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

Abstract

The present disclosure provides an immunoconjugate comprising an antibody comprising an antigen-binding fragment that specifically binds to an epitope in mesothelin, an N-glycan-binding domain, and an N-glycan, a linker that links the N-glycan, and payload A and payload B, which may be the same or different, each attached to the linker. Pharmaceutical compositions and methods for treating cancer comprising the immunoconjugate are also provided in the present disclosure.

Description

[Technical Field]

[0001]

[0001] The present disclosure relates to anti-mesothelin antibody-drug conjugates, wherein the glycoprotein contained therein comprises one or more trimannosyl cores. The present disclosure also relates to methods of treating diseases, such as cancer, in a subject in need thereof, comprising administering the anti-mesothelin antibody-drug conjugate to the subject. [Background technology]

[0002]

[0002] Antibody-drug conjugates (ADCs) typically contain an anti-cancer drug (e.g., a cytotoxin) attached to an antibody that specifically targets a marker, such as a tumor marker. The antibody tracks these markers in the body and attaches itself to the surface of cancer cells. Binding of the antibody to the target marker (antigen) generates a signal in the tumor cell, causing the ADC to be internalized. After the ADC is internalized, a cytotoxic drug can be released, killing the cancer cell. Specific targeting can reduce the side effects of the drug.

[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 biliary tract cancer, lung cancer, and epithelial mesothelioma. Therefore, mesothelin is a promising diagnostic / therapeutic target.

[0004]

[0004] Many antibodies against mesothelin have been developed, including SS1P (an anti-mesothelin immunotoxin composed of a targeting antibody fragment genetically fused to a cleaved fragment of Pseudomonas exotoxin A), anetumab (a monoclonal antibody), and anetumab ravtansine (an antibody-drug conjugate), but there remains a need for improved therapeutic agents using anti-mesothelin antibodies. Summary of the Invention

[0005]

[0005] The present disclosure relates to antibody-drug conjugates comprising anti-mesothelin antibodies and their use in therapy.

[0006]

[0006] One aspect of the present disclosure relates to an immunoconjugate. An immunoconjugate according to one embodiment of the present disclosure comprises: An antibody comprising an antigen-binding fragment that specifically binds to an epitope in mesothelin, an N-glycan-binding domain, and an N-glycan having the structure of formula (1): [ka] (In the formula, “ * " represents a bond or a protecting group); " * " represents a bond, " in the N-glycan * a linker connecting each of Payload A and payload B, which may be the same or different, independently conjugated to a linker.

[0007]

[0007] In some embodiments of the present disclosure, the antibody is a monoclonal antibody, a humanized antibody, a human antibody, an antibody Fab fragment, F(ab')2, an Fv fragment or Fc fragment from a cleaved antibody, an scFv-Fc fragment, a minibody, a diabody, or an scFv.

[0008] In some embodiments of the present disclosure, the antigen-binding fragment comprises a complementarity determining region (CDR) of a heavy chain variable region and a complementarity determining region of a light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3 regions, and the complementarity determining region of the light chain variable region comprises CDRL1, CDRL2, and CDRL3 regions; the CDRH1 region comprises the amino acid sequence of SEQ ID NO: 1; the CDRH2 region comprises the amino acid sequence of SEQ ID NO: 2; the CDRH3 region comprises the amino acid sequence of SEQ ID NO: 3; the CDRL1 region comprises the amino acid sequence of SEQ ID NO: 4; the CDRL2 region comprises the amino acid sequence of SEQ ID NO: 5; and the CDRL3 region comprises the amino acid sequence of SEQ ID NO: 6. In one embodiment of the present disclosure, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In one embodiment of the present 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 the present 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.

[0009] In some embodiments of the present disclosure, the antibody comprises a heavy chain constant region, and the N-glycan binding domain is located in the heavy chain constant region.

[0010] In some embodiments of the present disclosure, the antibody comprises two N-glycans.

[0011]

[0011] Particular embodiments of effective amounts of immunoconjugates of the present disclosure are in the range of 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.

[0012] In some embodiments of the present disclosure, the linker is selected from the group consisting of a linear or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamine, arylamine group, ether, ester, amide, carbamate, carbonate, Formula (3) to Formula (7), disulfide-containing linker, acid labile linker, photolabile linker, peptidase labile linker, and esterase labile linker, or a combination thereof, having 2 to 20 carbon atoms.

[0013] In some embodiments of the present disclosure, payload A and payload B are independently selected from a therapeutic agent and a label.

[0014]

[0014] Examples of therapeutic agents include, but are not limited to, antimetabolites, alkylating agents, alkylating-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, mitotic inhibitors, topoisomerase inhibitors, proteasome inhibitors, radioisotopes, and isotope chelators. Examples of specific compounds that are therapeutic agents include, but are not limited to, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoids, duocarmycin-hydroxybenzamide azaindole (DUBA), diethylenetriamine-N,N,N',N'',N''-pentaacetate (DTPA), exatecan, and Dxd2.

[0015]

[0015] Examples of labels include, but are not limited to, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radioactive labels, enzyme labels, and positron emitters.

[0016] An example of a protecting group is azide.

[0017]

[0017] One aspect of the present disclosure relates to a pharmaceutical composition comprising the immunoconjugate described above and a pharmaceutically acceptable carrier.

[0018]

[0018] One aspect of the present disclosure relates to a method for treating cancer. The method according to one embodiment of the present disclosure may include administering to a subject in need of cancer treatment a therapeutically effective amount of the immunoconjugate described above.

[0019] In some embodiments of the present disclosure, the cancer is a mesothelin-expressing cancer, including, but not limited to, ovarian cancer, mesothelioma, pancreatic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, biliary tract cancer, colorectal cancer, endometrial cancer, and breast cancer.

[0020]

[0020] Those skilled in the art will understand that the therapeutically effective amount depends on many factors, such as the patient's condition, age, disease state, route of administration, etc., and that such an effective amount can be determined in routine practice based on these factors without undue experimentation.

[0021]

[0021] Other aspects of the present disclosure will become apparent from the following description. [Brief explanation of the drawings]

