Method for preparing homogeneous antibody-drug conjugate and kit

Abstract

A method for preparing a homogeneous antibody-drug conjugate and a related kit. The method comprises the following steps: 1) under a reaction condition 1, placing an antibody, a disaccharide linker, an endoglycosidase in a buffer solution for incubation to obtain an antibody intermediate; and 2) under a reaction condition 2, incubating a small molecule drug-linker and the antibody intermediate obtained in step 1) in a buffer solution to obtain an antibody-drug conjugate. The optimized method can provide a homogeneous antibody-drug conjugate, solving the technical problems in the prior art of inconsistent kinetic properties, poor stability, and a narrow therapeutic window caused by the heterogeneity of drug conjugates.

Description

A method and kit for preparing homogenized antibody-drug conjugates Technical Field

[0001] This application relates to the field of medicinal chemistry, specifically to a method and kit for preparing homogenized antibody-drug conjugates. Background Technology

[0002] Antibody-drug conjugates (hereinafter also referred to as "ADCs") selectively deliver drugs to tumor tissue and kill cancer cells by binding highly cytotoxic drugs to antibodies that bind to antigens that can be expressed on the surface of cancer cells (Beck, A.; Goetsch, L.; Dumontet, C.; Corvaia, N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat. Rev. Drug. Discov. 2017, 16, 315-337.). Most marketed or clinically and non-clinically investigated antibody-drug conjugates (ADCs) bind to cytotoxic drugs via non-specific, random conjugation to cysteine ​​or lysine residues. This results in heterogeneous ADCs with varying antigen affinities, aggregation properties, serum half-lives, or other limitations (Strop, P.; Delaria, K.; Foletti, D.; Witt, JM; Hasa-Moreno, A.; Poulsen, K.; Casas, MG; Dorywalska, M.; Farias, S.; Pios, A.; et al. Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading. Nat. Biotechnol. 2015, 33, 694-696.). For example, a conventional IgG antibody contains approximately 80 lysine residues and 4 pairs of interchain disulfide bonds (which open with a reducing agent to produce 8 cysteine ​​residues) and can bind to drugs via a chemical reaction. However, the conjugation process via lysine residues is random, and the number of drugs conjugated and the conjugation site are uncertain. While conjugation using cysteine ​​residues can yield homogeneous antibody-drug conjugates by reacting to all eight sites, antibody-drug conjugates with a drug loading of eight typically exhibit higher toxicity and a narrower therapeutic window. Furthermore, not all antibodies and drugs are suitable for this method of conjugation; the hydrophobicity of the drug itself often leads to excessive antibody aggregation. In antibody-drug conjugate compositions with fewer than eight drug binding sites, the number of drugs bound by a single antibody is uneven, resulting in inconsistent kinetic properties, poor stability, and a narrow therapeutic window. Therefore, it is desirable to develop a method based on glycosidases for site-specific conjugation using the N-297 site of antibodies, where the number of drugs bound can be optimized by controlling reaction conditions during antibody-drug conjugate generation to form homogeneous antibody-drug conjugates.

[0003] IgG monoclonal antibodies typically have a conserved N-glycosylation site at Asn-297 in the Fc region. Glycosylation is a highly abundant and structurally complex type of post-translational modification of proteins, exhibiting strong macroscopic and microscopic heterogeneity. Endo-N-acetyl glucosaminidase (ENGase) can be used to prune all heterogeneous N-glycans on IgG antibodies, retaining only the first GlcNAc or Fuc-GlcNAc at the glycosylation site of IgGs. Then, the activated glycan in the form of oxazoline can be transferred to GlcNAc or Fuc-GlcNAc in a manner that produces β-1,4 bonds, resulting in more homogeneous glycosylated antibodies (Wang LX, Chemoenzymatic Glycoengineering of Intact IgG Antibodies for Gain of Functions. J. Am. Chem. Soc. 2012, 134(29), 12308-12318.). Summary of the Invention

[0004] Purpose of the invention

[0005] One of the technical objectives of this invention is to develop an optimized method for preparing homogeneous antibody-drug conjugates based on glycoside endonucleases, especially based on the glycoside endonuclease mutant Endo-S2M3.

[0006] Another technical objective of this invention is to develop a kit for preparing homogeneous antibody-drug conjugates.

[0007] Summary of the Invention

[0008] On one hand, the present invention provides a method for preparing a homogeneous antibody-drug conjugate based on the glycoside endonuclease Endo-S2M3 with the amino acid sequence SEQ ID No.: 1, the method comprising the following steps:

[0009] 1) Under reaction conditions 1, the antibody, the disaccharide linker with an oxazoline linker (represented by Formula I), and the glycoside endonuclease Endo-S2M3 were incubated in a buffer solution to obtain an antibody intermediate modified with the disaccharide linker; and

[0010] 2) Under reaction conditions 2, a small molecule drug-linker of Formula II, dissolved in an organic solvent, is added to the modified antibody intermediate obtained in step 1), and the mixture is incubated in a buffer solution to obtain the antibody-drug conjugate.

[0011] In Formula I, the G ring represents a monosaccharide molecule selected from galactose, N-acetylgalactose, mannose and glucose, which is linked to the 4 position of the cyclized N-acetyl-D-glucosamine at the 1,2 position by a glycosidic bond, wherein the glycosidic bond is a 1,4 glycosidic bond, a 2,4 glycosidic bond or a 3,4 glycosidic bond.

[0012] ZYX- represents a substituent on the G ring. The substitution position of ZYX- is any position except position 1 of the G ring of the monosaccharide molecule. For example, the substitution position of ZYX- is position 2, 3, 4, 5 or 6 of the G ring of the monosaccharide molecule, preferably position 5.

[0013] In the structure ZYX-, ZY- may or may not exist.

[0014] When ZY- is absent, X is an aldehyde group, -NH2, -CH2-NH2, -COOH, -N3, or -CH2-N3;

[0015] When ZY- is present, X is selected from the following groups: -CH2-, -CH2-O-, -CH2-Se-, -CO-NH-, -ON=CH-, -CONH-N=CH-, -NHCH2-, -CH=CH- or not present, and the following structures:

[0016] Y is a bivalent or multivalent connector that connects X and Z.

[0017] Preferably, Y is selected from the following groups: -(CH2)m-(CH-w)n-, -(CH2-CH2-O)m-(CH-w)n-, -NH-,

[0018] Where m and n are independently selected from integers between 0 and 30, and w is a hydrogen atom or a polyethylene glycol structure of different lengths;

[0019] Z is selected from the following reactive groups: azide residues, aldehyde residues, thiol residues, alkyne residues, olefin residues, halogen residues, tetrazine residues, nitroketone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boric acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues, isonitrile residues, stearone residues, conjugated diene residues, cycloalkyne residues, and cycloalkene residues;

[0020] Alternatively, Z can be selected from the following groups:

[0021] ELD

[0022] Formula II

[0023] In Formula II, E is the corresponding group that undergoes an orthogonal reaction with the azide group, and it is selected from straight-chain alkynyl groups, DBCO-type structures, and BCN-type structures.

[0024] L represents the connector.

[0025] D indicates a cytotoxic or cell-inhibiting drug.

