Antibody-drug conjugates containing anti-mesothelin antibodies and their uses

Abstract

This disclosure provides an immunoconjugant comprising an antibody, said antibody comprising an antigen-binding fragment specifically binding to an antigenic determinant in mesothelin, an N-glycan-binding domain, and an N-glycan; a connector linked to said N-glycan; and payloads A and B conjugated to said connector, respectively; wherein said payloads A and said payloads B may be the same or different. This disclosure also provides a pharmaceutical composition comprising said immunoconjugant and a method for treating cancer.

Description

Technical Field

[0001] This disclosure relates to an anti-mesothelin antibody-drug conjugate, wherein the glycoprotein contained in the conjugate comprises one or more trimannosyl cores. This disclosure also relates to a method of treating a disease such as cancer in an individual of need, comprising administering the anti-mesothelin antibody-drug conjugate to the individual. Background Technology

[0002] Antibody-drug conjugates (ADCs) typically contain an anticancer drug (e.g., a cytotoxic agent) coupled to an antibody that specifically targets a biomarker (e.g., a tumor marker). The antibody tracks these biomarkers in the body and attaches itself to the surface of cancer cells. The binding between the antibody and the target biomarker (antigen) triggers a signal in the tumor cells, which then internalize the ADC. After ADC internalization, the cytotoxic drug is released and kills the cancer cells. Due to the specific targeting, the drug's side effects may be reduced.

[0003] Mesothelin (MSLN) is a tumor differentiation antigen that is overexpressed in several human tumors, including mesothelioma, pancreatic cancer, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma, cholangiocarcinoma, extrahepatic cholangiocarcinoma, lung cancer, and epithelioid mesothelioma. Therefore, mesothelin is a promising diagnostic / therapeutic target.

[0004] Although many antibodies against mesothelin are under development, such as SS1P (an anti-mesothelin immunotoxin composed of a fusion of a targeting antibody fragment with a truncated fragment of the Pseudomonas exotoxin A gene), anetumab (a monoclonal antibody), and anetumab ravtansin (an antibody-drug conjugate), there is still a need for modified therapeutic agents against mesothelin antibodies. Summary of the Invention

[0005] This disclosure relates to antibody-drug conjugates containing anti-mesothelin antibodies and their use in therapy.

[0006] One aspect of this disclosure relates to immune conjugates. An immune conjugate according to one embodiment of this disclosure includes:

[0007] An antibody comprising an antigen-binding fragment that specifically binds to an antigenic determinant in mesothelin, an N-glycan binding domain, and an N-glycan having the structure of formula (1).

[0008]

[0009] Where “*” represents a bond or protecting group;

[0010] The connector, which connects to each * in the N-glycan when the * represents a bond; and

[0011] Payloads A and B are independently conjugate with the connector; where payloads A and B may be the same or different.

[0012] In some embodiments disclosed herein, the antibody is a monoclonal antibody, a humanized antibody, a human antibody, an antibody Fab fragment derived from a lysed antibody, an F(ab')2 fragment, an Fv fragment or an Fc fragment, a scFv-Fc fragment, a microantibody, a bifunctional antibody, or a scFv.

[0013] In some embodiments disclosed herein, the antigen-binding fragment includes a complementarity-determining region (CDR) of the heavy chain variable region and a complementarity-determining region of the light chain variable region, wherein the complementarity-determining region of the heavy chain variable region includes CDRH1, CDRH2, and CDRH3 regions, and the complementarity-determining region of the light chain variable region includes CDRL1, CDRL2, and CDRL3 regions; wherein the CDRH1 region includes the amino acid sequence of SEQ ID NO:1; the CDRH2 region includes the amino acid sequence of SEQ ID NO:2; the CDRH3 region includes the amino acid sequence of SEQ ID NO:3; the CDRL1 region includes the amino acid sequence of SEQ ID NO:4; the CDRL2 region includes the amino acid sequence of SEQ ID NO:5; and the CDRL3 region includes the amino acid sequence of SEQ ID NO:6. In one embodiment disclosed herein, the antibody includes a heavy chain variable region including the amino acid sequence of SEQ ID NO:7 and a light chain variable region including the amino acid sequence of SEQ ID NO:8. In one embodiment of this disclosure, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10. In one embodiment of this disclosure, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:12.

[0014] In some embodiments disclosed herein, the antibody comprises a heavy chain constant region, and an N-glycan binding domain is located within the heavy chain constant region.

[0015] In some embodiments disclosed herein, the antibody comprises two N-glycans.

[0016] Some examples of effective amounts of the immunoconjugate disclosed herein are those ranging from about 0.01 mg / kg to 800 mg / kg, 0.05 mg / kg to 600 mg / kg, 0.1 mg / kg to 500 mg / kg, 0.5 mg / kg to 400 mg / kg, 1 mg / kg to 300 mg / kg, 5 mg / kg to 200 mg / kg, 10 mg / kg to 100 mg / kg, 15 mg / kg to 80 mg / kg, 20 mg / kg to 60 mg / kg, and 25 mg / kg to 50 mg / kg.

[0017] In some embodiments disclosed herein, the connector is selected from the group consisting of: straight-chain or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, heteroaromatic, alkoxy, acyl, alkylamino, aromaticamino, ether, ester, amide, carbamate, carbonate, formula (3) to (7), connectors containing disulfide bonds, acid unstable connectors, light unstable connectors, peptidase unstable connectors and esterase unstable connectors or combinations thereof.

[0018] In some embodiments disclosed herein, payload A and payload B are independently selected from therapeutic agents and labels.

[0019] Examples of therapeutic agents include, but are not limited to, antimetabolites, alkylating agents, alkylating agents-like substances, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotics, topoisomerase inhibitors, proteasome inhibitors, radioisotopes, and isotope chelators. Specific examples of compounds used in therapeutic agents include, but are not limited to, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoids, duocarmycin-hydroxybenzamide azaindole (DUBA), diethyltriamine-N,N,N',N",N"-pentaacetic acid (DTPA), exatecan, and Dxd2.

[0020] Examples of labeling include, but are not limited to, fluorescent labeling, chromogenic labeling, electron-dense labeling, chemiluminescent labeling, radioactive labeling, enzyme labeling, and positron emitters.

[0021] One example of a protecting group is an azide group.

[0022] One aspect of this disclosure relates to a pharmaceutical composition comprising the aforementioned immunoconjugate and a pharmaceutically acceptable carrier.

[0023] One aspect of this disclosure relates to a method for treating cancer. According to one embodiment of this disclosure, the method may include administering a therapeutically effective amount of the aforementioned immune conjugate to an individual requiring cancer treatment.

[0024] In some embodiments disclosed herein, the cancer is a cancer that expresses mesothelin. Examples of cancer include, but are not limited to, ovarian cancer, mesothelioma, pancreatic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, bile duct cancer, colorectal cancer, endometrial cancer, and breast cancer.

[0025] Those familiar with this technique should understand that the effective therapeutic dose depends on many factors, such as the patient's condition, age, disease state, and route of administration, and that such an effective dose can be determined in routine practice based on these factors without excessive experimentation.

[0026] Other aspects of this disclosure will become apparent from the following description.

[0027] Simple Explanation of the Diagram

[0028] Figure 1A and 1B The sequence alignment of the anti-mesothelin antibody is shown.

[0029] Figure 2 A humanized version of the vector construct representing the mouse variable region, IMGT, and 4D5 variable region is presented.

[0030] Figure 3 This demonstrates the affinity of mesothelin for antibodies.

[0031] Figure 4 This demonstrates the kinetic analysis using BIAcore's SS1 and HuSS1 antibodies.

[0032] Figure 5 Demonstrates the ELISA binding affinity of the detected ADC.

[0033] Figure 6 The results of ADC internalization are shown.

[0034] Figure 7 Demonstrates quality analysis of ADC DCBPR2002-4 (DBCO-vc-MMAE).

[0035] Figure 8 The results present the pharmacokinetic profile of the ADC DCBPR2002-4 (DBCO-vc-MMAE).

[0036] Figure 9 Tumor growth curves of male NOD SCID mice implanted with KLM-1 are shown.

[0037] Figure 10 The changes in body weight of male NOD SCID mice implanted with KLM-1 are shown.

[0038] Figure 11 Tumor growth curves of male NOD SCID mice implanted with KLM-1 are shown.

[0039] Figure 12 The changes in body weight of male NOD SCID mice implanted with KLM-1 are shown.

[0040] Figure 13 Tumor growth curves of male NOD SCID mice implanted with KLM-1 are shown.

[0041] Figure 14 The changes in body weight of male NOD SCID mice implanted with KLM-1 are shown.

[0042] Figure 15 Tumor growth curves of female NOD SCID mice implanted with OVCAR-3 are shown.

[0043] Figure 16 The changes in body weight of female NOD SCID mice implanted with OVCAR-3 are shown.

[0044] Figure 17 Tumor growth curves of female NOD SCID mice implanted with OVCAR-3 are shown.

[0045] Figure 18 The changes in body weight of female NOD SCID mice implanted with OVCAR-3 are shown.

[0046] Figure 19 Tumor growth curves of female NOD SCID mice implanted with OVCAR-3 are shown.

[0047] Figure 20 The changes in body weight of female NOD SCID mice implanted with OVCAR-3 are shown.

[0048] Figures 21A to 20Demonstration of the preparation of immunoconjugates. A: Preparation of DCBPR2002-2Az. B: Preparation of DCBPR2002-4Az. C: Preparation of DCBPR2002-4(DBCO-vc-MMAE). D: Preparation of DCBPR2002-4(DBCO-S-DM1). E: Preparation of DCBPR2002-4(DBCO-vc-seco DUBA). F: Preparation of DCBPR2002-4(DBCO-PEG4-vc-PAB-MMAF). G: Preparation of DCBPR2002-4(DBCO-DTPA). H: Preparation of DCBPR2002-4(DBCO-PEG3-vc-exinotecan). I: Preparation of DCBPR2002-4(DBCO-PEG3-GGFG-exinotecan). J: Preparation of DCBPR2002-4 (DBCO-PEG12-GGFG-exinotecan). K: Preparation of DCBPR2002-4 (DBCO-PEG3-GGFG-DXd2). L: Preparation of DCBPR2002-4 (DBCO-PEG12-GGFG-DXd2). M: Preparation of DCBPR2002-4 (BCN-PEG3-VC-PAB-MMAE). N: Preparation of DCBPR2002-4 (BCN-PEG12-GGFG-exinotecan). O: Preparation of DCBPR2002-4 (BCN-PEG3-GGFG-exinotecan). P: Preparation of DCBPR2002-4 (BCN-PEG12-GGFG-DXd2). Q: Preparation of DCBPR2002-4 (DBCO-PEG3-2(PEG3-VC-PAB-MMAE)). R: Preparation of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco DUBA). S: Preparation of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-S-DM1). T: Preparation of DCBPR2002-2(DBCO-vc-secoDUBA)-2(DBCO-S-DM1). Detailed Implementation

[0049] It should be noted that, as used herein and in the claims of the appended patent applications, the singular forms “a / an” and “the” include a plurality of indicators unless the context clearly specifies otherwise. Similarly, the terms “a / an,” “one or more,” and “at least one” are used interchangeably herein. It should also be noted that the terms “comprising,” “including,” and “having” are used interchangeably.

[0050] Unless otherwise defined, all scientific or technical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains. Any methods and materials similar to or equivalent to those described herein may be understood and used by one of ordinary skill in the art to practice this disclosure.

[0051] It must be noted that, as used in this specification and the appended claims, the singular forms “a / an” and “the” include a plural of indicators unless the context clearly specifies otherwise. Therefore, unless the context requires otherwise, singular terms should include plural terms and plural terms should include singular terms.

[0052] The term "and / or" is used to refer to two things or either of the two things mentioned.

[0053] As used herein, the term "immunoconjugate" refers to a polypeptide molecule comprising at least one effector moiety (such as payloads A and B) and an antibody. In some embodiments, the immunoconjugate comprises no more than one effector moiety. A particular immunoconjugate according to this disclosure consists essentially of an effector moiety and an antibody bound by one or more linkers.

[0054] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., mesothelin). The term "antibody" includes immunoglobulin molecules and their multimers (e.g., IgM), which contain four polypeptide chains linked by disulfide bonds, namely two heavy (H) chains and two light (L) chains. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V). H The heavy chain constant region comprises three domains, namely C... H1 C H2 and C H3 Each light chain contains a light chain variable region (abbreviated as LCVR or V in this document). L The light chain constant region contains a domain (C) and the light chain constant region. L1 V H District and V L The region can be further subdivided into highly variable regions, called complementary determinant regions (CDRs), which are interspersed with more conservative regions, called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In the different embodiments disclosed herein, the FRs of the anti-α-toxin antibody (or its antigen-binding portion) may be identical to the human germline sequence or may be naturally or artificially modified. The common amino acid sequence may be defined based on the side-by-side analysis of two or more CDRs.

[0055] As used herein, the term "monoclonal antibody" is not limited to antibodies produced via fusion tumor technology. A monoclonal antibody is derived from a single strain by any means available or known in this technology, including any eukaryotic, prokaryotic, or phage strain.

[0056] The “humanized” form of a non-human antibody is a chimeric immunoglobulin containing a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody will contain substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin and all or substantially all of the FR regions are the FR regions of the human immunoglobulin sequence.

[0057] As used herein, the term “complementarity-determining region” (CDR) refers to a non-continuous antigenic combination site found within the variable region of a heavy-chain or light-chain polypeptide. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes overlaps or subsets of amino acid residues when compared with each other.

[0058] As used herein, the term "antigen-binding fragment" of an antibody and its analogues include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0059] As used herein, the term "mesothelin" refers to the 40 kDa protein mesothelin, which is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) bond and has a 31 kDa detached fragment at its N-terminus called megakaryocyte enhancer factor (MPF). Both fragments contain N-glycosylation sites. Preferably, the term refers to human mesothelin and its naturally occurring cleavage regions, such as those found on cell membranes (e.g., cancer cell membranes). Specifically, the mesothelin fragment contains the N-terminal region of mesothelin.

[0060] As used in this article, the term "antigen determinant" refers to the site on the antigen to which the antibody binds.

[0061] As used herein, the term “N-glycan” refers to an N-linked oligosaccharide, such as an oligosaccharide attached to an asparagine residue of a polypeptide via an asparagine-N-acetylglucosamine bond. N-glycans share a common pentasaccharide core of Man3GlcNAc2 (“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers to N-acetyl; GlcNAc refers to N-acetylglucosamine). The term “trimannose core” used with respect to N-glycans also refers to the structure Man3GlcNAc2 (“Man3”). N-glycans vary in the number of branches (antennae) that include peripheral sugars (such as trehalose and sialic acid) added to the Man3 core structure.

[0062] As used herein, the term "pharmaceutical composition" refers to a formulation or preparation containing an active ingredient having biological or pharmacological activity and a pharmaceutically acceptable carrier. Pharmaceutical compositions may be in the form of solutions, suspensions, tablets, powders, pellets, beads, granules, microspheres, capsules, pills, etc.

[0063] The term "treatment / treating / treat" generally refers to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease, condition, or its symptoms, and therapeutic in terms of partial or complete cure of a disease, condition, and / or symptoms attributable to it. As used herein, "treatment" encompasses any treatment of a disease in mammals, preferably humans, and includes (1) inhibiting the development of an individual's disease, condition, or its symptoms, or (2) alleviating or improving an individual's disease, condition, or its symptoms.

[0064] As used herein, the term "individual" means any animal that may benefit from administration of a compound or composition as disclosed herein. In some embodiments, an individual is a mammal, such as a human, primate, dog, cat, horse, cow, pig, rodent, such as a rat or mouse. Typically, the mammal is a human.

[0065] As used herein, the term "effective amount" for an active ingredient means the amount of the ingredient that is sufficient to provide the desired function. As will be noted below, the precise amount required will vary from person to person, depending on their disease state, physical condition, age, sex, species, weight, the specific characteristics of the composition, and the formulation. Dosing regimens can be adjusted to induce an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally as indicated by the urgency of the treatment situation. Therefore, it is impossible to specify an exact "effective amount." However, an appropriate effective amount can be determined by someone generally skilled in this art using only routine experimental methods.

