Coupled drug as well as preparation method and application thereof

Abstract

The invention provides a coupling drug and a preparation method thereof, and relates to the field of antibody drugs. The structure of the coupling drug provided by the invention is as shown in the following formula I, X is an antibody or fusion protein containing an Fc fragment, GlcNAc is N-acetylglucosamine, Man is mannose, Fuc is fucose, Gal is galactose, Fucc * is a fucose derivative and contains Dxd; and m is 1-4. Compared with DS-8201, the antibody coupling drug prepared by the invention has better hydrophilicity, stability and plasma stability. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drugs, and in particular to an antibody conjugate and a preparation method and application thereof. Background Art

[0002] Antibody drug conjugate (ADC) is a class of highly effective drugs that combine chemotherapy and immunotherapy. ADC consists of an antibody (Antibody), a cytotoxin molecule (ADC Cytotoxin, payload), and a linker that connects the two. It was first proposed by German scientist / doctor Paul Ehrlich more than 100 years ago. Paul Ehrlich likened ADC to a "magic bullet" because it can specifically identify cancer cells without harming the organism (with little toxic side effects), and uses the targeting of antibodies and the toxicity of payloads to achieve targeted killing of tumors. It has very important applications in the field of medicine.

[0003] Currently, ADCs based on topoisomerase 1 inhibitors have achieved good results, especially a series of antibody-drug conjugates prepared using MC-GGFG-Dxd with a DAR (drug to antibody ratio) value close to 8. The structure of MC-GGFG-Dxd is as follows:

[0004]

[0005] The most representative one is DS-8201 (Enhertu, Daiichi Sankyo) (patent CN201380053256.2). Although DS-8201 has achieved good efficacy, it still has safety issues such as side effects and toxicity. This is closely related to factors such as the hydrophilicity and stability of antibody-drug conjugates. Therefore, it is still necessary to further develop ADCs with better properties by further improving the hydrophilicity, plasma stability, and efficacy of ADCs.

[0006] Patent CN 115916802 A provides a site-specific protein conjugate and a preparation method thereof. The protein conjugate comprises a protein and an oligosaccharide, wherein the oligosaccharide comprises The protein conjugate and its preparation method have the characteristics of site specificity and high homogeneity. However, the patent does not disclose the structure and preparation scheme of the conjugate based on topoisomerase 1 inhibitor (such as Dxd) with high DAR value (such as DAR8), nor does it characterize the performance of the above conjugate.

[0007] Based on this, the present invention provides an antibody-drug conjugate and a preparation method thereof. Through the preparation method of the present invention, a high DAR value (such as DAR8) protein (such as antibody) conjugate based on glycosyl conjugation and topoisomerase 1 inhibitor can be prepared site-specifically and efficiently. And the antibody conjugate prepared by the present invention (such as trastuzumab-G2F-Fuc*) has better hydrophilicity, thermal stability and plasma stability than the antibody conjugate prepared by MC-GGFG-Dxd (such as DS-8201). Summary of the invention

[0008] In view of the above problems, the present invention provides a coupled drug and a preparation method thereof.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] In one aspect, the present invention provides a conjugated drug having a structure shown in the following formula I:

[0011]

[0012] Wherein, X is an antibody or fusion protein comprising an Fc fragment, and m is 1-4.

[0013] GlcNAc is N-acetylglucosamine, Man is mannose, Fuc is fucose, Gal is galactose, and Fuc* is a fucose derivative, and has the following structure:

[0014]

[0015] Among them, Sp1 is

[0016] SP2 is n is any integer from 1 to 4;

[0017] SP3 is n' is any integer from 1 to 4;

[0018] Sp4 is n" is any integer from 1 to 4, and t is any integer from 0 to 1;

[0019] Cn1 is

[0020] Cn2 is

[0021] Preferably, the GlcNAc near the X-terminus on the left side of the structure (core GlcNAc) is linked to an asparagine (Asn or N) residue on the Fc fragment in the X structure. Preferably, the asparagine on the Fc fragment is located at position 297, and the Fc fragment is numbered according to the Kabat numbering system. Preferably, the GlcNAc near the X-terminus on the left side of the structure is linked to an asparagine (Asn or N) residue on the Fc fragment in the X structure.

[0022] Preferably, the Fuc* can be linked to the terminal GlcNAc via an α1,3 glycosidic bond.

[0023] Preferably, the Gal can be linked to the terminal GlcNAc via a β-1,4 glycosidic bond.

[0024] Preferably, the Fuc can be linked to the core GlcNAc via an α1,6 glycosidic bond.

[0025] Preferably, m is 3 or 4; more preferably, m is 4.

[0026] Preferably, when t=0, Cn1 and Cn2 are When t=1, Cn1 is Cn2 is Or Cn1 is Cn2 is

[0027] Preferably, when t=0, Cn1 and Cn2 are For example, when t = 0, Cn1 and Cn2 are At this time, Fuc* can correspond to the D series structure described in the embodiment. For example, when t=0, Cn1 and Cn2 are Sp1 is SP2 is n=1; SP3 is n'=1;Sp4 is When n”=1, the structure corresponding to Fuc* is D-P1 / P1-P1-GGFG-Dxd.

[0028] Preferably, when t=1, Cn1 is Cn2 is Or Cn1 is Cn2 is For example, when t = 1, Cn1 is Cn2 is Or Cn1 is At this time, Fuc* can correspond to the N series structure described in the embodiment. For example, when t=0, Cn1 and Cn2 are Sp1 is SP2 is n=1; SP3 is n'=1;Sp4 is When n”=1, the structure corresponding to Fuc* is N-P1 / P1-P1-GGFG-Dxd.

[0029] Preferably, the Fuc* can be selected from the following structures:

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] Further preferably, the structure of the Fuc* can be selected from the following structures:

[0036]

[0037]

[0038]

[0039] More preferably, the structure of the Fuc* can be selected from the following structures:

[0040]

[0041] On the other hand, in the conjugated drug of the present invention, X is trastuzumab or rituximab. Preferably, X comprises the light chain sequence shown in sequence 1 and the heavy chain sequence shown in sequence 2 (trastuzumab), or comprises the light chain sequence shown in sequence 3 and the heavy chain sequence shown in sequence 4 (rituximab). The C-terminal lysine of the heavy chain sequence shown in sequence 2 and the heavy chain sequence shown in sequence 4 may fall off by itself during the fermentation and assembly of the antibody, and the sequence with the C-terminal lysine still belongs to sequence 2 and sequence 4.

[0042] On the other hand, the conjugated drug of the present invention can be a mixture. Preferably, the proportion of the conjugated drug with m being 4 in the conjugated drug mixture is >75%. That is, the proportion of the conjugate of DAR8-DXd in the conjugated mixture is >75%. For example, the DAR value of Fuc* in the conjugated mixture (the average ratio of Fuc* to the antibody or fusion protein with Fc fragment) is >3, that is, the DAR value of Dxd in the conjugated mixture (the average ratio of Dxd to the antibody or fusion protein with Fc fragment) is >6.

[0043] On the other hand, the conjugated drug of the present invention is a drug for treating or alleviating cancer, characterized in that the drug for treating or alleviating cancer comprises all the conjugated drugs mentioned above in the specification.

[0044] On the other hand, the present invention provides a method for preparing the above-mentioned antibody-drug conjugate, comprising the following steps: a) treating a compound having the following formula II The structured protein reacts with Fuc*-Q in the presence of fucosyltransferase to obtain the target conjugated drug, wherein Q in the Fuc*-Q is a diphosphate ribonucleoside selected from the group consisting of guanosine diphosphate (GDP), uridine diphosphate (UDP) and / or cytidine diphosphate (CDP). Preferably, Fuc*-Q is Fuc*-GDP (also represented by GDP-Fuc*).

[0045] On the other hand, the present invention provides a method for preparing the protein of formula II, characterized in that the protein of formula II The protein of the structure shown in the figure is prepared by the following steps Pre-a): a protein having the following formula III The structured protein is reacted with UDP-Gal (UDP-galactose) in the presence of β1,4 galactosyltransferase or a functional variant thereof, wherein m' is 0-4.

[0046] In certain embodiments of the method of the present application, the fucosyltransferase is α1,3-fucosyltransferase or a functional variant or fragment thereof. Preferably, the fucosyltransferase is derived from bacteria. Preferably, the fucosyltransferase is derived from Helicobacter pylori. Preferably, the fucosyltransferase is derived from Helicobacter pylori 26695.

[0047] In certain embodiments of the methods of the present application, the fucosyltransferase comprises a catalytically active region and 1 to 4 heptapeptide repeats, the catalytically active region comprises the amino acid sequence shown in Sequence 5, and the heptapeptide repeats comprise the amino acid sequence shown in Sequence 6. Preferably, the fucosyltransferase comprises a catalytically active region and 2 heptapeptide repeats. Preferably, the fucosyltransferase comprises the amino acid sequence shown in Sequence 7. In certain embodiments of the methods of the present application, the fucosyltransferase comprises the amino acid sequence shown in Sequence 7 and a tag sequence. Preferably, the fucosyltransferase comprises the amino acid sequence shown in Sequence 8.

[0048] In certain embodiments of the method of the present application, step a) is characterized in that the reaction temperature is 0-37° C. Preferably, the reaction temperature is 25-37° C. Preferably, the reaction temperature is 25-30° C.

[0049] In certain embodiments of the method of the present application, step a) is characterized in that the reaction pH is 6-9. Preferably, the reaction pH is 7-8.

[0050] In certain embodiments of the method of the present application, step a) is characterized in that it is necessary to 2+ under the conditions of existence.

[0051] In certain embodiments of the methods of the present application, β-1,4 galactosyltransferase and its functional variants comprise bovine β1,4-galactosyltransferase, human β1,4-galactosyltransferase or its functional variants or fragments. Preferably, β-1,4 galactosyltransferase comprises a wild-type human β(1,4)-GalT1 fragment and a Y285L mutated human β(1,4)-GalT1. Preferably, β-1,4 galactosyltransferase comprises the amino acid sequence shown in Sequence 9. Preferably, the β-1,4 galactosyltransferase comprises the β-1,4 galactosyltransferase and the tag sequence described in the present application. Preferably, the β-1,4 galactosyltransferase comprises the amino acid sequence shown in any one of Sequences 9 and 10.

[0052] In certain embodiments of the method of the present application, step Pre-a) is characterized in that the reaction temperature is 0-37° C. Preferably, the reaction temperature is 25-37° C.

[0053] In certain embodiments of the method of the present application, step Pre-a) is characterized in that the reaction pH is 5-9. Preferably, the reaction pH is 5.5-8.

[0054] In certain embodiments of the method of the present application, step Pre-a) is characterized in that it is necessary to 2+ Reaction under the conditions.

[0055] On the other hand, the present invention provides a method for preparing the conjugated drug mixture, characterized in that it comprises the following steps: pre-a) containing a structure shown in the following formula III: The protein mixture is reacted with UDP-galactose in the presence of β1,4 galactosyltransferase or a functional variant thereof to obtain a protein having a structure shown in the following formula II: A protein mixture, m'=0-4, comprising formula II The proportion of the protein with the structure in the protein mixture is >75%; and a) reacting the protein obtained by the above reaction with Fuc*-Q in the presence of fucosyltransferase to obtain the target conjugated drug mixture, wherein the Fuc*-Q is a diphosphate ribonucleoside selected from the following group: guanosine diphosphate (GDP), uridine diphosphate (UDP) and / or cytidine diphosphate (CDP). The fucosyltransferase is α1,3-fucosyltransferase or its functional variant or fragment. Preferably, Fuc*-Q is Fuc*-GDP. Preferably, the fucosyltransferase is derived from Helicobacter pylori 26695.