[0026] In a specific embodiment, the disaccharide linker is selected from the following structures:

[0027] Wherein, l, m, and n are independent integers from 0 to 30, preferably, where l, m, and n are independent integers from 0 to 24, and where l, m, and n are independent integers from 0 to 12.

[0028] Each 'l' independently represents an integer from 0 to 24. For example, 'l' represents 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0029] Each n independently represents an integer from 0 to 24. For example, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0030] Each m represents an integer from 0 to 24 independently. For example, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0031] In a specific embodiment, the disaccharide linker is selected from the following structures:

[0032] Where l, m, and n are independent integers from 0 to 30.

[0033] In a specific embodiment, the disaccharide linker is selected from the following structures:

[0034] In a specific embodiment, in the small molecule drug-linker shown in Formula II,

[0035] L is preferably selected from -(CH2)a-(OCH2 CH2)b-(NHCO)n-(CH2)c-, or selected from the following groups:

[0036] V and W are bifunctional linkers, including structures with lysine and propargylglycine as bifunctional linkers. For example, L is selected from:

[0037] Wherein, a, b, c, d and e are each independently selected from integers between 0 and 30, m and n are 0 or 1, R3 and R4 are each independently selected from CH3-, (CH3)2CH-, PhCH2, NH2(CH2)4-, NH2CONH(CH2)3-, and R is selected from PEG structures of different lengths of azidable monosaccharides, disaccharides, oligosaccharides or azid groups or combinations of PEG and chain or cyclic monosaccharides, disaccharides or oligosaccharides, wherein the oligosaccharides include branched oligosaccharide chains;

[0038] Represents the connection site;

[0039] For example, the L is -Lys (0-30) -PEG (0-30) -GGFG-; or Lys (0-30) -PEG (0-30) -VC-PAB-; where L is -PEG4-vc-PAB- or -PEG4-GGFG-.

[0040] In a specific embodiment, in the small molecule drug-linker shown in Formula II,

[0041] D is selected from the following:

[0042] (1) Microtubule inhibitors / disruptors: for example, but not limited to, auristatin class (e.g., MMAE or MMAF), maytansin derivatives (e.g., DM1, DM2, DM4), tubulosynins, cryptomycins, antimitotic EG5 inhibitors (e.g., spindle kinesin KSP inhibitors).

[0043] (2) DNA damaging agents: for example, but not limited to, pyrrolobenzodiazepines (e.g., pyrrolo[2,1-c][1,4]benzodiazepine (PBD)), ducarmycin, indolinobenzodiazepine; Duocarmycins; Calicheamicins;

[0044] (3) Topoisomerase inhibitors: for example, but not limited to, camptothecins (e.g., eczetidine and its derivative Dxd);

[0045] (4) Others: apoptosis inducers (Bcl-xL inhibitors), thailanstatin and its analogues, amatoxins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, carbamycin.

[0046] In a specific embodiment, the small molecule drug linker is selected from the following:

[0047] In a specific embodiment, the small molecule drug linker is selected from the following:

[0048] In a specific embodiment, the antibody is an antibody having an N-glycosylation site. More preferably, the antibody is a dual-antenna antibody having an N-glycosylation site. Most preferably, the antibody is IgG with a conserved N-glycosylation site at N297 in the Fc region.

[0049] For example, the antibody may be a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody from different species.

[0050] For example, the antibody is a human antibody, a mouse antibody, or a chimeric antibody.

[0051] For example, the antibody is IgG1, IgG2, or IgG4.

[0052] For example, the target sites of the antibody are selected from HER2, Claudin 18.2, EGFR, TROP2, c-Met, NECTIN4, CD276, HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR 1. DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG(5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BlyS, IL23, α4β7, IL-4R-α, HAE, FGF23, and IL6R; preferably, the target of the antibody is selected from VEGF, HER2, CD20, TROP2, EGFR, and PD-1.

[0053] Specifically, the antibodies include bevacizumab, trastuzumab, rituximab, pertuzumab, panitumumab, toripalimab, nivolumab, vidictetumab, and saxituzumab.

[0054] In a specific embodiment, in step 1), the reaction conditions 1 include: in the reaction system, the antibody concentration is 0.1-50 mg / mL, the molar equivalent of the disaccharide linker relative to the antibody is 10-200, the buffer pH is 5.5-7.5, the amount of glycosidase Endo-S2M3 relative to the amount of antibody is 1-500 μg / mg, the incubation temperature is 4-37℃, and the incubation time is 0.5-48 hours.

[0055] In a specific embodiment, under reaction condition 1,

[0056] The antibody concentration is 2-30 mg / mL, for example 5-20 mg / mL, such as 2, 5, 8, 10, 15, 20 mg / mL; the molar equivalent of the disaccharide linker is 30-100, for example 30, 60, 100; the pH of the buffer is 6.0-7.5, preferably 6.25-7.25, such as 6.25, 6.50, 6.75, 7.00, 7.25; the amount of glycosidase relative to the amount of antibody is 2-320 μg / mg, preferably 2, 5, 20, 160, 200 μg / mg; the incubation temperature is 10-37℃, preferably 15-30℃, preferably 15, 20, 22, 25, 28, 30℃; the incubation time is 2-24 hours, preferably 2-8 hours, such as 2, 4, 6, 8, 12, 24.

[0057] In a specific embodiment, in step 2), the reaction conditions 2 include: the use equivalent of the small molecule drug-linker is 2-20, the incubation temperature is 15-30℃, and the incubation time is 0.5-48 hours.

[0058] In a specific embodiment, under reaction condition 2,

[0059] The dosage equivalent of the small molecule drug-linker is 3-11, preferably 3, 4, 5, 6, 7, 8, 9, 10, or 11; the incubation temperature is 20-28°C, such as 20, 22, 25, or 28°C; and the incubation time is 0.5-24 hours, preferably 0.5-2.5 hours, such as 0.5, 1, 1.5, 2, or 2.5 hours. In step 2, the reaction is less affected by pH, therefore the reaction can be carried out under pH 4-8 conditions.

[0060] In a specific implementation, in steps 1) and 2), the buffer system is selected from: phosphate buffer, histidine buffer, and acetate buffer system.

[0061] In a specific implementation, after incubation in step 1), excess enzymes and substrates in the antibody intermediate are removed by Protein A column chromatography; after incubation in step 2), excess drug intermediates are removed by a 30kDa ultrafiltration tube.

[0062] In a specific embodiment, in step 1), the reaction conditions 1 include: an antibody concentration of 5 mg / mL, a molar equivalent of 30 for the monoazidobiose linker, a buffer pH of 6.5, a glycosidase Endo-S2M3 concentration of 20 μg / mg, an incubation temperature of 22°C, and an incubation time of 2 hours.

[0063] In step 2), the reaction conditions 2 include: the use equivalent of the small molecule drug-linker is 6, the incubation temperature is 22°C, and the incubation time is 60 min.

[0064] In a specific embodiment, in step 1), the reaction conditions 1 include: an antibody concentration of 5 mg / mL, a molar equivalent of 30 for the monoazidobiose linker, a buffer pH of 6.5, a glycosidase Endo-S2M3 concentration of 200 μg / mg, an incubation temperature of 22°C, and an incubation time of 2 hours.