[0066] As used herein, the term "medically acceptable" means, within the bounds of reasonable medical judgment, a compound, substance, composition, and / or dosage form suitable for contact with individual (human or non-human animal) tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit / risk ratio. In the sense of compatibility with other components of the formulation, each carrier, excipient, etc., must also be "acceptable." Suitable carriers, excipients, etc., can be found in standard medical texts.

[0067] The immunoconjugates disclosed herein can be formulated with a "carrier". As used herein, a "carrier" includes any solvent, dispersion medium, mordant, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delay agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharmaceutically active substances is well known in this art. For example, a pharmaceutical combination may be specifically formulated for administration in solid or liquid form, including forms suitable for: (1) oral administration, such as drenching (aqueous or non-aqueous solutions or suspensions), lozenges, sugar-coated pills, capsules, pellets, tablets (e.g., tablets targeted for buccal, sublingual and systemic absorption), large pills, powders, granules, pastes applied to the tongue; (2) non-enteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection in the form of, for example, sterile solutions or suspensions or sustained-release formulations; (3) topical application, such as application to the skin in the form of creams, lotions, gels, ointments or controlled-release patches or sprays; (4) intravaginal or rectal, such as in the form of pessaries, creams, suppositories or foams; (5) sublingual; (6) ocular; (7) transdermal; (8) mucosal; or (9) nasal.

[0068] Antibody-drug conjugates (ADCs) are a class of therapeutic agents in which a drug (or payload) is attached to an antibody or its antigen-binding fragment. The antibody in an ADC binds to a selected target (typically a cellular target), thereby delivering the drug to the vicinity of the target and producing a highly selective therapeutic effect. One example of an ADC may be an antibody targeting a protein expressed on cancer cells, and the payload may be a cytotoxic agent. One embodiment of this disclosure relates to an immunoconjugate (antibody-drug conjugate) containing an anti-mesothelin antibody or its binding fragment and two or more payloads. In one embodiment of this disclosure, the immunoconjugate comprises...

[0069] An antibody comprising an antigen-binding fragment that specifically binds to an antigenic determinant in mesothelin, an N-glycan binding domain, and an N-glycan having the structure of formula (1).

[0070]

[0071] Where “*” represents a bond or protecting group;

[0072] The connector, which connects to each * in the N-glycan when the * represents a bond; and

[0073] Payloads A and B are independently conjugate with the connector; where payloads A and B may be the same or different.

[0074] In one embodiment of this disclosure, the N-glycan, the connector, and the payloads A and B have the structure of formula (2):

[0075]

[0076] In another embodiment of this disclosure, payload A and payload B may be the same or different.

[0077] According to embodiments disclosed herein, anti-mesothelin antibodies or their binding fragments can recognize and bind to mesothelin or fragments thereof. The term "antibody" is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" also means, herein, human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to mesothelin. The term "antibody" means including intact antibodies and fragments of antibodies, such as antibody Fab fragments, F(ab')2, Fv fragments, or Fc fragments from lysed antibodies, scFv-Fc fragments, microantibodies, bifunctional antibodies, or scFv. Furthermore, the term includes genetically engineered derivatives of antibodies. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in this art.

[0078] The antibodies described herein contain an N-glycan binding domain. In one embodiment, the antibody includes a heavy chain constant region, and the N-glycan binding domain is located within the heavy chain constant region. In some embodiments, the antibody as described contains a C-terminal region in the Fc region. H2 The heavy chain of the constant domain contains N-glycosylated asparagine residues. In one embodiment, the antibody comprises two heavy chains and two N-glycans, each N-glycan being bound to one heavy chain. Specifically, the first GlcNAc (GlcNAc) in the N-glycan as shown in formula (1) 1 () binds to antibodies.

[0079] In one embodiment of this disclosure, the antigen-binding fragment of antibody DCBPR2002 includes a complementarity-determining region (CDR) of the heavy chain variable region and a complementarity-determining region of the light chain variable region. The complementarity-determining region of the heavy chain variable region includes CDRH1, CDRH2, and CDRH3 regions, and the complementarity-determining region of the light chain variable region includes CDRL1, CDRL2, and CDRL3 regions. The CDRH1 region contains the amino acid sequence of SEQ ID NO:1; the CDRH2 region contains the amino acid sequence of SEQ ID NO:2; the CDRH3 region contains the amino acid sequence of SEQ ID NO:3; the CDRL1 region contains the amino acid sequence of SEQ ID NO:4; the CDRL2 region contains the amino acid sequence of SEQ ID NO:5; and the CDRL3 region contains the amino acid sequence of SEQ ID NO:6.

[0080] In one embodiment of this disclosure, the antibody is a mouse antibody. A mouse anti-mesothelin antibody strain SS1 has been developed for cancer treatment in clinical trials. Antibody SS1 comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:7 and a light chain variable region containing the amino acid sequence of SEQ ID NO:8, as disclosed in US7081518 B1.

[0081] SS1 has been observed to induce potent immunogenicity and anti-drug antibodies in patients. Therefore, humanization of SS1 is a necessary and critical step for further drug development. For the preparation of humanized SS1 4D5 (HdSS1), the human receptor framework is selected from clinically validated frameworks. In one embodiment disclosed herein, the antibody HdSS1 comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:9 and a light chain variable region containing the amino acid sequence of SEQ ID NO:10.

[0082] In some embodiments disclosed herein, the antibody comprises human reproductive VL and VH sequences having the highest degree of homology with the mAb SS1 framework region. Specifically, the humanized antibody HuSS1 (DCBPR2002) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:12, as shown in Figure 1.

[0083] The sequences are listed in Table 1.

[0084] Table 1

[0085]

[0086] As used in this article, "GlcNAc" 1 “GlcNAc” 2 “GlcNAc” 3"and "GlcNAc 4 "These represent GlcNAc sugars at different locations in the tentacle-shaped glycan portion."

[0087] As used in this article, This indicates a trimannose structure containing three mannoses, of which the first mannose (Mann) 1 ) is linked to GlcNAc sugar; and the second and third mannoses (Man) 2 and Man 3 ) via α-1,3 and α-1,6 glycosidic bonds with Man 1 connect.

[0088] As used in this article, "-(Fuc)" 0-1 "This indicates that trehalose is present depending on the situation, and when it is present, only one trehalose is present."

[0089] The N-glycans described herein have the structure of formula (1). The synthesis methods of the N-glycans, linkers, and payloads A and B as shown in formula (2) can be found at least in WO2018 / 126092A1.

[0090] As used in this article, "-(CH2)" 0-8 -” indicates that -CH2- may or may not be present, and when present, it can independently be 1, 2, 3, 4, 5, 6, 7 or 8 -CH2- groups.

[0091] In some embodiments, the linker has a functional group capable of linking a conjugating agent and a payload. Examples of such linkers include, but are not limited to, non-cleavable linkers and cleavable linkers. In some embodiments, non-cleavable linkers include, but are not limited to, straight-chain or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, heteroaromatic, alkoxy, acyl, alkylamine, or aromaticamine groups having 2 to 20 carbon atoms. In some embodiments, cleavable linkers include, but are not limited to, linkers containing disulfide bonds, acid-labile linkers, photolabile linkers, peptidase-labile linkers, and esterase-labile linkers having 2 to 20 carbon atoms. Examples of linkers include, but are not limited to, straight-chain or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, heteroaromatic, alkoxy, acyl, alkylamine, aromaticamine, ether, ester, amide, carbamate, carbonate, formulas (3) to (7), linkers containing disulfide bonds, acid-labile linkers, photolabile linkers, peptidase-labile linkers, and esterase-labile linkers having 2 to 20 carbon atoms. In the above examples, more than one of them can be used simultaneously in any order.

[0092]

[0093] In equations (3) and (4):

[0094] R 1 Independently selected from the following groups: hydrogen, halogens, -OR 5 -NO2, -CN, -S(O)2R 5 C1-C 24 Alkyl, C6-C 24 (Hetero)aromatic group, C7-C 24 Alkyl (hetero)aromatic groups and C7-C 24 (Hetero)aryl groups, and

[0095] Wherein, alkyl, (hetero)aromatic, alkyl(hetero)aromatic and (hetero)aryl groups are substituted as appropriate.

[0096] Two substituents R 1 They can be linked together to form cycloalkyl or heteroaromatic substituents, and

[0097] R 5 Independently selected from the following groups of constituents: hydrogen, halogens, C1-C 24 Alkyl, C6-C 24 (Hetero)aromatic group, C7-C 24 Alkyl (hetero)aromatic groups and C7-C 24 (Hetero)aryl alkyl;

[0098] X is C(R) 1 )2, O, S or NR 2 , where R 2 For R 1 ; a is 0, 1, 2, 3, 4, 5, 6, 7, or 8; a' is 0, 1, 2, 3, 4, 5, 6, 7, or 8; and a + a' < 10; and

[0099] L is selected from the group consisting of: straight-chain or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, heteroaromatic, alkoxy, acyl, alkylamino, aromaticamino, ether, ester, amide, carbamate, carbonate, disulfide-containing linker, acid unstable linker, light unstable linker, peptidase unstable linker, and esterase unstable linker or a combination thereof having 2 to 20 carbon atoms.

[0100]

[0101] In equation (5):

[0102] R 1 And L is as defined in equations (3) and (4);

[0103] R 3 Independently selected from the following groups of constituents: hydrogen, halogens, C1-C 24 Alkyl, C6-C 24(Hetero)aromatic group, C7-C 24 Alkyl (hetero)aromatic groups and C7-C 24 (Hetero)aryl alkyl;

[0104] R 4 It is selected from the following groups: hydrogen, halogens, C1-C 24 Alkyl, C6-C 24 (Hetero)aromatic group, C7-C 24 Alkyl (hetero)aromatic groups and C7-C 24 (Hetero)aryl group, wherein the alkyl group is doped with one or more heteroatoms selected from the group consisting of O, N and S, wherein the alkyl group, (hetero)aromatic group, alkyl(hetero)aromatic group and (hetero)aryl group are independently substituted as appropriate.

[0105]

[0106] In equations (6) and (7), L is defined as in equations (3) and (4).

[0107] In some embodiments, when the immunoconjugate is used to treat an individual's disease, payloads A and B may therefore be therapeutic agents independently. The therapeutic agent may be a cell growth inhibitor or a cytotoxic agent or an isotope chelator having a corresponding radioactive isotope. Examples of cell growth inhibitors or cytotoxic agents include, but are not limited to, antimetabolites (e.g., fluorouracil (5-FU), fluorouridine (5-FUdR), methotrexate, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase, gemcitabine, capecitabine, azathioprine, and cytosine). methotrexate, trimethoprim, pyrimethamine, or pemetrexed; alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, mitomycin C, cyclophosphamide, mechlorodiethylamine). Rethamine, uramustine, dibromomannitol, tetranitrate, procarbazine, altretamine, mitozolomide, or temozolomide; alkylating agents (e.g., cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or triplatin); DNA minor groove alkylating agents (e.g., duocarmycins such as CC-1065 and any analogues or derivatives thereof);Pyrrolobenzodiazapenes or any analogues or derivatives thereof; anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, or valrubicin); antibiotics (e.g., dactinomycin, bleomycin, mithramycin, antramycin, streptozotocin, gramicidin D). D) Mitomycins (e.g., mitomycin C); calicheamicins; antimitotic agents (including, for example, maytansinoids) (such as DM1, DM3, and DM4), auristatins (including, for example, monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF)), dolastatins, cryptophycins, vincaalkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), taxanes (e.g., paclitaxel, docetaxel, or novel taxanes), tubulysins, and colchicine. chicines); topoisomerase inhibitors (e.g., irinotecan, topotecan, camptothecin, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, rubitecan, SN38, DXd, DXd2, etoposide, teniposide, amsacrine, or mitoxantrone); HDAC inhibitors (e.g., vorinostat, romidepsin, chidamide, panobinostat, or belinostat);Proteasome inhibitors (e.g., peptidyl boronic acids); and radioactive isotopes, such as At; 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 or 213 P 32 and radioactive isotopes of Lu, including Lu 177 Examples of isotope chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), 1,4,7,10-tetra(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (THP), triethylenetetraamine-N,N,N',N",N"',N"'-hexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetra(methylenephosphonate) (DOTP), and mercaptoacetyltriglycine (MAG3).

[0108] Specifically, the therapeutic agents used in this article include monomethylolpropamine E, monomethylolpropamine F, maytansine, docamycin-hydroxybenzamide azaindole, diethyltriamine-N,N,N',N",N"-pentaacetate, ethitecan, or Dxd2.

[0109] In some embodiments, when the immunoconjugate is used for detection, payloads A and B may be independently labeled. Labels include, but are not limited to, labels or portions thereof that are directly detectable (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions thereof that are indirectly detected, such as enzymes or ligands, for example via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, the radioisotope P. 32 C 14 I 125 H 3 and I 131 Fluorescein, such as rare earth chelates or luciferin and its derivatives; rhodamine and its derivatives; dansyl; umbelliferone; luciferase, such as firefly luciferase and bacterial luciferase; luciferin; 2,3-dihydrotannin Diketones; wasabi peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucoamylase; lysozyme; glycooxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as uricase and xanthine oxidase, coupled to enzymes (such as HRP, lactoperoxidase, or microperoxidase) using hydrogen peroxide dye precursors; biotin / avidin; spin labeling; phage labeling; stable free radicals and similar labels. In another embodiment, the label is a positron emitter. Positron emitters include, but are not limited to, Ga 68 F 18 Cu 64 Y 86 ,Br 76 Zr 89 and I 124 .

[0110] In some embodiments disclosed herein, the immunoconjugate may not be fully loaded with payloads A and B and the linker, and the N-glycan may bind directly to the protecting group. The protecting group may be further replaced by a therapeutic agent or label. One example of a protecting group is an azide group.

[0111] One embodiment of this disclosure relates to a pharmaceutical composition comprising the immunoconjugate of this disclosure and a pharmaceutically acceptable carrier.

[0112] One embodiment of this disclosure relates to a method of treating a disease or condition using the immunoconjugate of this disclosure. The disease may be cancer. Specifically, the cancer is a mesothelin-expressing cancer. "Mesothelin-expressing cancer" means any cancer having cells that express mesothelin. Mesothelin is generally expressed in solid tumors, including solid tumors associated with the lung, pleura, ovary, breast, stomach, bile duct, uterus, and thymus. Therefore, examples of mesothelin-expressing cancers include, but are not limited to, ovarian cancer, mesothelioma, pancreatic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, bile duct cancer, colorectal cancer, endometrial cancer, and breast cancer. Specifically, the cancer is ovarian cancer.

[0113] In some embodiments, based on in vivo pharmacokinetic profiles, immune conjugates exhibit relatively stable conjugation linkages compared to randomly conjugated immune conjugates.

[0114] In some embodiments, in xenograft animal models, immunoconjugates with N-glycans have shown better efficacy than random conjugate immunoconjugates.

[0115] The embodiments disclosed herein will be illustrated by the following specific examples. Those skilled in the art will understand that these examples are for illustrative purposes only and that other modifications and variations are possible without departing from the scope of this disclosure.

[0116] Example

[0117] Unless otherwise specified, each 1 ¹H NMR data were obtained at 500 MHz. Unless otherwise specified, the abbreviations used herein are as follows: Az: azide; Bu: butyl; Bn: benzyl; BOC: tributoxycarbonyl; BOP: benzotriazol-1-yloxytris / dimethylaminophosphonium hexafluorophosphate; DBCO: dibenzocyclooctyneyl; DCC: dicyclohexylcarbodiimide; DCM: dichloromethane; DIPEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide; DMAP: 4-dimethylaminopyridine Pyridine; EDC: 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride; EtOAc: ethyl acetate; eq.: equivalent; GlcNAc: N-acetylglucosamine; GlcNAz: azido-N-acetylglucosamine; HBTU: 3-[bis(dimethylamino)methylonyl]-3H-benzotriazole-1-oxide hexafluorophosphate; benzotriazole hexafluorophosphate tetramethyl pyruvate; HOBt: hydroxybenzotriazole; HOSu: N- Hydroxysuccinimide; HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; hexafluorophosphate azirzotriazole tetramethyl ginuronide; LAH: lithium aluminum hydride; MeOH: methanol; MES: 4-morpholinoethanesulfonic acid; MGAT-1: mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine transferase; MGAT-2: mannose β-1,2-N-acetylglucosamine transferase (α-1,6-)-glycoprotein; MHz: megahertz; MMAE: monomethyloripatetin E; MS (ES): mass spectrometer-electron spray; NMP: N-methylpyrrolidone; Ph: phenyl; Pr: propyl; TEA: triethylamine; TFA: trifluoroacetic acid; THF: tetrahydrofuran; TLC: thin-layer chromatography; Tetrakis: tetra(triphenylphosphine)palladium; UDP: uridine diphosphate.