[0022] [Figure 1A] Figures 1A and 1B show sequence alignments of anti-mesothelin antibodies. [Figure 1B] Figures 1A and 1B show sequence alignments of anti-mesothelin antibodies. [Figure 2] FIG. 2 shows the vector constructs expressing the murine variable regions, the humanized version of IMGT, and the 4D5 variable regions. [Figure 3] FIG. 3 shows the affinity of mesothelin for antibodies. [Figure 4] FIG. 4 shows kinetic analysis of SS1 and HuSS1 antibodies using BIAcore. [Figure 5] Figure 5 shows the ELISA binding affinities of the detected ADCs. [Figure 6] FIG. 6 shows the results of ADC internalization. [Figure 7]FIG. 7 shows the mass spectrometry of ADC DCBPR2002-4 (DBCO-vc-MMAE). [Figure 8] Figure 8 shows the results of the pharmacokinetic profile of ADC DCBPR2002-4 (DBCO-vc-MMAE). [Figure 9] FIG. 9 shows tumor growth curves in male NOD SCID mice implanted with KLM-1. [Figure 10] FIG. 10 shows the changes in body weight of male NOD SCID mice transplanted with KLM-1. [Figure 11] FIG. 11 shows tumor growth curves in male NOD SCID mice implanted with KLM-1. [Figure 12] FIG. 12 shows the changes in body weight of male NOD SCID mice transplanted with KLM-1. [Figure 13] FIG. 13 shows tumor growth curves in male NOD SCID mice implanted with KLM-1. [Figure 14] FIG. 14 shows the changes in body weight of male NOD SCID mice transplanted with KLM-1. [Figure 15] FIG. 15 shows tumor growth curves in female NOD SCID mice implanted with OVCAR-3. [Figure 16] FIG. 16 shows the weight change of female NOD SCID mice transplanted with OVCAR-3. [Figure 17] FIG. 17 shows tumor growth curves in female NOD SCID mice implanted with OVCAR-3. [Figure 18] FIG. 18 shows the weight change of female NOD SCID mice transplanted with OVCAR-3. [Figure 19] FIG. 19 shows tumor growth curves in female NOD SCID mice implanted with OVCAR-3. [Figure 20] FIG. 20 shows the weight change of female NOD SCID mice transplanted with OVCAR-3. [Figure 21A]Figures 21A-20 show the preparation of immunoconjugates: A: Preparation of DCBPR2002-2Az. [Figure 21B] Figures 21A-20 show the preparation of immunoconjugates. B: Preparation of DCBPR2002-4Az. [Figure 21C] Figures 21A-20 show the preparation of immunoconjugates. C: Preparation of DCBPR2002-4 (DBCO-vc-MMAE). [Figure 21D] Figures 21A-20 show the preparation of immunoconjugates. D: Preparation of DCBPR2002-4 (DBCO-S-DM1). [Figure 21E] Figures 21A-20 show the preparation of immunoconjugates. E: Preparation of DCBPR2002-4 (DBCO-vc-seco DUBA). [Figure 21F] Figures 21A-20 show the preparation of immunoconjugates: F: DCBPR2002-4 (DBCO-PEG4-vc-PAB-MMAF). [Figure 21G] 21A-20 show the preparation of immunoconjugates. G: Preparation of DCBPR2002-4 (DBCO-DTPA). [Figure 21H] Figures 21A-20 show the preparation of immunoconjugates. H: Preparation of DCBPR2002-4 (DBCO-PEG3-vc-exatecan). [Figure 21I] Figures 21A-20 show the preparation of immunoconjugates. I: Preparation of DCBPR2002-4 (DBCO-PEG3-GGFG-Exatecan). [Figure 21J] Figures 21A-20 show the preparation of immunoconjugates. J: Preparation of DCBPR2002-4 (DBCO-PEG12-GGFG-Exatecan). [Figure 21K] 21A-20 show the preparation of immunoconjugates. K: Preparation of DCBPR2002-4 (DBCO-PEG3-GGFG-DXd2). [Figure 21L]Figures 21A-20 show the preparation of immunoconjugates. L: Preparation of DCBPR2002-4 (DBCO-PEG12-GGFG-DXd2). [Figure 21M] 21A-20 show the preparation of immunoconjugates. M: Preparation of DCBPR2002-4 (BCN-PEG3-VC-PAB-MMAE). [Figure 21N] Figures 21A-20 show the preparation of immunoconjugates. N: Preparation of DCBPR2002-4 (BCN-PEG12-GGFG-Exatecan). [Figure 21O] Figures 21A-20 show the preparation of immunoconjugates. O: Preparation of DCBPR2002-4 (BCN-PEG-GGGF-Exatecan). [Figure 21P] 21A-20 show the preparation of immunoconjugates. P: Preparation of DCBPR2002-4 (BCN-PEG12-GGFG-DXd2). [Figure 21Q] 21A-20 show the preparation of immunoconjugates. Q: Preparation of DCBPR2002-4 (DBCO-PEG3-2(PEG3-VC-PAB-MMAE)). [Figure 21R] Figures 21A-20 show the preparation of immunoconjugates. R: Preparation of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco DUBA). [Figure 21S] 21A-20 show the preparation of immunoconjugates. S: Preparation of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-S-DM1). [Figure 21T] Figures 21A-20 show the preparation of immunoconjugates. T: Preparation of DCBPR2002-2 (DBCO-vc-seco DUBA)-2 (DBCO-S-DM1). DETAILED DESCRIPTION OF THE INVENTION

[0023]

[0043] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0024]

[0044] 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 invention belongs. Any methods and materials similar or equivalent to those described herein can be understood and used by one of ordinary skill in the art to practice the present invention.

[0025]

[0045] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0026]

[0046] The term "and / or" is used to refer to both or either of two mentioned items.

[0027]

[0047] 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 certain embodiments, an immunoconjugate comprises no more than one effector moiety. Certain immunoconjugates according to the invention consist essentially of one effector moiety and one antibody joined by one or more linkers.

[0028]

[0048] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., mesothelin). The term "antibody" encompasses immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V H The heavy chain constant region comprises a C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (herein referred to as LCVR or V L The light chain constant region comprises one domain (C L1 ) included. V H and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of an anti-alpha toxin antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0029]

[0049] The term "monoclonal antibody," as used herein, is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies may be obtained from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art.

[0030]

[0050] "Humanized" forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.

[0031]

[0051] The term "complementarity-determining region" (CDR), as used herein, refers to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Department 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), and the definitions encompass overlapping or subsets of amino acid residues when compared against each other.

[0032]

[0052] As used herein, the term "antigen-binding fragment" of an antibody and like terms include any naturally occurring, enzymatically derived, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0033]

[0053] As used herein, the term "mesothelin" refers to a 40 kDa protein, mesothelin, anchored to cell membranes by glycosylphosphatidylinositol (GPI) linkages and shed from a 31 kDa amino-terminal fragment called megakaryocyte potentiating factor (MPF). Both fragments contain N-glycosylation sites. Preferably, the term refers to human mesothelin and its naturally cleaved portions, for example, expressed on cell membranes, e.g., cancer cell membranes. In particular, the mesothelin fragment includes the N-terminal region of mesothelin.

[0034]

[0054] As used herein, the term "epitope" refers to a site on an antigen to which an antibody binds.

[0035]

[0055] As used herein, the term "N-glycan" refers to an N-linked oligosaccharide, e.g., attached to an asparagine residue of a polypeptide via an asparagine-N-acetylglucosamine linkage. N-glycans share a common pentasaccharide core, Man3GlcNAc2 ("Man" refers to mannose; "Glc" refers to glucose; "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 differ with respect to the number of branches (antennae) containing peripheral sugars (e.g., fucose and sialic acid) that are added to the Man3 core structure.

[0036]

[0056] The term "pharmaceutical composition" as used herein refers to a formulation or preparation containing a biologically or pharmacologically active ingredient and a pharmaceutically acceptable carrier. The pharmaceutical composition may be in the form of a solution, suspension, tablet, powder, pellet, bead, granule, microsphere, capsule, pill, etc.

[0037]

[0057] The terms "treatment," "treating," and "treat" generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease, disorder, or its symptoms are completely or partially prevented, or therapeutic, in that a disease, disorder, and / or its resulting symptoms are partially or completely cured. As used herein, "treatment" covers the treatment of a disease in a mammal, preferably a human, and includes (1) inhibiting the progression of the disease, disorder, or its symptoms in a subject, or (2) alleviating or ameliorating the disease, disorder, or its symptoms in a subject.