[0056] In certain embodiments of the method of the present application, the fucosyltransferase comprises a catalytically active region and 1 to 4 heptapeptide repeats, the catalytically active region comprises the amino acid sequence shown in sequence 5, and the heptapeptide repeats comprise the amino acid sequence shown in sequence 6. Preferably, the fucosyltransferase comprises a catalytically active region and 2 heptapeptide repeats. Preferably, the fucosyltransferase comprises the amino acid sequence shown in sequence 7. In certain embodiments of the method of the present application, the fucosyltransferase comprises the amino acid sequence shown in sequence 7 and a tag sequence. Preferably, in certain embodiments of the method of the present application, step a) is characterized in that the reaction temperature is 0-37°C. Preferably, the reaction temperature is 25-37°C. Preferably, the reaction temperature is 25-30°C.

[0057] In certain embodiments of the method of the present application, step a) is characterized in that the reaction pH is 6-9. Preferably, the reaction pH is 7-8.

[0058] In certain embodiments of the method of the present application, step a) is characterized in that it is necessary to 2+ under the conditions of existence.

[0059] In certain embodiments of the methods of the present application, β-1,4 galactosyltransferase and its functional variants comprise bovine β1,4-galactosyltransferase, human β1,4-galactosyltransferase or its functional variants or fragments. Preferably, β-1,4 galactosyltransferase comprises a wild-type human β(1,4)-GalT1 fragment and a Y285L mutated human β(1,4)-GalT1. Preferably, β-1,4 galactosyltransferase comprises the amino acid sequence shown in any one of Sequence 9 and Sequence 10. Preferably, the β-1,4 galactosyltransferase comprises the β-1,4 galactosyltransferase and the tag sequence described in the present application. Preferably, the β-1,4 galactosyltransferase comprises the amino acid sequence shown in any one of Sequences 9, 10 and 11.

[0060] In certain embodiments of the method of the present application, step Pre-a) is characterized in that the reaction temperature is 0-37° C. Preferably, the reaction temperature is 25-37° C.

[0061] In certain embodiments of the method of the present application, step Pre-a) is characterized in that the reaction pH is 5-9. Preferably, the reaction pH is 5.5-8.

[0062] In certain embodiments of the method of the present application, step Pre-a) is characterized in that it is necessary to 2+ Reaction under the conditions.

[0063] In another aspect, a compound Fuc*-Q and its corresponding salt form, wherein the Fuc*-Q is selected from the following structures:

[0064]

[0065]

[0066]

[0067] Compared with the prior art, the present invention has the following beneficial effects: the antibody conjugate prepared by the present invention (such as trastuzumab-G2F-Fuc*) has better hydrophilicity, thermal stability and plasma stability than the antibody conjugate prepared by MC-GGFG-Dxd (such as DS-8201).

[0068] In the present application, the term "antibody" generally refers to a protein or antigen-binding fragment thereof that can recognize and bind to a specific antigen produced by the immune system. The term antibody is used in the present application in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., multispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. Antibodies also include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. In the present application. The term "antibody" generally includes complete antibodies, but also includes antibody fragments, such as antibody Fab fragments, (Fab')2, Fv fragments or Fc fragments from cracked antibodies, scFv-Fc fragments, minibodies, single-domain antibodies (also referred to as nanobodies, nanobodies), multispecific antibodies (diabodies), affibodies (affibodies) or scFv. In addition, the term "antibody" also includes antibodies and / or derivatives of antibodies engineered or genetically modified. In some embodiments, antibodies are referred to as immunoglobulins and include various classes and isotypes, such as IgA (IgA1 and IgA2), IgD, IgE, IgM and IgG (IgG1, IgG3 and IgG4), etc. In the present application, the term "antibody" may include polyclonal antibodies and monoclonal antibodies and functional fragments thereof. Antibodies include modified or derived antibody variants that retain the ability to specifically bind to epitopes. Antibodies can selectively bind to target antigens or epitopes. In the present application, antibodies can be from any source, such as mice or humans, including chimeric antibodies thereof, for example, antibodies can be humanized.

[0069] In the present application, the term "Fc region" generally refers to the C-terminal region of the heavy chain of an immunoglobulin, which can be produced by papain digestion of a complete antibody. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain and / or a hinge region. In the present application, the term "Fc region" includes any polypeptide (or a nucleic acid encoding such a polypeptide), regardless of its production method. As used herein, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially uniform antibody population, i.e., except for possible natural mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts, the individual antibodies constituting the population are identical.

[0070] In the present application, the term "Fc-fusion protein" generally refers to a protein consisting of the Fc domain of IgG transgenically linked to a peptide or protein of interest.

[0071] In the present application, the term "nucleotide" generally refers to a molecule consisting of a nucleobase, a pentose (ribose or 2-deoxyribose) and one, two or three phosphate groups. Without a phosphate group, the nucleobase and the sugar constitute a nucleoside. Therefore, nucleotides are also referred to as nucleoside monophosphates, nucleoside diphosphates or nucleoside triphosphates. The nucleobase can be adenine, guanine, cytosine, uracil or thymine. Examples of nucleotides include diphosphate ribonucleotides, such as uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP).

[0072] In this application, the terms "GlcNAc" or "N-acetylglucosamine" are used interchangeably and generally refer to the amide derivative of the monosaccharide glucose.

[0073] In the present application, the term "humanized antibody" generally refers to an antibody that contains some or all CDRs from non-human animals, and the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

[0074] In the present application, the terms "α1,3 glycosidic bond" and "β1,4 glycosidic bond" refer to the connection mode between sugars. For example, galactose can be connected to the C4 position of GlcNAc at its C1 position through a β1,4 glycosidic bond. For example, Fuc* can be connected to the C3 position of GlcNAc at its C1 position through an α1,3 glycosidic bond.

[0075] Unless otherwise specified herein or the context clearly indicates the contrary, the terms "a", "an", "the", "said", and similar terms used in the context of the present invention (especially in the context of the claims) should be understood to include both the singular and the plural.

[0076] In the present application, a "functional variant" of a parent polypeptide or protein generally has substantial or significant sequence identity or similarity to a parent polypeptide or protein, and the functional variant retains at least a portion of the function of the parent polypeptide or protein. For example, the functional variant of an enzyme retains enzymatic activity to a degree similar to the parent enzyme, to the same degree, or to a higher degree. With respect to a parent polypeptide or protein, a functional variant can be, for example, a sequence identity with it of about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more in amino acid sequence.

[0077] In the present application, "functional fragment" of a parent polypeptide or protein generally refers to following peptide or polypeptide (including but not limited to enzyme), it comprises at least 5 continuous amino acid residues, at least 10 continuous amino acid residues, at least 15 continuous amino acid residues, at least 20 continuous amino acid residues, at least 25 continuous amino acid residues, at least 40 continuous amino acid residues, at least 50 continuous amino acid residues, at least 60 continuous amino acid residues, at least 70 continuous amino acid residues, at least 80 continuous amino acid residues, at least 90 continuous amino acid residues, at least 100 continuous amino acid residues, at least 125 continuous amino acid residues, at least 150 continuous amino acid residues, at least 175 continuous amino acid residues, at least 200 continuous amino acid residues, at least 250 continuous amino acid residues or at least 350 continuous amino acid residues of a parent polypeptide or protein, wherein the functional fragment has at least a portion of the function of its parent polypeptide or protein. For example, "functional fragment" of a parent enzyme retains enzymatic activity to a degree similar to the parent enzyme, to the same degree or to a higher degree.

[0078] In the present application, antibodies or fusion proteins containing Fc fragments have different glycoforms due to differences in glycosylation of the Fc region. G0(F) 0,1 Type lacks two galactoses at the ends of the diantennary chain. G1(F) 0,1 There is a galactose at the end of the double antenna chain. 0,1 In G0F, G1F and G2F, the core fucose is linked to the core GlcNAc via an α1,6 glycosidic bond. Both G1F and G2F structures contain a disaccharide N-acetyllactosamine (LacNAc, Galβ1,4GlcNAc) unit. For example, antibody-G2F usually refers to an antibody with the G2F glycoform.

[0079]

[0080] In the present application, the term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partial or complete cure of a disease and / or side effects caused by the disease. "Treatment" as used in the present application encompasses any treatment of a disease in a mammal (particularly a human), and includes preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; inhibiting the disease, i.e., preventing its development; alleviating the disease, i.e., causing the regression of the disease.

[0081] Compared with the prior art, the present invention has the following beneficial effects:

[0082] The present invention provides an antibody-drug conjugate and a preparation method thereof. Through the preparation method of the present invention, a high DAR value (such as DAR8) protein (such as antibody) conjugate based on glycosyl conjugation and topoisomerase 1 inhibitor can be prepared site-specifically and efficiently. In addition, the antibody conjugate prepared by the present invention (such as trastuzumab-G2F-Fuc*) has better hydrophilicity, thermal stability and plasma stability than the antibody conjugate prepared by MC-GGFG-Dxd (such as DS-8201). BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The mass spectrometry characterization spectra of trastuzumab, trastuzumab-G2F, rituximab, and rituximab-G2F;

[0084] Figure 2 HIC analysis of trastuzumab-G2F after reaction with GDP-K-P2 / P4-P4-GGFG-Dxd and GDP-D-P2 / 4-P4-GGFG-Dxd for 8 h (A) and 24 h (B);

[0085] Figure 3 HIC analysis diagram after trastuzumab-G2F reacted with GDP-D-P2 / P8-P4-GGFG-Dxd, GDP-D-P2 / P4-P4-GGFG-Dxd, GDP-D-P1 / P2-P4-GGFG-Dxd and GDP-D-P1 / P1-P4-GGFG-Dxd for 24 hours;

[0086] Figure 4Figure 2 is the HIC analysis of trastuzumab-G2F reacting with GDP-D-P2 / P4-P1-GGFG-Dxd, GDP-D-P2 / P4-P2-GGFG-Dxd, and GDP-D-P2 / P4-P4-GGFG-Dxd for 24 hours (A); Figure 2 is the HIC analysis of trastuzumab-G2F reacting with GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-D-P1 / P1-P2-GGFG-Dxd, and GDP-D-P1 / P1-P4-GGFG-Dxd for 24 hours (B); -D-P1 / P2-P1-GGFG-Dxd, GDP-D-P1 / P2-P2-GGFG-Dxd, and GDP-D-P1 / P2-P4-GGFG-Dxd after 24h of reaction (C); HIC analysis of trastuzumab-G2F reacting with GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-D-P1 / P1-C2-GGFG-Dxd, GDP-D-P1 / P2-P1-GGFG-Dxd, and GDP-D-P1 / P2-C2-GGFG-Dxd for 8h and 24h, respectively (D)

[0087] Figure 5 HIC analysis of trastuzumab-G2F reacting with GDP-N-P1 / P1-P1-GGFG-Dxd, GDP-N-P1 / P2-P1-GGFG-Dxd, GDP-D-P1 / P1-P1-GGFG-Dxd and GDP-D-P1 / P2-P1-GGFG-Dxd for 24 h (A) and 8 h (B);