[0065] In step 2), the reaction conditions 2 include: the use equivalent of the small molecule drug-linker is 6, the incubation temperature is 22°C, and the incubation time is 60 min.

[0066] On the other hand, the present invention provides an antibody intermediate generated by step 1) of the method described above.

[0067] On the other hand, the present invention provides antibody-drug conjugates prepared by the method described above.

[0068] In a specific embodiment, the number of drugs bound in the antibody-drug conjugate is 1.4 to 2.0, preferably 1.5 to 2.0, preferably 1.6 to 2.0, preferably 1.7 to 2.0, more preferably 1.8 to 2.0, and more preferably 1.9 to 2.0.

[0069] In another aspect, the present invention provides a kit for preparing homogeneous antibody-drug conjugates, the kit comprising at least:

[0070] v. antibody;

[0071] vi. The disaccharide linker shown in Formula I above;

[0072] vii. Endo-S2M3 glycosidase; and

[0073] viii. Small molecule drug linker as shown in formula II above.

[0074] In a specific embodiment, the kit also includes reagents for preparing buffer solutions. Beneficial effects

[0075] This invention provides a chemical enzymatic method for preparing homogenized antibody-drug conjugates via the N-297 glycosylation site. This application describes a two-step method for preparing ADCs, optimizing the reaction parameters in each step. The antibody-drug conjugates prepared according to this invention contain 1.4 to 2.0 drug-binding units, preferably 1.5 to 2.0, more preferably 1.6 to 2.0, more preferably 1.7 to 2.0, and can further reach 1.8 to 2.0, or even 1.9 to 2.0, resulting in a more homogenized conjugate product.

[0076] In summary, the optimized method of this application can provide a homogeneous antibody-drug conjugate, which effectively solves the technical problems of inconsistent kinetic properties, poor stability, and narrow therapeutic window caused by the heterogeneity of the conjugated drug in the prior art. Attached Figure Description

[0077] Figure 1 shows the results of glycan hydrolysis at the N-glycosylation site of trastuzumab by Endo-S2 and S2M3. Figure A shows the hydrolysis of the glycan at the N-glycosylation site of the antibody after co-incubation with the two glycosylases and trastuzumab for 1 hour, and Figure B shows the hydrolysis of the glycan at the N-glycosylation site of the antibody after co-incubation with the two glycosylases and trastuzumab for 4 hours.

[0078] Figure 2: Shows the effect of the amount of Endo-S2 and S2M3 enzymes on the transglycosylation activity of Az-LacNAc-ox antibody. Detailed Implementation

[0079] The technical content of the present invention will be described in detail below through specific embodiments, so that those skilled in the art can better understand the present invention.

[0080] the term

[0081] In this document, the term "about" when used in conjunction with a numeric value means to cover a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value. The term is also intended to cover values ​​within ±1%, ±0.5%, or ±0.1% of the specified numeric value.

[0082] In this document, the terms "comprising" or "including" mean including the stated elements, integers, or steps, or groups of elements, integers, or steps, but do not exclude any other elements, integers, or steps, or other groups of elements, integers, or steps. When the terms "comprising" or "including" are used herein, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps. For example, when referring to a polypeptide / protein that "comprising" a specific sequence, it is also intended to cover polypeptides / proteins consisting of that specific sequence.

[0083] As used herein, the term “antibody” (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein. As used herein, “human antibody” refers to an antibody naturally occurring in the human body, a functional fragment thereof, or a humanized antibody, i.e., a genetically engineered antibody in which a portion (e.g., the frame region or Fc region) is derived from a naturally occurring human antibody.

[0084] The terms "disaccharide linker," "disaccharide substrate," or "disaccharide oxazoline substrate" are used interchangeably herein and refer to a disaccharide molecule having an oxazoline-substituted GlcNAc at the reducing end of its sugar chain. When disaccharide linkers are used for sugar remodeling for drug discovery purposes, disaccharide linkers having human or human-compatible sugar chains are preferred, as they cause few problems when applied to humans. Such sugar chains are those known not to exhibit antigenicity in the human body.

[0085] The term "small molecule drug" refers to low-molecular-weight organic compounds capable of regulating biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics.

[0086] In this document, the term “DAR” and “number of bound drugs” are used interchangeably and refer to the ratio of other molecules (e.g., disaccharide molecules or small molecule drugs) conjugated to a protein molecule (e.g., an antibody) having an N-glycosylation site as described herein to the protein molecule (e.g., an antibody) having an N-glycosylation site. In some embodiments for preparing antibodies with site-reconstructed homoglycans, the DAR value refers to the ratio of disaccharide molecules conjugated to the Fc region of a protein molecule (e.g., an antibody) with site-reconstructed homoglycans as described herein to the protein molecule (e.g., an antibody) with the Fc region of site-reconstructed homoglycans as described herein. In embodiments of antibody-drug conjugates (ADCs), the DAR value refers to the ratio of a small molecule drug portion conjugated to the Fc region of a protein molecule (e.g., an antibody) with the Fc region of site-reconstructed homoglycans as described herein to the protein molecule (e.g., an antibody) with the Fc region of site-reconstructed homoglycans as described herein. In some embodiments, DAR is calculated as the average DAR of the molecular population in the product, i.e., the total proportion of disaccharide molecules or small molecule drugs conjugated to, for example, the antibody moiety described herein, in the product, as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC). This DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the present invention is, for example, 1.4-2.0, such as 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, and any range of values ​​with two of these values ​​as endpoints.

[0087] In this application, "DBCO-type structure" and "BCN-type structure" refer to structures containing a DBCO or BCN core that can undergo a click reaction with an azide group, respectively.

[0088] Unless otherwise specified, the glycoside endonucleases, antibodies, and disaccharide substrates involved in this invention are commercially available.

[0089] The monoazidobiose substrate structure used below is as follows:

[0090] [Structure I]N3-NH-LacNAc-ox

[0091] [Structure II] N3-ON=CH-LacNAc-ox

[0092] The small molecule drug-linker structures used below are as follows:

[0093] [Structure III]DBCO-PEG4-vc-PAB-MMAE

[0094] [Structure IV]DBCO-PEG4-GGFG-Dxd

[0095] Expression and purification of glycoside endonuclease Endo-S2M3

[0096] The target gene (SEQ ID No.: 2) was ligated into the pET22b(+) vector to construct a plasmid. The plasmid was then transformed into *E. coli* DH5α competent cells for cloning. 1 μL of the constructed plasmid (10 ng / μL) was transformed into 50 μL of *E. coli* DH5α competent cells and incubated on ice for 30 min. The cells were then heat-shocked at 42°C for 90 sec, and then transferred to ice and incubated for 3–5 min. 500 μL of antibiotic-free LB medium was added, mixed well, and the cells were incubated at 37°C with shaking at 220 rpm for 1–2 h. Subsequently, 200–300 μL of the bacterial culture was spread onto LB agar plates containing ampicillin and incubated overnight at 37°C. The next day, single clones were picked and cultured in 3–5 mL of LB medium. OD was then calculated. 600 When the bacterial culture reaches 0.6–0.8, 50 μL of the bacterial culture is transferred to a sequencing company for Sanger sequencing.