[0038]

[0058] As used herein, the term "subject" refers to any animal that can benefit from the administration of a compound or composition disclosed herein. In some embodiments, the subject is a mammal, such as a human, a primate, a dog, a cat, a horse, a cow, a pig, or a rodent, such as a rat or a mouse. Typically, the mammal is a human.

[0039]

[0059] The term "effective amount" of an active ingredient provided herein refers to an amount of the ingredient sufficient to provide the desired modulation of a desired function. As noted below, the exact amount required will vary from subject to subject, depending on the subject's disease state, physical condition, age, sex, species, and weight, the specific identity and formulation of the composition, etc. The administration regimen can be adjusted to induce the optimal therapeutic response. For example, several divided doses can be administered daily, or the dosage can be proportionally reduced as indicated by the requirements of the therapeutic situation. Therefore, it is not possible to specify an exact "effective amount." However, an appropriate effective amount can be determined by one skilled in the art using only routine experimentation.

[0040]

[0060] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (either a human or non-human animal) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical textbooks.

[0041]

[0061] The immunoconjugates of the present disclosure may be formulated with a "carrier." As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharmaceutical active substances is well known in the art. For example, the pharmaceutical combination may be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., buccal, sublingual, and those targeted for systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation; (3) topical application, e.g., as a cream, lotion, gel, ointment, or controlled-release patch or spray applied to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, suppository, or foam; (5) sublingual administration; (6) ophthalmic administration; (7) transdermal administration; (8) transmucosal administration; or (9) nasal administration.

[0042]

[0062] 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 bringing the drug into the vicinity of the target and resulting in a highly selective therapeutic effect. An example of an ADC may be an antibody that targets a protein expressed on cancer cells, and the payload may be a cytotoxic drug. One embodiment of the present disclosure relates to an immunoconjugate (antibody-drug conjugate) comprising an anti-mesothelin antibody or binding fragment thereof and two or more payloads. In one embodiment of the present disclosure, the immunoconjugate comprises: an antigen-binding fragment that specifically binds to an epitope in mesothelin, an antibody comprising an N-glycan-binding domain and an N-glycan having the structure of Formula (1): [ka] During the ceremony, `` * " represents a bond or a protecting group; " * " represents a bond, " in the N-glycan * a linker connecting each of Payload A and payload B, which may be the same or different, independently conjugated to a linker.

[0043]

[0063] In one embodiment of the present disclosure, the N-glycan, linker, and payloads A and B have the structure of formula (2): [ka]

[0044]

[0064] In further embodiments of the present disclosure, payload A and payload B are the same or different.

[0045]

[0065] According to embodiments of the present disclosure, anti-mesothelin antibodies or binding fragments thereof can recognize and bind to mesothelin or its fragments. As used herein, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" as used herein is also intended to encompass human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to mesothelin. The term "antibody" encompasses whole antibodies, as well as antibody fragments, such as antibody Fab fragments, F(ab')2 fragments, Fv or Fc fragments from truncated antibodies, scFv-Fc fragments, minibodies, diabodies, or scFv fragments. Furthermore, the term encompasses genetically engineered derivatives of antibodies. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art.

[0046]

[0066] The antibodies described herein comprise an N-glycan binding domain. In one embodiment, the antibodies comprise a heavy chain constant region, and the N-glycan binding domain is located in the heavy chain constant region. In some embodiments, the antibodies described comprise a C of the Fc region. H2 The constant domain has N-glycosylation of asparagine residues in the heavy chain. In one embodiment, the antibody comprises two heavy chains and two N-glycans, each N-glycan attached to one heavy chain. In particular, the first GlcNAc in the N-glycan shown in formula (1) (GlcNAc 1 ) is bound to the antibody.

[0047]

[0067] In one embodiment of the present disclosure, an antigen-binding fragment of antibody DCBPR2002 comprises the complementarity determining regions (CDRs) of the heavy chain variable region and the complementarity determining regions of the light chain variable region, wherein the complementarity determining regions of the heavy chain variable region comprise CDRH1, CDRH2, and CDRH3 regions, and the complementarity determining regions of the light chain variable region comprise CDRL1, CDRL2, and CDRL3 regions; the CDRH1 region comprises the amino acid sequence of SEQ ID NO: 1; the CDRH2 region comprises the amino acid sequence of SEQ ID NO: 2; the CDRH3 region comprises the amino acid sequence of SEQ ID NO: 3; the CDRL1 region comprises the amino acid sequence of SEQ ID NO: 4; the CDRL2 region comprises the amino acid sequence of SEQ ID NO: 5; and the CDRL3 region comprises the amino acid sequence of SEQ ID NO: 6.

[0048]

[0068] In one embodiment of the present disclosure, the antibody is a murine antibody. The murine anti-mesothelin antibody clone SS1 has been developed for cancer treatment in clinical trials. Antibody SS1 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8, as disclosed in U.S. Patent No. 7,081,518.

[0049]

[0069] SS1 has been observed to induce strong immunogenicity and anti-drug antibodies in patients. Therefore, humanization of SS1 is an essential and important step for further drug development. For the preparation of humanized SS1 4D5 (HdSS1), a human acceptor framework was selected from clinically validated frameworks. In one embodiment of the present disclosure, antibody HdSS1 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.

[0050]

[0070] In some embodiments of the present disclosure, the antibodies comprise human germline VL and VH sequences with the highest degree of homology to the mAb SS1 framework regions. In particular, 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 Figures 1A and 1B.

[0051]

[0071] The sequences are shown in Table 1.

[0052]

[0072] Table 1 [Table 1]

[0053]

[0073] As used herein, "GlcNAc 1 ", "GlcNAc 2 ", "GlcNAc 3 " and "GlcNAc 4 " each represent a GlcNAc sugar at a different position on the antennary glycan moiety.

[0054]

[0074] As used herein, " [ka] " represents a trimannose structure containing three mannoses, and the first mannose (Man 1 ) is linked to the GlcNAc sugar; the second and third mannose (Man 2 and Man 3 ) are linked to Man via α-1,3 and α-1,6 glycosidic bonds, respectively. 1 is linked to.

[0055]

[0075] As used herein, "-(Fuc) 0-1 " denotes that a fucose sugar is optionally present, and if present, that there is only one fucose sugar.

[0056]

[0076] The N-glycans described herein have the structure of Formula (1). The process for synthesizing the N-glycans, linkers, payloads A and B shown in Formula (2) can be found at least in WO 2018 / 126092.

[0057]

[0077] As used herein, "-(CH)0-8 "-" indicates that -CH2- may or may not be present, and if present, may independently be 1, 2, 3, 4, 5, 6, 7, or 8 -CH2- groups.