[0088] Figure 6 For trastuzumab-G2F-D-P2 / P8-P4-GGFG-Dxd, trastuzumab-G2F-D-P2 / P2-P2-GGFG-Dxd, trastuzumab-G2F-D-P1 / P2-P2-GGFG-Dxd, trastuzumab-G2F-D-P1 / P1-P2-GGFG-Dxd, trastuzumab-G2F- HIC analysis of D-P1 / P2-P1-GGFG-Dxd, trastuzumab-G2F-D-P1 / P1-P1-GGFG-Dxd, trastuzumab-G2F-N-P1 / P2-P1-GGFG-Dxd, trastuzumab-G2F-N-P1 / P1-P1-GGFG-Dxd, DS-8201 and trastuzumab;

[0089] Figure 7for rituximab and GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-D-P1 / P1-C2-GGFG-Dxd, GDP-D-P1 / P1-P2-GGFG-Dxd, GDP-D-P1 / P1-P4-GGFG-Dxd, GDP-D-P2 / P2-P4-GGFG-Dxd, GDP-D-P2 / P4-P4-GGFG-Dxd, GDP-D-P2 / P8-P4-GGFG-Dxd, GDP-D-P2 / P2-P2-GGFG-Dxd, and GDP-N- HIC analysis of P1 / P1-P1-GGFG-Dxd after 24h reaction and rituximab control (A); HIC analysis of rituximab after 8h reaction with GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-D-P1 / P1-C2-GGFG-Dxd, GDP-N-P1 / P1-P1-GGFG-Dxd, GDP-D-P2 / P2-P4-GGFG-Dxd, GDP-D-P2 / P4-P4-GGFG-Dxd and GDP-K-P2 / P4-P4-GGFG-Dxd (B);

[0090] Figure 8 Mass spectrometry analysis of trastuzumab-G2F-D-P1 / P1-P1-GGFG-Dxd, trastuzumab-G2F-N-P1 / P1-P1-GGFG-Dxd, trastuzumab-G2F-D-P2 / P2-P2-GGFG-Dxd and trastuzumab-G2F-D-P2 / P4-P4-GGFG-Dxd prepared by the “one-pot method”;

[0091] Fig. 9 SEC analysis spectra of DS-8201, trastuzumab-G2F-D-P2 / P2-P2-GGFG-Dxd, trastuzumab-G2F-D-P1 / P1-P1-GGFG-Dxd, trastuzumab-G2F-N-P1 / P1-P1-GGFG-Dxd and trastuzumab-G2F-D-P2 / P4-P4-GGFG-Dxd before and after heat treatment

[0092] Fig.10 This is a graph of the in vitro human plasma stability HIC analysis of DS-8201, trastuzumab-G2F-D-P2 / P2-P2-GGFG-Dxd, trastuzumab-G2F-D-P1 / P1-P1-GGFG-Dxd, and trastuzumab-G2F-N-P1 / P1-P1-GGFG-Dxd.

[0093] Fig.11The in vitro cell killing activities of different doses of DS-8201, trastuzumab-G2F-D-P1 / P1-P1-GGFG-Dxd, trastuzumab-G2F-N-P1 / P1-P1-GGFG-Dxd and trastuzumab-G2F-D-P2 / P2-P2-GGFG-Dxd against NCI-N87 (Her2+) cell line, BT-474 (Her2+) cell line and MDA-MB-231 (Her2-) cell line, respectively.

[0094] Fig.12 The in vivo tumor inhibitory effects of different doses of DS-8201, trastuzumab, trastuzumab-G2F-D-P1 / P1-P1-GGFG-Dxd and trastuzumab-G2F-N-P1 / P1-P1-GGFG-Dxd on the NCI-N87 (Her2+) xenograft model in nude mice (BALB / c). DETAILED DESCRIPTION

[0095] Basic Experimental Examples

[0096] Unless otherwise specified, the chemical reagents used in this experiment were purchased from J&K, MacLean, Aladdin or Aicon.

[0097] Experimental Example 1 GDP-D-P2 / P4

[0098]

[0099] Compound 2 (2.2 g, 12.63 mmol) was dissolved in 10 mL of dimethyl sulfoxide, and 1.75 mL of triethylamine was added under stirring; Compound 1 (1.41 g, 6.32 mmol) was dissolved in 10 mL of dimethyl sulfoxide, and added dropwise to the above system for 2 hours, and the reaction was allowed to proceed overnight at room temperature. The mixture was separated and purified by semi-preparative HPLC to obtain a light yellow oil 3 (920.5 mg, 45.9%). HRMS (ESI + ) Theoretical value C 13 H 27 N5O4(M+H + )318.2136, measured value 318.2128.

[0100] Compound 3 (190.3 mg, 0.6 mmol) was dissolved in 3 mL of dimethyl sulfoxide, and 0.42 mL of triethylamine and NHS-PEG4-Az (azide-tetraethylene glycol-succinimide ester) (232.9 mg, 0.6 mmol) (Xi'an Kangfuno) dissolved in 3 mL of dimethyl sulfoxide were added under stirring, and the reaction was carried out at room temperature for about 5 h. The mixture was separated and purified by semi-preparative HPLC to obtain a white solid 4 (182 mg, 51.4%). HRMS (ESI -)C 24 H 46 N8O9(MH + )Theoretical value is 589.3315, measured value is 589.3312.

[0101] The white solid 4 (182 mg, 0.31 mmol) was dissolved in 10 mL of dichloromethane, and 10 mL of trifluoroacetic acid was added under stirring. The reaction was carried out at room temperature for about 1 h and monitored by TLC. The solvent was spin-dried without further purification to obtain the crude product 5. HRMS (ESI - )C 19 H 38 N8O7(MH + ) Theoretical value 489.2791, measured value 489.2784. The crude product 5 was dissolved in 10 mL of tetrahydrofuran, and 43 μL of triethylamine and succinic anhydride (93.1 mg, 0.93 mmol) were added under stirring. The reaction was allowed to proceed overnight at room temperature, and the product was separated and purified by semi-preparative HPLC to obtain a white solid 6 (81.1 mg, 44.3% for two steps). HRMS (ESI - )C 23 H 42 N8O 10 (MH + )Theoretical value is 589.2951, measured value is 589.2943.

[0102] Compound 6 (81.1 mg, 0.14 mmol) was dissolved in 5 mL of dichloromethane, and NHS (N-hydroxysuccinimide) (48.3 mg, 0.42 mmol) and EDC·HCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) (161.0 mg, 0.84 mmol) were added under stirring. The reaction was carried out at room temperature for about 3 h, monitored by TLC, and separated and purified by semi-preparative high performance liquid chromatography to obtain a white solid 7 (85.0 mg, 88.3%). HRMS (ESI - )C 27 H 45 N9O 12 (M+Cl - )Theoretical value: 722.2882, measured value: 722.2887.

[0103] GFAm was synthesized according to the method reported in patent CN 115916802A.

[0104] Dissolve GFAm (72.5 mg, 0.12 mmol) in 2.5 mL of water, add sodium bicarbonate (20.2 mg, 0.24 mmol) and compound 7 (85.0 mg, 0.12 mmol) dissolved in tetrahydrofuran. Reaction was carried out at room temperature for about 5 h, and TLC was monitored. Semi-preparative HPLC was used to separate and purify the product to obtain white solid GDP-D-P2 / P4 (67.8 mg, 48.0%). HRMS (ESI - )C 39 H 66 N 14 O 24 P2(M-2H + ) / 2Theoretical value is 587.1852, and the measured value is 587.1865.

[0105] Experimental Example 2 GDP-D-P1 / P1

[0106]

[0107] Compound 8 (520 mg, 4.0 mmol) was dissolved in 10 mL of dimethyl sulfoxide, and 556 μL of triethylamine was added under stirring; Compound 1 (446 mg, 2.0 mmol) was dissolved in 10 mL of dimethyl sulfoxide and added dropwise to the above system for 2 hours, and the reaction was allowed to proceed overnight at room temperature. Semi-preparative HPLC was used to separate and purify the product to obtain a light yellow oil 9 (169 mg, 30.9%).

[0108] Compound 9 (169 mg, 0.62 mmol) was dissolved in 5 mL of dimethyl sulfoxide, and 0.43 mL of triethylamine and NHS-PEG1-Az (azido-polyethylene glycol-succinimide ester) (190 mg, 0.744 mmol) (Xi'an Kangfuno) dissolved in 5 mL of dimethyl sulfoxide were added under stirring, and the reaction was allowed to proceed overnight at room temperature. The compound was separated and purified by semi-preparative high performance liquid chromatography to obtain a white solid 10 (173.7 mg, 67.6%).

[0109] Compound 10 (173.7 mg, 0.42 mmol) was dissolved in 10 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added under stirring. The reaction was allowed to proceed overnight at room temperature and monitored by TLC. The solvent was spin-dried without further purification to obtain crude product 11. Crude product 11 was dissolved in 6 mL of N,N-dimethylformamide, and succinic anhydride (120 mg, 1.2 mmol) was added under stirring. The reaction was allowed to proceed overnight at room temperature, and the mixture was separated and purified by semi-preparative HPLC to obtain white solid 12 (107 mg, 61.5% for two steps).

[0110] Compound 12 (107 mg, 0.26 mmol) was dissolved in 15 mL of dichloromethane, and NHS (90 mg, 0.78 mmol) and EDC·HCl (299 mg, 1.56 mmol) were added under stirring. The reaction was carried out at room temperature for about 1 h, monitored by TLC, extracted three times with dichloromethane, and the organic layer was taken, dried over anhydrous sodium sulfate, and spin-dried to obtain crude product 13. 6.25 mL of water, sodium bicarbonate aqueous solution (2.5 mL, 200 mM), GFAm aqueous solution (3.75 mL, 100 mM), tetrahydrofuran 7.5 mL, and compound 13 (dissolved in 5 mL of tetrahydrofuran) were mixed in sequence and reacted overnight at room temperature. The white solid GDP-D-P1 / P1 (37.1 mg, 14.3% two steps) was obtained by separation and purification by semi-preparative HPLC. HRMS (ESI - )C 31 H 50 N 14 O 20 P2(MH + )Theoretical value is 999.2728, measured value is 999.2738.

[0111] Experimental Example 3 GDP-D-P1 / P2

[0112]

[0113] Compound 9 (70.5 mg, 0.26 mmol) was dissolved in 5 mL of dimethyl sulfoxide, and 0.18 mL of triethylamine and NHS-PEG2-Az (azido-diethylene glycol-succinimide ester) (117 mg, 0.39 mmol) (Xi'an Kangfuno) were added under stirring, and the reaction was allowed to proceed overnight at room temperature. The compound was separated and purified by semi-preparative HPLC to obtain a white solid 14 (71.7 mg, 60.2%).

[0114] Compound 14 (71.7 mg, 0.16 mmol) was dissolved in 5 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added under stirring. The reaction was carried out at room temperature for about 1.5 h and monitored by TLC. The solvent was spin-dried without further purification to obtain crude product 15. Crude product 15 was dissolved in 3 mL of N,N-dimethylformamide, and succinic anhydride (45.7 mg, 0.46 mmol) was added under stirring. The reaction was carried out at room temperature overnight, and the mixture was separated and purified by semi-preparative high performance liquid chromatography to obtain white solid 16 (52.9 mg, 72.1% two steps).