[0118] Example 1: Humanization of anti-mesothelin SS1 mAb

[0119] Selection of human V-region architectural sequence:

[0120] By using the mouse monoclonal antibody SS1 (whose sequence is disclosed in US 7,081,518 B1) as the parental antibody, the SS1 mAb CDR sequence is described according to the Kabat definition. Figure 1A (SEQ ID NO:7 and 8)

[0121] For the preparation of humanized SS1 4D5 (HdSS1), the human receptor framework was selected from clinically validated frameworks. The human heavy and light chain framework sequences in VH subgroup III (IGHV3-66*04) and VLκ subgroup I (IGKV1-39*01) have been clinically validated and have been successfully used in many humanized antibodies.

[0122] like Figure 1A As shown, the sequence of the heavy chain framework region of IGHV3-66*04 differs from the sequence in mAb SS1 by 35 amino acids (underlined residues), which corresponds to 42.68% (35 / 82 of the total residues in the framework region) of variation. Additionally, the sequence of the light chain framework region of IGKV1-39*01(VL) differs from the sequence in mAb SS1 by 25 amino acids (underlined residues), which corresponds to 30.86% (25 / 81 of the total residues in the framework region) of variation.

[0123] For the preparation of humanized SS1 IMGT (HuSS1), information was obtained from the IMGT database (International Immunogenetics Information). The human germline VL and VH sequences with the highest degree of homology to the mAb SS1 framework region were identified. Homology searches can be performed using BLAST or similar methods. In these studies, the human germline genes IGHV1-2*02 (VH) and IGVK3-11*01 (VL) were identified as the VH and VL sequences most homologous to the corresponding heavy and light chain framework sequences in mAb SS1, respectively.

[0124] like Figure 1B As shown, the sequence of the heavy chain framework region of IGHV1-2*02 differs from that in mAb SS1 by 25 amino acids (underlined residues), which corresponds to a variation of 30.49% (25 / 82 of the total residues in the framework region). Figure 1B As shown, the sequence of the light chain framework region of IGVK3-11*01(VL) differs from that in mAb SS1 by 27 amino acids (underlined residues), which corresponds to a variation of 33.33% (27 / 81 of the total residues in the framework region).

[0125] These two pairs of light and heavy chain sequences (hum 4D5 and hum IMGT) are examples of constructing humanized antibodies against human mesothelin. (SEQ ID NO: 9, 10, 11 and 12)

[0126] Example 2: Binding Affinity Analysis of Humanized Antibodies

[0127] Performance of full-length antibodies

[0128] To confirm the changes in affinity of mouse antibodies after humanization, variable regions of the humanized light chain and humanized heavy chain, respectively, were directly generated using nucleotide synthesis methods. The mouse variable regions, and the humanized versions of IMGT (SEQ ID NO: 11 and 12) and 4D5 (SEQ ID NO: 9 and 10) variable regions were then selectively colonized into... Figure 2 The human Fc chimeric antibody expression vector pTCAE8, as shown, was introduced into host cells to prepare recombinant antibody expression cells. FreeStyle293 cells (manufactured by Invitrogen) were used as the host cells for expression.

[0129] The following procedure is used to transfect the vector constructed in this way into a 30 ml volume of FreeStyle. TM 293 cells were in a cell suspension. During transfection, the cells remained in FreeStyle. TM 293 expression medium. Approximately 24 hours before transfection, FreeStyle... TM 293 cells at 2×10 6 Transfer 15 ml of culture medium per cell per ml. Place the flask in a 37°C incubator containing 8% CO2. Then, dilute 37.5 μg of plasso DNA in 1.5 ml of sterile 150 mM NaCl, for a total volume of 1.5 ml. In another tube, dilute 37.5 μl of PEI (2.0 mg / ml) in 1.5 ml of sterile 150 mM NaCl. Allow the DNA and PEI solutions to stand at room temperature for 5 minutes. Gently mix the solutions by inverting the tube, and then allow the tube to stand at room temperature for approximately 10–20 minutes. Add the DNA-PEI mixture to F293 cells, and incubate the transfected cells on a oscillator platform at 135–150 rpm in a 37°C, 8% CO2 incubator for 4 hours. Then, add an equal volume of fresh culture medium, for a total volume of 30 ml, and culture the cells for 5–7 days. The cells are then harvested for antibody purification and quantification.

[0130] The collected supernatant was filtered through a 0.2-micron filter (manufactured by Millpore) to remove contaminants. The culture supernatant containing the antibody was subjected to affinity purification using protein A (manufactured by Millpore), 1.5M glycine / NaOH buffer, 3M NaCl (pH 9.0) as the absorption buffer, and 0.2M glycine / HCl buffer (pH 2.5) as the dissociation buffer. The pH of the dissociation was adjusted to approximately 6.0–7.0 by adding 1M Tris / HCl buffer (pH 9.0). The prepared antibody solution was replaced with PBS using a dialysis membrane (10,000MW cutoff, manufactured by Spectrum Laboratories) and sterilized by filtration through a 0.22-micron membrane filter (manufactured by Millpore) to produce purified antibody. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm, based on an optimal density conversion of 1.45, equal to 1 mg / ml.

[0131] Antibody binding affinity is measured by ELISA.

[0132] ELISA discs were coated with 1–2 μg / 100 μl mesothelin protein per well. The wells were washed three times with PBS and blocked for 2 hours at 37°C with 300 μl of 5% MPBS per well. After washing with PBS, the wells were incubated with serially diluted mesothelin antibody in 5% MPBS at 37°C for 1.5 hours. The culture discs were washed, and goat multi-line anti-human IgG-HRP antibody (1:10,000) (Jackson Immuno Research) was added to each well. Absorbance was measured as described above, and antibody binding affinity was calculated using Prism software (GraphPad) via nonlinear regression.

[0133] In cases of high variability in the scaffold region, HdSS1(HH), generated by transplanting a CDR sequence from mAb SS1 into the IGHV3-66*04 and IGVK1-39*01 sequences, showed a much lower affinity for mesothelin (KD = 2.61E-08M) (for comparison, mAb SS1, KD = 8.36E-11M). Figure 3 ), (see Table 2 below).

[0134] Compared to HdSS1, HuSS1(HH), generated by transplanting a CDR sequence from mAb SS1 into the IGHV1-2*02 and IGVK3-11*01 sequences, exhibits relatively good affinity for mesothelin (KD = 4.99E-11M) even with high variability in the framework region (for comparison, mAb SS1, KD = 8.36E-11M). Figure 3 (See Table 2 below).

[0135] These results indicate that the IGHV1-2*02 heavy chain framework region and the IGVK3-11*01 light chain framework region are superior at tolerating a relatively high degree of variation without affecting the CDR region configuration.

[0136] Table 2

[0137] ELISA KD(M) SS1 MM 8.36E-11 HdSS1 HH 2.61E-08 HuSS1-HH 4.99E-11

[0138] Affinity measurement and dynamic analysis using BIAcore

[0139] To understand the differences in binding kinetics between individual antibodies, surface plasma resonance (SPR) measurements were performed using a BIAcore T200 (Cytiva Inc.) as previously described (Karlsson and Falt, (1997) J. Immunol Methods 200:121-133). Following the supplier's instructions, the carboxymethylated dextran biosensor chip (CM5, Cytiva Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Mesothelin was diluted to 5 μg / ml with 10 mM sodium acetate at pH 4.0 and then injected at a flow rate of 10 μL / min to obtain approximately 1500 reactive units (RU) of coupled protein, followed by injection of 1 M ethanolamine to block unreacted groups. For kinetic measurements, serially diluted anti-mesothelin mAb (0.3125 nM to 40 nM) was injected at 30 μL / min into HBS-EP+Biacore running buffer provided by the manufacturer (Cytiva Inc.) at 25 °C. The mesothelin binding reaction was corrected by subtracting the reaction from the blank flow cell. The association rate (kon or ka) and dissociation rate (koff or kd) were calculated using a simple one-to-one Langmuir binding model, with individual fittings of kon and koff (Cytiva™ Biacore Insight Evaluation Software).

[0140] The results are displayed in Figure 4 And as shown in Table 3 (below). The kon and koff of the chimeric SS1 mAb binding to mesothelin were 3.415E6 and 1.194E-5, respectively, and the KD was 3.496E-12 mol / L. The kon and koff of HuSS1mAb (IMGT version) binding to mesothelin were 4.493E6 and 1.124E-5, respectively, and the KD was 2.501E-12.

[0141] according to Figure 4The results showed that the humanized antibody HuSS1 (DCBPR2002) can recognize human mesothelin protein, and after humanization, the IMGT version has an affinity similar to that of the mouse SS1 antibody and has good affinity, with a KD value of approximately 2.501E-12.

[0142] Table 3

[0143] ka(1 / Ms) kd(1 / s) KD(M) Rmax(RU) <![CDATA[Chi 2 (RU 2 )]]> SS1 3.415E+6 1.194E-5 3.496E-12 218.2 20.3 HuSS1 4.493E+6 1.124E-5 2.501E-12 180.6 17.9

[0144] Example 3: Preparation of Trimannosyl-DCBPR2002 (DCBPR2002-TM)

[0145] To remove the galactose and sialic acid fractions of the N-glycan from DCBPR2002, 10 mg of DCBPR2002 was treated with 20 μl of β1,4-galactosidase (NEB, PO745L, 8 units / µL) and 5 μl of α2-3,6,8-neuraminidase (NEB, PO720L, 50 units / µL) in 1X GlycoBuffer (NEB, 1 mL total volume) at 37°C for 24 hours. Another 10 μl of β1,4-galactosidase (NEB, PO745L, 8 units / µL) was added to the reaction mixture, and the reaction was continued at 37°C for another 24 hours to obtain G0F / G0 antibody samples. The antibody samples were purified using rProtein ASepharose Fast Flow (GE Healthcare, 17-1279-02). After purification, the antibody samples were analyzed by reduced mass chromatography.

[0146] Preparation of DCBPR2002-2Az Figure 21A )

[0147] MGAT-1 transfers UDP-azido-N-acetylglucosamine to one of the terminal mannose molecules in each arm of the trimannosyl core protein. To confirm this phenomenon in the antibody, trimannosyl-DCBPR2002 (5 mg) and UDP-GlcNAz (final concentration 2.5 mg) were incubated in 1000 μl of 1X buffer SP (25 mM MES (4-morpholinoethanesulfonic acid), 10 mM MnCl2, pH 6.5) at 37 °C for 16 h in the presence of MGAT-1 (0.1 mg; R&D, 8334-GT or homemade). The product DCBPR2002-2Az was analyzed by folded mass chromatography.

[0148] Preparation of DCBPR2002-4Az Figure 21B )

[0149] Trimannosyl-DCBPR2002 (5 mg) and UDP-GlcNAz (2.5 mg) were incubated in 800 μl of 1X buffer SP (25 mM MME S, 10 mM MnCl2, pH 6.5) in the presence of rabbit MGAT-1 (0.2 mg) and rat MGAT-2 (0.05 mg) at 37 °C for 16 hours. After incubation, the reaction product DCBPR2002-4Az was analyzed by reduced mass chromatography and intact mass chromatography.

[0150] Example 4: Preparation of DBCO-vc-MMAE (Compound 5)

[0151] Synthesis of Compound 3

[0152]

[0153] A mixture of DBCO-CO2H(1) (200 mg, 1 eq), EDC (226 mg, 3 eq), and HOSu (376 mg, 3 eq) was dissolved in dichloromethane (5 mL) and stirred at room temperature for 3 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure to give compound 2, which was not further purified.

[0154] DIPEA (170 mg, 2 eq) was added to a mixture of compound 2 (1 eq) and 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionic acid (174 mg, 1.2 eq) in dichloromethane (5 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with dichloromethane and 1N HCl (aq). The organic layer was then washed with brine and dried over MgSO4. The residue was purified by column chromatography with methanol / dichloromethane to give compound 3 (54% yield) as a brown liquid. 1H NMR (600MHz, DMSO) δ7.69(dd,J=7.7,1.3Hz,1H),7.63(d,J=7.4Hz,1H),7.53-7.44(m,3H),7.39(td,J=7.5,1.6Hz ,1H),7.35(td,J=7.5,1.3Hz,1H),7.30(dd,J=7.4,1.6Hz,1H),5.04(d,J=14.0Hz,1H),3.62(d,J=14.0Hz,1H),3.5 8(t,J=6.4Hz,2H),3.48-3.43(m,8H),3.29(dd,J=5.9,2.3Hz,2H),3.13-3.04(m,2H),2.61-2.55(m,1H),2.42(t,J = 6.4Hz, 2H), 2.24 (dt, J = 15.5, 7.8Hz, 1H), 2.00 (ddd, J = 15.4, 8.2, 5.7Hz, 1H), 1.76 (ddd, J = 16.3, 8.0, 5.7Hz, 1H). LC-MS(ESI):m / z[C 28 H 32 The calculated value of [N₂O₇] is 509.2 [M+1]. + The experimental value was 509.2 [M+1]. + .

[0155] Synthesis of DBCO-vc-MMAE (Compound 5)

[0156]

[0157] DIPEA (145 mg, 2 eq) was added to a mixture of compound 3 (286 mg, 1 eq), vc-MMAE (compound 4) (630 mg, 1.1 eq), and HATU (428 mg, 2 eq) in DCM:DMF 2:1 (6 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give compound 5 (71% yield) as a pale yellow solid. 1H NMR(600MHz,MeOD)δ7.66(d,J=7.4Hz,1H),7.61(d,J=3.5Hz,3H),7.51-7.44(m, 3H),7.42-7.37(m,3H),7.33(dt,J=15.5,8.2Hz,5H),7.26(d,J=7.3Hz,1H),7.2 3(t,J=7.4Hz,1H),5.23-5.07(m,3H),4.71-4.48(m,4H),4.29-4.15(m,4H),3.7 5-3.69(m,3H),3.63-3.54(m,8H),3.46-3.39(m,3H),3.36(s,4H),3.30(d,J=16 .5Hz,3H),3.26-3.23(m,2H),3.23-3.16(m,2H),3.12(s,2H),2.96(dd,J=17.0, 10.0Hz,3H),2.75-2.68(m,1H),2.57-2.46(m,4H),2.38(dt,J=15.0,7.5Hz,1H) ,2.28-2.04(m,5H),2.04-1.66(m,8H),1.66-1.49(m,4H),1.45(d,J=29.4Hz,2H ), 1.19 (dd, J=6.6, 3.0Hz, 3H), 1.15 (dd, J=12.8, 6.8Hz, 3H), 1.03-0.70 (m, 24H).

[0158] Example 5: Preparation of DBCO-S-DM1 (Compound 11)

[0159] Synthesis of Compound 7

[0160]

[0161] 1,2-Di(pyridin-2-yl)disulfide (1.83 g, 2 eq) was added to a solution of 4-mercaptobutyric acid (0.5 g, 1 eq) in methanol (10 mL). The mixture was stirred overnight at room temperature. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography with hexane / ethyl acetate to give colorless liquid compound 6 (14% yield). 1H NMR (600MHz, CDCl3) δ8.49 (ddd, J=4.8, 1.8, 0.9Hz, 1H), 7.72 (dt, J=8.1, 1.0Hz, 1H), 7.69-7.61 (m, 1H) ,7.12(ddd,J=7.3,4.9,1.1Hz,1H),2.88(t,J=7.1Hz,2H),2.53(t,J=7.2Hz,2H),2.07(p,J=7.2Hz,2H).