[0058]

[0078] In some embodiments, the linker has a function that can connect the conjugator and the 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, linear or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamine, or arylamine groups having 2 to 20 carbon atoms. In some embodiments, cleavable linkers include, but are not limited to, disulfide-containing linkers, acid-labile linkers, photolabile linkers, peptidase-labile linkers, and esterase-labile linkers. Examples of linkers include, but are not limited to, linear or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamine, arylamine groups, ethers, esters, amides, carbamates, carbonates, Formulas (3) to (7), disulfide-containing linkers, acid-labile linkers, photolabile linkers, peptidase-labile linkers, and esterase-labile linkers having 2 to 20 carbon atoms. Multiple of the above examples may be used simultaneously in any order. [ka]

[0059]

[0079] In equations (3) and (4): R 1 are independently hydrogen, halogen, -OR 5 , -NO2, -CN, -S(O)2R 5 , C1~C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, The alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted; Two substituents R 1 may be linked together to form a fused cycloalkyl or fused (hetero)arene substituent; R 5 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; X is C(R 1 )2, O, S or NR 2 and R 2 is 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; L is selected from the group consisting of linear or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamine, arylamine groups, ethers, esters, amides, carbamates, carbonates, disulfide-containing linkers, acid-labile linkers, photolabile linkers, peptidase-labile linkers, and esterase-labile linkers having 2 to 20 carbon atoms, or combinations thereof. [ka]

[0060]

[0080] In equation (5): R 1 and L is as defined in Equation (3) and Equation (4); R 3are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; R 4 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N and S, and the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are independently optionally substituted. [ka]

[0061]

[0081] In formulas (6) and (7), L is as defined in formulas (3) and (4).

[0062]

[0082] In some embodiments, when the immunoconjugate is used for the treatment of a disease in a subject, payloads A and B can therefore independently be therapeutic agents, which can be cytostatic or cytotoxic agents or isotope chelators bearing the corresponding radioisotopes. Examples of cytostatic or cytotoxic agents include antimetabolites (e.g., fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase, gemcitabine, capecitibine, azathioprine, cytosine methotrexate, trimethoprim, pyrimethamine, or pemetrexed); alkylating agents (e.g., cerphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, mitomycin C, cyclophosphamide, mechlorethamine, uramustine, 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 analogs or derivatives thereof; pyrrolobenzodiazapenes, or any analogs or derivatives thereof); anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, or barbicin); antibiotics (e.g., dactinomycin, bleomycin, mithramycin, anthramycin, streptozotocin, gramicidin D, mitomycins (e.g., mitomycin C); calicheamicin;Mitotic inhibitors (including, for example, maytansinoids (such as DM1, DM3, and DM4), auristatins (including, for example, monomethylaurostatin E (MMAE) and monomethylaurostatin F (MMAF)), dolastatins, cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorubine), taxanes (e.g., paclitaxel, docetaxel, or novel taxanes), tubulysin, and colchicine); thiourea isomerase inhibitors (e.g., irinotecan, topotecan, camptothecin, ciratecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, rubitecan, SN38, DXd, DXd2, etoposide, teniposide, amisacrine, or mitocron; HDAC inhibitors (e.g., vorinostat, romidepsin, chidamide, panobinostat, or belinostat); proteasome inhibitors (e.g., peptidylboronic acid); and At; 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 or 213 , P 32 and Lu 177 Examples of isotope chelators include, but are not limited to, radioactive isotopes of Lu, including: 1,4,7,10-tetraazacyclododecane-N,N,N',N'',N''-pentaacetate (DTPA), 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetate (DOTA), 1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (THP), triethylenetetraamine-N,N,N',N'',N''',N'''-hexaacetate (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetrakis(methylenephosphonate) (DOTP), and mercaptoacetyltriglycine (MAG3).

[0063]

[0083] In particular, the therapeutic agent used herein is monomethylauristatin E, monomethylauristatin F, maytansinoid, duocarmycin-hydroxybenzamide azaindole, diethylenetriamine-N,N,N',N'',N''-pentaacetate, exatecan, or Dxd2.

[0064]

[0084] In some embodiments, when an immunoconjugate is used for detection, payloads A and B can independently be labels. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radioactive labels, etc.), and moieties that are indirectly detected, for example, by enzymatic reaction or molecular interaction, such as an enzyme or ligand. Exemplary labels include the radioisotope P 32 , C 14 , I 125 , H 3 , and I 131 Examples of suitable positron emitters include, but are not limited to, rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and other fluorophores; luciferases, e.g., firefly luciferase and bacterial luciferase; luciferin; 2,3-dihydrophthaledione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucoamylase; lysozyme; saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as uricase and xanthine oxidase, conjugated to enzymes that use hydrogen peroxide to oxidize dye precursors, such as HRP, lactoperoxidase, or microperoxidase; biotin / avidin; spin labels; bacteriophage labels; stable free radicals; and the like. In another embodiment, the label is a positron emitter. Positron emitters include Ga 68 , F 18 , Cu 64 , Y 86 , Br 76 , Zr 89 , and I 124These include, but are not limited to:

[0065]

[0085] In some embodiments of the present disclosure, the immunoconjugate may not be fully loaded with payloads A and B and a linker, and the N-glycan may be directly attached with a protecting group. The protecting group may be further substituted with a therapeutic agent or label. One example of a protecting group is azide.

[0066]

[0086] One embodiment of the present disclosure relates to a pharmaceutical composition comprising an immunoconjugate of the present disclosure and a pharmaceutically acceptable carrier.

[0067]

[0087] One embodiment of the present disclosure relates to a method of treating a disease or disorder using an immunoconjugate of the present disclosure. The disease can be cancer. In particular, the cancer is a mesothelin-expressing cancer. "Mesothelin-expressing cancer" refers to all cancers whose cells express mesothelin. Mesothelin is generally expressed in solid tumors, including tumors associated with the lung, pleura, ovary, breast, stomach, bile duct, uterus, and thymus. Thus, 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, biliary tract cancer, colorectal cancer, endometrial cancer, and breast cancer. In particular, the cancer is ovarian cancer.

[0068]

[0088] In some embodiments, the immunoconjugates exhibit relatively more stable conjugation bonds than random conjugation immunoconjugates, based on their in vivo pharmacokinetic profiles.

[0069]

[0089] In some embodiments, immunoconjugates with N-glycans demonstrate superior efficacy in xenograft animal models compared to random conjugation immunoconjugates.

[0070]

[0090] Embodiments of the present disclosure are described using the following specific examples, but those skilled in the art will recognize that these examples are for illustrative purposes only and that other modifications and variations are possible without departing from the scope of the present disclosure. [Example]

[0071]

[0091] Unless otherwise noted, each 1H NMR data were obtained at 500 MHz. Abbreviations used herein, unless otherwise specified, are as follows: Az: azide; Bu: butyl; Bn: benzyl; BOC: t-butyloxycarbonyl; BOP: benzotriazol-1-yloxytri / dimethylamino-phosphonium hexafluorophosphate; DBCO: dibenzocyclooctyne group; DCC: dicyclohexylcarbodiimide; DCM: dichloromethane; DIPEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide; DMAP: 4-dimethylamino EDC: 1-(3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride; EtOAc: ethyl acetate; eq.: equivalent(s); GlcNAc: N-acetylglucosamine; GlcNAz: azido-N-acetylglucosamine; HBTU: 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazole-1-oxide hexafluorophosphate; hexafluorophosphate benzotriazole tetramethyluronium; HOBt: hydroxybenztriazole; HOSu: N- Hydroxysuccinimide; HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; hexafluorophosphate azabenzotriazole tetramethyluronium; LAH: lithium aluminum hydride; MeOH: methanol; MES: 4-morpholineethanesulfonic acid; MGAT-1: mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase; MGAT-2: Mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase; MHz: megahertz; MMAE: monomethylauristatin E; MS(ES): mass spectrometry-electrospray; NMP: N-methylpyrrolidinone; Ph: phenyl; Pr: propyl; TEA: triethylamine; TFA: trifluoroacetic acid; THF: tetrahydrofuran; TLC: thin-layer chromatography; Tetrakis: tetrakis(triphenylphosphine)palladium; UDP: uridine diphosphate.