[0115] Compound 16 (52.9 mg, 0.12 mmol) was dissolved in 10 mL of dichloromethane, and NHS (39.7 mg, 0.35 mmol) and EDC·HCl (132.3 mg, 0.69 mmol) were added under stirring. The mixture was reacted overnight at room temperature, monitored by TLC, extracted three times with dichloromethane, and the organic layer was taken, dried over anhydrous sodium sulfate, and spin-dried to obtain crude product 17. 0.005 mL of water, aqueous sodium bicarbonate solution (1.15 mL, 200 mM), aqueous GFAm solution (1.73 mL, 100 mM), 0.575 mL of tetrahydrofuran, and crude product 17 (dissolved in 2.3 mL of tetrahydrofuran) were mixed in sequence and reacted overnight at room temperature. The mixture was separated and purified by semi-preparative HPLC to obtain white solid GDP-D-P1 / P2 (22 mg, 17.6% in two steps). HRMS (ESI - )C 33 H 54 N 14 O 21 P2(MH + )Theoretical value is 1043.2990, measured value is 1043.3069.

[0116] Experimental Example 4 GDP-D-P2 / P8

[0117]

[0118] Compound 2 (63.4 mg, 0.2 mmol) was dissolved in 5 mL of dimethyl sulfoxide, and 0.14 mL of triethylamine and NHS-PEG8-Az (azido-octapolyethylene glycol-succinimide ester) (112.8 mg, 0.2 mmol) (Xi'an Kangfuno) were added under stirring, and the reaction was allowed to proceed overnight at room temperature. The compound was separated and purified by semi-preparative HPLC to obtain a white solid 18 (102.6 mg, 67.0%).

[0119] Compound 18 (102.6 mg, 0.134 mmol) was dissolved in 10 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added under stirring. The reaction was carried out at room temperature for about 3 h and monitored by TLC. The solvent was spin-dried without further purification to obtain crude product 19. Crude product 19 was dissolved in 5 mL of N,N-dimethylformamide, and succinic anhydride (32.1 mg, 0.32 mmol) was added under stirring. The reaction was carried out at room temperature overnight, and the mixture was separated and purified by semi-preparative high performance liquid chromatography to obtain a white solid 20 (67.7 mg, 44.2% two steps).

[0120] Compound 20 (67.7 mg, 0.09 mmol) was dissolved in 5 mL of dichloromethane, and NHS (30.4 mg, 0.26 mmol) and EDC·HCl (101.2 mg, 0.53 mmol) were added under stirring. The reaction was carried out at room temperature for about 2 h, monitored by TLC, extracted three times with dichloromethane, and the organic layer was taken, dried over anhydrous sodium sulfate, and spin-dried to obtain crude product 21. 5 mL of water, sodium bicarbonate aqueous solution (0.9 mL, 200 mM), GFAm aqueous solution (1.8 mL, 100 mM), N,N-dimethylformamide 5 mL, and crude product 21 (dissolved in 1.8 mL of tetrahydrofuran) were mixed in sequence and reacted overnight at room temperature. The white solid GDP-D-P2 / P8 (62.4 mg, 51.3% two steps) was obtained by separation and purification by semi-preparative HPLC. HRMS (ESI - )C 47 H 82 N 14 O 28 P2(MH + )Theoretical value is 1351.4825, measured value is 1051.5030.

[0121] Experimental Example 5 GDP-D-P2 / P2

[0122]

[0123] Compound 3 (71 mg, 0.28 mmol) was dissolved in 5 mL of dimethyl sulfoxide, and 0.195 mL of triethylamine and NHS-PEG2-Az (azido-diethylene glycol-succinimide ester) (102 mg, 0.34 mmol) (Xi'an Kangfuno) were added under stirring, and the reaction was allowed to proceed overnight at room temperature. The compound was separated and purified by semi-preparative HPLC to obtain a white solid 22 (111.6 mg, 79.4%).

[0124] Compound 4 (84.9 mg, 0.17 mmol) was dissolved in 5 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added under stirring. The reaction was carried out at room temperature for about 2 h and monitored by TLC. The solvent was spin-dried without further purification to obtain crude product 23. Crude product 23 was dissolved in 5 mL of N,N-dimethylformamide, and succinic anhydride (60 mg, 0.6 mmol) was added under stirring. The reaction was carried out at room temperature overnight, and the mixture was separated and purified by semi-preparative high performance liquid chromatography to obtain white solid 24 (70.1 mg, 82.1% two steps).

[0125] Compound 24 (70.1 mg, 0.14 mmol) was dissolved in 10 mL of dichloromethane, and NHS (48.5 mg, 0.42 mmol) and EDC·HCl (160.1 mg, 0.84 mmol) were added under stirring. The reaction was carried out at room temperature for about 3.5 h, monitored by TLC, extracted three times with dichloromethane, and the organic layer was taken, dried over anhydrous sodium sulfate, and spin-dried to obtain crude product 25. 2.75 mL of water, sodium bicarbonate aqueous solution (1.1 mL, 200 mM), GFAm aqueous solution (1.65 mL, 100 mM), tetrahydrofuran 3.3 mL, and crude product 25 (dissolved in 2.2 mL of tetrahydrofuran) were mixed in sequence and reacted overnight at room temperature. The white solid GDP-D-P2 / P2 (23 mg, 15.1% two steps) was obtained by separation and purification by semi-preparative HPLC. HRMS (ESI - )C 35 H 58 N 14 O 22 P2(MH + )Theoretical value is 1087.3253, measured value is 1087.3302.

[0126] Experimental Example 6 GDP-N-P1 / P1

[0127]

[0128] Compound 8 (260 mg, 2.0 mmol) was dissolved in 5 mL of dimethyl sulfoxide, and 0.278 mL of triethylamine was added under stirring; Compound 1 (193 mg, 1.0 mmol) was dissolved in 5 mL of dimethyl sulfoxide and added dropwise to the above system for 2 hours, and the reaction was allowed to proceed overnight at room temperature. The mixture was separated and purified by semi-preparative HPLC to obtain a light yellow oil (119.2 mg, 49.0%).

[0129] Compound 27 (119.2 mg, 0.49 mmol) was dissolved in 6 mL of N,N-dimethylformamide, and succinic anhydride (147 mg, 1.47 mmol) was added under stirring. The mixture was reacted at room temperature overnight, and separated and purified by semi-preparative HPLC to obtain a white solid 28 (149 mg, 88.6%).

[0130] Compound 28 (149 mg, 0.43 mmol) was dissolved in 7 mL of dichloromethane, and NHS (148 mg, 1.29 mmol) and EDC·HCl (493 mg, 5.58 mmol) were added under stirring. The mixture was reacted overnight at room temperature, monitored by TLC, extracted three times with dichloromethane, and the organic layer was taken, dried over anhydrous sodium sulfate, and spin-dried to obtain crude product 29. 11.6 mL of water, aqueous sodium bicarbonate solution (4 mL, 200 mM), aqueous GFAm solution (4.4 mL, 100 mM), 12 mL of tetrahydrofuran, and crude product 29 (dissolved in 8 mL of tetrahydrofuran) were mixed in sequence and reacted overnight at room temperature. The mixture was separated and purified by semi-preparative HPLC to obtain white solid GDP-N-P1 / P1 (104.2 mg, 26.1% two steps). HRMS (ESI - )C 28 H 45 N 13 O 19 P2(MH + )Theoretical value is 928.2357, measured value is 928.2375.

[0131] Experimental Example 7 GDP-N-P1 / P2

[0132]

[0133] Compound 8 (260 mg, 2.0 mmol) was dissolved in 5 mL of dimethyl sulfoxide, and 0.278 mL of triethylamine was added under stirring; Compound 1 (237 mg, 1.0 mmol) was dissolved in 5 mL of DMSO and added dropwise to the above system for 2 hours, and reacted at room temperature overnight. The mixture was separated and purified by semi-preparative HPLC to obtain a light yellow oil 31 (120 mg, 41.8%).

[0134] Compound 31 (120 mg, 0.42 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and succinic anhydride (126 mg, 1.26 mmol) was added under stirring. The mixture was reacted at room temperature overnight, and separated and purified by semi-preparative HPLC to obtain a white solid 32 (147.3 mg, 90.6%).

[0135] Compound 32 (147.3 mg, 0.38 mmol) was dissolved in 15 mL of dichloromethane, and NHS (131.2 mg, 1.14 mmol) and EDC·HCl (437.1 mg, 2.28 mmol) were added under stirring. The mixture was reacted overnight at room temperature, monitored by TLC, extracted three times with dichloromethane, and the organic layer was taken, dried over anhydrous sodium sulfate, and spin-dried to obtain crude product 33. 11 mL of water, aqueous sodium bicarbonate solution (3.8 mL, 200 mM), aqueous GFAm solution (4.18 mL, 100 mM), 11.4 mL of tetrahydrofuran, and crude product 33 (dissolved in 7.6 mL of tetrahydrofuran) were mixed in sequence and reacted overnight at room temperature. The mixture was separated and purified by semi-preparative HPLC to obtain white solid GDP-N-P1 / P2 (49.4 mg, 13.4% in two steps). HRMS (ESI - )C 30 H 49 N 13 O 20 P2(MH + )Theoretical value is 972.2619, measured value is 972.2800.

[0136] Experimental Example 8 GDP-K-P2 / P4

[0137]

[0138] Compound 34 (212 mg, 0.5 mmol) was dissolved in 10 mL of N,N-dimethylformamide, and 0.261 mL of N,N-diisopropylethylamine and NHS-PEG4-Az (azide-tetraethylene glycol-succinimide ester) (233 mg, 0.6 mmol) were added under stirring. The mixture was reacted at room temperature overnight, and separated and purified by semi-preparative HPLC to obtain a white solid 35 (176 mg, 50.5%).

[0139] Compound 35 (176 mg, 0.25 mmol) was dissolved in 9 mL of N,N-dimethylformamide, and 1.1 mL of piperidine was added under stirring. The mixture was reacted at room temperature for about 3 h and monitored by TLC. The mixture was separated and purified by semi-preparative HPLC to obtain a white solid 36 (91.9 mg, 77.3%).

[0140] Compound 36 (91.9 mg, 0.2 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and 0.084 mL of triethylamine and NHS-PEG2-Az (azido-diethylene glycol-succinimide ester) (72 mg, 0.24 mmol) were added under stirring. The mixture was reacted at room temperature overnight, and separated and purified by semi-preparative HPLC to obtain a white solid 37 (108.6 mg, 82.2%).

[0141] Compound 37 (108.6 mg, 0.16 mmol) was dissolved in 7 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid was added under stirring. The mixture was reacted at room temperature overnight, and the solvent was dried by spin drying without further purification to obtain crude product 38. Crude product 38 was dissolved in 7 mL of dichloromethane, and NHS (55.2 mg, 0.48 mmol) and EDC·HCl (184 mg, 0.96 mmol) were added under stirring. The mixture was reacted overnight at room temperature, monitored by TLC, extracted three times with dichloromethane, and the organic layer was taken, dried over anhydrous sodium sulfate, and dried by spin drying to obtain crude product 39. 4.35 mL of water, sodium bicarbonate aqueous solution (1.5 mL, 200 mM), GFAm aqueous solution (1.65 mL, 100 mM), tetrahydrofuran 4.5 mL, and crude product 39 (dissolved in 3 mL of tetrahydrofuran) were mixed in sequence and reacted overnight at room temperature. Semi-preparative HPLC separation and purification gave GDP-K-P2 / P4 (87.2 mg, 45.8% in three steps) as a white solid. HRMS (ESI - )C 40 H 68 N 14 O 24 P2(MH + )Theoretical value is 1189.3933, measured value is 1189.4068.

[0142] Experimental Example 9 Prop-PEG4-GGFG-Dxd

[0143]

[0144] The synthesis of GGFG-COOH refers to patent EP3677589A1.

