibody drug conjugate is produced using the above process, and the uniformity of the antibody drug conjugate is higher than that produced by the conventional conjugation process. Specifically, in the ADC prepared by the process of the present invention, the content of D1 is usually more than 65wt%, preferably more than 75wt%, while in the ADC prepared by the conventional bisthiol conjugation process, the content of D1 is usually less than 50wt%.

[0057] The process of the present invention meets the need for preparing low DAR value conjugate drugs due to the narrow therapeutic window or special mechanism of action of special loads such as highly active toxin compounds and immune agonists, further broadening the application field of ADC.

[0058] In a third aspect, the present invention provides use of the DAR1 antibody-drug conjugate described in the first aspect in preparing a pharmaceutical preparation for treating tumors.

[0059] 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.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] (1) The present invention provides a metal-assisted coupling technology that controls disulfide bond reduction and sulfhydryl oxidation through chemical reactions. It does not require protein engineering, enzyme engineering, or payload modification. After reacting with a disulfhydryl-reactive payload, DAR1 ADC with improved uniformity can be produced. The technology is simple to operate and low in cost.

[0062] (2) In the present invention, transition metal ions are selective in disulfide bond reduction and oxidation, and after adding an effective amount of a metal chelator (such as EDTA) and an excess of a load that can react with a dithiol group (such as a bismaleimide-linked drug, etc.), a DAR1 ADC is formed. The high content of D1 (the portion of the ADC with DAR1 of 1) in the obtained ADC improves the uniformity of the ADC.

[0063] (3) By using the process of the present invention to produce antibody drug conjugates, the uniformity of the antibody drug conjugates is higher than that produced by conventional conjugation processes. Specifically, in the ADC prepared by the process of the present invention, the content of D1 is usually more than 65wt%, preferably more than 75wt%, while in the ADC prepared by the conventional bisthiol conjugation process, the content of D1 is usually less than 50wt%.

[0064] (4) The process of the present invention also meets the need to prepare conjugates with low DAR values, such as DAR1 products, due to the specificity of the therapeutic window and mechanism of action of super-toxin conjugates, thereby further broadening the application field of ADC. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 It is the HIC of Trastuzumab-STB-VC-PAB-MMAE conjugate prepared by the method of the present invention.

[0066] Figure 2 is the HIC of Trastuzumab-STB-VC-PAB-MMAE conjugate prepared by traditional methods.

[0067] Figure 3 is the HIC of Daratumumab-STB-VC-PAB-MMAE conjugate prepared by the method of the present invention.

[0068] Figure 4 is the HIC of Daratumumab-STB-VC-PAB-MMAE conjugate prepared by conventional methods.

[0069] Figure 5 It is the HIC of Basiliximab-STB-VC-PAB-MMAE conjugate prepared by the method of the present invention.

[0070] Figure 6 It is the HIC of Basiliximab-STB-VC-PAB-MMAE conjugate prepared by traditional method.

[0071] Figure 7 It is the HIC of Trastuzumab-Bismaleimide-VC-PAB-MMAE conjugate prepared by the method of the present invention.

[0072] Figure 8 It is the HIC of Trastuzumab-Bismaleimide-VC-PAB-MMAE conjugate prepared by traditional methods.

[0073] Fig. 9 is the HIC of Trastuzumab-DBM-MMAF conjugate prepared by the method of the present invention.

[0074] Fig.10 is the HIC of Trastuzumab-DBM-MMAF conjugate prepared by conventional methods.

[0075] Fig.11 is the HIC of Trastuzumab-DBM-MMAF conjugate prepared by different methods of the present invention.

[0076] Fig.12This is the chemical structure diagram of STB-VC-PAB-MMAE.

[0077] Fig.13 This is the chemical structure diagram of Bismaleimide-VC-PAB-MMAE.

[0078] Fig.14 This is the chemical structure diagram of DBM-MMAF.

[0079] Fig.15 It is a reaction schematic diagram of the present invention. DETAILED DESCRIPTION

[0080] 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.