[0072]

[0092] Example 1 Humanization of Anti-Mesothelin SS1 mAb

[0073]

[0093] Selection of human V region framework sequences:

[0094] Using the murine monoclonal antibody SS1, whose sequence is disclosed in US Pat. No. 7,081,518, as the parent antibody, the SS1 mAb CDR sequences according to Kabat's definition are set forth in FIG. 1A (SEQ ID NOs: 7 and 8).

[0074]

[0095] For the preparation of humanized SS1 4D5 (HdSS1), a human acceptor framework was selected from clinically validated frameworks: VH subgroup III, IGHV3-66. * 04 and VLκ subgroup I, IGKV1-39 * The human heavy and light chain framework sequences of O1 have been clinically validated and have also been used successfully in many humanized antibodies.

[0075]

[0096] As shown in Figure 1A, IGHV3-66 * The sequence of the 04 heavy chain framework region differs from that of mAb SS1 by 35 amino acids (underlined residues), which corresponds to a variation of 42.68% (35 residues out of a total of 82 residues in the framework region). * The sequence of the O1 (VL) light chain framework region differs from that of mAb SS1 by 25 amino acids (underlined residues), which corresponds to a variation of 30.86% (25 residues out of a total of 81 residues in the framework region).

[0076]

[0097] For the preparation of humanized SS1 IMGT (HuSS1), human germline VL and VH sequences with the highest homology to the mAb SS1 framework region were identified from the IMGT database (International Immunogenetics Information System®). Homology searches can be performed using BLAST or similar methods. These studies revealed that the human germline genes IGHV1-2 * 02(VH) and IGVK3-11 * 01(VL) was identified as the VH and VL sequences most homologous to the corresponding heavy and light chain framework sequences of mAb SS1, respectively.

[0077]

[0098] As shown in Figure 1B, IGHV1-2 * The sequence of the 02 heavy chain framework region differs from that of mAb SS1 by 25 amino acids (underlined residues), which corresponds to a variation of 30.49% (25 residues out of a total of 82 residues in the framework region). * The sequence of the O1 (VL) light chain framework region differs from that of mAb SS1 by 27 amino acids (underlined residues), which corresponds to a variation of 33.33% (27 residues out of a total of 81 residues in the framework region).

[0078]

[0099] These two pairs of light and heavy chain sequences (hum 4D5 and hum IMGT) were used as examples for the construction of humanized antibodies against human mesothelin (SEQ ID NOS: 9, 10, 11, and 12).

[0079]

[0100] Example 2 Binding Affinity Analysis of Humanized Antibodies

[0080]

[0101] Full-length antibody expression

[0102] To confirm the change in affinity after humanizing the mouse antibody, the humanized light and heavy chain variable regions of the IMGT and 4D5 versions were directly generated by nucleotide synthesis. As shown in Figure 2, the mouse variable regions, the humanized versions of IMGT (SEQ ID NOS: 11 and 12), and the 4D5 (SEQ ID NOS: 9 and 10) variable regions were subcloned into the human Fc chimeric antibody expression vector pTCAE8 and introduced into host cells to prepare recombinant antibody-expressing cells. FreeStyle293 cells (Invitrogen) were used as host cells for expression.

[0081]

[0103] The following procedure is used to transfect the vector thus constructed into a 30 ml volume of a suspension of FreeStyle™ 293 cells. During transfection, the cells can be maintained in FreeStyle™ 293 Expression Medium. Approximately 24 hours prior to transfection, FreeStyle™ 293 cells are transfected into 15 ml of 2 x 10 6 The cells were passaged at 1000kJ / ml. The flask(s) were placed in an incubator at 37°C with 8% CO2. Next, 37.5 μg of plasmid DNA was diluted in 1.5 ml of sterile 150 mM NaCl to a total volume of 1.5 ml. In a separate tube, 37.5 μl of PEI (2.0 mg / ml) was diluted in 1.5 ml of sterile 150 mM NaCl. The DNA and PEI solution was allowed to stand at room temperature for 5 minutes. The solution was gently mixed by inversion of the tube, and then left at room temperature for 10–20 minutes. The DNA-PEI mixture was added to F293 cells, and the transfected cells were incubated for 4 hours on an orbital shaker platform rotating at 135–150 rpm in an incubator at 37°C with 8% CO2. An equal volume of fresh culture medium was then added to bring the total volume to 30 ml, and the cells were cultured for 5–7 days. The cells were then harvested for antibody purification and quantification.

[0082]

[0104] The collected supernatant was filtered through a 0.2 μm filter (Millipore) to remove contaminants. The antibody-containing culture supernatant was affinity purified using Protein A (Millipore), 1.5 M glycine / NaOH buffer, and 3 M NaCl (pH 9.0) as the absorption buffer, and 0.2 M glycine / HCl buffer (pH 2.5) as the elution buffer. The eluted fraction was adjusted to approximately pH 6.0–7.0 by adding 1 M Tris / HCl buffer (pH 9.0). The prepared antibody solution was substituted with PBS using a dialysis membrane (10,000 MW cutoff, Spectrum Laboratories) and sterilized by filtration through a 0.22 μm pore-size membrane filter (Millipore) to obtain purified antibody. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm and converted based on an optimal density of 1.45, which is equivalent to 1 mg / ml.

[0083]

[0105] Measurement of antibody binding affinity by ELISA

[0106] ELISA plates were coated with 1–2 μg / 100 μl of mesothelin protein per well. The wells were washed three times with PBS and blocked with 300 μl of 5% MPBS per well for 2 hours at 37°C. After washing with PBS, the wells were incubated with mesothelin antibody serially diluted in 5% MPBS for 1.5 hours at 37°C. The plates were washed, and goat polyclonal anti-human IgG-HRP antibody (1:10,000) (Jackson ImmunoResearch) was added to each well. Absorbance was measured as described above, and antibody binding affinity was calculated by nonlinear regression using Prism software (GraphPad).

[0084]

[0107] The CDR sequences of mAb SS1 were cloned into IGHV3-66 * 04 and IGVK1-39 *HdSS1(HH), generated by grafting onto the 01 sequence, has a higher degree of variation in the framework regions and exhibits a much lower affinity for mesothelin (KD = 2.61E-08 M) (for comparison, KD = 8.36E-11 M for mAB SS1) (Figure 3), (Table 2 below).

[0085]

[0108] In contrast to HdSS1, the CDR sequences of mAb SS1 were cloned into IGHV1-2 * 02 and IGVK3-11 * HuSS1(HH), generated by grafting onto the 01 sequence, has a high degree of variation in the framework regions but has relatively good affinity for mesothelin (KD = 4.99E-11 M) (for comparison, KD = 8.36E-11 M for mAB SS1) (Figure 3) (Table 2 below).

[0086]

[0109] These results suggest that IGHV1-2 * O2 heavy chain framework region and IGVK3-11 * This suggests that the O1 light chain framework regions are good at tolerating a relatively high degree of variation without affecting the conformation of the CDR regions.