[0162] DIPEA (145 mg, 2 eq) was added to a mixture of compound 6 (286 mg, 1 eq), vc-MMAE(4) (630 mg, 1.1 eq), and HATU (428 mg, 2 eq) in DCM (5 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give compound 7 (71% yield) as a pale yellow solid. 1H NMR (600MHz, DMSO) δ7.18 (d, J=1.5Hz, 1H), 6.90 (s, 1H), 6.62-6.56 (m, 1H), 6.56-6.52 (m, 1H), 5.94 (s, 1H), 5.56 (dd, J= 14.8,9.0Hz,1H),5.31(q,J=6.8Hz,1H),4.52(dd,J=12.0,2.7Hz,1H),4.06(t,J=12.3Hz,1H),3.92(s,3H),3.52-3.45( m,2H),3.25(s,3H),3.13(s,3H),2.93-2.78(m,4H),2.72(s,3H),2.21(t,J=7.2Hz,2H),2.04(dd,J=14.4,2.4Hz,1H),1 .69(p,J=7.4Hz,2H),1.59(s,3H),1.50-1.40(m,2H),1.14(dd,J=28.7,6.6Hz,6H),0.97(d,J=6.4Hz,6H),0.78(s,3H).

[0163] Synthesis of Compound 9

[0164]

[0165] HBTU (0.92 g, 1.5 eq) and DIPEA (0.57 mL, 2 eq) were added to a mixture of compound 1 (0.5 g, 1 eq) and compound 8 (0.62 g, 1.3 eq) in DMF (8 mL). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was extracted with ethyl acetate and water. The organic layer was then washed with brine and dried over MgSO4. The residue was purified by column chromatography with methanol / dichloromethane to give an orange liquid compound 9 (76% yield). LC-MS (ESI): m / z [C 32 H 41 The calculated value of N3O7 is 579.68 [M+1]. + The experimental value was 479.95 [M+1]. + .

[0166] Synthesis of Compound 10

[0167] TFA (2.85 mL) was added to a solution of compound 9 (0.72 g, 1 eq) in dichloromethane (15 mL) under ice bath conditions. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give compound 10 as a brown solid (69% yield). NMR (600MHz, DMSO) δ7.78-7.74(m,2H),7.69-7.66(m,1H),7.62(d,J=7.4Hz,1H),7.52-7.45(m,2H),7.40-7 .33(m,2H),7.30(dd,J=7.4,1.2Hz,1H),5.03(d,J=14.1Hz,1H),3.62(d,J=14.0Hz,1H),3.57-3.53(m,6H),3 .49-3.44(m,6H),3.30(td,J=6.0,1.8Hz,2H),3.12-3.04(m,2H),2.96(dd,J=10.7,5.5Hz,2H),2.59(ddd,J =24.2,9.8,4.7Hz,1H),2.23(dt,J=15.4,7.6Hz,1H),2.04-1.96(m,1H),1.76(ddd,J=16.4,8.0,5.8Hz,1H). LC-MS(ESI):m / z[C 27 H 33 The calculated value of N3O5 is 479.57 [M+1]. + The experimental value was 480.1 [M+1]. + .

[0168] Synthesis of DBCO-S-DM1 (Compound 11)

[0169]

[0170] HBTU (77 mg, 1.5 eq) and DIPEA (0.047 mL, 2 eq) were added to a mixture of compound 7 (70 mg, 1 eq) and compound 10 (100 mg, 0.9 eq) in DMF (7 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with ethyl acetate and water. The organic layer was then washed with brine and dried over MgSO4. The residue was purified by column chromatography with methanol / dichloromethane to give compound 11 (DBCO-S-DM1) as an orange solid (54% yield).1H NMR (600 MHz, DMSO) δ 7.86 (t, J = 5.6 Hz, 1H), 7.76 (t, J = 5.6 Hz, 1H), 7.70 - 7.66 (m, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.52 - 7.43 (m, 2H), 7.40 - 7.32 (m, 2H), 7.29 (d, J = 7.4 Hz, 1H), 7.17 (s, 1H), 6.90 (s, 1H), 6.63 - 6.56 (m, 1H), 6.55 - 6.52 (m, 1H), 5.94 (s, 1H), 5.56 (dd, J = 14.8, 9.0 Hz, 1H), 5.31 (q, J = 6.8 Hz, 1H), 5.02 (d, J = 14.0 Hz, 1H), 4.52 (dd, J = 12.1, 2.7 Hz, 1H), 4.06 (t, J = 12.3 Hz, 1H), 3.93 (d, J = 8.8 Hz, 3H), 3.61 (d, J = 14.0 Hz, 1H), 3.47 (s, 9H), 3.37 (t, J = 5.9 Hz, 2H), 3.29 (td, J = 5.9, 2.2 Hz, 2H), 3.24 (s, 3H), 3.17 (dt, J = 11.6, 9.2 Hz, 3H), 3.12 (s, 2H), 3.10 - 3.02 (m, 2H), 2.88 (ddd, J = 15.9, 12.2, 5.3 Hz, 2H), 2.84 - 2.81 (m, 1H), 2.81 - 2.78 (m, 1H), 2.71 (s, 2H), 2.60 - 2.53 (m, 2H), 2.53 - 2.51 (m, 5H), 2.48 - 2.44 (m, 3H), 2.23 (dt, J = 15.5, 7.8 Hz, 1H), 2.07 (td, J = 7.0, 2.7 Hz, 2H), 2.00 (ddd, J = 15.3, 12.4, 8.4 Hz, 2H), 1.76 (ddd, J = 16.4, 7.9, 5.7 Hz, 1H), 1.71 - 1.65 (m, 2H), 1.59 (s, 2H), 1.50 - 1.41 (m, 2H), 1.24 (dd, J = 6.9, 5.8 Hz, 2H), 1.17 (d, J = 6.8 Hz, 3H), 1.12 (d, J = 6.4 Hz, 3H), 0.84 (ddd, J = 13.1, 9.9, 6.7 Hz, 1H), 0.78 (s, 2H). LC-MS (ESI): m / z [C. 66 H 85 ClN6O 16 S2] Calculated value 1317.99 [M + 1] + , Experimental value 1299.41 [M - 18] + .

[0171] Example 6: Preparation of DBCO-vc-seco DUBA (Compound 20)

[0172] Synthesis of Compound 12

[0173] Compound 12 was prepared as described by Beusker, PH (Mol. Pharmaceutics 2015, 12, 1813-1835).

[0174] Synthesis of Compound 14

[0175]

[0176] Under ice bath conditions, bis(4-nitrophenyl) carbonate (0.858 g, 2 eq) and trimethylamine (0.983 mL, 5 eq) were added to a solution of compound 12 (0.805 g, 1 eq) in 40 mL of THF. The reaction mixture was stirred at room temperature for 8 hours, and then compound 13 (1.85 g, 5 eq) was added to the reaction mixture under ice bath conditions. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the organic solvent was removed under reduced pressure. The organic layer was then washed with brine and dried over MgSO4. The residue was purified by column chromatography with methanol / dichloromethane to give compound 14 (21.7% yield). 1H NMR (600MHz, DMSO) δ10.33(s,1H),9.47(s,1H),8.70(s,1H),8.40-8.31(m,1H),7.99(d,J=8.8Hz,2H),7.74(d,J=9.6Hz ,2H),7.58(d,J=11.7Hz,1H),7.42(s,1H),7.36(s,1H),7.18(d,J=8.8Hz,2H),5.30(s,2H),5.16(t,J=4.2Hz,1H),4.69 -4.61(m,2H),4.47(d,J=2.7Hz,1H),3.83(d,J=11.8Hz,1H),3.80-3.64(m,3H),3.64-3.60(m,1H),3.60-3.51(m,4H),3 .52-3.43(m,3H),3.41(s,4H),3.33(s,1H),2.96-2.92(m,1H),2.88-2.81(m,3H),2.81-2.73(m,2H),1.47-1.21(m,9H). LC-MS(ESI):m / z[C 44 H 51 ClN6O 10 Calculated value: 859.36 [M+1] + The experimental value was 859.7 [M+1]. + .

[0177] Synthesis of Compound 15

[0178]

[0179] Under ice bath conditions, DIPEA (0.34 mL, 2 eq) was added to a mixture of compound 1 (0.3 g, 1 eq), HBTU (0.56 g, 1.5 eq), and 2-(2-aminoethoxy)ethanol-1-ol (0.12 g, 1.2 eq) in DMF (4 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with ethyl acetate and water. The organic layer was then washed with brine and dried over MgSO4. The residue was purified by column chromatography with methanol / dichloromethane to give compound 15 (70% yield). 1H NMR(600MHz,MeOD)δ7.55(d,J=7.4Hz,1H),7.51-7.47(m,1H),7.38-7.33(m,3H),7.24(dtd ,J=22.1,7.5,1.2Hz,2H),7.14(dd,J=7.5,1.4Hz,1H),5.02(t,J=10.9Hz,1H),3.66-3.56( m,2H),3.56-3.50(m,2H),3.39-3.36(m,2H),3.36-3.28(m,2H),3.16-3.11(m,2H),2.59(d t,J=16.4,7.6Hz,1H),2.25(dt,J=15.1,7.5Hz,1H),2.10-2.01(m,1H),1.90-1.82(m,1H). LC-MS(ESI):m / z[C 23 H 24 The calculated value of N₂O₄ is 392.45 [M+1]. + The experimental value was 393.39 [M+1]. + .

[0180] Synthesis of Compound 16

[0181] Under an inert atmosphere, bis(4-nitrophenyl) carbonate (0.47 g, 3 eq) and DIPEA (0.2 mL, 3 eq) were added to a solution of compound 15 (0.2 g, 1 eq) in DMF / CH2Cl2 (6 / 2 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with ethyl acetate and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvents were combined and removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give compound 16 (70% yield). 1H NMR (600MHz, CDCl3) δ8.30-8.27(m,2H),7.70(d,J=7.5Hz,1H),7.53-7.50(m,1H),7.44-7.38(m,5H),7.37(dd ,J=7.5,1.4Hz,1H),7.32(td,J=7.5,0.8Hz,1H),7.27(d,J=1.2Hz,1H),5.17(d,J=13.9Hz,1H),4.43(t,J=4.6 Hz,2H),3.81-3.72(m,2H),3.69(d,J=13.9Hz,1H),3.57-3.44(m,2H),3.43-3.32(m,2H),2.83(ddd,J=16.9,8 .6,5.9Hz,1H),2.45(ddd,J=14.7,8.6,5.8Hz,1H),2.21(dt,J=15.2,6.1Hz,1H),1.97(dt,J=17.0,6.1Hz,1H). LC-MS(ESI):m / z[C 30 H 27 The calculated value of [N3O8] is 557.55 [M+1]. + The experimental value was 558.58 [M+1]. + .

[0182] Synthesis of Compound 18

[0183] DIPEA (0.134 mL, 2.2 eq) was added to a mixture of compound 16 (0.2 g, 1 eq), compound 17 (0.2 g, 1.5 eq), and HOBt (0.11 g, 2.2 eq) in DMF (5 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with ethyl acetate and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvents were combined and removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give compound 18 (36% yield).

[0184] Synthesis of Compound 19

[0185] Under an inert atmosphere, bis(4-nitrophenyl) carbonate (0.08 g, 3 eq) and DIPEA (0.043 mL, 3 eq) were added to a solution of compound 18 (0.05 g, 1 eq) in DMF (3 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with ethyl acetate and water. Subsequently, the organic layer was washed with brine and dried over MgSO4. The organic solvents were combined and removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give compound 19 (33% yield).

[0186] Synthesis of DBCO-vc-seco DUBA (Compound 20)

[0187]

[0188] Under ice bath conditions, TFA (11.2 mL, 3 eq) was added to a solution of compound 14 (0.263 g, 1 eq) in CH2Cl2 (11.2 mL). The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was dissolved in DMF (7.6 mL). Under ice bath conditions, compound 19 (0.324 g, 1.1 eq) and TEA (0.21 mL, 5 eq) were added to the mixture. The reaction mixture was stirred at room temperature overnight. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was extracted with dichloromethane and water. Subsequently, the organic layer was washed with brine and dried over MgSO4. The residue was purified by column chromatography with methanol / dichloromethane to give compound 20 (DBCO-vc-seco DUBA) (55.2% yield).

[0189] Example 7: Synthesis of DBCO-DTPA (Compound 22)

[0190]

[0191] Synthesis of Compound 21

[0192] HOSu (226 mg, 3 eq) was added to a mixture of DBCO-CO2H(1) (200 mg, 1 eq) and EDC (376 mg, 3 eq) in dichloromethane (5 mL). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was extracted with DCM and water. Subsequently, the organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure to give a pale yellow liquid.

[0193] The yellow liquid was added dropwise to a solution of ethylenediamine in dichloromethane over 20 minutes. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was diluted with DCM and NaHCO3. 3(aq.)Extraction was performed. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give a yellow oil (yield: 58.9%). 1 H NMR (600MHz, MeOD) δ7.63 (d, J=7.4Hz, 1H), 7.61-7.56 (m, 1H), 7.47-7.41 (m, 3H), 7.32 (dtd, J= 23.9,7.5,1.2Hz,2H),7.23(dd,J=7.5,1.3Hz,1H),5.08(d,J=14.0Hz,1H),3.63(d,J=14.0Hz,1 H),3.14(dtd,J=19.7,13.5,6.2Hz,2H),2.74(ddd,J=16.6,8.0,6.8Hz,1H),2.68-2.54(m,2H), 2.31(ddd,J=14.8,8.0,6.6Hz,1H), 2.16(dt,J=15.2,6.5Hz,1H), 1.95(dt,J=16.7,6.4Hz,1H). LC-MS(ESI):m / z[C 21 H 21 The calculated value of [N3O2] is 348.16 [M+1]. + The experimental value was 348.03 [M+1]. + .

[0194] Synthesis of DBCO-DTPA (Compound 22)

[0195] Commercially available DTPA (26 mg, 1.1 eq) was added to a solution of compound 21 (13 mg, 1 eq.) in H2O:DMF 3:1 (3 mL). The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure to give a yellow solid DBCO-DTPA (20 mg). LC-MS (ESI): m / z [C 43 H 49 N7O 12 S] Calculated value: 888.32 [M+1] + The experimental value was 888.47 [M+1]. + .

[0196] Example 8: Synthesis of DBCO-PEG3-vc-exinotecan (Compound 25)

[0197]

[0198] Synthesis of Compound 23

[0199] DIPEA (0.082 mL, 2.5 eq) was added to a mixture of ezeticosine mesylate (0.11 g, 1.1 eq) and Fmoc-vc-PAB-PNP (0.144 g, 1 eq) in DMF (3 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the DMF was removed under reduced pressure. The residue was washed with diethyl ether and dichloromethane to give 0.2 g of a gray solid (compound 23), which was not further purified.

[0200] Synthesis of Compound 24

[0201] Diethylamine (0.082 mL, 2.5 eq) was added to a solution of compound 23 in DMF (3 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the DMF was removed under reduced pressure. The residue was washed with diethyl ether and dichloromethane to give 0.14 g of a black solid (compound 24), which was not further purified. 1 H NMR (600MHz, DMSO) δ10.18(s,1H),8.45(s,1H),8.08(d,J=9.0Hz,1H),7.96( s,1H),7.79(d,J=10.8Hz,1H),7.61(d,J=8.4Hz,2H),7.38(d,J=8.4Hz,2H),7 .32(s,1H),6.55(s,1H),6.03(t,J=5.6Hz,1H),5.46(s,2H),5.45(s,2H),5.3 4-5.22(m,3H),5.14-5.04(m,2H),4.54-4.45(m,1H),4.13(dt,J=6.9,6.2Hz, 1H),3.28-3.21(m,1H),3.19-3.15(m,1H),3.15-3.07(m,1H),3.06-3.00(m,1 H),2.98-2.93(m,1H),2.39-2.35(m,3H),2.25-2.18(m,1H),2.18-2.11(m,1H ),2.04-1.98(m,1H),1.93-1.83(m,2H),1.74-1.67(m,1H),1.64-1.56(m,1H) ,1.50-1.42(m,1H),1.42-1.34(m,1H),0.96-0.91(m,3H),0.91-0.85(m,6H). LC-MS(ESI):m / z[C 43 H 49 The calculated value of FN8O9 is 841.36 [M+1]. + The experimental value was 841.34 [M+1]. + .