[0097] Once the sequencing results are confirmed to be correct, the plasmid is transformed into E. coli BL21(DE3) competent cells, and IPTG is used to induce the expression of glycoside endonucleases. 1 μL of plasmid (10 ng / μL) is transformed into 50 μL of E. coli BL21(DE3) competent cells and incubated on ice for 30 min. A heat shock at 42°C for 90 sec is performed, followed by incubation on ice for 3–5 min. 500 μL of antibiotic-free LB medium is added, mixed well, and the cells are incubated at 37°C with shaking at 220 rpm for 1–2 h. Subsequently, 200–300 μL of the bacterial culture is spread onto LB agar plates containing ampicillin and incubated overnight at 37°C. The next day, single colonies are picked and expanded into 3–5 mL of LB medium. OD... 600 When the concentration reaches 0.6–0.8, IPTG is added to the bacterial culture to a final concentration of 0.5 mM. The culture is then incubated overnight at 20°C and 200 rpm. After expression, the cells are centrifuged at 4500 rpm for 20 min, and the supernatant is discarded. The cells are resuspended in 30 mL of phosphate buffer (0.01 M, pH 7.4), sonicated, and then centrifuged at 12000 rpm for 15 min to collect the supernatant for protein purification.

[0098] The overexpressed glycoside endonuclease Endo-S2M3 was purified using a nickel-ion affinity chromatography column. The column bed was equilibrated with 5 column volumes of phosphate-buffered saline (0.01 M, pH 7.4), and the supernatant from the lysed culture was then loaded into the column, repeated three times to ensure the target protein bound to the column packing material. Elution was then performed with 10 column volumes of 50 mM, 100 mM, and 250 mM imidazole, and the eluent was collected in separate tubes.

[0099] The eluent was subjected to polyacrylamide gel electrophoresis to determine the location of the sample, then concentrated by ultrafiltration and the buffer was replaced with phosphate buffer (0.01M, pH 7.4).

[0100] Protein concentration was determined using the UV280 method, and molecular weight was identified using LC-MS.

[0101] The amino acid sequence of the final expressed glycoside endonuclease Endo-S2M3 is SEQ ID No.: 1.

[0102] General Operation 1

[0103] Methods for generating antibody-drug conjugates include:

[0104] (i) Incubate the antibody, monoazidobiose substrate (structure I or structure II above), and glycoside endonuclease Endo-S2M3 in a buffer solution to obtain an antibody intermediate modified with an azide group; and

[0105] (ii) The small molecule drug-linker (structure III or structure IV above) and the antibody intermediate with azide group modification obtained in step (i) are incubated in the organic reagent DMSO and histidine buffer to prepare the antibody-drug conjugate. The conjugation efficiency is evaluated by RP-HPLC.

[0106] Materials and reagents

[0107] The DAR value of the conjugated antibody was measured by RP-HPLC and calculated using the following formula:

[0108] DAR value=[Intensity(H0 ADC)×0+Intensity(H1 ADC)×1+Intensity(H2 ADC)×2]÷[Intensity(H0 ADC)+Intensity(H1 ADC)+Intensity(H2 ADC)]×2

[0109] The RP-HPLC operating conditions are as follows:

[0110] Sample pretreatment: Add 1 mol / L DTT (final concentration of DTT is 0.1 mol / L) to the sample solution, and dilute the sample solution to 2 mg / mL with 8 mol / L guanidine hydrochloride.

[0111] Instrument: Agilent 1260.

[0112] Manufacturer: Agilent.

[0113] Column: Agilent, PLRP-S 4.6x250mm, 8μm

[0114] Mobile phase A: 0.1% TFA in H2O solution

[0115] Mobile phase B: 0.1% TFA in ACN

[0116] Flow rate: 0.8 ml / min

[0117] Column temperature: 80℃

[0118] Detection wavelength: 280nm

[0119] Loading capacity: 25 μL (50 μg)

[0120] The gradient is as follows:

[0121]

[0122] LC-MS detection method:

[0123] Conditions for the LC method:

[0124] Instrument: SCIEX ExionLC

[0125] Column: ACQUITYUPLC BEH

[0126] SEC 1.7μm, 4.6×300mm

[0127] Mobile phase: A: 0.1% FA, 0.05% TFA, 25% ACN in H2O

[0128] Injection volume: 10 μL

[0129] Column temperature: 25℃

[0130] Flow rate: 200 μL / min

[0131] Gradient: Isotropic

[0132] The DAR value of the conjugated antibody was measured by LC-MS and calculated using the following formula:

[0133] DAR value=[Intensity(H0 ADC)×0+Intensity(H1 ADC)×1+Intensity(H2 ADC)×2]÷[Intensity(H0 ADC)+Intensity(H1 ADC)+Intensity(H2 ADC)]×2

[0134] Below, in Test Examples 1-2, the hydrolytic and transglycosylation activities of the glycosidases Endo-S2M and Endo-S2 were tested, and the experimental results of the two were compared.

[0135] Test Example 1. Antibody Glycosylation and Hydrolysis Activity Assay for Endo S2M3 Glycosylase

[0136] Trastuzumab (5 mg / mL) was added to phosphate buffer (pH 7.0), followed by the addition of glycosidase (100 ng / mg, Endo-S2 (SEQ ID NO: 3) as a control). The mixture was incubated at 30°C for 1 hour and 4 hours, and samples were analyzed by LC-MS. The results are shown in Figure 1. The results indicate that both Endo-S2 and Endo S2M3 can hydrolyze the glycans of wild-type antibodies, with S2M3 exhibiting better hydrolytic activity than Endo-S2.

[0137] Test Example 2: Assay of antibody transglycosylation activity of Endo-S2 and S2M3 enzymes against Az-LacNAc-ox substrate

[0138] Az-LacNAc-ox (0.5 mM, structural formula shown below) was added to phosphate buffer (pH 6.5). Trastuzumab (5 mg / mL) and glycoside endonuclease (Endo-S2 or S2M3, 0.02-2.4 mg / mL) were then added sequentially to the above reaction system. The mixture was incubated at 30°C for 2 hours and then analyzed by LC-MS.

[0139] The specific design is shown in the table below:

[0140] Reaction conditions for the antibody transglycosylation activity of Endo-S2 and S2M3 enzyme amounts on Az-LacNAc-ox substrate.

[0141] As shown in the table above, antibody transglycosylation activity was tested at Endo-S2 or S2M3 enzyme concentrations of 4, 20, 40, 80, 160, 240, 320, and 480 μg / mg (enzyme / antibody concentration). The trastuzumab concentration was 5 mg / mL, the sugar substrate Az-LacNAc-ox concentration was 0.5 mM, and the reaction was carried out in a 50 mM phosphate buffer system at pH 6.5. After reacting at 30°C for 2 hours, LC-MS analysis was performed, as shown in Figure 2.