[0087]

[0110] Table 2 [Table 2]

[0088]

[0111] Affinity measurements and kinetic analysis using BIAcore

[0112] To determine the differences in binding kinetics between individual antibodies, surface plasmon resonance (SPR) measurements were performed using a BIAcore T200 (Cytiva Inc.) as previously described (Karlsson & Falt, (1997) J. Immunol Methods 200:121-133). Carboxymethylated dextran biosensor chips (CM5, Cytiva Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Mesothelin protein was diluted to 5 μg / ml in 10 mM sodium acetate, pH 4.0, and then injected at a flow rate of 10 μL / min. After the bound protein reached approximately 1500 response units (RU), unreacted groups were blocked by injection of 1 M ethanolamine. For kinetic measurements, two-fold serial dilutions of anti-mesothelin mAb (0.3125 nM to 40 nM) were injected in HBS-EP+ Biacore running buffer provided by the manufacturer (Cytiva Inc.) at a flow rate of 30 μL / min at 25°C. The binding response to mesothelin protein was corrected by subtracting the response from a blank flow cell. The association rate (k or k) and dissociation rate (k or k) were calculated using a simple one-to-one Langmuir binding model, fitting k and k separately (Cytiva™ Biacore Insight Evaluation Software).

[0089]

[0113] The results are shown in Figure 4 and Table 3 (lower panel). The k and k of the chimeric SS1 mAb binding to mesothelin were 3.415E6 and 1.194E-5, respectively, with a K of 3.496E-12 mol / L. The k and k of the HuSS1mAb (IMGT version) binding to mesothelin were 4.493E6 and 1.124E-5, respectively, with a K of 2.501E-12.

[0090]

[0114] The results in Figure 4 suggest that the humanized antibody HuSS1 (DCBPR2002) can recognize the human mesothelin protein, and that the affinity of the humanized IMGT version is similar to that of the murine SS1 antibody, with a favorable affinity of approximately 2.501E-12.

[0091]

[0115] Table 3 [Table 3]

[0092]

[0116] Example 3 Preparation of trimannosyl-DCBPR2002 (DCBPR2002-TM)

[0093]

[0117] To remove galactose and sialic acid moieties from N-glycans from DCBPR2002, 10 mg of DCBPR2002 was treated with 20 μl of β1,4-galactosidase (NEB, P0745L, 8 units / μl) and 5 μl of α2-3,6,8 neuraminidase (NEB, P0720L, 50 units / μl) in 1× GlycoBuffer (NEB, total volume 1 mL) at 37°C for 24 hours. An additional 10 μl of β1,4-galactosidase (NEB, P0745L, 8 units / μl) was added to the reaction mixture, and the reaction was continued for another 24 hours at 37°C to obtain the G0F / G0 antibody sample. The antibody sample was purified using rProtein A Sepharose Fast Flow (GE Healthcare, 17-1279-02). After purification, the antibody sample was subjected to reduced mass chromatographic analysis.

[0094]

[0118] Preparation of DCBPR2002-2Az (Figure 21A)

[0119] MGAT-1 transfers UDP-azido-N-acetylglucosamine to one of the terminal mannose residues of each arm of the trimannosyl core protein. To confirm this phenomenon with antibodies, trimannosyl-DCBPR2002 (5 mg) and UDP-GlcNAz (final concentration: 2.5 mg) were incubated in 1000 μl of 1× buffer SP (25 mM MES (4-morpholineethanesulfonic acid), 10 mM MnCl2, pH 6.5) in the presence of MGAT-1 (0.1 mg; R&D, 8334-GT or in-house preparation) at 37°C for 16 h. The resulting product, DCBPR2002-2Az, was subjected to reduced pressure mass chromatographic analysis.

[0095]

[0120] Preparation of DCBPR2002-4Az (Figure 21B)

[0121] Trimannosyl-DCBPR2002 (5 mg) and UDP-GlcNAz (2.5 mg) in 800 μl of 1× buffer SP (25 mM MES, 10 mM MnCl, pH 6.5) were incubated 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 subjected to reduced mass chromatography and intact mass chromatography.

[0096]

[0122] Example 4: Preparation of DBCO-vc-MMAE (Compound 5)

[0097]

[0123] Synthesis of compound 3 [ka]

[0098]

[0124] A mixture of DBCO-COH (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 h. 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 MgSO. The organic solvent was removed under reduced pressure to give compound 2 without further purification.

[0099]

[0125] To a mixture of compound 2 (1 eq) and 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoic acid (174 mg, 1.2 eq) in dichloromethane (5 mL) was added DIPEA (170 mg, 2 eq). The reaction mixture was stirred at room temperature overnight. After the reaction was complete, the reaction mixture was extracted with dichloromethane and 1 N HCl (aq). The organic layer was then washed with brine and dried over MgSO. The residue was purified by column chromatography using methanol / dichloromethane to give compound 3 as a brown liquid (54% yield). 1 H NMR (600 MHz, DMSO) δ 7.69 (dd, J = 7.7, 1.3 Hz, 1H), 7.63 (d, J = 7.4 Hz, 1H), 7.53 -7.44 (m, 3H), 7.39 (td, J = 7.5, 1.6 Hz, 1H), 7.35 (td, J = 7.5, 1.3 Hz, 1H),7.30 (dd, J = 7.4, 1.6 Hz, 1H), 5.04 (d, J = 14.0 Hz, 1H), 3.62 (d, J = 14.0Hz, 1H), 3.58 (t, J = 6.4 Hz, 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 = 6.4 Hz, 2H),2.24 (dt, J = 15.5, 7.8 Hz, 1H), 2.00 (ddd, J = 15.4, 8.2, 5.7 Hz, 1H), 1.76(ddd, J = 16.3, 8.0, 5.7 Hz, 1H).LC-MS(ESI): m / z [C28 H 32 N2O7] calculated value 509.2 [M +1] + , Actual measured value 509.2[M +1] + .

[0100]

[0126] Synthesis of DBCO-vc-MMAE (compound 5) [ka]

[0101]

[0127] 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 2:1 DCM:DMF (6 mL) was added DIPEA (145 mg, 2 eq). The mixture was stirred at room temperature for 1 h. 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 MgSO. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography using methanol / dichloromethane to give compound 5 as a pale yellow solid (71% yield). 1H NMR (600 MHz, MeOD) δ 7.66 (d, J = 7.4 Hz, 1H), 7.61 (d, J = 3.5 Hz, 3H), 7.51 - 7.44 (m,3H), 7.42 - 7.37 (m, 3H), 7.33 (dt, J = 15.5, 8.2 Hz, 5H), 7.26 (d, J = 7.3 Hz,1H), 7.23 (t, J = 7.4 Hz, 1H), 5.23 - 5.07 (m, 3H), 4.71 - 4.48 (m, 4H), 4.29 -4.15 (m, 4H), 3.75 - 3.69 (m, 3H), 3.63 - 3.54 (m, 8H), 3.46 - 3.39 (m, 3H), 3.36 (s, 4H), 3.30 (d, J = 16.5 Hz, 3H), 3.26 - 3.23 (m, 2H), 3.23 - 3.16 (m,2H), 3.12 (s, 2H), 2.96 (dd, J = 17.0, 10.0 Hz, 3H), 2.75 - 2.68 (m, 1H), 2.57- 2.46 (m, 4H), 2.38 (dt, J = 15.0, 7.5 Hz, 1H), 2.28 - 2.04 (m, 5H), 2.04 -1.66 (m, 8H), 1.66 - 1.49 (m, 4H), 1.45 (d, J = 29.4 Hz, 2H), 1.19 (dd, J =6.6, 3.0 Hz, 3H), 1.15 (dd, J = 12.8, 6.8 Hz, 3H), 1.03 - 0.70 (m, 24H).