[0202] Synthesis of Compound 25

[0203] DIPEA (13.7 μL, 3 eq) was added to a mixture of compound 3 (13 mg, 1 eq), compound 24 (33 mg, 1.5 eq), and HATU (30 mg, 3 eq) in DCM:DMF 2:1 (3 mL). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was extracted with DCM and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give compound 25 as a pale yellow solid (66.7% yield). 1 H NMR (600MHz, DMSO) δ10.00(s,1H),8.69(s,1H),8.47(d,J=8.9Hz,1H),8.14(d,J=7.7Hz,1H),8 .07(t,J=9.0Hz,1H),7.88(d,J=8.8Hz,1H),7.82-7.74(m,2H),7.68(dd,J=7.6,1.0Hz,1H),7. 64-7.60(m,2H),7.52-7.43(m,3H),7.37(d,J=8.5Hz,2H),7.35-7.27(m,2H),6.54(s,1H),5.9 9(t,J=7.1Hz,1H),5.45(s,1H),5.43(s,2H),5.32-5.27(m,2H),5.10-5.06(m,1H),5.02(d,J= 14.1Hz,1H),4.38(dd,J=13.0,7.7Hz,1H),4.26-4.20(m,1H),3.64-3.56(m,3H),3.51-3.41(m ,7H),3.31-3.19(m,4H),3.18-2.91(m,6H),2.62(dt,J=3.6,1.8Hz,1H),2.61-2.54(m,1H),2. 40-2.33(m,4H),2.27-2.11(m,3H),2.03-1.92(m,2H),1.91-1.81(m,2H),1.80-1.65(m,3H),1 .63-1.59(m,1H),1.59-1.54(m,1H),1.52-1.40(m,2H),1.38-1.32(m,1H),0.91-0.79(m,9H). LC-MS(ESI):m / z[C 71 H 79 FN 10 O 15 Calculated value: 1331.57 [M+1] +The experimental value was 1331.72 [M+1]. + .

[0204] Example 9: Synthesis of DBCO-PEG3-GGFG-exinotecan (Compound 29)

[0205]

[0206] Synthesis of Compound 27

[0207] Commercially available Boc-GGFG-OH (compound 26) (415 mg, 1 eq) was added to a mixture of EDCI (273 mg, 1.5 eq) and HOSu (164 mg, 1.5 eq) in dichloromethane (18 mL). The mixture was stirred at room temperature for 3.5 hours. The reaction mixture was added dropwise to a mixed DMF solution of ezeticosine mesylate (343 mg, 0.83 eq) and triethylamine (0.2 mL, 1.5 eq). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give a pale yellow solid, compound 27 (507 mg, 63% yield). LC-MS (ESI): m / z [C 44 H 48 FN7O 10 Calculated value: 853.91 [M+1] + The experimental value was 854.35 [M+1]. + 875.91 [M+Na] + The experimental value was 875.52 [M+Na]. + .

[0208] Synthesis of Compound 28

[0209] Trifluoroacetic acid (4 mL) was added to a solution of compound 27 (507 mg, 1 eq) in dichloromethane (4 mL). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was washed with dichloromethane to give compound 28 (378 mg, 85% yield) as a yellow solid. LC-MS (ESI): m / z [C 39 H 40 The calculated value of FN7O8 is 753.79 [M+1]. + The experimental value was 754.18 [M+1]. + .

[0210] Synthesis of Compound 29

[0211] DIPEA (0.18 mL, 20 eq) was added to a solution of compound 28 (40 mg, 1 eq) in DMF (1 mL). The reaction mixture was stirred in an ice bath for 15 minutes. The reaction mixture was then added dropwise to a mixed DMF solution (1 mL) of compound 3 (48 mg, 1.2 eq) and HBTU (30 mg, 1.5 eq). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give compound 29 (44 mg, 67% yield) as a pale yellow solid. LC-MS (ESI): m / z [C 67 H 70 FN9O 14 Calculated value: 1244.34 [M+1] + The experimental value was 1244.56 [M+1]. + 1266.34 [M+Na] + The experimental value was 1266.83 [M+Na]. + .

[0212] Example 10: Synthesis of DBCO-PEG12-GGFG-exinotecan (compound 31)

[0213]

[0214] Synthesis of Compound 30

[0215] DIPEA (0.7 mL, 2 eq) was added to a mixture of compound 2 and NH2-PEG12-COOH (1217 mg, 1.0 eq) in dichloromethane / DMF (8 mL / 8 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give the viscous liquid DBCO-PEG12-GGFG-exinotecan (compound 31) (401 mg, 23% yield). LC-MS (ESI): m / z [C 46 H 68 N2O 16 Calculated value: 905.05 [M] + The experimental value was 905.53 [M]. + .

[0216] Synthesis of Compound 31

[0217] DIPEA (0.14 mL, 20 eq) was added to a mixture of compound 28 (30 mg, 1 eq) in DMF (1 mL). The reaction mixture was stirred in an ice bath for 15 minutes. The reaction mixture was then added dropwise to a mixture of compound 30 (43 mg, 1.2 eq) and HBTU (23 mg, 1.5 eq) in DMF (1 mL). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol / dichloromethane to give compound 31 (17 mg, 26% yield) as a yellow solid. LC-MS (ESI): m / z [C 85 H 106 FN9O 23 Calculated value: 1640.82 [M+1] + The experimental value was 1641.07 [M+1]. + .

[0218] Example 11 Synthesis of DBCO-PEG3-GGFG-DXd2 (Compound 36)

[0219]

[0220] Synthesis of Compound 32

[0221] HOSu (170 mg, 1.5 eq) was added to a mixture of N-(tributoxycarbonyl)-4-aminobutyric acid (200 mg, 1.0 eq) and EDCI (283 mg, 1.5 eq) in DCM (5 mL). The reaction mixture was stirred for 2 hours at room temperature under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was added to a mixture of ezeticosine mesylate (434 mg, 0.83 eq) and Et3N (0.21 mL, 1.5 eq) in DMF (5 mL). The reaction mixture was stirred for 12 hours at room temperature. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography to give compound 32 (402 mg, 79% yield) as a yellow solid. LC-MS (ESI): m / z C 33 H 37 FN4O7[M+H] + Calculated value: 621.26, Experimental value: 621.01.

[0222] Synthesis of Compound 33

[0223] Compound 32 was added to a 1 / 1 (9.5 mL / 9.5 mL) mixture of DCM and TFA. The reaction mixture was stirred for 2 hours at room temperature under a nitrogen atmosphere. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 33 (23 mg, 69% yield) as a yellow solid. LC-MS (ESI): m / z C 28 H 29 FN4O5[M+H] + Calculated value: 521.21, Experimental value: 521.09. 1 H NMR(600MHz,DMSO)δ8.53(d,J=8.7Hz,1H),7.82(d,J=10.8Hz,1H),7.32(s,3H),6 .56(s,1H),5.65-5.53(m,1H),5.43(s,2H),5.25(d,J=18.7Hz,1H),5.15(d,J=18 .7Hz,1H),3.17(t,J=6.0Hz,2H),2.81(t,J=7.6Hz,2H),2.42-2.37(m,3H),2.26( t,J=7.1Hz,2H),2.14(d,J=5.3Hz,2H),1.99-1.70(m,4H),0.87(t,J=7.3Hz,3H).

[0224] Synthesis of Compound 34

[0225] Compound 26 (252 mg, 1.3 eq) was added to a mixture of EDCI (104 mg, 1.5 eq) and HOSu (77 mg, 1.5 eq) in DCM (9 mL). The reaction mixture was stirred for 2 hours at room temperature under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was added to a mixture of compound 33 (231 mg, 1.0 eq) and Et3N (0.1 mL, 1.5 eq). The reaction mixture was stirred for 12 hours at room temperature under a nitrogen atmosphere. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 34 (153 mg, 28% yield) as a yellow solid. LC-MS (ESI): m / z C 48 H 55 FN8O 11 [M+H] + Calculated value: 939.4, Experimental value: 939.68.

[0226] Synthesis of Compound 35

[0227] Compound 34 was added to a 1 / 1 (3 mL / 3 mL) mixture of DCM and TFA. The reaction mixture was stirred for 2 hours at room temperature under a nitrogen atmosphere. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 35 (90 mg, 65% yield) as a yellow solid. LC-MS (ESI): m / z C 43 H 47 FN8O9[M+H] + Calculated value: 839.35, Experimental value: 839.22.

[0228] Synthesis of DBCO-PEG3-GGFG-DXd2 (Compound 36)

[0229]

[0230] Compound 35 (20 mg, 1 eq) was added to a mixture of compound 3 (13.3 mg, 1.1 eq), DIPEA (0.083 mL, 20 eq), and HBTU (13.6 mg, 1.5 eq) in DMF (2 mL). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 1.5 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 36 (18 mg, 58% yield) as a yellow solid. LC-MS (ESI): m / z C 71 H 77 FN 10 O 15 [M+H] + Calculated value: 1329.43, experimental value: 1329.69.

[0231] Example 12 Synthesis of DBCO-PEG12-GGFG-DXd2 (Compound 37)

[0232]

[0233] Compound 35 (20 mg, 1 eq) was added to a mixture of compound 30 (19 mg, 0.9 eq), DIPEA (0.083 mL, 20 eq), and HBTU (13.6 mg, 1.5 eq) in DMF (2 mL). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 1.5 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 37 (6.4 mg, 17% yield) as a yellow solid. LC-MS (ESI): m / z C 89 H 113 FN 10 O 24[M+H] + Calculated value: 1726.9, Experimental value: 1726.6.

[0234] Example 13 Synthesis of BCN-PEG3-VC-PAB-MMAE (Compound 40)

[0235]

[0236] Synthesis of Compound 39

[0237] DIPEA (0.5 mL, 3 eq) was added to a mixture of commercially available compound 38 (300 mg, 1 eq) and NH2-PEG3-COOH (273 mg, 1.3 eq) in dichloromethane / DMF (3 mL / 3 mL). The reaction mixture was stirred at room temperature for 18 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give a viscous liquid compound 39 (235 mg, 62% yield). LC-MS (ESI): m / z [C 20 H 31 NO7] Calculated value: 397.47 [M+1] + The experimental value was 397.39 [M+1]. + 420.47 [M+Na] + The experimental value was 420.07 [M+Na]. + .

[0238] Synthesis of Compound 40

[0239] DIPEA (69 μL, 4 eq) was added to a mixture of compound 39 (41 mg, 1 eq), compound 4 (135 mg, 1.2 eq), and HATU (57 mg, 1.5 eq) in DMF (3 mL). The reaction mixture was stirred at room temperature for 18 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 40 (41 mg, 27% yield) as a yellow solid. LC-MS (ESI): m / z [C 78 H 123 N 11 O 18 Calculated value: 1502.90 [M+1] + The experimental value was 1503.13 [M+1]. + 1524.90 [M+Na] + The experimental value was 1525.43 [M+Na]. + .

[0240] Example 14 Synthesis of BCN-PEG12-GGFG-exinotecan (compound 42)

[0241]

[0242] Synthesis of Compound 41

[0243] DIPEA (0.5 mL, 3 eq) was added to a mixture of commercially available compound 38 (300 mg, 1 eq) and NH2-PEG12-COOH (587 mg, 1.0 eq) in dichloromethane / DMF (4 mL / 4 mL). The reaction mixture was stirred at room temperature for 18 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give a viscous liquid compound 41 (598 mg, 79% yield). LC-MS (ESI): m / z [C 38 H 67 NO 16 Calculated value: 793.95 [M+1] + The experimental value was 794.25 [M+1]. + .

[0244] Synthesis of BCN-PEG12-GGFG-Exitane (Compound 42)

[0245] DIPEA (0.14 mL, 20 eq) was added to a mixture of compound 28 (30 mg, 1 eq), compound 41 (38 mg, 1.2 eq), and HBTU (23 mg, 1.5 eq) in DMF (2 mL). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 42 (10.9 mg, 18% yield) as a yellow solid. LC-MS (ESI): m / z [C 77 H 105 FN8O 23 Calculated value: 1529.72 [M+1] + The experimental value was 1530.9 [M+1]. + .

[0246] Example 15 Synthesis of BCN-PEG3-GGFG-exinotecan (compound 43)

[0247]

[0248] DIPEA (0.14 mL, 20 eq) was added to a mixture of compound 28 (30 mg, 1 eq), compound 39 (19 mg, 1.2 eq), and HBTU (23 mg, 1.5 eq) in DMF (2 mL). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 43 (7.4 mg, 16% yield) as a yellow solid. LC-MS (ESI): m / z [C 59 H 69 FN8O 14 Calculated value: 1133.24 [M+1] + The experimental value was 1133.63 [M+1]. + .

[0249] Example 16 Synthesis of BCN-PEG12-GGFG-DXd2 (Compound 44)

[0250]

[0251] DIPEA (0.14 mL, 20 eq) was added to a mixture of compound 35 (34 mg, 1 eq), compound 39 (29 mg, 0.9 eq), and HBTU (23 mg, 1.5 eq) in DMF (2 mL). The reaction mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 44 (19 mg, 32% yield) as a yellow solid. LC-MS (ESI): m / z C 81 H 112 FN9O 24 [M+H] + Calculated value: 1615.8, experimental value: 1615.42.

[0252] Example 17 Synthesis of DBCO-PEG3-2(PEG3-VC-PAB-MMAE) (Compound 48)

[0253]

[0254] Synthesis of Compound 45

[0255] Compound 3 (490 mg, 1 eq) was added to a mixture of EDC (552 mg, 3 eq) and HOSu (333 mg, 3 eq) in anhydrous dichloromethane / anhydrous DMF (2.4 mL / 2.4 mL). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 18 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure to give a viscous liquid compound 45 (856 mg), which did not require further purification.

[0256] Synthesis of Compound 47

[0257] DIPEA (621.7 mg, 5 eq) was added to a mixture of compound 46 (856 mg) and NH-bis(PEG3-CO2H) (574 mg, 1.4 eq) in dichloromethane / DMF (4.8 mL / 4.8 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give a viscous liquid compound 47 (485 mg, 54% yield). LC-MS (ESI): m / z [C 46 H 65 N3O 16 Calculated value: 916.03 [M] + The experimental value was 916.3 [M]. + . 1H NMR (600MHz, DMSO) δ12.17(s,2H),7.77(t,J=5.6Hz,1H),7.68(dd,J=7.7,1.3Hz,1H),7.62(d,J=7.3Hz ,1H),7.48(m,3H),7.36(m,2H),7.30(dd,J=7.4,1.4Hz,1H),5.02(d,J=14.1Hz,1H),3.61-3.56(m,8H), 3.54-3.32(m,39H),3.31-3.26(m,2H),3.16(d,J=4.9Hz,1H),3.13-3.03(m,3H),2.63-2.54(m,4H),2.4 3(td,J=6.3,2.7Hz,4H),2.23(dt,J=15.5,7.7Hz,1H),1.99(m,1H),1.75(m,1H),1.24(d,J=5.9Hz,9H).

[0258] Synthesis of DBCO-PEG3-2(PEG3-VC-PAB-MMAE) (Compound 48)

[0259] DIPEA (22 mg, 3.2 eq) was added to a mixture of compound 47 (49 mg, 1 eq), compound 4 (72 mg, 1.2 eq), and HBTU (51 mg, 2.5 eq) in DMF (0.43 mL). The reaction mixture was stirred at room temperature for 24 hours. After the reaction was complete, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give a viscous liquid compound 48 (41 mg). LC-MS (TOF): m / z [C 162 H 249 N 23 O 38 Calculated value: 3126.9 [M] + The experimental value was 1042.95 [M]. 3+ 1563.92 [M] 2+ .

[0260] Example 18 Preparation of DCBPR2002-4 (DBCO-vc-MMAE) Figure 21C )

[0261] Preparation of MES pH 6.5 buffer: 4.881 g of free MES acid (2-morpholinoethanesulfonic acid, CAS 4432-31-9) was suspended in 750 mL of dH2O. The pH was adjusted to 6.5 using 10 N NaOH (aq). Subsequently, distilled water was added to the suspension until the volume reached 1 L.

[0262] 5.78 mL of DBCO-vc-MMAE (10 mM in DMSO) was slowly added to a solution of DCBPR2002-4Az (34 mL, 2.5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in sodium citrate pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-vc-MMAE). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 3.89.