[0081] 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.

[0082] Example 1

[0083] This example prepares a Trastuzumab-STB-VC-PAB-MMAE conjugate, and performs homogeneity testing on the conjugate. The specific preparation steps of the Trastuzumab-STB-VC-PAB-MMAE conjugate are as follows:

[0084] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl2 (STBK6550, Sigma-Aldrich) (0.15 mM) were added to a solution of Trastuzumab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.04 mM, in phosphate buffer, pH 7, 40 mM), and the reaction mixture was allowed to stand at 12°C overnight.

[0085] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.26 mM) was introduced and the reaction was continued at 12° C. for 1 hour. Then, 0.08 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.

[0086] (3) Add EDTA (5 mM) to capture Zn 2+, STB-VC-PAB-MMAE (from Syntabio, 0.15 mM) in DMA (dimethylacetamide, commercially available from AldrichSigma) was introduced simultaneously, and the reaction was continued at 12°C for 3 hours.

[0087] (4) Use an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to replace the product buffer with its storage buffer (20 mM histidine buffer, pH 5.5) to remove excess small molecule impurities in the reaction system.

[0088] Comparative Example 1

[0089] This comparative example provides a conventional preparation method of Trastuzumab-STB-VC-PAB-MMAE conjugate as a control group. The specific preparation steps of the conventional preparation method are as follows:

[0090] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.08 mM) was added to a solution of Trastuzumab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.04 mM, in phosphate buffer, pH 7, 40 mM) and the reaction mixture was allowed to react at 37°C for 2 hours.

[0091] (2) STB-VC-PAB-MMAE (from Syntabio, 0.15 mM) in DMA (dimethylacetamide, commercially available from Aldrich Sigma) was added, and the reaction was continued at 12°C for 1 hour.

[0092] (3) The product buffer was replaced with its storage buffer (20 mM histidine buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0093] The purified ADC was characterized and the results are shown in Table 1.

[0094] Table 1

[0095]

[0096] From Table 1, Figure 1 , Figure 2 It can be seen that, compared with the known solutions, the preparation method of the present invention can increase the DAR1 ratio in this antibody.

[0097] Example 2

[0098] This example prepares Daratumumab-STB-VC-PAB-MMAE conjugate, and performs homogeneity detection on the conjugate. The specific preparation steps of Daratumumab-STB-VC-PAB-MMAE conjugate are as follows:

[0099] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl2 (STBK6550, Sigma-Aldrich) (0.15 mM) were added to a solution of Daratumumab (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) and the reaction mixture was allowed to stand at 12°C overnight.

[0100] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.26 mM) was introduced and the reaction was continued at 12° C. for 1 hour. Then, 0.08 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.

[0101] (3) Add EDTA (5 mM) to capture Zn 2+ , STB-VC-PAB-MMAE (from Syntabio, 0.15 mM) in DMA (dimethylacetamide, commercially available from AldrichSigma) was introduced simultaneously, and the reaction was continued at 12°C for 3 hours.

[0102] (4) Use an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to replace the product buffer with its storage buffer (20 mM histidine buffer, pH 5.5) to remove excess small molecule impurities in the reaction system.

[0103] Comparative Example 2

[0104] This comparative example provides a conventional preparation method of Daratumumab-STB-VC-PAB-MMAE conjugate as a control group. The specific preparation steps of the conventional preparation method are as follows:

[0105] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.08 mM) was added to a solution of Daratumumab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.04 mM, in phosphate buffer, pH 7, 40 mM) and the reaction mixture was allowed to react at 37°C for 2 hours.

[0106] (2) STB-VC-PAB-MMAE (from Syntabio, 0.15 mM) in DMA (dimethylacetamide, commercially available from Aldrich Sigma) was added, and the reaction was continued at 12°C for 1 hour.

[0107] (3) The product buffer was replaced with its storage buffer (20 mM histidine buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0108] The purified ADC was characterized and the results are shown in Table 2.