[0145] Prop-PEG4-GGFG-COOH. To a solution of GGFG-COOH dissolved in 5 mL of N,N-dimethylformamide (98.4 mg, 0.23 mmol), add 0.2 mL of N,N-diisopropylethylamine and Prop-PEG4-OSu (propynyl-tetraethylene glycol-acrylic acid succinimide ester) (Xi'an Kangfuno) (99.6 mg, 0.28 mmol). Stir overnight at room temperature and monitor by thin layer chromatography. Separate and purify by semi-preparative HPLC to obtain a white solid product (114.8 mg, 75%). HRMS (ESI - )C 30 H 43 N5O 12 (MH + )Theoretical value is 664.2835, measured value is 664.2808.

[0146] Prop-PEG4-GGFG-Dxd. To a solution of Prop-PEG4-GGFG-COOH dissolved in 5 mL of N,N-dimethylformamide (66.6 mg, 0.1 mmol), 0.1 mL of N,N-diisopropylethylamine, exitecan mesylate (Zhejiang Huida Biological) (53.1 mg, 0.1 mmol) and PyBOP (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (104.9 mg, 0.2 mmol) were added. The reaction was stirred at room temperature for 2 h and monitored by thin layer chromatography. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (70.6 mg, 65%). HRMS (ESI + )C 54 H 63 F8O 15 (M+Na + )Theoretical value is 1105.4289, measured value is 1105.4255.

[0147] Experimental Example 10 Prop-PEG2-GGFG-Dxd

[0148]

[0149] Prop-PEG2-GGFG-COOH. To 4mL N, N-dimethylformamide dissolved GGFG-COOH solution (80mg, 0.19mmol), add 0.098mL N, N-diisopropylethylamine and Prop-PEG2-OSu (propynyl-diethylene glycol-acrylic acid succinimide ester) (Xi'an Kangfuno) (59mg, 0.22mmol). Stir overnight at room temperature and monitor by thin layer chromatography. Separate and purify by semi-preparative high performance liquid chromatography to obtain a white solid product (58mg, 52.9%).

[0150] Prop-PEG2-GGFG-Dxd. To a solution of Prop-PEG2-GGFG-COOH (58 mg, 0.1 mmol) dissolved in 4 mL of N,N-dimethylformamide was added 0.079 mL of N,N-diisopropylethylamine, exitecan mesylate (60 mg, 0.14 mmol) and PyBOP (117 mg, 0.22 mmol). Stir overnight at room temperature and monitor by thin layer chromatography. Separate and purify by semi-preparative HPLC to obtain a light yellow solid product (68 mg, 68.4%). HRMS (ESI + )C 50 H 55 F8O 13 (M+Na +)Theoretical value is 995.3945, measured value is 995.4025.

[0151] Experimental Example 11 Prop-PEG1-GGFG-Dxd

[0152]

[0153] Prop-PEG1-GGFG-COOH. GGFG-COOH (196.7 mg, 0.465 mmol), N,N-dimethylformamide (10 mL), Prop-PEG1-OSu (propynyl-polyethylene glycol-acrylic acid succinimide ester) (Xi'an Kangfuno) (157 mg, 0.697 mmol) and N,N-diisopropylethylamine (243 μL, 1.394 mmol) were added to the reaction bottle in sequence. Stir overnight at room temperature, separate and purify by semi-preparative high performance liquid chromatography to obtain an oily product (61 mg, 23.7%).

[0154] Prop-PEG1-GGFG-Dxd. Prop-PEG1-GGFG-COOH (61 mg, 114 μmol), exitecan mesylate (60 mg, 114 μmol), PyBOP (119 mg, 228 μmol), N,N-dimethylformamide (10 mL) and N,N-diisopropylethylamine (81 μL, 456 μmol) were added to the reaction bottle in sequence. Stir overnight at room temperature and separate and purify by semi-preparative HPLC to obtain a yellow oily product (56 mg, 51.4%). MS (ESI + ):C 48 H 52 F8O 12 (M+H + )Theoretical value is 951.4, measured value is 951.6.

[0155] Experimental Example 12 Prop-C2-GGFG-Dxd

[0156]

[0157] Prop-C2-GGFG-COOH. GGFG-COOH (80.7 mg, 0.191 mmol), Prop-C2-OSu (5-hexynoic acid-succinimide ester) (Xi'an Kangfuno) (60 mg, 0.287 mmol), N, N-dimethylformamide (5 mL) and N, N-diisopropylethylamine (100 μL, 0.574 mmol) were added to the reaction bottle in sequence. Stir overnight at room temperature, separate and purify by semi-preparative high performance liquid chromatography to obtain an oily product (36 mg, 36.5%).

[0158] Prop-C2-GGFG-Dxd. Prop-C2-GGFG-COOH (36 mg, 69.6 μmol), exitecan mesylate (37 mg, 69.6 μmol), PyBOP (72 mg, 139.2 μmol), N,N-dimethylformamide (4 mL) and N,N-diisopropylethylamine (48 μL, 278.4 μmol) were added to the reaction bottle in sequence. Stir overnight at room temperature, separate and purify by semi-preparative HPLC to obtain a yellow-brown oily product (44 mg, 58.4%). MS (ESI + )C 48 H 52 F8O 11 (M+H + )Theoretical value is 935.4, measured value is 935.5.

[0159] Experimental Example 13 GDP-D-P2 / P4-P4-GGFG-Dxd

[0160]

[0161] To 300 μL GDP-D-P2 / P4 (25 mM aqueous solution) were added 1050 μL water and 1140 μL dimethyl sulfoxide, 15 μL copper sulfate (100 mM aqueous solution), 60 μL BTTP (4-[[Bis[[1-(1,1-dimethylethyl)-1H-1,2,3-triazol-4-yl]methyl]amino]methyl]-1H-1,2,3-triazol-1-propanol 3-(4-((Bis((1-(tert-butyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propan-1-ol) (50 mM aqueous solution), 360 μL Prop-PEG4-GGFG-Dxd (50 mM aqueous solution) in sequence. DMSO solution) and 75 μL sodium ascorbate (100 mM aqueous solution), stirred at room temperature for 5 h, monitored by TLC, and then 2 mM BCS (2,9-dimethyl-4,7-diphenyl-1,10-o-phenanthroline disulfonic acid disodium salt) was added to quench the reaction. The product was separated and purified by semi-preparative HPLC to obtain a white solid product (16.8 mg, 67.6%). HRMS (ESI - )C 147 H 192 F2N 30 O 54 P2(M-2H + ) / 2theoretical value 1670.6283, theoretical value 1670.6271.

[0162] Experimental Example 14 GDP-D-P2 / P4-P1-GGFG-Dxd

[0163]

[0164] To 50 μL GDP-D-P2 / P4 (50 mM aqueous solution), 520 μL water and 750 μL dimethyl sulfoxide, 20 μL copper sulfate (50 mM aqueous solution), 40 μL TBTA (tri[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) (50 mM dimethyl sulfoxide solution), 600 μL Prop-PEG1-GGFG-Dxd (12.5 mM dimethyl sulfoxide solution) and 100 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (4.3 mg, 55.9%). HRMS (ESI - )C 135 H 168 F2N 30 O 48 P2(M-2H + ) / 2theoretical value 1538.0479, theoretical value 1538.0491.

[0165] Experimental Example 15 GDP-D-P2 / P4-C2-GGFG-Dxd

[0166]

[0167] To 100 μL GDP-D-P2 / P4 (50 mM aqueous solution), 2 mL of water and 2 mL of dimethyl sulfoxide, 96 μL of copper sulfate (50 mM aqueous solution), 192 μL of TBTA (50 mM dimethyl sulfoxide solution), 960 μL of Prop-C2-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 480 μL of sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (8.9 mg, 58.5%). HRMS (ESI - )C 135 H 168 F2N 30 O 46 P2(M-2H + ) / 2theoretical value 1521.5513, theoretical value 1521.5533.

[0168] Experimental Example 16 GDP-D-P2 / P4-P2-GGFG-Dxd

[0169]

[0170] To 50 μL GDP-D-P2 / P4 (50 mM aqueous solution), 520 μL water and 750 μL dimethyl sulfoxide, 20 μL copper sulfate (50 mM aqueous solution), 40 μL TBTA (50 mM aqueous solution), 300 μL Prop-PEG1-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 100 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (2.7 mg, 34.1%). HRMS (ESI - )C 139 H 176 F2N 30 O 50 P2(M-2H + ) / 2theoretical value 1582.0741, theoretical value 1582.0711.

[0171] Experimental Example 17 GDP-D-P1 / P1-P4-GGFG-Dxd

[0172]

[0173] To 50 μL GDP-D-P1 / P1 (50 mM aqueous solution), 515 μL water and 750 μL dimethyl sulfoxide, 10 μL copper sulfate (50 mM aqueous solution), 20 μL TBTA (50 mM aqueous solution), 300 μL Prop-PEG4-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 50 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (3.8 mg, 48.0%). HRMS (ESI - )C 139 H 176 F2N 30 O 50 P2(M-2H + ) / 2theoretical value 1582.0741,theoretical value 1582.0741.

[0174] Experimental Example 18 GDP-D-P1 / P1-P1-GGFG-Dxd

[0175]

[0176] To 50 μL GDP-D-P1 / P1 (50 mM aqueous solution), 515 μL water and 750 μL dimethyl sulfoxide, 10 μL copper sulfate (50 mM aqueous solution), 20 μL TBTA (50 mM aqueous solution), 300 μL Prop-PEG1-GGFG-Dxd (25 mM DMSO solution) and 50 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (3.0 mg, 41.4%). HRMS (ESI - )C 127 H 152 F2N 30 O 44 P2(M-2H + ) / 2Theoretical value is 1449.9955, theoretical value is 1450.0012.

[0177] Experimental Example 19 GDP-D-P1 / P1-C2-GGFG-Dxd

[0178]

[0179] Add 0.25 mL of water and 0.25 mL of dimethyl sulfoxide to a 5 mL centrifuge tube and start stirring. Then add 50 μL of GDP-D-P1 / P1 (50 mM aqueous solution), 30 μL of copper sulfate (50 mM aqueous solution), 60 μL of TBTA (50 mM dimethyl sulfoxide solution), 300 μL of Prop-C2-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 150 μL of sodium ascorbate (50 mM aqueous solution) in sequence, and then add 0.25 mL of dimethyl sulfoxide, and stir the reaction at room temperature overnight. Separate and purify by semi-preparative HPLC to obtain a light yellow solid product (2.7 mg, 37.6%). HRMS (ESI - )C 127 H 150 F2N 30 O 42 P2(M-2H + ) / 2Theoretical value is 1434.0006, theoretical value is 1433.9970.

[0180] Experimental Example 20 GDP-D-P1 / P1-P2-GGFG-Dxd

[0181]

[0182] Add 0.25 mL of water and 0.25 mL of dimethyl sulfoxide to a 5 mL centrifuge tube and start stirring. Then add 50 μL of GDP-D-P1 / P1 (50 mM aqueous solution), 30 μL of copper sulfate (50 mM aqueous solution), 60 μL of TBTA (50 mM dimethyl sulfoxide solution), 300 μL of Prop-PEG2-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 150 μL of sodium ascorbate (50 mM aqueous solution) in sequence, and then add 0.25 mL of dimethyl sulfoxide, and stir the reaction at room temperature overnight. Separate and purify by semi-preparative HPLC to obtain a light yellow solid product (1.3 mg, 17.3%). HRMS (ESI - )C 131 H 158 F2N 30 O 46 P2(M-2H + ) / 2Theoretical value is 1494.0217, theoretical value is 1494.0151.