[0263] Example 19 Preparation of DCBPR2002-4 (DBCO-S-DM1) Figure 21D )

[0264] 4.48 mL of DBCO-S-DM1 (10 mM in DMA) was slowly added to a solution of DCBPR2002-4Az (11.2 mL, 2.5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C for 6 hours under argon atmosphere. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with 30 kDa NMWL in sodium citrate pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-S-DM1). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: ~4.

[0265] Example 20: Preparation of DCBPR2002-4 (DBCO-vc-seco DUBA) Figure 21E )

[0266] 0.4 mL of DBCO-vc-seco DUBA (10 mM in DMA) and 1.2 mL of DMA were slowly added to a solution of DCBPR2002-4Az (4 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 20 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in sodium citrate pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-vc-seco DUBA). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: ~4.

[0267] Example 21 Preparation of DCBPR2002-4 (DBCO-PEG4-vc-PAB-MMAF) Figure 21F )

[0268] DBCO-PEG4-VC-PAB-MMAF is a commercially available connector - payload.

[0269] 0.4 mL of DBCO-PEG4-VC-PAB-MMAF (10 mM in DMSO) and 0.4 mL of DMSO were slowly added to a solution of DCBPR2002-4Az (4 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with 30 kDa NMWL in sodium citrate pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-PEG4-vc-PAB-MMAF). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: ~4.

[0270] Example 22 Preparation of DCBPR2002-4 (DBCO-DTPA) Figure 21G )

[0271] 0.24 mL of DBCO-DTPA (10 mM in ddH2O) was slowly added to a solution of DCBPR2002-4Az (2.4 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in sodium citrate pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-DTPA). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: ~4.

[0272] Example 23 Preparation of DCBPR2002-4 (DBCO-PEG3-vc-exinotecan) Figure 21H )

[0273] 0.04 mL of DBCO-PEG3-VC-exinotecan (10 mM in DMA) and 0.12 mL of DMA were slowly added to a solution of DCBPR2002-4Az (0.4 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in MES pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-PEG3-VC-exinotecan). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 3.77.

[0274] Example 24 Preparation of DCBPR2002-4 (DBCO-PEG3-GGFG-exinotecan) Figure 21I )

[0275] 0.02 mL of DBCO-PEG3-GGFG-exinotecan (10 mM in DMA) and 0.06 mL of DMA were slowly added to a solution of DCBPR2002-4Az (0.2 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in MES pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-PEG3-GGFG-exinotecan). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 3.21.

[0276] Example 25 Preparation of DCBPR2002-4 (DBCO-PEG12-GGFG-exinotecan) Figure 21J )

[0277] 0.02 mL of DBCO-PEG12-GGFG-exinotecan (10 mM in DMA) and 0.02 mL of DMA were slowly added to a solution of DCBPR2002-4Az (0.1 mL, 10 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in MES pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-PEG12-GGFG-exinotecan). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 3.91.

[0278] Example 26 Preparation of DCBPR2002-4 (DBCO-PEG3-GGFG-DXd2) Figure 21K )

[0279] 0.02 mL of DBCO-PEG3-GGFG-DXd2 (10 mM in DMA) and 0.06 mL of DMA were slowly added to a solution of DCBPR2002-4Az (0.213 mL, 4.7 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in MES pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-branched PEG3-GGFG-exinotecan). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 3.12.

[0280] Example 27 Preparation of DCBPR2002-4 (DBCO-PEG12-GGFG-DXd2) Figure 21L )

[0281] 0.02 mL of DBCO-PEG12-GGFG-DXd2 (10 mM in DMA) and 0.06 mL of DMA were slowly added to a solution of DCBPR2002-4Az (0.213 mL, 4.7 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in MES pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-PEG12-GGFG-DX8951). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 3.52.

[0282] Example 28 Preparation of DCBPR2002-4 (BCN-PEG3-VC-PAB-MMAE) Figure 21M )

[0283] 0.0067 mL of BCN-PEG3-VC-PAB-MMAE (10 mM in DMSO) and 0.0333 mL of DMSO were slowly added to a solution of DCBPR2002-4Az (0.2 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in MES pH 6.5 buffer to obtain DCBPR2002-4 (BCN-PEG3-VC-PAB-MMAE). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 2.67.

[0284] Example 29 Preparation of DCBPR2002-4 (BCN-PEG12-GGFG-exinotecan) Figure 21N )

[0285] 0.02 mL of BCN-PEG12-GGFG-exinotecan (10 mM in DMA) and 0.01 mL of DMA were slowly added to a solution of DCBPR2002-4Az (0.05 mL, 10 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 42 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in MES pH 6.5 buffer to obtain DCBPR2002-4 (BCN-PEG12-GGFG-exinotecan). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 3.51.

[0286] Example 30: Preparation of DCBPR2002-4 (BCN-PEG3-GGFG-exinotecan) Figure 21O )

[0287] 0.01 mL of BCN-PEG3-GGFG-exinotecan (10 mM in DMA) and 0.01 mL of DMA were slowly added to a solution of DCBPR2002-4Az (0.05 mL, 10 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in MES pH 6.5 buffer to obtain DCBPR2002-4 (BCN-PEG3-GGFG-exinotecan). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 3.73.

[0288] Example 31: Preparation of DCBPR2002-4 (BCN-PEG12-GGFG-DXd2) Figure 21P )

[0289] 0.02 mL of BCN-PEG12-GGFG-DXd2 (10 mM in DMA) and 0.06 mL of DMA were slowly added to a solution of DCBPR2002-4Az (0.08 mL, 10 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in MES pH 6.5 buffer to obtain DCBPR2002-4 (DBCO-PEG12-GGFG-DXd2). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 3.51.

[0290] Example 32 Preparation of DCBPR2002-4 (DBCO-PEG3-2 (PEG3-VC-PAB-MMAE)) Figure 21Q )

[0291] 0.02 mL of DBCO-branched-chain-PEG-VC-MMAE-B (10 mM in DMA) and 0.113 mL of DMA were slowly added to a solution of DCBPR2002-4Az (0.333 mL, 3 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with 30 kDa NMWL in MES pH 6.5 buffer to obtain DCBPR2002-4(DBCO-PEG3-2(PEG3-VC-PAB-MMAE)). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: 5.68.

[0292] Example 33 Preparation of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco DUBA) Figure 21R )

[0293] Synthesis of DCBPR2002-2 (DBCO-vc-MMAE)

[0294] 2.04 mL of DBCO-vc-MMAE (10 mM in DMSO) was slowly added to a solution of DCBPR2002-2Az (12 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 20 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with 30 kDa NMWL in sodium citrate pH 6.5 buffer to obtain DCBPR2002-trimannosyl-2 (DBCO-vc-MMAE). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: ~2.

[0295] DCBPR2002-2 (Connector-Payment)-2Az General Synthesis

[0296] 5 mg of DCBPR2002-2 (linker-load) and UDP-GlcNAz (2.5 mg) were incubated in 1000 μl of 1X buffer SP (25 mM MES, 10 mM MnCl2, pH 6.5) in the presence of rat MGAT-2 (0.05 mg) at 37 °C for 16 h. After the reaction, the antibody product was purified by Amicon Ultra-15 centrifugation and filtration to obtain DCBPR2002-2 (linker-load), in which two active GlcNAz molecules were attached to the remaining terminal mannose in the heavy chain. The product was analyzed by folded mass chromatography.

[0297] Synthesis of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco DUBA)

[0298] 4.08 mL of DBCO-vc-seco DUBA (10 mM in DMA) was slowly added to a solution of DCBPR2002-2(DBCO-vc-MMAE)-2Az (10.2 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with 30 kDa NMWL in sodium citrate pH 6.5 buffer to obtain DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco DUBA). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: ~4.

[0299] Example 34 Preparation of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-S-DM1) Figure 21S )

[0300] Synthesis of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-S-DM1)

[0301] 2.08 mL of DBCO-S-DM1 (10 mM in DMA) was slowly added to a solution of DCBPR2002-2(DBCO-vc-MMAE)-2Az (5.2 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with 30 kDa NMWL in sodium citrate pH 6.5 buffer to obtain DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-S-DM1). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: ~4.

[0302] Example 35 Preparation of DCBPR2002-2(DBCO-vc-seco DUBA)-2(DBCO-S-DM1) Figure 21T )

[0303] Synthesis of DCBPR2002-2 (DBCO-vc-seco DUBA)

[0304] 7.2 mL of DBCO-vc-seco DUBA (10 mM in DMSO) was slowly added to a solution of DCBPR2002-2Az (18 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 20 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in sodium citrate pH 6.5 buffer to obtain DCBPR2002-2 (DBCO-vc-seco DUBA). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: ~2. Synthesis of DCBPR2002-2 (DBCO-vc-seco DUBA)-2 (DBCO-S-DM1)

[0305] 2.4 mL of DBCO-S-DM1 (10 mM in DMA) was slowly added to a solution of DCBPR2002-2(DBCO-vc-secoDUBA)-2Az (6 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C under argon atmosphere for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifuge with a 30 kDa NMWL filter in sodium citrate pH 6.5 buffer to obtain DCBPR2002-2(DBCO-vc-secoDUBA)-2(DBCO-S-DM1). The drug-to-antibody ratio (DAR) of the ADC was measured by LC-MS: ~4.

[0306] Example 36 SDS-PAGE

[0307] The ADCs disclosed herein can be analyzed using techniques known in this field, such as SDS-PAGE and HPLC. For example, solutions of anti-MSLN mAb and anti-MSLN ADC can be analyzed by using 4-12% non-reducing and reducing SDS-PAGE gels followed by Kumas Brilliant Blue staining.

[0308] Example 37: Payload Coupling Analysis

[0309] Assessing the drug-to-antibody ratio (DAR) is crucial for monitoring the effective conjugation efficiency of target antibodies. The DAR can influence the therapeutic efficacy of anti-MSLN ADC products. Liquid chromatography-mass spectrometry (LC-MS) is the preferred method for determining the DAR and drug load distribution of lysine-linked antibody-drug conjugates (ADCs). Peak area percentage represents the relative distribution of a specific drug-loaded ADC species. A weighted average DAR is then calculated using the peak area percentage information and the number of drug loads.

[0310] Figure 7 This illustration shows an example of the quality analysis of the disclosed ADC (DCBPR2002-4(DBCO-vc-MMAE)), indicating the distribution of various numbers of drugs attached to the antibody, with the most abundant species having four drugs attached to the antibody. The mean drug-to-antibody ratio (DAR) in this sample is 4.07.

[0311] Example 38 ELISA Binding Affinity

[0312] Add 100 μL of a 1 μg / mL mesothelin-containing spread buffer to each well of the culture dish and spread it onto the wells. Seal the culture dish and incubate overnight at 4°C. Aspirate from the wells and wash three times with 300 μL of PBST (0.05% Tween 20). Block the wells by adding 200 μL of PBS-5% skim milk and incubate at 37°C for 1 hour. Aspirate from the wells and wash three times with 300 μL / well of PBST (0.05% Tween 20). Add 100 μL of 400 ng ADC sample diluted in PBS to each well and then incubate the culture dish at 37°C for 1 hour. Aspirate from the wells and wash three times with 300 μL of PBST (0.05% Tween 20). Add 50 μL of anti-human κ light chain (1:5000) to each well and incubate the culture dish at 37°C for 1 hour. Aspirate from each well and wash three times with 300 μL / well PBST (0.05% Tween 20). Add 100 μL TMB to each well and incubate at room temperature for 15 minutes. Stop color development by adding 100 μL 1N HCl. Measure the absorbance of the culture dish using an ELISA reader at 450-650 nm. Data presented in [link to data]. Figure 5 middle.

[0313] DCBPR2002 Kd=9.243e-011; DCBPR2002-4(DBCO-vc-MMAE)Kd=1.329e-010; DCBPR2002-4(DBCO-s-DM1) Kd=1.449e-010; DCBPR2002-4(DBCO-vc-seco-DUBA) Kd=9.747e-011; DCBPR2002-2(DBCO-vc -MMAE)-2(DBCO-s-DM1)Kd=1.355e-010; DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco-D UBA) Kd = 1.580e-010; and DCBPR2002-2 (DBCO-vc-seco-DUBA)-2 (DBCO-s-DM1) Kd = 7.315e-011.

[0314] Example 39 Binding kinetics of anti-mesothelin ADCs

[0315] By means of surface plasma resonance ( (T100, Biacore, Inc., Piscataway, NJ) to determine the kinetic constants of the interaction between anti-mesothelin ADCs and mesothelin. The flow cell of the CM5 chip used approximately 10,000 reaction units (RU) of anti-human IgG-Fc. 10 mM glycine at pH 5.0 was used for fixation at 10 μL / min for 600 seconds. 10 μg / mL anti-mesothelin antibody and anti-mesothelin ADC diluted in TBS were captured on a CM5 chip at 10 μL / min. Four concentrations (3.7 to 100 nM) of recombinant human mesothelin protein and zero concentration (flow buffer) were recorded at 100 μL / min for 3 minutes in PBS containing 1 mM CaCl2. The dissociation of the complex was measured at 10 minutes. The water surface was regenerated by injecting 3 M MgCl2 and 3 mM EGTA at 10 μL / min for 60 seconds. T100 Evaluation Software The curves obtained after subtracting the reference and buffer signals were incorporated into a 1:1 Langmuir binding model. Ka, Kd, ​​and KD are shown in Table 4. Kinetic analysis showed that the anti-mesothelin antibody and the anti-mesothelin ADC had similar ka(on) and kd(off) rates.

[0316] Table 4

[0317]

[0318] Example 40: In vitro cytotoxicity study (KLM-1 and OVCAR-3)

[0319] Pancreatic cancer cell line KUNK-1 was grown in RPMI 1640 Medium (ATCC modified) supplemented with 10% fetal bovine serum. Ovarian cancer cell line OVCAR-3 was grown in RPMI 1640 Medium (ATCC modified) supplemented with 20% fetal bovine serum. KUNK-1 and OVCAR-3 cell lines were maintained in a humidified incubator at 37°C in a 5% CO2 atmosphere. Cells were collected and seeded in 96-well plates (4,000 cells per well) one day prior to treatment. On the second day, cells were treated with a 3-fold serially diluted toxic payload and ADC. Each treatment was performed at eight triplicate data points. After 72 hours of treatment, cells were analyzed according to the manufacturer's instructions. Cell viability was assessed using the Promega kit. At the end of culture, luminescence was measured using the SpectraMax i3x Molecular Devices multimodal detection platform. Cytotoxicity of the compounds was evaluated compared to cells treated with 0.05% PBS (ADC) or 0.05% DMSO (toxic payload). IC50 was calculated by fitting a four-parameter logarithmic equation to the viability data using GraphPadprism 5.0 software. 50 Values. The results are shown in Table 5.

[0320] Table 5: IC of Toxic Payload and ADC 50 value

[0321]

[0322]

[0323] Example 41 Internalization Analysis

[0324] KLM-1 or OVCAR3 cells were treated with trypsin, then harvested and resuspended in FAC buffer. Control: Secondary Ab anti-human IgG PE (1:200) was added to KLM-1 or OVCAR3 cells. Cells were incubated at 4°C for 0, 0.5, 2, 5, and 24 hours, followed by washing with 1 mL FACS buffer. The supernatant was discarded. Test group: KLM-1 or OVCAR3 cells were pre-incubated on ice for 60 minutes with 10 μg / mL FACS buffer containing trimannosyl anti-mesothelin ADC, washed three times with FACS buffer, and then incubated at 37°C for 0, 0.5, 2, 5, and 24 hours. Cells were analyzed by flow cytometry (BDLSRFortessa), and the results are presented in [image / image / etc.]. Figure 6 middle.

[0325] Example 42 In vivo PK

[0326] This study used the Meso Scale Discovery (MSD) electrochemiluminescence (ECL) method to perform pharmacokinetic analysis of DCBPR2002-4 (DBCO-vc-MMAE) in BALB / c mouse and rat samples. MSD analysis measures both conjugated and unconjugated antibodies. As shown in this example or total antibody analysis, the culture dish was coated with goat anti-human IgG, which captures all humanized antibodies (conjugated and unconjugated). For conjugated antibody analysis, the culture dish was coated with antibodies against the payload (drug), such as anti-MMAE antibodies.