[0109] Table 2

[0110]

[0111] From Table 2, Figure 3 , Figure 4 It can be seen that, compared with the known solutions, the preparation method of the present invention can increase the DAR1 ratio in this antibody.

[0112] Example 3

[0113] This example prepares Basiliximab-STB-VC-PAB-MMAE conjugate, and performs homogeneity detection on the conjugate. The specific preparation steps of Basiliximab-STB-VC-PAB-MMAE conjugate are as follows:

[0114] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl2 (STBK6550, Sigma-Aldrich) (0.15 mM) were added to a solution of Basiliximab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) and the reaction mixture was allowed to stand at 12°C overnight.

[0115] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.26 mM) was introduced and the reaction was continued at 12° C. for 1 hour. Then, 0.08 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.

[0116] (3) Add EDTA (5 mM) to capture Zn 2+, STB-VC-PAB-MMAE (purchased from Syntabio, 0.15 mM) in DMA (dimethylacetamide, commercially available from AldrichSigma) was introduced simultaneously, and the reaction was continued at 12°C for 3 hours.

[0117] (4) Use an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to replace the product buffer with its storage buffer (20 mM histidine buffer, pH 5.5) to remove excess small molecule impurities in the reaction system.

[0118] Comparative Example 3

[0119] This comparative example provides a conventional preparation method of Basiliximab-STB-VC-PAB-MMAE conjugate as a control group. The specific preparation steps of the conventional preparation method are as follows:

[0120] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.08 mM) was added to a solution of Basiliximab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.04 mM, in phosphate buffer, pH 7, 40 mM) and the reaction mixture was allowed to react at 37° C. for 2 hours.

[0121] (2) STB-VC-PAB-MMAE (from Syntabio, 0.15 mM) in DMA (dimethylacetamide, commercially available from Aldrich Sigma) was added, and the reaction was continued at 12°C for 1 hour.

[0122] (3) The product buffer was replaced with its storage buffer (20 mM histidine buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0123] The purified ADC was characterized and the results are shown in Table 3.

[0124] Table 3

[0125]

[0126] From Table 3, Figure 5 , Figure 6 It can be seen that, compared with the known solutions, the preparation method of the present invention can increase the DAR1 ratio in this antibody.

[0127] Example 4

[0128] This example prepares Trastuzumab-BISMALEIMIDE-VC-PAB-MMAE conjugate, and performs homogeneity detection on the conjugate. The specific preparation steps of Trastuzumab-BISMALEIMIDE-VC-PAB-MMAE conjugate are as follows:

[0129] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl2 (STBK6550, Sigma-Aldrich) (0.15 mM) were added to a solution of Trastuzumab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) and the reaction mixture was allowed to stand at 12°C overnight.

[0130] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.26 mM) was introduced and the reaction was continued at 12° C. for 1 hour. Then, 0.08 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.

[0131] (3) Add EDTA (5 mM) to capture Zn 2+ , BISMALEIMIDE-VC-PAB-MMAE (self-synthesized from XDC, 0.088 mM) in DMA (dimethylacetamide, commercially available from AldrichSigma) was introduced simultaneously, and the reaction was continued at 12°C for 3 hours.

[0132] (4) Use an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to replace the product buffer with its storage buffer (20 mM histidine buffer, pH 5.5) to remove excess small molecule impurities in the reaction system.

[0133] Comparative Example 4

[0134] This comparative example provides a conventional preparation method of Trastuzumab-BISMALEIMIDE-VC-PAB-MMAE conjugate as a control group. The specific preparation steps of the conventional preparation method are as follows:

[0135] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.08 mM) was added to a solution of Trastuzumab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.04 mM, in phosphate buffer, pH 7, 40 mM) and the reaction mixture was allowed to react at 37°C for 2 hours.

[0136] (2) BISMALEIMIDE-VC-PAB-MMAE (self-synthesized from XDC, 0.15 mM) in DMA (dimethylacetamide, commercially available from Aldrich Sigma) was added, and the reaction was continued at 12°C for 1 hour.