[0183] Experimental Example 21 GDP-D-P1 / P2-P4-GGFG-Dxd

[0184]

[0185] To 100 μL GDP-D-P1 / P2 (50 mM aqueous solution), 1030 μL water and 750 μL dimethyl sulfoxide, 20 μL copper sulfate (50 mM aqueous solution), 40 μL TBTA (50 mM aqueous solution), 400 μL Prop-PEG4-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 100 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (5.0 mg, 31.2%). HRMS (ESI - )C 134 H 180 F2N 30 O 51 P2(M-2H + ) / 2Theoretical value is 1604.0873, theoretical value is 1604.0854.

[0186] Experimental Example 22 GDP-D-P1 / P2-P1-GGFG-Dxd

[0187]

[0188] To 50 μL GDP-D-P1 / P2 (50 mM aqueous solution), 520 μL water and 750 μL dimethyl sulfoxide, 20 μL copper sulfate (50 mM aqueous solution), 40 μL TBTA (50 mM aqueous solution), 600 μL Prop-PEG1-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 100 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (4.2 mg, 57.1%). HRMS (ESI - )C 129 H 155 F2N 30 O 45 P2(M-2H + ) / 2Theoretical value is 1471.5047, theoretical value is 1471.4996.

[0189] Experimental Example 23 GDP-D-P1 / P2-C2-GGFG-Dxd

[0190]

[0191] To 50 μL GDP-D-P1 / P2 (50 mM aqueous solution), 515 μL water and 750 μL dimethyl sulfoxide, 10 μL copper sulfate (50 mM aqueous solution), 20 μL TBTA (50 mM aqueous solution), 600 μL Prop-C2-GGFG-Dxd (12.5 mM dimethyl sulfoxide solution) and 50 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (2.9 mg, 39.8%). HRMS (ESI - )C 129 H 155 F2N 30 O 43 P2(M-2H + ) / 2theoretical value 1455.5098, theoretical value 1455.5062.

[0192] Experimental Example 24 GDP-D-P1 / P2-P2-GGFG-Dxd

[0193]

[0194] Add 0.5 mL of water and 0.5 mL of dimethyl sulfoxide to a 5 mL centrifuge tube and start stirring. Then add 100 μL of GDP-D-P1 / P2 (50 mM aqueous solution), 40 μL of copper sulfate (50 mM aqueous solution), 80 μL of TBTA (50 mM DMSO solution), 600 μL of Prop-PEG2-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 200 μL of sodium ascorbate (50 mM aqueous solution) in sequence, and then add 0.5 mL of dimethyl sulfoxide, and stir the reaction at room temperature overnight. Separate and purify by semi-preparative HPLC to obtain a light yellow solid product (6 mg, 39.5%). HRMS (ESI - )C 133 H 162 F2N 30 O 47 P2(M-2H + ) / 2Theoretical value is 1516.0348, theoretical value is 1516.0279.

[0195] Experimental Example 25 GDP-D-P2 / P8-P4-GGFG-Dxd

[0196]

[0197] To 100 μL GDP-D-P2 / P8 (50 mM aqueous solution), 1030 μL water and 750 μL dimethyl sulfoxide, 20 μL copper sulfate (50 mM aqueous solution), 40 μL TBTA (50 mM aqueous solution), 400 μL Prop-C2-GGFG-Dxd (50 mM dimethyl sulfoxide solution) and 100 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (13.2 mg, 75.1%). HRMS (ESI - )C 155 H 208 F2N 30 O 58 P2(M-2H + ) / 2Theoretical value is 1758.6807, theoretical value is 1758.6820.

[0198] Experimental Example 26 GDP-D-P2 / P2-P2-GGFG-Dxd

[0199]

[0200] Add 0.5 mL of water and 0.5 mL of dimethyl sulfoxide to a 5 mL centrifuge tube and start stirring. Then add 100 μL of GDP-D-P2 / P2 (50 mM aqueous solution), 60 μL of copper sulfate (50 mM aqueous solution), 120 μL of TBTA (50 mM dimethyl sulfoxide solution), 600 μL of Prop-PEG2-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 300 μL of sodium ascorbate (50 mM aqueous solution) in sequence, and then add 0.5 mL of dimethyl sulfoxide, and stir the reaction at room temperature overnight. Separate and purify by semi-preparative HPLC to obtain a light yellow solid product (7.3 mg, 47.4%). HRMS (ESI - )C 135 H 166 F2N 30 O 48 P2(M-2H + ) / 2theoretical value 1538.0479, theoretical value 1538.0528.

[0201] Experimental Example 27 GDP-N-P1 / P1-P1-GGFG-Dxd

[0202]

[0203] To 150 μL GDP-N-P1 / P1 (50 mM aqueous solution), 990 μL water and 1230 μL dimethyl sulfoxide, 60 μL copper sulfate (50 mM aqueous solution), 120 μL TBTA (50 mM aqueous solution), 900 μL Prop-PEG1-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 300 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (4.7 mg, 22.1%). HRMS (ESI - )C 124 H 147 F2N 29 O 43 P2(M-2H + ) / 2Theoretical value is 1414.4769, theoretical value is 1414.4800.

[0204] Experimental Example 28 GDP-N-P1 / P2-P1-GGFG-Dxd

[0205]

[0206] To 100 μL GDP-N-P1 / P2 (25 mM aqueous solution), 520 μL water and 750 μL dimethyl sulfoxide, 20 μL copper sulfate (50 mM aqueous solution), 40 μL TBTA (50 mM aqueous solution), 600 μL Prop-PEG1-GGFG-Dxd (12.5 mM dimethyl sulfoxide solution) and 100 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (3.2 mg, 44.6%). HRMS (ESI - )C 126 H 150 F2N 29 O 44 P2(M-2H + ) / 2theoretical value 1435.9861, theoretical value 1435.9824.

[0207] Experimental Example 29 GDP-K-P2 / P4-P4-GGFG-Dxd

[0208]

[0209] To 75 μL GDP-K-P2 / P4 (50 mM aqueous solution), 495 μL water and 615 μL dimethyl sulfoxide, 30 μL copper sulfate (50 mM aqueous solution), 60 μL TBTA (50 mM aqueous solution), 450 μL Prop-PEG4-GGFG-Dxd (25 mM dimethyl sulfoxide solution) and 150 μL sodium ascorbate (50 mM aqueous solution) were added in sequence, and the reaction was stirred at room temperature overnight and monitored by TLC. The product was separated and purified by semi-preparative HPLC to obtain a light yellow solid product (5.1 mg, 40.5%). HRMS (ESI - )C1 48 H 194 F2N 30 O 54 P2(M-2H + ) / 2theoretical value 1677.1344, theoretical value 1677.1393.

[0210] Experimental Example 30 Construction, expression and purification of HpFT-2HR

[0211] HpFT-2HR (sequence 8) was constructed, expressed and purified according to the method reported in patent WO2023 / 097604.

[0212] Experimental Example 31 Construction, expression and purification of Hβ1,4-GalT1(Y285L)

[0213] The nucleic acid sequence encoding Hβ1,4-GalT1 (Uniprot accession number P15291) Y285L (which contains an N-terminal fused 8×His tag, three GGGGS repeats, and a catalytically active region at positions 63-398, and its amino acid sequence is shown in SEQ ID NO: 10) was synthesized and inserted into the PTT5 vector (Shanghai Langjing) through the EcoRI and BamHI restriction sites. FreeStyle 293F cells were grown to approximately 2.5×10 6 The cells were grown at a density of 10 cells / mL and transfected by adding 1 μg / mL of expression plasmid DNA and 2.2 μg / mL of polyethyleneimine (linear 25 kDa PEI, Polysciences, Inc, Warrington, Pa) directly to the suspension culture. 24 hours after transfection, the culture was diluted 1:1 with Freestyle 293 expression medium containing 4.4 mM valproic acid (final concentration of 2.2 mM), and protein production was continued at 37 ° C for another 4 to 5 days. The supernatant of the fermentation broth was collected and purified with Ni-NTA filler (GE Health). Finally, it was dialyzed into storage buffer (25 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 5% glycerol).

[0214] Example

[0215] Example 1 Synthesis of Antibody-G2F

[0216] The antibodies (trastuzumab (Herceptin, Roche) or rituximab (Rituxan, Roche)) (15 mg / mL) were incubated with UDP-galactose (final concentration 5 mM, Shanghai Baosen Biotechnology Co., Ltd., China), Hβ1,4-GalT1 (Y285L) (sequence 10, final concentration 0.7 mg / mL) and MnCl2 (final concentration 5 mM) in 50 mM Tris-HCl buffer (pH 7.0) at 30 °C for 40 h. The modified antibodies were then purified with protein A resin to obtain antibody-G2F. Mass spectrometric characterization of trastuzumab, trastuzumab-G2F, rituximab and rituximab-G2F is shown in Figure 2. Figure 1 shown.

[0217] Example 2 Comparison of the coupling rate and hydrophilicity of the coupling products prepared by reacting different series of GDP-Fuc* with antibody-G2F

[0218] The antibody-G2F (final concentration 8 mg / mL) prepared in Example 1 and GDP-Fuc* (final concentration 1 mM, respectively, include the following structures: D-P1 / P1-P1-GGFG-Dxd, D-P1 / P1-C2-GGFG-Dxd, D-P1 / P1-P2-GGFG-Dxd, D-P1 / P1-P4-GGFG-Dxd, D-P1 / P2-P1-GGFG-Dxd, D-P1 / P2-C2-GGFG-Dxd, D-P1 / P2-P2-GGFG-Dxd, D-P1 / P2-P4-GGFG-Dxd, D-P2 / The reaction was terminated by incubating the samples with 100 mM Tris-HCl buffer (pH 7.5) and HpFT-2HR (final concentration 0.7 mg / mL) at 30°C for 8 hours and 24 hours respectively, and then removing the substrate molecules through a desalting column. The modified antibody was purified using protein A resin to obtain the antibody-G2F-Fuc* conjugate.

[0219] The resulting antibody-G2F-Fuc* conjugate was compared for hydrophilicity and reaction efficiency by hydrophobic chromatography on a TSKgel Butyl-NPR column (4.6 mm×35 mm, 2.5 μm; TOSOH) on an Agilent 1260 HPLC system. The hydrophobic chromatography conditions were as follows: (1) Buffer A: 20 mM sodium phosphate, 1.5 M ammonium sulfate (pH 6.9); (2) Buffer B: 75% (v / v) 26.7 mM sodium phosphate (final concentration 20 mM), 25% (v / v) isopropanol (pH 6.9); (3) Flow rate: 0.4 mL / min; (4) Gradient: from 100% buffer A to 100% buffer B (within 1-13 min); and (5) Column temperature was 25°C.

[0220] Example 2.1 Comparison of the preparation efficiency of antibody conjugates and the hydrophilicity of the obtained conjugated products between the GDP-K series and the GDP-D series.

[0221] Reference Example 2 Antibody conjugates were prepared using trastuzumab-G2F, GDP-K-P2 / P4-P4-GGFG-Dxd, and GDP-D-P2 / P4-P4-GGFG-Dxd.