[0102]

[0128] Example 5 Preparation of DBCO-S-DM1 (Compound 11)

[0103]

[0129] Synthesis of compound 7

change

[0104]

[0130] To a solution of 4-mercaptobutanoic acid (0.5 g, 1 eq) in methanol (10 mL) was added 1,2-di(pyridin-2-yl)disulfane (1.83 g, 2 eq). The mixture was stirred at room temperature overnight. After the reaction was completed, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography using hexane / ethyl acetate to give compound 6 as a colorless liquid (yield 14%). 1 H NMR (600 MHz, CDCl3) δ 8.49 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 7.72 (dt, J = 8.1, 1.0 Hz,1H), 7.69 - 7.61 (m, 1H), 7.12 (ddd, J = 7.3, 4.9, 1.1 Hz, 1H), 2.88 (t, J =7.1 Hz, 2H), 2.53 (t, J = 7.2 Hz, 2H), 2.07 (p, J = 7.2 Hz, 2H).

[0105]

[0131] 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) was added DIPEA (145 mg, 2 eq). The mixture was stirred at room temperature for 1 h. 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 MgSO. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography using methanol / dichloromethane to give compound 7 as a pale yellow solid (71% yield). 1H NMR (600 MHz, DMSO) δ 7.18 (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, 9.0 Hz, 1H), 5.31 (q, J = 6.8 Hz, 1H), 4.52 (dd, J = 12.0,2.7 Hz, 1H), 4.06 (t, J = 12.3 Hz, 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 = 7.2 Hz,2H), 2.04 (dd, J = 14.4, 2.4 Hz, 1H), 1.69 (p, J = 7.4 Hz, 2H), 1.59 (s, 3H),1.50 - 1.40 (m, 2H), 1.14 (dd, J = 28.7, 6.6 Hz, 6H), 0.97 (d, J = 6.4 Hz, 6H),0.78 (s, 3H).

[0106]

[0132] Synthesis of compound 9 [ka]

[0107]

[0133] To a mixture of compound 1 (0.5 g, 1 eq) and compound 8 (0.62 g, 1.3 eq) in DMF (8 mL) was added HBTU (0.92 g, 1.5 eq) and DIPEA (0.57 mL, 2 eq). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, 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 using methanol / dichloromethane to give compound 9 as an orange liquid (76% yield). LC-MS (ESI): m / z [C 32 H 41 N3O7] calculated value 579.68 [M +1] + , Actual value 479.95[M +1] + .

[0108]

[0134] Synthesis of compound 10

[0135] To a solution of compound 9 (0.72 g, 1 eq) in dichloromethane (15 mL) was added TFA (2.85 mL) under ice bath. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was completed, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography using methanol / dichloromethane to give compound 10 as a brown solid (yield 69%). H NMR (600 MHz, DMSO) δ 7.78 - 7.74 (m, 2H),7.69 - 7.66 (m, 1H), 7.62 (d, J = 7.4 Hz, 1H), 7.52 - 7.45 (m, 2H), 7.40 - 7.33(m, 2H), 7.30 (dd, J = 7.4, 1.2 Hz, 1H), 5.03 (d, J = 14.1 Hz, 1H), 3.62 (d, J= 14.0 Hz, 1H), 3.57 - 3.53 (m, 6H), 3.49 - 3.44 (m, 6H), 3.30 (td, J = 6.0,1.8 Hz, 2H), 3.12 - 3.04 (m, 2H), 2.96 (dd, J = 10.7, 5.5 Hz, 2H), 2.59 (ddd, J= 24.2, 9.8, 4.7 Hz, 1H), 2.23 (dt, J = 15.4, 7.6 Hz, 1H), 2.04 - 1.96 (m, 1H),1.76 (ddd, J = 16.4, 8.0, 5.8 Hz, 1H). LC-MS(ESI): m / z [C 27 H 33 N3O5] calculated value 479.57 [M +1] + , Actual value 480.1 [M +1] + .

[0109]

[0136] Synthesis of DBCO-S-DM1 (compound 11) [ka]

[0110]

[0137] To a mixture of compound 7 (70 mg, 1 eq) and compound 10 (100 mg, 0.9 eq) in DMF (7 mL) was added HBTU (77 mg, 1.5 eq) and DIPEA (0.047 mL, 2 eq). The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was extracted with ethyl acetate and water. The organic layer was then washed with brine and dried over MgSO. The residue was purified by column chromatography using 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 = 5.6Hz, 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 = 14.0Hz, 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 = 6.4Hz, 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 ClNO 16 S2] calculated value 1317.99 [M +1] + , Actual measured value 1299.41 [M-18] + .

[0111]

[0138] Example 6 Preparation of DBCO-vc-seco DUBA (Compound 20)

[0112]

[0139] Synthesis of compound 12

[0140] Compound 12 was prepared as described by Beusker, PH (Mol. Pharmaceutics 2015, 12, 1813-1835).

[0113]

[0141] Synthesis of compound 14 [ka]

[0114]

[0142] To a solution of compound 12 (0.805 g, 1 eq) in THF (40 mL) was added bis(4-nitrophenyl)carbonate (0.858 g, 2 eq) and trimethylamine (0.983 mL, 5 eq) under ice bath. 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. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the organic solvent was removed under reduced pressure. The organic layer was then washed with brine and dried over MgSO. The residue was purified by column chromatography using methanol / dichloromethane to give compound 14 (yield 21.7%). 1H NMR (600 MHz, DMSO) δ 10.33 (s, 1H), 9.47(s, 1H), 8.70 (s, 1H), 8.40 - 8.31 (m, 1H), 7.99 (d, J = 8.8 Hz, 2H), 7.74 (d,J = 9.6 Hz, 2H), 7.58 (d, J = 11.7 Hz, 1H), 7.42 (s, 1H), 7.36 (s, 1H), 7.18(d, J = 8.8 Hz, 2H), 5.30 (s, 2H), 5.16 (t, J = 4.2 Hz, 1H), 4.69 - 4.61 (m,2H), 4.47 (d, J = 2.7 Hz, 1H), 3.83 (d, J = 11.8 Hz, 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 ClNO 10 ] calculated value 859.36 [M +1] + , Actual value 859.7 [M +1] + .

[0115]

[0143] Synthesis of compound 15 [ka]

[0116]

[0144] To a mixture of compound 1 (0.3 g, 1 eq), HBTU (0.56 g, 1.5 eq), and 2-(2-aminoethoxy)ethan-1-ol (0.12 g, 1.2 eq) in DMF (4 mL) was added DIPEA (0.34 mL, 2 eq) under ice bath. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was extracted with ethyl acetate and water. The organic layer was then washed with brine and dried over MgSO. The residue was purified by column chromatography using methanol / dichloromethane to give compound 15 (70% yield). 1H NMR (600 MHz, MeOD) δ 7.55 (d, J = 7.4Hz, 1H), 7.51 - 7.47 (m, 1H), 7.38 - 7.33 (m, 3H), 7.24 (dtd, J = 22.1, 7.5,1.2 Hz, 2H), 7.14 (dd, J = 7.5, 1.4 Hz, 1H), 5.02 (t, J = 10.9 Hz, 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 (dt, J = 16.4, 7.6 Hz, 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 N2O4] calculated value 392.45 [M +1] + , Actual value 393.39 [M +1] + .