[0327] Mice were administered a dose of 3 mg / kg via tail vein. Blood samples were subsequently collected at different time points to determine the concentration of DCBPR2002-4 (DBCO-vc-MMAE) in mice using the MESO QuickPlex SQ 120 method. Phoenix was used. TM WinNonlin program version 6.3 analyzes pharmacokinetic parameters of DCBPR2002-4 (DBCO-vc-MMAE) using non-compartmental analysis.

[0328] Table 6 summarizes the results of the PK study. Total antibody MSD analysis: measurement of conjugated and non-conjugated antibodies. Conjugated antibody MSD analysis: measurement of conjugated antibodies only. The in vivo half-life of DCBPR2002-4 (DBCO-vc-MMAE) is approximately 87.2 hours. This is because the higher degree of linker protein hydrolysis observed in mice compared to other species can be attributed to carboxylesterase 1C, and the valine-citrulline linker on our ADC is the acceptor of this enzyme.

[0329] In vivo pharmacokinetic studies were designed to compare the connector-load stability of the trimannosyl-conjugated and cysteine-conjugated synthetic ADC (Adcetris). Rats were administered DCBPR2002-4 (DBCO-vc-MMAE) and Adcetris via tail vein at a dose of 5 mg / kg. Serum samples were subsequently obtained at different time points, and the concentrations of DCBPR2002-4 (DBCO-vc-MMAE) and Adcetris in rats were determined using the MESOQuickPlex SQ 120 method. Phoenix [likely referring to a specific pharmacokinetic study or method] was used. TM The WinNonlin program version 6.3 uses non-compartmental analysis to analyze the pharmacokinetic parameters of DCBPR2002-4 (DBCO-vc-MMAE) and Adcetris.

[0330] Table 7 summarizes the results of the PK study. Total antibody MSD analysis: Measured conjugated and non-conjugated antibodies. Conjugated antibody MSD analysis: Measured only conjugated antibodies. The in vivo half-life of DCBPR2002-4 (DBCO-vc-MMAE) for total antibodies was 194 ± 35.0 hours; the half-life of conjugated antibodies for DCBPR2002-4 (DBCO-vc-MMAE) was 148 ± 8.14 hours and for Adcetris was 182 ± 10.9 hours. Figure 8 )

[0331] Table 6

[0332]

[0333] Table 7

[0334]

[0335] A comparison of the pharmacokinetic profiles of total and conjugated antibodies of DCBPR2002-4 (DBCO-vc-MMAE) and Adcetris revealed that the difference between the total and conjugated antibody profiles was closer to that of DCBPR2002-4 (DBCO-vc-MMAE) than that of Adcetris. In vivo results indicated that the proposed trimannosyl conjugation differed from cysteine ​​conjugation (Adcetris) in terms of the stability of the conjugated linker-load.

[0336] Example 43: Xenograft Model of Anti-MSLN ADC (Pancreatic Cancer)

[0337] The purpose of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002-lysine-DBCO-vc-MMAE (a peptide linker of DBCO-vc-MMAE with an azido-activated lysine) and DCBPR2002-4 (DBCO-vc-MMAE) in a male NOD SCID mouse KLM-1 human pancreatic cancer xenograft model.

[0338] Recombinants containing test compound DCBPR2002 lysine-DBCO-vc-MMAE, test compound DCBPR2002-4 (DBCO-vc-MMAE), and corresponding mediators were prepared by diluting the stock solution with 25 mM sodium citrate buffer (pH 6.5). The recombinants were administered intravenously (IV) to mice once a week for three weeks.

[0339] KLM-1 cells were cultured as a monolayer in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C under a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice a week by treatment with trypsin-EDTA. Cells growing in the exponential growth phase were harvested, counted, and used for tumor inoculation.

[0340] Male NOD SCID mice aged 6-7 weeks were purchased from Lesco Biotechnology Co., Ltd. (Taiwan Province, China) and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility with a 12-hour light / 12-hour dark cycle at 19-25℃. The animals had free access to rodent pellet food and water.

[0341] KLM-1 cells were subcutaneously (SC) implanted into the right abdomen of male NOD SCID mice (containing 4 × 10⁻⁶ cells). 6 0.1 mL of a 1:1 PBS / Matrelle mixture per mouse (0.1 mL per cell). When the average tumor volume reached approximately 200 mm²... 3Mice were randomly divided into 3 groups (N=6 per group). Each of the following was administered intravenously once a week for 3 weeks: a mediator, DCBPR2002-lysine-DBCO-vc-MMAE (15 mg / kg), and DCBPR2002-4 (DBCO-vc-MMAE) (15 mg / kg).

[0342] Tumor volume, body weight, mortality rate, and signs of significant toxicity were monitored and recorded three times a week for 28 days. Tumor volume (mm) 3 ) Measure the tumor volume three times a week using a caliper and calculate it according to the following formula: Tumor volume = (w 2 ×l) / 2, where w = tumor width and l = tumor diameter (mm). Tumor growth inhibition (TGI) percentage was calculated using the following formula: TGI% = [1 - (T / C)] × 100%, where T and C represent the mean tumor volume of the treatment group and control group, respectively. A TGI (%) value ≥ 58% was considered significant antitumor activity. One-way ANOVA followed by Dunnett's test was used to compare the catalyst and test substance treatment groups. A p-value < 0.05 was considered statistically significant. Animals were weighed three times per week until the study was completed.

[0343] Figure 9 Tumor growth curves of KLM-1-implanted male NOD SCID mice are presented. Each of the test substances, DCBPR2002-lysine-DBCO-vc-MMAE (15 mg / kg) and DCBPR2002-4 (DBCO-vc-MMAE) (15 mg / kg), was administered intravenously once weekly for 3 weeks. A tumor growth inhibition (TGI) ≥58% compared to the mordant group was considered significant antitumor activity (#). One-way ANOVA followed by Dunnett's test was used to compare the mordant and test substance treatment groups. *P < 0.05 was considered significant. 15 mg / kg DCBPR2002-4 (DBCO-vc-MMAE) significantly reduced KLM-1 tumor growth from day 7 to day 28. 15 mg / kg DCBPR2002-lysine-DBCO-vc-MMAE did not show significant antitumor activity.

[0344] Figure 10 This study demonstrates the changes in body weight in male NOD SCID mice implanted with KLM-1. The test substances DCBPR2002-lysine-DBCO-vc-MMAE (15 mg / kg) and DCBPR2002-4 (DBCO-vc-MMAE) (15 mg / kg) were administered intravenously once weekly for 3 weeks. No weight loss was observed throughout the experiment.

[0345] Example 44: Xenograft Model of Anti-MSLN ADC (Pancreatic Cancer)

[0346] The purpose of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002 and DCBPR2002-4 (DBCO-vc-MMAE) in a male NOD SCID mouse KLM-1 human pancreatic cancer xenograft model.

[0347] Mice were prepared by diluting the stock solution with 25 mM sodium citrate buffer (pH 6.5) to create formulations containing test substance DCBPR2002, test substance DCBPR2002-4 (DBCO-vc-MMAE), and their respective mediators. The formulations were administered intravenously (IV) to mice once a week for three weeks.

[0348] KLM-1 cells were cultured as a monolayer in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C under a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice a week by treatment with trypsin-EDTA. Cells growing in the exponential growth phase were harvested, counted, and used for tumor inoculation.

[0349] Male NOD SCID mice aged 6-7 weeks were purchased from Lesco Biotechnology Co., Ltd. (Taiwan Province, China) and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility with a 12-hour light / 12-hour dark cycle at 19-25℃. The animals had free access to rodent pellet food and water.

[0350] KLM-1 cells were subcutaneously (SC) implanted into the right abdomen of male NOD SCID mice (containing 4 × 10⁻⁶ cells). 6 0.1 mL of a 1:1 PBS / Matrelle mixture per mouse (0.1 mL per cell). When the average tumor volume reached 200 mmHg... 3 Mice were randomly divided into 4 groups (N=6 per group). Each of the following was administered intravenously once a week for 3 weeks: a mediator, DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg) and a naked antibody (DCBPR2002, 30 mg / kg).

[0351] Tumor volume, body weight, mortality rate, and signs of significant toxicity were monitored and recorded three times a week for 28 days. Tumor volume (mm) 3 ) Measure the tumor volume three times a week using a caliper and calculate it according to the following formula: Tumor volume = (w 2×l) / 2, where w = width of the tumor and l = diameter of the tumor (mm). Tumor growth inhibition (TGI) percentage was calculated using the following formula: TGI% = [1 - (T / C)] × 100%, where T and C represent the mean tumor volume of the treatment group and control group, respectively. A TGI (%) value ≥ 58% was considered significant antitumor activity. One-way ANOVA followed by a Dunnett test was used to compare the mordant and test substance treatment groups. A p-value < 0.05 was considered statistically significant. Animals were weighed three times per week until the study was completed.

[0352] Figure 11 Tumor growth curves of KLM-1-implanted male NOD SCID mice are presented. Each of the test substances, DCBPR2002 (30 mg / kg) and DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg), was administered intravenously once weekly for 3 weeks. A tumor growth inhibition (TGI) ≥58% compared to the mordant group was considered significant antitumor activity (#). One-way ANOVA followed by Dunnett's test was used to compare the mordant and test substance treatment groups. *P < 0.05 was considered significant. DCBPR2002-4 (DBCO-vc-MMAE) at 15 and 30 mg / kg significantly reduced KLM-1 tumor growth from day 7 to day 28. DCBPR2002 at 30 mg / kg did not show significant antitumor activity.

[0353] Figure 12 This study demonstrates the changes in body weight in male NOD SCID mice implanted with KLM-1. The test substance DCBPR2002 (30 mg / kg) and DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg) were administered intravenously once weekly for 3 weeks. No weight loss was observed throughout the experiment.

[0354] Example 45: Xenograft Model of Anti-MSLN ADC (Pancreatic Cancer)

[0355] The purpose of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002-4(DBCO-vc-MMAE), DCBPR2002-4(DBCO-vc-seco-DUBA), DCBPR2002-4(DBCO-s-DM1), DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco-DUBA), DCBPR2002-2(DBCO-vc-seco-DUBA)-2(DBCO-s-DM1), and DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-s-DM1) in a male NOD SCID mouse KLM-1 human pancreatic cancer xenograft model.

[0356] The stock solutions were diluted with 25 mM sodium citrate buffer (pH 6.5) to prepare formulations containing test substance DCBPR2002-4 (DBCO-vc-MMAE) (15 mg / kg), test substance DCBPR2002-4 (DBCO-vc-seco-DUBA) (15 mg / kg), test substance DCBPR2002-4 (DBCO-s-DM1) (15 mg / kg), test substance DCBPR2002-2 (DBCO-vc-MMAE)-2 (DBCO-vc-seco-DUBA) (15 mg / kg), test substance DCBPR2002-2 (DBCO-vc-MMAE)-2 (DBCO-s-DM1) (15 mg / kg), test substance DCBPR2002-2 (DBCO-vc-seco-DUBA)-2 (DBCO-s-DM1) (15 mg / kg) and corresponding mediators. The preparations were administered intravenously (IV) once a week for three weeks.

[0357] KLM-1 cells were cultured as a monolayer in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C under a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice a week by treatment with trypsin-EDTA. Cells growing in the exponential growth phase were harvested, counted, and used for tumor inoculation.

[0358] Male NOD SCID mice aged 6-7 weeks were purchased from Lesco Biotechnology Co., Ltd. (Taiwan Province, China) and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility with a 12-hour light / 12-hour dark cycle at 19-25℃. The animals had free access to rodent pellet food and water.

[0359] KLM-1 cells were subcutaneously (SC) implanted into the right abdomen of male NOD SCID mice (containing 4 × 10⁻⁶ cells). 60.1 mL of a 1:1 PBS / Matrelle mixture per mouse (0.1 mL per cell). When the average tumor volume reached 300 mmHg... 3 Mice were randomly divided into 7 groups (N=6 per group). Each of the following drugs was administered intravenously at 15 mg / kg: DCBPR2002-4(DBCO-vc-MMAE), DCBPR2002-4(DBCO-vc-seco-DUBA), DCBPR2002-4(DBCO-s-DM1), DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco-DUBA), DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-s-DM1), and DCBPR2002-2(DBCO-vc-seco-DUBA)-2(DBCO-s-DM1), once a week for 3 weeks.

[0360] Tumor volume, body weight, mortality rate, and signs of significant toxicity were monitored and recorded three times a week for 28 days. Tumor volume was measured three times a week using a caliper and calculated using the following formula: Tumor volume = (w 2 ×l) / 2, where w = width of the tumor and l = diameter of the tumor (mm). Tumor growth inhibition (TGI) percentage was calculated using the following formula: TGI% = [1 - (T / C)] × 100%, where T and C represent the mean tumor volume of the treatment group and control group, respectively. A TGI (%) value ≥ 58% was considered significant antitumor activity. One-way ANOVA followed by a Dunnett test was used to compare the mordant and test substance treatment groups. A p-value < 0.05 was considered statistically significant. Animals were weighed three times per week until the study was completed.

[0361] Figure 13Tumor growth curves of KLM-1-implanted male NOD SCID mice are shown. Each of the test substances DCBPR2002-4 (DBCO-vc-MMAE), DCBPR2002-4 (DBCO-vc-seco-DUBA), DCBPR2002-4 (DBCO-s-DM1), DCBPR2002-2 (DBCO-vc-MMAE)-2 (DBCO-s-DM1), DCBPR2002-2 (DBCO-vc-MMAE)-2 (DBCO-vc-seco-DUBA), and DCBPR2002-2 (DBCO-vc-seco-DUBA)-2 (DBCO-s-DM1) was administered intravenously at 15 mg / kg once weekly for 3 weeks. A tumor growth inhibition (TGI) ≥58% compared to the catalyst group was considered significant antitumor activity (#). One-way ANOVA followed by Dunnett's test was used to compare the catalyst and test group. *P < 0.05 was considered statistically significant. All anti-MSLN ADCs showed significant antitumor activity. The efficacy ranking was: DCBPR2002-4(DBCO-vc-seco-DUBA) = DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco-DUBA)-2(DBCO-s-DM1) > DCBPR2002-4(DBCO-vc-MMAE) > DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-s-DM1) > DCBPR2002-4(DBCO-s-DM1).

[0362] Figure 14 This study demonstrates the changes in body weight in male NOD SCID mice implanted with KLM-1. Each of the following test substances, DCBPR2002-4 (DBCO-vc-MMAE), DCBPR2002-4 (DBCO-vc-seco-DUBA), DCBPR2002-4 (DBCO-s-DM1), DCBPR2002-2 (DBCO-vc-MMAE)-2 (DBCO-s-DM1), DCBPR2002-2 (DBCO-vc-MMAE)-2 (DBCO-vc-seco-DUBA), and DCBPR2002-2 (DBCO-vc-seco-DUBA)-2 (DBCO-s-DM1), was administered intravenously at 15 mg / kg once weekly for 3 weeks. No weight loss was observed in the treatment groups.

[0363] Example 46: Xenograft Model of Anti-MSLN ADC (Ovarian Cancer)

[0364] The purpose of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002-4 (DBCO-vc-MMAE) and DCBPR2002-4 (DBCO-vc-seco-DUBA) in a female NOD SCID mouse OVCAR-3 human ovarian cancer xenograft model.

[0365] Prepare formulations containing test substance DCBPR2002-4 (DBCO-vc-MMAE), test substance DCBPR2002-4 (DBCO-vc-seco-DUBA), and corresponding mediators by diluting the stock solution with 25 mM sodium citrate buffer (pH 6.5). Administer each formulation intravenously (IV) once a week for three weeks.

[0366] OVCAR-3 cells were cultured as a monolayer in RPMI-1640 medium supplemented with 20% fetal bovine serum at 37°C in a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice weekly with trypsin-EDTA treatment. Cells in the exponential growth phase were harvested, counted, and used for tumor inoculation.

[0367] Female NOD SCID mice aged 6-7 weeks were purchased from Lesco Biotechnology Co., Ltd. (Taiwan Province, China) and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility with a 12-hour light / 12-hour dark cycle at 19-25℃. The animals had free access to rodent pellet food and water.