[0137] (3) The product buffer was replaced with its storage buffer (20 mM histidine buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0138] The ADC submitted for purification was characterized and the results are shown in Table 4.

[0139] Table 4

[0140]

[0141] From Table 4, Figure 7 , Figure 8 It can be seen that, compared with the known solutions, the preparation method of the present invention can increase the DAR1 ratio in this antibody.

[0142] Example 5

[0143] This example prepares a Trastuzumab-DBM-MMAF conjugate, and performs homogeneity testing on the conjugate. The specific preparation steps of the Trastuzumab-DBM-MMAF conjugate are as follows:

[0144] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.3 mM) and ZnCl2 (STBK6550, Sigma-Aldrich) (0.15 mM) were added to a solution of Trastuzumab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) and the reaction mixture was allowed to stand at 12°C overnight.

[0145] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.26 mM) was introduced and the reaction was continued at 12° C. for 1 hour. Then, 0.08 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.

[0146] (3) Add EDTA (5 mM) to capture Zn 2+ , DBM-MMAF (commercially available from XDC-CZ, 0.15 mM) in DMA (dimethylacetamide, commercially available from AldrichSigma) was introduced simultaneously, and the reaction was continued at 4°C for 3 hours.

[0147] (4) Use an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to replace the product buffer with its storage buffer (20 mM histidine buffer, pH 5.5) to remove excess small molecule impurities in the reaction system.

[0148] Comparative Example 5

[0149] This comparative example provides a conventional preparation method of Trastuzumab-DBM-MMAF conjugate as a control group. The specific preparation steps of the conventional preparation method are as follows:

[0150] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.08 mM) was added to a solution of Trastuzumab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.04 mM, in phosphate buffer, pH 7, 40 mM) and the reaction mixture was allowed to react at 37°C for 2 hours.

[0151] (2) DBM-MMAF (self-synthesized from XDC, 0.15 mM) in DMA (dimethylacetamide, commercially available from Aldrich Sigma) was added, and the reaction was continued at 12°C for 1 hour.

[0152] (3) The product buffer was replaced with its storage buffer (20 mM histidine buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0153] The purified ADC was characterized and the results are shown in Table 5.

[0154] Table 5

[0155]

[0156] From Table 5, Fig. 9 , Fig.10 It can be seen that, compared with the known solutions, the preparation method of the present invention can increase the DAR1 ratio in this antibody.

[0157] Example 6

[0158] This example uses different processes to prepare Trastuzumab-DBM-MMAF conjugates, and performs homogeneity testing on the conjugates. The specific preparation steps of Trastuzumab-DBM-MMAF conjugates are as follows:

[0159] (1) TCEP (C4706-2G, Sigma-Aldrich) (0.1 mM) and ZnCl2 (STBK6550, Sigma-Aldrich) (0.15 mM) were added to a solution of Trastuzumab (prepared by WuXi Biologics according to the published corresponding protein sequence by a standard method for preparing monoclonal antibodies, 0.03 mM, in phosphate buffer, pH 7, 40 mM) and the reaction mixture was allowed to stand at 12°C overnight.

[0160] (2) DTNB (D8130-500MG, Sigma-Aldrich) (0.06 mM) was introduced and the reaction was continued at 12° C. for 1 hour. Then, 0.08 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.

[0161] (3) Add EDTA (5 mM) to capture Zn 2+ , DBM-MMAF (from XDC-CZ, 0.15 mM) in DMA (dimethylacetamide, commercially available from AldrichSigma) was introduced simultaneously, and the reaction was continued at 4°C for 3 hours.

[0162] (4) Use an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to replace the product buffer with its storage buffer (20 mM histidine buffer, pH 5.5) to remove excess small molecule impurities in the reaction system.

[0163] The purified ADC was characterized and the results are shown in Table 6.

[0164] Table 6

[0165]

[0166] From Table 6, Fig.11 , Fig.10It can be seen that, compared with the known solutions, the preparation method of the present invention can increase the DAR1 ratio in this antibody.