[0142] The results showed that under low enzyme concentrations (approximately <50 μg / mg), both wild-type Endo-S2 and the mutant enzyme S2M3 positively promoted the DAR value. Under higher enzyme concentrations, Endo-S2 showed a decreasing DAR value with increasing enzyme concentration, while S2M3 exhibited a longer plateau period, maintaining a high DAR value across a wide range (approximately 100-500 μg / mg). This indicates that the mutant enzyme S2M3 has more stable transglycosylation activity compared to Endo-S2. In summary, under the above process conditions, S2M3 achieves stable results only at higher enzyme concentrations. Therefore, further development of this process is needed to explore an efficient and uniform preparation process under lower enzyme concentrations.

[0143] Example 1: Preparation of antibody-drug conjugates using Endo-S2M3 enzyme and monoazidobisaccharide oxazoline substrate

[0144] (1) Preparation of antibody-drug conjugates: The general procedure is followed as follows:

[0145] Trastuzumab antibody 5 mg / mL was added to 25 mM His buffer (pH 6.5) with 30, 60, and 100 molar equivalents (see Table 1) of monoazidobiose substrate (structure I, N3-NH-LacNAc-ox), followed by the addition of Endo-S2M3 glycosidase 160 and 320 μg / mg (see Table 1). The mixture was incubated at 22 °C for 2 hours to obtain antibody intermediates modified with azido groups. Excess enzymes and substrates in the antibody intermediates were removed by Protein A column chromatography.

[0146] Six molar equivalents of a small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) were used with the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography. The mixture was incubated in 10% (v / v) DMSO in 25 mM His buffer (pH 6.5) at 22°C for 2 hours. Excess drug intermediate was removed using a 30 kDa ultrafiltration tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0147] (2) Characteristic Evaluation

[0148] Table 1 Summary of Feature Evaluation Results

[0149] (3) Results

[0150] When the concentration of monoazidobiose substrate is 30-100 molar equivalents, the number of bound drugs in ADC compositions prepared using enzyme Endo-S2M3 at 160-320 μg / mg can reach more than 1.8 as detected by RP-HPLC.

[0151] Example 2: Optimization of disaccharide modification reaction time for the preparation of antibody-drug conjugates using Endo-S2M3 enzyme and monoazidodisaccharide oxazoline substrate.

[0152] (1) Preparation of antibody-drug conjugates

[0153] Trastuzumab antibody 5 mg / mL and 30 molar equivalents of monoazidobiose substrate (structure I, N3-NH-LacNAc-ox) were added to 25 mM His buffer (pH 6.5), followed by the addition of Endo-S2M3 glycosidase 160 μg / mg. The mixture was incubated at 22°C for 0.5–8 hours to obtain an antibody intermediate modified with an azide group. Excess enzyme and substrate in the antibody intermediate were removed by Protein A column chromatography.

[0154] Six molar equivalents of a small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) were used with the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography. The mixture was incubated in 10% (v / v) DMSO in 25 mM His buffer (pH 6.5) at 22°C for 2 hours. Excess drug intermediate was removed using a 30 kDa ultrafiltration tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0155] (2) Characteristic Evaluation

[0156] Table 2 Summary of Characteristic Evaluation Results

[0157] The number of bound drugs in the ADC composition was measured by RP-HPLC.

[0158] (3) Results

[0159] In ADC compositions prepared by this method using enzyme S2M3 with a monoazidobiose substrate, the number of bound drugs gradually increases with the progress of the disaccharide modification reaction and reaches a plateau. Between 2 and 8 hours, the number of antibody-bound drugs can remain above 1.6.

[0160] Therefore, the disaccharide modification reaction time in the prepared ADC composition is not less than 2 hours, preferably 2 to 8 hours, and preferably 2 hours in order to improve efficiency.

[0161] Example 3: pH optimization of the disaccharide modification reaction for preparing antibody-drug conjugates using Endo-S2M3 enzyme and monoazidodisaccharide oxazoline substrate.

[0162] (1) Preparation of antibody-drug conjugates

[0163] Trastuzumab antibody 5 mg / mL was mixed with 30 molar equivalents of monoazidobiose substrate (structure I, N3-NH-LacNAc-ox) in 25 mM His buffer (pH 5.50, 5.75, 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50), and then Endo-S2M3 glycosidase 160 μg / mg was added. The mixture was incubated at 22 °C for 4 hours to obtain an antibody intermediate modified with an azide group. Excess enzyme and substrate in the antibody intermediate were removed by Protein A column chromatography.

[0164] Six molar equivalents of a small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) were used with the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography. The mixture was incubated in 10% (v / v) DMSO in 25 mM His buffer (pH 6.5) at 22°C for 2 hours. Excess drug intermediate was removed using a 30 kDa ultrafiltration tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0165] (2) Characteristic Evaluation

[0166] Table 3 Summary of Characteristic Evaluation Results

[0167] The number of bound drugs in the ADC composition was measured by RP-HPLC.

[0168] (3) Results

[0169] In ADC compositions prepared by this method using enzyme S2M3 with a monoazidobiose substrate, the number of bound drugs is significantly affected by the reaction pH. The preferred reaction pH is 6.25 to 7.25, where the number of bound drugs can be maintained above 1.7; more preferably, it is 6.50 to 7.00, where the number of bound drugs can be maintained above 1.8.

[0170] Example 4: Modification reaction for preparing antibody-drug conjugates using Endo-S2M3 enzyme and monoazidobisaccharide oxazoline substrate; antibody concentration optimization.

[0171] (1) Preparation of antibody-drug conjugates

[0172] Trastuzumab antibody 1-15 mg / mL and 40 molar equivalents of monoazidobiose substrate (structure IIN3-ON=CH-LacNAc-ox) were added to 25 mM His buffer (pH 6.5), followed by the addition of Endo-S2M3 glycosidase 160 μg / mg. The mixture was incubated at 22 °C for 4 hours to obtain an antibody intermediate modified with an azide group. Excess enzyme and substrate in the antibody intermediate were removed by Protein A column chromatography.

[0173] Six molar equivalents of a small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) were used with the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography. The mixture was incubated in 10% (v / v) DMSO in 25 mM His buffer (pH 6.5) at 22°C for 2 hours. Excess drug intermediate was removed using a 30 kDa ultrafiltration tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0174] (2) Characteristic Evaluation

[0175] Table 4 Summary of Characteristic Evaluation Results

[0176] The number of bound drugs in the ADC composition was measured by RP-HPLC (see Table 5).

[0177] (3) Results

[0178] In ADC compositions prepared using enzyme S2M3 with a monoazidobiose substrate, the number of bound drugs increases with increasing antibody concentration in the modification reaction and reaches a plateau. Within a reaction concentration range of 2-15 mg / mL, changing the reaction concentration has no significant effect on the number of bound drugs, and the number of bound drugs is consistently above 1.6. Therefore, an antibody reaction concentration greater than 2 mg / mL is preferred, preferably 2-15 mg / mL. Furthermore, when the antibody concentration is 5-15 mg / mL, the number of antibody-bound drugs can be maintained above 1.8.

[0179] Example 5: Optimization of Small Molecule Drug-Linker Dosage for the Preparation of Antibody-Drug Conjugates Using Endo-S2M3 Enzyme and Monoazidobisaccharide Oxazoline Substrate

[0180] (1) Preparation of antibody-drug conjugates

[0181] 10 mg / mL of trastuzumab antibody and 40 molar equivalents of monoazidobiose substrate (structure IIN3-ON=CH-LacNAc-ox) were added to 25 mM Hmis (pH 6.5) buffer, followed by the addition of 160 μg / mg of Endo-S2M3 glycosidase. The mixture was incubated at 22 °C for 4 hours to obtain an antibody intermediate modified with an azide group. Excess enzyme and substrate in the antibody intermediate were removed by Protein A column chromatography.