[0368] OVCAR-3 cells were subcutaneously (SC) implanted into the right abdomen of female NOD SCID mice (containing 1×10⁻⁶ cells). 7 0.2 mL of a 1:1 PBS / Matrelle mixture per mouse (0.2 mL per cell). When the average tumor volume reached 300 mmHg... 3 Mice were randomly divided into 4 groups (N=6 per group). Each of the following was administered intravenously once a week for 3 weeks: a cartonator, DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg) and DCBPR2002-4 (DBCO-vc-seco-DUBA) (15 mg / kg).

[0369] Tumor volume is measured three times a week using a caliper and estimated using the following formula: Tumor volume = (w 2×l) / 2, where w = width of the tumor and l = diameter of the tumor (mm). Tumor growth inhibition (TGI) percentage was calculated using the following formula: TGI% = [1 - (T / C)] × 100%, where T and C represent the mean tumor volume of the treatment group and control group, respectively. A TGI (%) value ≥ 58% was considered significant antitumor activity. One-way ANOVA followed by a Dunnett test was used to compare the mordant and test substance treatment groups. A p-value < 0.05 was considered statistically significant. Animals were weighed three times per week until the study was completed.

[0370] Figure 15 Tumor growth curves of OVCAR-3-implanted female NOD SCID mice are presented. The test agents DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg) and DCBPR2002-4 (DBCO-vc-seco-DUBA) (15 mg / kg) were administered intravenously once weekly for 3 weeks. A tumor growth inhibition (TGI) ≥58% compared to the mordant group was considered significant antitumor activity (#). One-way ANOVA followed by Dunnett's test was used to compare the mordant and test agent treatment groups. *P < 0.05 was considered statistically significant. DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg) and DCBPR2002-4 (DBCO-vc-seco-DUBA) (15 mg / kg) significantly reduced OVCAR-3 tumor growth, with TGI (%) values ​​>90%.

[0371] Figure 16 This study demonstrates the changes in body weight in female NOD SCID mice implanted with OVCAR-3. Each of the test substances, DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg) and DCBPR2002-4 (DBCO-vc-seco-DUBA) (15 mg / kg), was administered intravenously once weekly for 3 weeks. No weight loss was observed in the treatment groups.

[0372] Example 47: Xenograft Model of Anti-MSLN ADC (Ovarian Cancer)

[0373] The purpose of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002-4 (DBCO-vc-MMAE) and DCBPR2002-TM in a female NODSCID mouse OVCAR-3 human ovarian cancer xenograft model.

[0374] Prepare formulations containing test substance DCBPR2002-4 (DBCO-vc-MMAE), test substance DCBPR2002-TM, and corresponding mediators by diluting the stock solution with 25 mM sodium citrate buffer (pH 6.5). Administer each formulation intravenously (IV) once a week for three weeks.

[0375] OVCAR-3 cells were cultured as a monolayer in RPMI-1640 medium supplemented with 20% fetal bovine serum at 37°C in a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice weekly with trypsin-EDTA treatment. Cells in the exponential growth phase were harvested, counted, and used for tumor inoculation.

[0376] Female NOD SCID mice aged 6-7 weeks were purchased from Lesco Biotechnology Co., Ltd. (Taiwan Province, China) and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility with a 12-hour light / 12-hour dark cycle at 19-25℃. The animals had free access to rodent pellet food and water.

[0377] OVCAR-3 cells were subcutaneously (SC) implanted into the right abdomen of female NOD SCID mice (containing 1×10⁻⁶ cells). 7 0.2 mL of a 1:1 PBS / Matrelle mixture per mouse (0.2 mL per cell). When the average tumor volume reached 300 mmHg... 3 Mice were randomly divided into 4 groups (N=5 per group). Each of the following was administered intravenously once a week for 3 weeks: a carton, DCBPR2002-4 (DBCO-vc-MMAE) (5 and 15 mg / kg) and DCBPR2002-TM (15 mg / kg).

[0378] Tumor volume is measured three times a week using a caliper and estimated using the following formula: Tumor volume = (w 2 ×l) / 2, where w = width of the tumor and l = diameter of the tumor (mm). Tumor growth inhibition (TGI) percentage was calculated using the following formula: TGI% = [1 - (T / C)] × 100%, where T and C represent the mean tumor volume of the treatment group and control group, respectively. A TGI (%) value ≥ 58% was considered significant antitumor activity. One-way ANOVA followed by a Dunnett test was used to compare the mordant and test substance treatment groups. A p-value < 0.05 was considered statistically significant. Animals were weighed three times per week until the study was completed.

[0379] Figure 17Tumor growth curves were displayed in OVCAR-3-implanted female NOD SCID mice. Each of the test substances, DCBPR2002-4 (DBCO-vc-MMAE) (5 and 15 mg / kg) and DCBPR2002-TM (15 mg / kg), was administered intravenously once weekly for 3 weeks. A tumor growth inhibition (TGI) ≥58% compared to the mordant group was considered significant antitumor activity (#). One-way ANOVA followed by Dunnett's test was used to compare the mordant and test substance treatment groups. *P < 0.05 was considered statistically significant. DCBPR2002-4 (DBCO-vc-MMAE) (5 and 15 mg / kg) significantly reduced OVCAR-3 tumor growth. DCBPR2002-TM at 15 mg / kg did not show antitumor activity.

[0380] Figure 18 This study demonstrates the changes in body weight in female NOD SCID mice implanted with OVCAR-3. Each of the test substances DCBPR2002-4 (DBCO-vc-MMAE) (5 and 15 mg / kg) and DCBPR2002-TM (15 mg / kg) was administered intravenously once weekly for 3 weeks. No weight loss was observed in the treatment groups.

[0381] Example 48: Xenograft Model of Anti-MSLN ADC (Ovarian Cancer)

[0382] The purpose of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002-4 (DBCO-vc-MMAE) in a female NOD SCID mouse OVCAR-3 human ovarian cancer xenograft model.

[0383] Prepare formulations containing test substance DCBPR2002-4 (DBCO-vc-MMAE) and the corresponding mediator by diluting the stock solution with 25 mM sodium citrate buffer (pH 6.5). Administer each formulation intravenously (IV) once a week for three weeks.

[0384] OVCAR-3 cells were cultured as a monolayer in RPMI-1640 medium supplemented with 20% fetal bovine serum at 37°C in a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice weekly with trypsin-EDTA treatment. Cells in the exponential growth phase were harvested, counted, and used for tumor inoculation.

[0385] Female NOD SCID mice aged 6-7 weeks were purchased from Lesco Biotechnology Co., Ltd. (Taiwan Province, China) and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility with a 12-hour light / 12-hour dark cycle at 19-25℃. The animals had free access to rodent pellet food and water.

[0386] OVCAR-3 cells were subcutaneously (SC) implanted into the right abdomen of female NOD SCID mice (containing 1×10⁻⁶ cells). 7 0.2 mL of a 1:1 PBS / Matrelle mixture per mouse (0.2 mL per cell). When the average tumor volume reached 300 mmHg... 3 Mice were randomly divided into 4 groups (N=5 per group). Each of the following was administered intravenously once a week for 3 weeks: a carboxin and DCBPR2002-4 (DBCO-vc-MMAE) (5, 10 and 15 mg / kg).

[0387] Tumor volume is measured three times a week using a caliper and estimated using the following formula: Tumor volume = (w 2 ×l) / 2, where w = width of the tumor and l = diameter of the tumor (mm). Tumor growth inhibition (TGI) percentage was calculated using the following formula: TGI% = [1 - (T / C)] × 100%, where T and C represent the mean tumor volume of the treatment group and control group, respectively. A TGI (%) value ≥ 58% was considered significant antitumor activity. One-way ANOVA followed by a Dunnett test was used to compare the mordant and test substance treatment groups. A p-value < 0.05 was considered statistically significant. Animals were weighed three times per week until the study was completed.

[0388] Figure 19 Tumor growth curves of OVCAR-3-implanted female NOD SCID mice are presented. Each of the test substances DCBPR2002-4 (DBCO-vc-MMAE) (5, 10, and 15 mg / kg) was administered intravenously once weekly for 3 weeks. A tumor growth inhibition (TGI) ≥58% compared to the mordant group was considered significant antitumor activity (#). One-way ANOVA followed by Dunnett's test was used to compare the mordant and test substance treatment groups. *P < 0.05 was considered statistically significant. DCBPR2002-4 (DBCO-vc-MMAE) (5, 10, and 15 mg / kg) significantly reduced OVCAR-3 tumor growth in a dose-dependent manner.

[0389] Figure 20 This study demonstrates changes in body weight in female NOD SCID mice implanted with OVCAR-3. Each of the test substance DCBPR2002-4 (DBCO-vc-MMAE) (5, 10, and 15 mg / kg) was administered intravenously once weekly for 3 weeks. No weight loss was observed in the treatment groups.

[0390] The above examples clearly illustrate the various methods for obtaining and characterizing the ADCs disclosed herein, as well as the effectiveness of the ADCs disclosed herein in treating cancer. Although the embodiments of this disclosure are illustrated by a limited number of examples, those skilled in the art will understand that other variations and modifications are possible without departing from the scope of this disclosure. Therefore, the scope of protection of this disclosure should be limited only by the appended claims. sequence list <110> Biotechnology Development Center Foundation <120> Antibody-drug conjugates containing anti-mesothelin antibodies and their uses <130> none <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> mice <400> 1 Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn 1 5 10 <210> 2 <211> 17 <212> PRT <213> mice <400> 2 Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg 1 5 10 15 Gly <210> 3 <211> 10 <212> PRT <213> mice <400> 3 Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr 1 5 10 <210> 4 <211> 10 <212> PRT <213> mice <400> 4 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mouse <400> 5 Asp Thr Ser Lys Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Mouse <400> 6 Gln Gln Trp Ser Lys His Pro Leu Thr 1 5 <210> 7 <211> 119 <212> PRT <213> Mouse <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 8 <211> 107 <212> PRT <213> Mouse <400> 8 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 9 <211> 119 <212> PRT <213> artificial sequence <220> <223> artificial antibody <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 10 <211> 107 <212> PRT <213> artificial sequence <220> <223> artificial antibody <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 11 <211> 119 <212> PRT <213> artificial sequence <220> <223> artificial antibody <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 12 <211> 107 <212> PRT <213> artificial sequence <220> <223> artificial antibody <400> 12 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105

Claims

1. An immune conjugate comprising: An antibody comprising an antigen-binding fragment that specifically binds to an antigenic determinant in mesothelin, an N-glycan binding domain, and an N-glycan having the structure of formula (1). Equation (1) Where "*" represents a bond or protecting group; A connector that connects to each of the asterisks in the N-glycan when the asterisk represents the bond; Payloads A and B, independently conjugate with the isoconnector; wherein payload A and payload B are the same or different; and The antibody comprises a heavy chain constant region, and the N-glycan binding domain is located within the heavy chain constant region; the antibody comprises two N-glycans; the antibody comprises a heavy chain variable region of the amino acid sequence SEQ ID NO: 11 and a light chain variable region of the amino acid sequence SEQ ID NO: 12; and the antibody is a monoclonal antibody, a humanized antibody, or an scFv-Fc fragment; and At least one of the effective loads A and B is a therapeutic agent, wherein the therapeutic agent is an antimetabolite, a DNA minor groove alkylating agent, a topoisomerase inhibitor, or an isotope chelating agent.

2. The immunoconjugate of claim 1, wherein the linker is selected from the group consisting of: straight-chain or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, heteroaromatic, alkoxy, acyl, alkylamino, aromaticamino, ether, ester, amide, carbamate, carbonate, formula (3) to (7), linkers containing disulfide bonds, acid unstable linkers, light unstable linkers, peptidase unstable linkers, and esterase unstable linkers or combinations thereof; Equation (3) Equation (4) In equations (3) and (4): R 1 is independently selected from the group consisting of hydrogen, halogen, -OR 5 , -NO2, -CN, -S(O)2R 5 , C1 - C 24 alkyl, C6 -C 24 (hetero)aromatic, C7 - C 24 alkyl(hetero)aromatic, and C7 - C 24 (hetero)aralkyl, and The alkyl, (hetero)aromatic, alkyl(hetero)aromatic and (hetero)aryl groups are substituted as appropriate. two substituents R 1 may be linked together to form an annelated cycloalkyl or annelated (hetero)arene substituent, and R 5 Independently selected from the following groups of constituents: hydrogen, halogens, C1-C 24 Alkyl, C6-C 24 (Hetero)aromatic group, C7-C 24 Alkyl (hetero)aromatic groups and C7-C 24 (Hetero)aryl alkyl; X is C(R) 1 )2, O, S or NR 2 , where R 2 For R 1 a is 0, 1, 2, 3, 4, 5, 6, 7, or 8; a' is 0, 1, 2, 3, 4, 5, 6, 7, or 8; and a + a' < 10; and L is selected from the group consisting of: straight-chain or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, heteroaromatic, alkoxy, acyl, alkylamino, aromaticamino, ether, ester, amide, carbamate, carbonate, disulfide-containing linker, acid unstable linker, light unstable linker, peptidase unstable linker, and esterase unstable linker or combinations thereof having 2 to 20 carbon atoms; Equation (5) In equation (5), R 1 Independently selected from the following groups: hydrogen, halogens, -OR 5 -NO2, -CN, -S(O)2R 5 C1 - C 24 Alkyl, C6-C 24 (Hetero)aromatic group, C7-C 24 Alkyl (hetero)aromatic groups and C7-C 24 (Hetero)aryl groups, and The alkyl, (hetero)aromatic, alkyl(hetero)aromatic and (hetero)aryl groups are substituted as appropriate. Two substituents R 1 They can be linked together to form cycloalkyl or heteroaromatic substituents, and R 5 Independently selected from the following groups of constituents: hydrogen, halogens, C1-C 24 Alkyl, C6-C 24 (Hetero)aromatic group, C7-C 24 Alkyl (hetero)aromatic groups and C7-C 24 (Hetero)aryl alkyl; L is selected from the group consisting of: straight-chain or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, heteroaromatic, alkoxy, acyl, alkylamino, aromaticamino, ether, ester, amide, carbamate, carbonate, disulfide-containing linker, acid unstable linker, light unstable linker, peptidase unstable linker, and esterase unstable linker or combinations thereof having 2 to 20 carbon atoms; R 3 Independently selected from the following groups of constituents: hydrogen, halogens, C1-C 24 Alkyl, C6-C 24 (Hetero)aromatic group, C7-C 24 Alkyl (hetero)aromatic groups and C7-C 24 (Hetero)aryl alkyl; and R 4 It is selected from the following groups: hydrogen, halogens, C1-C 24 Alkyl, C6-C 24 (Hetero)aromatic group, C7-C 24 Alkyl (hetero)aromatic, C7-C 24 (Hetero)aryl group, wherein the alkyl group is heteroatomated with one or more heteroatoms selected from the group consisting of O, N and S, wherein the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)aryl group are independently substituted as appropriate; and Equation (6) Equation (7) In equations (6) and (7), L is selected from the group consisting of: straight-chain or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, heteroaromatic, alkoxy, acyl, alkylamino, aromaticamino, ether, ester, amide, carbamate, carbonate, disulfide-containing linker, acid unstable linker, light unstable linker, peptidase unstable linker, and esterase unstable linker or a combination thereof having 2 to 20 carbon atoms.

3. The immune conjugate of claim 1, wherein the other of the payloads A and B is a marker.

4. The immunoconjugate of claim 1, wherein the therapeutic agent is monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), maytansinoids, duocarmycin-hydroxybenzamide azaindole (DUBA), diethyltriamine-N,N,N',N",N"-pentaacetic acid (DTPA), exatecan, or Dxd2.

5. The immunoconjugate of claim 3, wherein the label is a fluorescent label, a chromogenic label, an electron-dense label, a chemiluminescent label, a radioactive label, an enzyme label, or a positron emitter.

6. The immunoconjugate of claim 1, wherein the protecting group is an azide group.

7. A pharmaceutical composition comprising an immunoconjugate as described in any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. Use of an immune conjugate as described in any one of claims 1 to 6, for the preparation of a medicament for treating cancer in an individual in need, wherein the cancer is ovarian cancer or pancreatic cancer expressing mesothelin.

9. The use as described in claim 8, wherein the cancer is ovarian cancer.

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