[0167] In summary, the thiol-reactive loading used in the present invention is conducive to the preparation of DAR1 ADC, and can produce DAR1 ADC with improved uniformity, and the thiol-reactive loading includes: STB-VC-PAB-MMAE ( Fig.12 )、Bismaleimide-VC-PAB-MMAE( Fig.13 )、DBM-MMAF( Fig.14 ); The reaction schematic diagram of the present invention is as follows Fig.15 As shown, the reduction of disulfide bonds and oxidation of sulfhydryl groups are controlled by chemical reactions, without the need for protein engineering, introduction of enzyme engineering, and modification of payloads; in the ADC prepared by the process of the present invention, the content of D1 is usually more than 65wt%, preferably more than 75wt%, whereas in the ADC prepared by the conventional disulfhydryl coupling process, the content of D1 is usually less than 50wt%; it can be seen that the present invention greatly improves the preparation efficiency of DAR1 antibody-drug conjugates and has important application value in the production of antibody-drug conjugates.

[0168] 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 DAR1 antibody-drug conjugate, characterized in that: The drug is connected to a thiol-reactive payload, and the thiol-reactive payload is coupled to the thiol of the Fab or hinge domain of the antibody; the thiol-reactive payload is a reactive group coupled to a dithiol; The drug is monomethyl auristatin E; the thiol-reactive payload is a reactive group containing a sulfhydryl bridge group.

2. The DAR1 antibody-drug conjugate according to claim 1, characterized in that: The antibody is selected from: any one of trastuzumab, datumumab or basiliximab; 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 linker is STB; Preferably, the bismaleimide linker is Bismaleimide; Preferably, the dibromomaleimide linker is DBM.

3. A method for preparing the DAR1 antibody-drug conjugate according to claim 1 or 2, characterized in that: The preparation method comprises: (S1) incubating the antibody with a reducing agent and a transition metal ion in a buffer system to selectively reduce interchain disulfide bonds in the antibody; (S2) selectively oxidizing the reduced thiol groups with an oxidant, and quenching the oxidation with diPPBS after the oxidation reaction; (S3) mixing the metal chelator, the thiol-reactive load and the antibody to perform a coupling reaction to generate a conjugate with a DAR value of 1; (S4) purifying small molecule impurities in the system to obtain DAR1 antibody-drug conjugate.

4. The method for preparing the DAR1 antibody-drug conjugate according to claim 3, characterized in that: (S1), the buffer system is selected from any one of Hepes buffer, histidine buffer, PBS buffer or MES buffer; the pH value of the buffer system is 5-8; 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 selected from Zn 2+ , Mn 2+ 、Ni 2+ , Fe 2+ , Fe 3+ , Ca 2+ or Cu 2+ ; preferably Zn 2+ .

5. The method for preparing the DAR1 antibody-drug conjugate according to claim 3 or 4, 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, the antibody incubation conditions are: standing at 0-37°C for 0.5-72h.

6. The method for preparing a DAR1 antibody-drug conjugate according to any one of claims 3 to 5, characterized in that: In (S2), the oxidant is selected from the group consisting of 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 dehydroascorbic acid or 5,5'-dithiobis(2-nitrobenzoic acid); 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 0.5-170h.

7. The method for preparing a DAR1 antibody-drug conjugate according to any one of claims 3 to 6, characterized in that: (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.

8. The method for preparing a DAR1 antibody-drug conjugate according to any one of claims 3 to 7, 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 molar ratio of the metal chelator to the antibody is (30-40):1; Preferably, in (S3), the molar ratio of the thiol-reactive payload 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.

9. The method for preparing a DAR1 antibody-drug conjugate according to any one of claims 3 to 8, characterized in that: In (S4), the purification method comprises any one of filtration, ion exchange, ultrafiltration or column chromatography, or a combination of at least two thereof.

10. Use of the DAR1 antibody-drug conjugate according to claim 1 or 2 in the preparation of a pharmaceutical preparation for treating tumors.

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