[0182] Using 3-11 molar equivalents of a small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) and the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography, the mixture was incubated in 10% (v / v) DMSO at 25 mM HIS buffer (pH 6.5) at 22°C for 2 hours. Excess drug intermediate was removed by ultrafiltration through a 30 kDa tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0183] (2) Characteristic Evaluation

[0184] Table 5 Summary of Characteristic Evaluation Results

[0185] The area values ​​of the number of drugs bound in the ADC composition were measured by RP-HPLC (see Table 5).

[0186] (3) Results

[0187] In ADC compositions prepared using enzyme S2M3 with a monoazidobiose substrate, the number of bound drugs has no significant effect on the amount of small molecule drug to linker when the drug-to-linker ratio is 3-11 equivalents, and is consistently above 1.8. Furthermore, the number of bound drugs stabilizes after exceeding 3 equivalents. Therefore, the drug-to-linker ratio in the prepared ADC composition should be greater than 3 equivalents, preferably 3 to 11 equivalents.

[0188] Example 6: Optimization of binding reaction time for the preparation of antibody-drug conjugates using Endo-S2M3 enzyme and monoazidobisaccharide oxazoline substrate.

[0189] (1) Preparation of antibody-drug conjugates

[0190] 10 mg / mL of trastuzumab antibody and 40 molar equivalents of monoazidobiose substrate (structure IIN3-ON=CH-LacNAc-ox) were added to 25 mM Hmis (pH 6.5) buffer, followed by the addition of 160 μg / mg of Endo-S2M3 glycosidase. The mixture was incubated at 22 °C for 4 hours to obtain an antibody intermediate modified with an azide group. Excess enzyme and substrate in the antibody intermediate were removed by Protein A column chromatography.

[0191] Five molar equivalents of a small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) were used with the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography. The mixture was incubated in 10% (v / v) DMSO in 25 mM His buffer (pH 6.5) at 22°C for 15-150 min. Excess drug intermediate was removed using a 30 kDa ultrafiltration tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0192] (2) Characteristic Evaluation

[0193] Table 6 Summary of Characteristic Evaluation Results

[0194] The number of bound drugs in the ADC composition was measured by RP-HPLC.

[0195] (3) Results

[0196] In the ADC compositions prepared by this method using enzyme S2M3 with a monoazidobiose substrate, the number of bound drugs had no significant effect on the number of bound drugs when the coupling reaction time was between 60 and 150 minutes, and remained above 1.8, stabilizing after 60 minutes. Therefore, in the prepared ADC compositions, a coupling reaction time greater than 60 minutes resulted in a number of bound drugs above 1.8, and more preferably, between 60 and 150 minutes.

[0197] Example 7: Enzyme Concentration Optimization for the Preparation of Antibody-Drug Conjugates Using Endo-S2M3 Enzyme and Monoazidobiose Oxazoline Substrate

[0198] (1) Preparation of antibody-drug conjugates

[0199] 10 mg / mL of trastuzumab antibody and 40 molar equivalents of monoazidobiose substrate (structure IIN3-ON=CH-LacNAc-ox) were added to 25 mM His buffer (pH 6.5), followed by the addition of 1-200 μg / mg of Endo-S2M3 glycosidase. The mixture was incubated at 22°C for 4 hours to obtain an antibody intermediate modified with an azide group. Excess enzyme and substrate in the antibody intermediate were removed by Protein A column chromatography.

[0200] Five molar equivalents of the small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) were used with the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography. The mixture was incubated in 10% (v / v) DMSO in 25 mM His buffer (pH 6.5) at 22°C for 120 min. Excess drug intermediate was removed using a 30 kDa ultrafiltration tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0201] (2) Characteristic Evaluation

[0202] Table 7 Summary of Characteristic Evaluation Results

[0203] The number of bound drugs in the ADC composition was measured by RP-HPLC.

[0204] (3) Results

[0205] In ADC compositions prepared by this method using enzyme S2M3 with a monoazidobiose substrate, the number of bound drugs has no significant effect on the number of bound drugs when the enzyme concentration is between 5 and 200 μg / mg, and is always above 1.7. Furthermore, using only 5 μg / mg of enzyme S2M3 is sufficient to achieve a drug-bound number of 1.8 or more. Therefore, the enzyme concentration in the prepared ADC composition should be no less than 5 μg / mg, preferably 5 to 200 μg / mg, more preferably 5 to 120 μg / mg, more preferably 10 to 50 μg / mg, and even more preferably 10 to 20 μg / mg.

[0206] Example 8: Preparation of antibody-drug conjugates using Endo-S2M3 enzyme and monoazidobiose oxazoline substrate. Optimization of antibody reaction concentration, monoazidobiose substrate amount, enzyme concentration, and reaction pH.

[0207] (1) Preparation of antibody-drug conjugates

[0208] Trastuzumab antibody at concentrations of 2, 10, and 20 mg / mL was added to 25 mM His buffer (pH 6.00, 6.50, and 7.00) with 10, 30, and 60 molar equivalents of monoazidobiose substrate (structure IIN3-ON=CH-LacNAc-ox). Endo-S2M3 glycosidase at concentrations of 2, 20, and 200 μg / mg was then added, and the mixture was incubated at 22°C for 4 hours to obtain antibody intermediates modified with azido groups. Excess enzyme and substrate in the antibody intermediates were removed by Protein A column chromatography.

[0209] Five molar equivalents of the small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) were used with antibody intermediate I (5 mg / mL) obtained in the previous step after Protein A column chromatography. The mixture was incubated in 10% (v / v) DMSO in 25 mM His buffer (pH 6.5) at 22°C for 120 min. Excess drug intermediate was removed using a 30 kDa ultrafiltration tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0210] (2) Characteristic Evaluation

[0211] Table 8 Summary of Characteristic Evaluation Results

[0212] The number of bound drugs in the ADC composition was measured by RP-HPLC.

[0213] (3) Results

[0214] In ADC compositions prepared using enzyme S2M3 with a monoazidobiose substrate, the pH range of 6.00-7.00 had no significant effect on the number of drugs bound. At antibody concentrations of 10-20 mg / mL, disaccharide substrate molar equivalents of 30-60, and enzyme concentrations of 20-200 μg / mg, the coupling efficiency increased with increasing antibody concentration and disaccharide substrate molar equivalents, consistently yielding binding products with efficiencies above 1.6.

[0215] Example 9: Preparation of antibody-drug conjugates using Endo-S2M3 enzyme and monoazidobisaccharide oxazoline substrate on various antibodies.

[0216] (1) Preparation of antibody-drug conjugates

[0217] Trastuzumab, bevacizumab, and vedictitumab 10 mg / mL were added to 25 mM His buffer (pH 6.5) along with 30 molar equivalents of monoazidobiose substrate (structure II N3-ON=CH-LacNAc-ox), followed by the addition of 20 μg / mg Endo-S2M3 glycoside endonuclease. The mixture was incubated at 22°C for 4 hours to obtain antibody intermediates modified with azido groups. Excess enzyme and substrate in the antibody intermediates were removed by Protein A column chromatography.