[0222] The purified product after 8 h of reaction was subjected to HIC analysis ( Figure 2 ). The results of HIC analysis showed that the DAR8-Dxd product produced by the GDP-K-P2 / P4-P4-GGFG-Dxd reaction for 8 hours (because one Fuc* carries two Dxd molecules, the DAR8-Dxd product corresponds to the DAR4-Fuc* product) accounted for 34% (calculated by peak area integration), and the DAR8-Dxd product produced by the GDP-D-P2 / P4-P4-GGFG-Dxd reaction for 8 hours accounted for 63%. The purified product after 24 hours of reaction was subjected to HIC analysis ( Figure 2 ). HIC analysis results showed that the DAR8-Dxd product produced by GDP-K-P2 / P4-P4-GGFG-Dxd reaction for 24h accounted for 70%, with a corresponding retention time t of 9.459min, while the DAR8-Dxd product produced by GDP-D-P2 / P4-P4-GGFG-Dxd reaction for 24h accounted for 96%, with a corresponding retention time of 9.473min. .

[0223] In summary, under the same other conditions, the GDP-D series has a greatly improved reaction rate and higher efficiency in the preparation of antibody conjugates compared to the GDP-K series, and it is easier to obtain uniform products. At the same time, the products prepared by the two are comparable in hydrophilicity.

[0224] Example 2.2 Comparison of the effects of different Sp2 and Sp3 structures on the hydrophilicity of prepared antibody conjugates in the GDP-D series

[0225] Reference Example 2 Antibody conjugates were prepared using trastuzumab-G2F and GDP-D-P2 / P8-P4-GGFG-Dxd, GDP-D-P2 / P4-P4-GGFG-Dxd, GDP-D-P1 / P2-P4-GGFG-Dxd and GDP-D-P1 / P1-P4-GGFG-Dxd.

[0226] The purified product after 24 h of reaction was subjected to HIC analysis ( Figure 3). The results of HIC analysis showed that the retention time of DAR8-Dxd in the antibody conjugates prepared by GDP-D-P2 / P8-P4-GGFG-Dxd, GDP-D-P2 / P4-P4-GGFG-Dxd, GDP-D-P1 / P2-P4-GGFG-Dxd and GDP-D-P1 / P1-P4-GGFG-Dxd (respectively: 9.714min, 9.473min, 9.234min, 9.163min) shortened successively, indicating that the hydrophilicity of the corresponding DAR8-DXd conjugates increased successively. The above results show that when other conditions are the same, the shorter the PEG chain in the Sp2 and Sp3 structures, the more hydrophilic the conjugates are. Therefore, it can be concluded that Sp2 and Sp3 are both When , the coupling product is relatively the most hydrophilic.

[0227] Example 2.3 Comparison of the effects of different Sp4 structures on the hydrophilicity of prepared antibody conjugates in the GDP-D series

[0228] Reference Example 2 Using trastuzumab-G2F and GDP-D-P2 / P4-P1-GGFG-Dxd, GDP-D-P2 / P4-P2-GGFG-Dxd, GDP-D-P2 / P4-P4-GGFG-Dxd, GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-D-P1 / P1-C2-GGFG-Dxd, GDP-D- Antibody conjugates were prepared using P1 / P1-P2-GGFG-Dxd, GDP-D-P1 / P1-P4-GGFG-Dxd, GDP-D-P1 / P2-P1-GGFG-Dxd, GDP-D-P1 / P2-C2-GGFG-Dxd, GDP-D-P1 / P2-P2-GGFG-Dxd and GDP-D-P1 / P2-P4-GGFG-Dxd.

[0229] The purified product after 24 h of reaction was subjected to HIC analysis ( Figure 4 ). The results of HIC analysis showed that the retention time of DAR8-Dxd in the antibody conjugates prepared from GDP-D-P2 / P4-P1-GGFG-Dxd, GDP-D-P2 / P4-P2-GGFG-Dxd and GDP-D-P2 / P4-P4-GGFG-Dxd (respectively 9.038min, 9.190min, 9.473min) was prolonged successively ( Figure 4Similarly, the retention time of DAR8-Dxd in the antibody conjugates prepared from GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-D-P1 / P1-P2-GGFG-Dxd and GDP-D-P1 / P1-P4-GGFG-Dxd (8.758 min, 8.913 min, 9.163 min, respectively) was successively prolonged ( Figure 4 Similarly, the retention time of DAR8-Dxd in the antibody conjugates prepared from GDP-D-P1 / P2-P1-GGFG-Dxd, GDP-D-P1 / P2-P2-GGFG-Dxd and GDP-D-P1 / P2-P4-GGFG-Dxd (8.831 min, 8.974 min, 9.234 min, respectively) was successively prolonged ( Figure 4 ). The above results show that when other conditions are the same, when Sp4 is a PEG chain, the shorter the structure, the more hydrophilic the coupling product.

[0230] In addition, the proportion of DAR8-Dxd in the coupling products of GDP-D-P1 / P1-P1-GGFG-Dxd and GDP-D-P1 / P1-C2-GGFG-Dxd, as well as GDP-D-P1 / P2-P1-GGFG-Dxd and GDP-D-P1 / P2-C2-GGFG-Dxd after 8h and 24h reaction was analyzed by HIC. The results of HIC analysis showed that the proportion of DAR8-Dxd products produced by GDP-D-P1 / P1-P1-GGFG-Dxd after 8h and 24h reaction (65% and 95%, respectively) was much better than the proportion of DAR8-Dxd products produced by GDP-D-P1 / P1-C2-GGFG-Dxd after 8h reaction (19% and 68%, respectively) ( Figure 4 ). Similarly, the DAR8-Dxd product percentages produced by GDP-D-P1 / P2-P1-GGFG-Dxd after 8h and 24h of reaction (55% and 94%, respectively) were much higher than those produced by GDP-D-P1 / P2-C2-GGFG-Dxd after 8h and 24h of reaction (19% and 71%, respectively) ( Figure 4 On the other hand, the DAR8-Dxd conjugate prepared by the GDP-D-P1 / P1-P1-GGFG-Dxd reaction (t = 8.758 min) was more hydrophilic than the DAR8-Dxd conjugate prepared by the GDP-D-P1 / P1-C2-GGFG-Dxd reaction (t = 8.826 min) ( Figure 4 ). It can be seen from this that when other conditions are the same, when Sp4 is a PEG chain (such as ), which is superior to alkyl chains (such as ).

[0231] Example 2.4 Comparison of the hydrophilicity of antibody conjugates prepared using GDP-N series and GDP-D series

[0232] The GDP-D series structure was further derived to produce the new GDP-N series structure.

[0233] Reference Example 2 utilizes trastuzumab-G2F and GDP-N-P1 / P1-P1-GGFG-Dxd, GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-N-P1 / P2-P1-GGFG-Dxd and GDP-D-P1 / P2-P1-GGFG-Dxd.

[0234] The purified product after 24 h of reaction was subjected to HIC analysis ( Figure 5 ). The results of HIC column analysis showed that the hydrophilicity of the DAR8-Dxd conjugate prepared from GDP-N-P1 / P1-P1-GGFG-Dxd (t=8.616min) was slightly better than that of the DAR8-Dxd conjugate prepared from GDP-D-P1 / P1-P1-GGFG-Dxd (t=8.758min). Similar conclusions also appeared in the comparison between GDP-N-P1 / P2-P1-GGFG-Dxd and GDP-D-P1 / P2-P1-GGFG-Dxd. The above results show that, under the same other conditions, the hydrophilicity of the antibody conjugate containing the N series structure is better than that of the antibody conjugate containing the D series structure.

[0235] In addition, for the above structures, the purified products after 8h and 24h of reaction were taken and analyzed by HIC to determine the proportion of DAR8-Dxd coupling products. The analysis results showed that the coupling products of DAR8-Dxd obtained by GDP-N-P1 / P1-P1-GGFG-Dxd after 8h and 24h of reaction (accounting for 55% and 94%, respectively) were close to those of GDP-D-P1 / P1-P1-GGFG-Dxd (accounting for 65% and 95%, respectively). Similarly, the coupling products of DAR8-Dxd obtained by GDP-N-P1 / P2-P1-GGFG-Dxd after 8h and 24h of reaction (accounting for 56% and 94%, respectively) were close to those of GDP-D-P1 / P2-P1-GGFG-Dxd (accounting for 55% and 94%, respectively).

[0236] In summary, the conjugate with N structure has better hydrophilicity than the conjugate with D structure.

[0237] Example 2.5 Comparison of the hydrophilicity of the trastuzumab-DAR8-Dxd conjugate prepared by the present invention and DS-8201.

[0238] The purified products after 24 h of reaction of GDP-D-P2 / P8-P4-GGFG-Dxd, GDP-D-P2 / P4-P4-GGFG-Dxd, GDP-D-P2 / P2-P2-GGFG-Dxd, GDP-D-P1 / P2-P2-GGFG-Dxd, GDP-D-P1 / P1-P2-GGFG-Dxd, GDP-D-P1 / P2-P1-GGFG-Dxd, GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-N-P1 / P2-P1-GGFG-Dxd, GDP-N-P1 / P1-P1-GGFG-Dxd, and DS-8201 (Dexit trastuzumab, Enhertu, Daiichi Sankyo) and trastuzumab were respectively used for HIC analysis ( Figure 6 ).

[0239] HIC analysis results showed that, except for the coupling products of GDP-D-P2 / P8-P4-GGFG-Dxd and GDP-D-P2 / P4-P4-GGFG-Dxd, the hydrophilicity of DAR8-Dxd in the coupling products obtained by other molecules was better than that of DS-8201. Among them, the DAR8-Dxd conjugate prepared by GDP-N-P1 / P1-P1-GGFG-Dxd had the best hydrophilicity.

[0240] Example 2.6 Comparison of the efficiency of preparing antibody conjugates on rituximab-G2F among GDP-K, GDP-D and GDP-N series and the hydrophilicity of the conjugated products.

[0241] Reference Example 2: Rituximab-G2F and GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-D-P1 / P1-C2-GGFG-Dxd, GDP-D-P1 / P1-P2-GGFG-Dxd, GDP-D-P1 / P1-P4-GGFG-Dxd, GDP-D-P2 / P2-P4-GGFG-Dxd, GDP-D-P2 / P4- Antibody conjugates were prepared using GDP-P4-GGFG-Dxd, GDP-D-P2 / P8-P4-GGFG-Dxd, GDP-D-P2 / P2-P2-GGFG-Dxd, GDP-K-P2 / P4-P4-GGFG-Dxd and GDP-N-P1 / P1-P1-GGFG-Dxd, and the retention time and ratio of the DAR8-Dxd product in the conjugates were analyzed by HIC ( Figure 7 ).

[0242] The preparation of antibody conjugates on rituximab using the above molecules follows the same rules as the preparation of antibody conjugates on trastuzumab, which indicates that the present invention is not limited to specific antibodies but has wide applicability.

[0243] Example 3 Preparation of Antibody-G2F-Fuc* Conjugates by One-Pot Method

[0244] The antibody (trastuzumab (Herceptin, Roche) (15 mg / mL) was mixed with UDP-galactose (final concentration 5 mM, Shanghai Baosen Biotechnology Co., Ltd., China), Hβ1,4-GalT1 (Y285L) (sequence 10, final concentration 0.7 mg / mL) and MnCl2 (final concentration 5 mM) and 50 mM Tris-HCl buffer (pH 7.0) was incubated at 30°C for 40 h. The reaction solution was then mixed with GDP-Fuc* (final concentration 1 mM, Fuc* included the following structures: D-P1 / P1-P1-GGFG-Dxd, DD-P2 / P2-P2-GGFG-Dxd, D-P2 / P4-P4-GGFG-Dxd, N-P1 / P1-P1-GGFG-Dxd), HpFT-2HR (sequence 8, final concentration 0.7 mg / mL), MgCl2 (final concentration 5 mM) and 50 mM Tris-HCl buffer (pH 7.5) Incubate at 30°C for 24 hours. The modified antibody was purified with protein A resin to obtain antibody-G2F-Fuc* conjugate, trastuzumab-G2F-D-P1 / P1-P1-GGFG-Dxd (DRA8-Dxd ratio 98%, HIC characterization), trastuzumab-G2F-D-P2 / P2-P2-GGFG-Dxd (DRA8-Dxd ratio 96%, HIC characterization), trastuzumab-D-P2 / P4-P4-GGFG-Dxd (DRA8-Dxd ratio 96%, HIC characterization), trastuzumab-N-P1 / P1-P1-GGFG-Dxd (DRA8-Dxd ratio 96%, HIC characterization). The mass spectrometry characterization of the above antibody conjugates is shown in the figure below. Figure 8 shown.