[0218] Five molar equivalents of a small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) were used with the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography. The mixture was incubated in 10% (v / v) DMSO in 25 mM His buffer (pH 6.5) at 22°C for 120 min. Excess drug intermediate was removed using a 30 kDa ultrafiltration tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0219] (2) Characteristic Evaluation

[0220] Table 9 Summary of Characteristic Evaluation Results

[0221] The number of bound drugs in the ADC composition was measured by RP-HPLC.

[0222] (3) Results

[0223] In the preparation of ADC compositions of different types of antibodies using the enzyme S2M3 with a monoazidobiose substrate, products with a drug binding number of 1.8 or more were obtained.

[0224] Example 10: Optimization of reaction temperature for preparing antibody-drug conjugates using Endo-S2M3 enzyme and monoazidobisaccharide oxazoline substrate.

[0225] (1) Preparation of antibody-drug conjugates

[0226] Trastuzumab 10 mg / mL was mixed with 30 molar equivalents of monoazidobiose substrate (structure II N3-ON=CH-LacNAc-ox) in 25 mM His buffer (pH 6.5), followed by the addition of Endo-S2M3 glycosidase 20 μg / mg. The reaction was carried out at 4°C, 10°C, 15°C, 22°C, 30°C, and 37°C for 4 hours to obtain an antibody intermediate modified with an azide group. Excess enzyme and substrate in the antibody intermediate were removed by Protein A column chromatography.

[0227] Five molar equivalents of a small molecule drug-linker (structure III, DBCO-PEG4-vc-PAB-MMAE) were used with the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography. The mixture was incubated in 10% (v / v) DMSO in 25 mM His buffer (pH 6.5) at 22°C for 120 min. Excess drug intermediate was removed using a 30 kDa ultrafiltration tube to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by RP-HPLC.

[0228] (2) Characteristic Evaluation

[0229] Table 10 Summary of Characteristic Evaluation Results

[0230] The number of bound drugs in the ADC composition was measured by RP-HPLC.

[0231] (3) Results

[0232] In ADC compositions prepared by this method using enzyme S2M3 with a monoazidobiose substrate, the reaction temperature has no significant effect on the number of drugs bound when it is between 15°C and 30°C. Therefore, the reaction temperature in the prepared ADC composition is preferably between 15°C and 30°C, more preferably between 20°C and 30°C.

[0233] Example 11: Preparation of antibody-drug conjugates using Endo-S2M3 enzyme, monoazidobisaccharide oxazoline substrate, and different small molecule drug-linkers.

[0234] (1) Preparation of antibody-drug conjugates

[0235] Trastuzumab and bevacizumab 10 mg / mL were added to 25 mM His buffer (pH 6.5) along with 30 molar equivalents of monoazidobiose substrate (structure II N3-ON=CH-LacNAc-ox), followed by the addition of 20 μg / mg Endo-S2M3 glycosidase. The mixture was incubated at 22 °C for 4 hours to obtain antibody intermediates modified with azido groups. Excess enzymes and substrates in the antibody intermediates were removed by Protein A column chromatography.

[0236] Five molar equivalents of the small molecule drug-linker (structure IV, DBCO-PEG4-GGFG-Dxd) were incubated with the antibody intermediate (5 mg / mL) obtained in the previous step after Protein A column chromatography at 22°C for 120 min in 10% (v / v) DMSO and 25 mM His buffer (pH 6.5). Excess drug intermediate was removed by ultrafiltration to prepare the HER2 antibody ADC. The conjugation efficiency was evaluated by LC-MS.

[0237] (2) Characteristic Evaluation

[0238] Table 11 Summary of Characteristic Evaluation Results

[0239] The number of bound drugs in the ADC composition was measured by LC-MS.

[0240] (3) Results

[0241] In ADC compositions prepared by this method using enzyme S2M3 with a monoazidobiose substrate, an ADC composition with an MS-DAR of 1.9-2.0 can be obtained.

[0242] In summary, this application provides a method for preparing antibody-drug conjugates based on Endo-S2M3. This method can efficiently prepare homogeneous antibody-drug conjugates with a DAR of 1.4 to 2.0, preferably 1.5 to 2.0, more preferably 1.6 to 2.0, more preferably 1.7 to 2.0, and further capable of reaching 1.8 to 2.0, or even 1.9 to 2.0.

Claims

1. A method for preparing a homogeneous antibody-drug conjugate based on the glycoside endonuclease Endo-S2M3 with the amino acid sequence SEQ ID No.: 1, the method comprising the following steps: 1) The antibody, the disaccharide linker with an oxazoline linker (represented by Formula I), and the glycosidase Endo-S2M3 were incubated in a buffer solution to obtain an antibody intermediate modified with the disaccharide linker; and 2) Add the small molecule drug-linker of Formula II, dissolved in an organic solvent, to the modified antibody intermediate obtained in step 1), and incubate in a buffer solution to obtain the antibody-drug conjugate. In Formula I, the G ring represents a monosaccharide molecule selected from galactose, N-acetylgalactose, mannose and glucose, which is linked to the 4 position of the cyclized N-acetyl-D-glucosamine at the 1,2 position by a glycosidic bond, wherein the glycosidic bond is a 1,4 glycosidic bond, a 2,4 glycosidic bond or a 3,4 glycosidic bond. ZYX- represents a substituent on the G ring. The substitution position of ZYX- is any position except position 1 of the G ring of the monosaccharide molecule. For example, the substitution position of ZYX- is position 2, 3, 4, 5 or 6 of the G ring of the monosaccharide molecule, preferably position 5. In the structure ZYX-, ZY- may or may not exist. When ZY- is absent, X is an aldehyde group, -NH2, -CH2-NH2, -COOH, -N3, or -CH2-N3; When ZY- is present, X is selected from the following groups: -CH2-, -CH2-O-, -CH2-Se-, -CO-NH-, -ON=CH-, -CONH-N=CH-, -NHCH2-, -CH=CH- or not present, and the following structures: Y is a bivalent or multivalent connector that connects X and Z. Preferably, Y is selected from the following groups: -(CH2)m-(CH-w)n-, -(CH2-CH2-O)m-(CH-w)n-, -NH-, Where m and n are independently selected from integers between 0 and 30, and w is a hydrogen atom or a polyethylene glycol structure of different lengths; Z is selected from the following reactive groups: azide residues, aldehyde residues, thiol residues, alkyne residues, olefin residues, halogen residues, tetrazine residues, nitroketone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boric acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues, isonitrile residues, stearone residues, conjugated diene residues, cycloalkyne residues, and cycloalkene residues; Alternatively, Z can be selected from the following groups: ELD Formula II In Equation II, E represents the corresponding group that undergoes an orthogonal reaction with the azide group, and is selected from straight-chain alkynyl groups, DBCO-type structures, and BCN-type structures. L represents the connector. D indicates a cytotoxic or cell-inhibiting drug.

2. The method according to claim 1, wherein, The disaccharide linker is selected from the following structures: Preferably, the disaccharide linker is selected from the following structures: Wherein, l, m, and n are each an independent integer from 0 to 30; preferably, l, m, and n are each an independent integer from 0 to 24; more preferably, l, m, and n are each an independent integer from 0 to 12. Each 'l' independently represents an integer from 0 to 24. For example, 'l' represents 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Each n independently represents an integer from 0 to 24. For example, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Each m independently represents an integer from 0 to 24. For example, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Most preferably, the disaccharide linker is selected from the following structures:

3. The method according to claim 1, wherein, In the small molecule drug-linker shown in Formula II L is selected from -(CH2)a-(OCH2CH2)b-(NHCO)n-(CH2)c-, or from the following groups: V and W are bifunctional linkers, containing structures with lysine and propargylglycine as bifunctional linkers. For example, L is selected from: Wherein, a, b, c, d and e are each independently selected from integers between 0 and 30, m and n are 0 or 1, R3 and R4 are each independently selected from CH3-, (CH3)2CH-, PhCH2, NH2(CH2)4-, NH2CONH(CH2)3-, and R is selected from PEG structures of different lengths of azidable monosaccharides, disaccharides, oligosaccharides or azid groups or combinations of PEG and chain or cyclic monosaccharides, disaccharides or oligosaccharides, wherein the oligosaccharides include branched oligosaccharide chains; Represents the connection site; For example, the L is -Lys (0-30) -PEG (0-30) -GGFG-; or Lys (0-30) -PEG (0-30) -VC-PAB-; where L is -PEG4-vc-PAB- or -PEG4-GGFG-; and / or In the small molecule drug-linker shown in Formula II D is selected from the following: (1) Microtubule inhibitors / disruptors: for example, auristatin class (e.g., MMAE or MMAF), maytansin derivatives (e.g., DM1, DM2, DM4), tubulosynins, cryptomycins, antimitotic EG5 inhibitors (e.g., spindle kinesin KSP inhibitors). (2) DNA damaging agents: for example, pyrrolobenzodiazepines (e.g., pyrrolo[2,1-c][1,4]benzodiazepines (PBD)), ducarmycin, indolinobenzodiazepine; Duocarmycins; Calicheamicins; (3) Topoisomerase inhibitors: for example, camptothecins (e.g., eczetidine and its derivative Dxd); (4) Others: apoptosis inducers (Bcl-xL inhibitors), thailanstatin and its analogues, amatoxins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, carbamycin; Preferably, the small molecule drug linker is selected from the following: Most preferably, the small molecule drug linker is selected from the following:

4. The method according to claim 1, wherein, The antibody is an antibody with an N-glycosylation site; more preferably, the antibody is a dual-antenna antibody with an N-glycosylation site; most preferably, the antibody is IgG with a conserved N-glycosylation site at N297 in the Fc region. For example, the antibody may be a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, or a therapeutic or functional antibody from different species. For example, the antibody is a human antibody, a mouse antibody, or a chimeric antibody. For example, the antibodies are IgG1, IgG2, and IgG4. For example, the target sites of the antibody are selected from HER2, Claudin 18.2, EGFR, TROP2, c-Met, NECTIN4, CD276, HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR 1. DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG(5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BlyS, IL23, α4β7, IL-4R-α, HAE, FGF23, and IL6R; preferably, the target of the antibody is selected from VEGF, HER2, CD20, TROP2, EGFR, and PD-1. Specifically, the antibodies include bevacizumab, trastuzumab, rituximab, pertuzumab, panitumumab, toripalimab, nivolumab, vidictetumab, and saxituzumab.

5. The method according to claim 1, wherein, In step 1), reaction conditions 1 include: in the reaction system, the antibody concentration is 0.1-50 mg / mL, the molar equivalent of the disaccharide linker relative to the antibody is 10-200, the buffer pH is 5.5-7.5, the amount of glycosidase Endo-S2M3 relative to the amount of antibody is 1-500 μg / mg, the incubation temperature is 4-37℃, and the incubation time is 0.5-48 hours; and Preferably, in reaction condition 1, The antibody concentration is 2-30 mg / mL, for example 5-20 mg / mL, such as 2, 5, 8, 10, 15, 20 mg / mL; the molar equivalent of the disaccharide linker is 30-100, for example 30, 60, 100; the pH of the buffer is 6.0-7.5, preferably 6.25-7.25, such as 6.25, 6.50, 6.75, 7.00, 7.25; the amount of glycosidase relative to the amount of antibody is 2-320 μg / mg, preferably 2, 5, 20, 160, 200 μg / mg, preferably 5-200 μg / mg; the incubation temperature is 15-37℃, preferably 15-30℃, preferably 15, 20, 22, 25, 28, 30℃; the incubation time is 2-24 hours, preferably 2-8 hours, such as 2, 4, 6, 8, 12, 24 hours.

6. The method according to claim 1, wherein, in, In step 2), the reaction conditions 2 include: the use equivalent of the small molecule drug-linker is 2-20, the incubation temperature is 15-30℃, and the incubation time is 0.5-48 hours; Preferably, in reaction condition 2, The use equivalent of the small molecule drug-linker is 3-11, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11; the incubation temperature is 20-28℃, such as 20, 22, 25, 28℃; the incubation time is 0.5-24 hours, preferably 0.5-2.5 hours, such as 0.5, 1, 1.5, 2, 2.5 hours.

7. The method according to claim 1, wherein, In steps 1) and 2), the buffer system is selected from: phosphate buffer, histidine buffer, acetate buffer system, and / or After incubation in step 1), excess enzymes and substrates in the antibody intermediate were removed by Protein A column chromatography; and After incubation in step 2), excess drug intermediates are removed using a 30kDa ultrafiltration tube.

8. The method according to claim 1, wherein, In step 1), reaction conditions 1 include: antibody concentration of 5 mg / mL, molar equivalent of the monoazidobiose linker of 30, buffer pH of 6.5, concentration of endoglycoside endonuclease Endo-S2M3 of 20 μg / mg, incubation temperature of 22°C, and incubation time of 2 hours. In step 2), reaction conditions 2 include: the use equivalent of the small molecule drug-linker is 6, the incubation temperature is 22°C, and the incubation time is 60 min; or In step 1), reaction conditions 1 include: antibody concentration of 5 mg / mL, molar equivalent of the monoazidobiose linker of 30, buffer pH of 6.5, concentration of endoglycoside endonuclease Endo-S2M3 of 200 μg / mg, incubation temperature of 22°C, and incubation time of 2 hours. In step 2), the reaction conditions 2 include: the use equivalent of the small molecule drug-linker is 6, the incubation temperature is 22°C, and the incubation time is 60 min.

9. An antibody intermediate generated by step 1) of the method as described in any one of claims 1-8.

10. An antibody-drug conjugate prepared by any one of claims 1-8.

11. A kit for preparing homogeneous antibody-drug conjugates, said kit comprising at least: i. Antibody; ii. The disaccharide linker represented by Formula I as defined in claim 1 or 2; iii. Endo-S2M3 glycoside endonuclease; and iv. The small molecule drug linker represented by Formula II as defined in claim 1 or 3.

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