[0245] Example 4 Comparison of thermal stability between the trastuzumab-G2F-Fuc* conjugate of the present invention and DS-8201.

[0246] The solution of trastuzumab-G2F-D-P1 / P1-P1-GGFG-Dxd, trastuzumab-G2F-D-P2 / P2-P2-GGFG-Dxd, trastuzumab-G2F-D-P2 / P4-P4-GGFG-Dxd, trastuzumab-G2F-N-P1 / P1-P1-GGFG-Dxd and DS-8201 was replaced with PBS (pH 7.2), the concentration was adjusted to 2 mg / mL, and incubated in a 55°C water bath for 18 h.

[0247] The samples before and after incubation were respectively passed through an XBridge Protein BEH SEC column ( 3.5 μm, 7.8 mm X 150 mm; Waters) was used to study the monomer and aggregate conditions by size exclusion chromatography on an Agilent 1260 HPLC system. The size exclusion chromatography conditions were as follows: (1) buffer: 20 mM sodium phosphate, 150 mM sodium chloride, 15% (v / v) isopropanol (pH 7.2); (2) flow rate: 0.6 mL / min.

[0248] The polymer conditions before and after incubation are as follows Fig. 9 The results showed that trastuzumab-D-G2F-P1 / P1-P1-GGFG-Dxd, trastuzumab-G2F-D-P2 / P2-P2-GGFG-Dxd, trastuzumab-G2F-D-P2 / P4-P4-GGFG-Dxd, and trastuzumab-G2F-N-P1 / P1-P1-GGFG-Dxd had lower aggregate content, and after heat treatment, had lower dimerization, multimerization, and fragmentation content than DS-8201 that was heat treated at the same time.

[0249] Example 5 In vitro plasma stability

[0250] Human plasma (Kejing Bio) was incubated with protein A resin for 30 minutes (volume ratio 5:1) and the supernatant was taken. The supernatant was incubated with protein A resin again for 30 minutes (volume ratio 5:1) and the supernatant was taken to remove IgG. The plasma after IgG removal was then sterilized by filtration through a 0.22 μM filter. DS-8201, as well as trastuzumab-D-P1 / P1-P1-GGFG-Dxd, trastuzumab-D-P2 / P2-P2-GGFG-Dxd and trastuzumab-N-P1 / P1-P1-GGFG-Dxd obtained in Example 3 were incubated with plasma respectively (ADC final concentration was 100 μg / mL). Samples were taken on days 0, 2, 4, 6, and 8, purified with protein A, and then analyzed by HIC. The HIC analysis method is as described in Example 3. The analysis results show that the peak shape corresponding to the antibody-G2F-Fuc* conjugate of the present invention does not change significantly with the increase of incubation time in plasma. In contrast, the peak shape corresponding to the DS-8201 conjugate shifts significantly with the increase of incubation time in plasma, and multiple new peaks appear. The above data show that the conjugate of the present invention is more stable in plasma than DS-8201 ( Fig.10 ).

[0251] Example 6 In vitro cytotoxic activity of trastuzumab-G2F-Fuc* conjugate against BT-474, NCI-N87, and MDA-MB-231 cells

[0252] BT-474 (Her2+) and NCI-N87 (Her2+) cells were cultured in RPMI 1640 medium containing 10% FBS (Gibco). MDA-MB-231 (Her2-) cells were cultured in DMEM (Gibco) containing 10% FBS (Gibco). Cells were plated in 96-well plates at 3000 cells per well and incubated at 37°C and 5% CO2 for 24 hours. After removing the culture medium, sample DS-8201 and trastuzumab-D-P1 / P1-P1-GGFG-Dxd, trastuzumab-D-P2 / P2-P2-GGFG-Dxd and trastuzumab-N-P1 / P1-P1-GGFG-Dxd obtained in Example 3 were added to the culture medium, respectively, to obtain a series of final concentrations for NCI-N87 and MDA-MB-231 cells (500nM, 200nM, 80nM, 20nM, 5nM, 1nM, 0.2nM, 0.05nM, 0.01nM, 0nM) and BT-474 cells (200nM, 80nM, 20nM, 5nM, 1nM, 0.2nM, 0.05nM, 0.01nM, 0.001nM, 0nM) and added to the plate, respectively. The cells were incubated at 37°C and 5% CO2 for 6 days and Luminescent Cell Viability Assay (Promega) was used to measure cell viability.

[0253] The trastuzumab-G2F-Fuc* conjugate showed high cytotoxicity against Her2-positive cell lines, but did not show high cytotoxicity against the Her2-negative cell line MDA-MB-231 ( Fig.11 ), on the BT-474 cell line, the trastuzumab-G2F-Fuc* conjugate showed better cell killing activity than DS-8201, and on the NCI-N87 cell line, the trastuzumab-G2F-Fuc* conjugate showed similar cell killing activity to DS-8201.

[0254] Example 7 Evaluation of in vivo tumor inhibition effect in xenograft tumor models

[0255] 1×10 6 NCI-N87 (Her2+) cells resuspended in 50% PBS (pH 7.4) and 50% Matrigel (BD) were inoculated into female BALB / c nude mice (4-5 weeks old). When the average tumor size reached 150-200 mm 3At 4 pm, PBS, trastuzumab (10 mg / kg), DS-8201 (10 mg / kg), DS-8201 (5 mg / kg), and trastuzumab-D-P1 / P1-P1-GGFG-Dxd (5 mg / kg), trastuzumab-N-P1 / P1-P1-GGFG-Dxd (5 mg / kg), and trastuzumab-N-P1 / P1-P1-GGFG-Dxd (10 mg / kg) obtained in Example 3 were injected into different groups (n=7 mice / group) once via tail vein. The tumor size and body weight of the mice were monitored twice a week during the entire study period ( Fig.12 ). The tumor volume was calculated according to the formula: tumor volume (mm 3 ) = π × major diameter × (minor diameter) 2 / 6. All animal studies were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee and were conducted at Hangzhou Medical College with an ethical review approval number of ZJCLA-IACUC-20010329.

[0256] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further explained below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without making creative work all belong to the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited. The technology and scientific terms used in the embodiments have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

Claims

1. A conjugated drug, characterized in that: Has the following structure: Wherein, X is an antibody or fusion protein comprising an Fc fragment; m is 1-4; GlcNAc is N-acetylglucosamine, Man is mannose, Fuc is fucose, Gal is galactose, and Fuc* is a fucose derivative, and has the following structure: Among them, Sp1 is SP2 is n is any integer from 1 to 4; SP3 is n' is any integer from 1 to 4; Sp4 is n" is any integer from 1 to 4, and t is any integer from 0 to 1; Cn1 is Cn2 is The Fuc* is connected to the terminal GlcNAc via an α1,3 glycosidic bond; The core GlcNAc is linked to an asparagine residue on the Fc fragment in X.

2. The conjugated drug according to claim 1, characterized in that: Sp1 is SP2 is n is 1 or 2; SP3 is n' is 1 or 2; Sp4 is n" is 1 or 2, t is 0 or 1; Cn1 is Cn2 is 3. The conjugated drug according to claim 2, characterized in that: When t = 0, Cn1 and Cn2 are When t=1, Cn1 is Cn2 is Or Cn1 is Cn2 is 4. The conjugated drug according to any one of claims 1 to 3, characterized in that: The structure of the Fuc* is selected from the following structures:

5. The conjugated drug according to claim 4, characterized in that: The structure of the Fuc* is selected from the following structures:

6. The conjugated drug according to claim 5, characterized in that: The structure of the Fuc* is selected from the following structures:

7. The conjugated drug according to claim 1, characterized in that: The m is 3-4.

8. The conjugated drug according to claim 7, characterized in that: The m is 4.

9. The conjugated drug according to claim 1, wherein X is trastuzumab or rituximab.

10. A mixture of conjugated drugs, comprising the conjugated drugs according to claim 8 or 9, wherein the proportion of the conjugated drugs in the conjugated drug mixture is > 75%.

11. A drug for treating or alleviating cancer, characterized in that: The drug for treating or alleviating cancer comprises the conjugated drug according to any one of claims 1 to 9.

12. The method for preparing a conjugated drug according to any one of claims 1 to 9, characterized in that: The following steps are included: a) The structured protein reacts with Fuc*-Q in the presence of an enzyme to obtain a target conjugated drug, wherein Q in the Fuc*-Q is selected from at least one of UDP, GDP, ADP, CDP and their corresponding salt forms.

13. The preparation method according to claim 12, characterized in that: The The protein of structure is prepared by the following steps Pre-a): The structured protein is reacted with UDP-galactose in the presence of β1,4 galactosyltransferase or a functional variant thereof, wherein m' is 0-4.

14. The preparation method according to claim 12, characterized in that: The enzyme is α1,3 fucosyltransferase or a functional variant thereof.

15. The preparation method according to claim 14, characterized in that: The α1,3 fucosyltransferase comprises a catalytic fragment sequence shown in sequence 5 and 1 to 4 heptapeptide repeat sequences shown in sequence 6.

16. The preparation method according to claim 12, characterized in that: The reaction temperature is 0-37°C.

17. The preparation method according to claim 12, characterized in that: The reaction pH is 6-9.

18. The preparation method according to claim 12, characterized in that: The Q in the Fuc*-Q is selected from GDP and its corresponding salt forms.

19. The method for preparing the drug-coupled mixture according to claim 10, characterized in that Contains the following steps: pre-a) will contain The protein mixture of the structure is reacted with UDP-galactose in the presence of β1,4-galactosyltransferase or its functional variant to obtain a A protein mixture of structure, m'=0-4, comprising The proportion of the structured protein in the protein mixture is >75%; a) reacting the protein obtained by the above reaction with Fuc*-Q in the presence of α1,3 fucosyltransferase to obtain the target conjugated drug mixture, wherein Q in the Fuc*-Q is selected from GDP and its corresponding salt form.

20. The preparation method according to claim 19, characterized in that: The α1,3 fucosyltransferase comprises a catalytic fragment sequence shown in sequence 5 and 1 to 4 heptapeptide repeat sequences shown in sequence 6.

21. The preparation method according to claim 12, characterized in that: The reaction temperature is 0-37°C, and the reaction pH is 6-9.

22. A compound Fuc*-Q and its corresponding salt form, wherein the Fuc*-Q is selected from the following structures: GDP-D-P1 / P1-P1-GGFG-Dxd, GDP-D-P1 / P2-P1-GGFG-Dxd, GDP-D-P1 / P2-P2-GGFG-Dxd, GDP-D-P1 / P1-P2-GGFG-Dxd, GDP-D-P2 / P2-P2-GGFG-Dxd, GDP-N-P1 / P1-P1-GGFG-Dxd, GDP-N-P1 / P2-P1-GGFG-Dxd

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