[0117]

[0145] Synthesis of compound 16

[0146] To a solution of compound 15 (0.2 g, 1 eq) in DMF / CHCl (6 / 2 mL) was added bis(4-nitrophenyl)carbonate (0.47 g, 3 eq) and DIPEA (0.2 mL, 3 eq) under an inert atmosphere. The reaction mixture was stirred at room temperature overnight. 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 MgSO. The organic solvents were combined and removed under reduced pressure. The residue was purified by column chromatography using methanol / dichloromethane to give compound 16 (70% yield). 1H NMR (600 MHz, CDCl3) δ 8.30 - 8.27 (m,2H), 7.70 (d, J = 7.5 Hz, 1H), 7.53 - 7.50 (m, 1H), 7.44 - 7.38 (m, 5H), 7.37(dd, J = 7.5, 1.4 Hz, 1H), 7.32 (td, J = 7.5, 0.8 Hz, 1H), 7.27 (d, J = 1.2 Hz,1H), 5.17 (d, J = 13.9 Hz, 1H), 4.43 (t, J = 4.6 Hz, 2H), 3.81 - 3.72 (m, 2H),3.69 (d, J = 13.9 Hz, 1H), 3.57 - 3.44 (m, 2H), 3.43 - 3.32 (m, 2H), 2.83 (ddd,J = 16.9, 8.6, 5.9 Hz, 1H), 2.45 (ddd, J = 14.7, 8.6, 5.8 Hz, 1H), 2.21 (dt, J= 15.2, 6.1 Hz, 1H), 1.97 (dt, J = 17.0, 6.1 Hz, 1H). LC-MS(ESI): m / z [C 30 H 27 N3O8] calculated value 557.55 [M +1] + , Actual value 558.58 [M +1] + .

[0118]

[0147] Synthesis of compound 18

[0148] 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) was added DIPEA (0.134 mL, 2.2 eq). The reaction mixture was stirred at room temperature overnight. 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 MgSO. The combined organic solvents were removed under reduced pressure. The residue was purified by column chromatography using methanol / dichloromethane to give compound 18 (36% yield).

[0119]

[0149] Synthesis of compound 19

[0150] To a solution of compound 18 (0.05 g, 1 eq) in DMF (3 mL) was added bis(4-nitrophenyl)carbonate (0.08 g, 3 eq) and DIPEA (0.043 mL, 3 eq) under an inert atmosphere. The reaction mixture was stirred at room temperature overnight. 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 MgSO. The organic solvents were combined and removed under reduced pressure. The residue was purified by column chromatography using methanol / dichloromethane to give compound 19 (33% yield).

[0120]

[0151] Synthesis of DBCO-vc-seco DUBA (compound 20) [ka]

[0121]

[0152] To a solution of compound 14 (0.263 g, 1 eq) in CHCl (11.2 mL) was added TFA (11.2 mL, 3 eq) under ice bath. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the organic solvent was removed under reduced pressure. The residue was dissolved in DMF (7.6 mL). Compound 19 (0.324 g, 1.1 eq) and TEA (0.21 mL, 5 eq) were added to the mixture under ice bath. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the organic solvent was removed under reduced pressure. The residue was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO. The residue was purified by column chromatography using methanol / dichloromethane to give compound 20 (DBCO-vc-seco DUBA) (55.2% yield).

[0122]

[0153] Example 7 Synthesis of DBCO-DTPA (Compound 22) [ka]

[0123]

[0154] Synthesis of compound 21

[0155] To a mixture of DBCO-COH (1) (200 mg, 1 eq), EDC (376 mg, 3 eq) in dichloromethane (5 mL) was added HOSu (226 mg, 3 eq). The mixture was stirred at room temperature for 2 h. 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 MgSO. The organic solvent was removed under reduced pressure to give a pale yellow liquid.

[0156] The yellow liquid was added dropwise to a solution of ethylenediamine in dichloromethane over 20 minutes. The mixture was stirred at room temperature overnight. After the reaction was complete, the reaction mixture was extracted with DCM and NaHCO3 (aq.). 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 using methanol / dichloromethane to give a yellow oil (yield: 58.9%). 1H NMR (600 MHz, MeOD) δ 7.63 (d, J = 7.4 Hz, 1H), 7.61 - 7.56 (m, 1H), 7.47 - 7.41 (m, 3H),7.32 (dtd, J = 23.9, 7.5, 1.2 Hz, 2H), 7.23 (dd, J = 7.5, 1.3 Hz, 1H), 5.08 (d,J = 14.0 Hz, 1H), 3.63 (d, J = 14.0 Hz, 1H), 3.14 (dtd, J = 19.7, 13.5, 6.2 Hz,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, 6.6 Hz, 1H), 2.16 (dt, J = 15.2, 6.5 Hz, 1H), 1.95 (dt, J = 16.7,6.4 Hz, 1H). LC-MS (ESI): m / z [C 21 H 21 N3O2] calculated value 348.16 [M +1] + , Actual value 348.03 [M +1] + .

[0157] Synthesis of DBCO-DTPA (compound 22)

[0158] To a solution of compound 21 (13 mg, 1 eq) in 3:1 HO:DMF (3 mL) was added commercially available DTPA (26 mg, 1.1 eq). The mixture was stirred at room temperature overnight. 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 MgSO. The organic solvent was removed under reduced pressure to give DBCO-DTPA (20 mg) as a yellow solid. LC-MS (ESI): m / z [C 43 H 49 N7O 12 S] calculated value 888.32 [M +1] + , Actual value 888.47 [M +1] + .

[0124]

[0159] Example 8 Synthesis of DBCO-PEG3-vc-exatecan (Compound 25) [ka]

[0125]

[0160] Synthesis of compound 23

[0161] To a mixture of exatecan mesylate (0.11 g, 1.1 eq) and Fmoc-vc-PAB-PNP (0.144 g, 1 eq) in DMF (3 mL) was added DIPEA (0.082 mL, 2.5 eq). The reaction mixture was stirred at room temperature overnight. After completion of the reaction, 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) without further purification.

[0162] Synthesis of compound 24

[0163] To a solution of compound 23 in DMF (3 mL) was added diethylamine (0.082 mL, 2.5 eq). The reaction mixture was stirred at room temperature overnight. After completion of the reaction, 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) without further purification. 1H NMR (600 MHz, DMSO) δ 10.18 (s, 1H), 8.45 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.96 (s,1H), 7.79 (d, J = 10.8 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz,2H), 7.32 (s, 1H), 6.55 (s, 1H), 6.03 (t, J = 5.6 Hz, 1H), 5.46 (s, 2H), 5.45(s, 2H), 5.34 - 5.22 (m, 3H), 5.14 - 5.04 (m, 2H), 4.54 - 4.45 (m, 1H), 4.13(dt, J = 6.9, 6.2 Hz, 1H), 3.28 - 3.21 (m, 1H), 3.19 - 3.15 (m, 1H), 3.15 -3.07 (m, 1H), 3.06 - 3.00 (m, 1H), 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 FN8O9] calculated value 841.36 [M +1] + , measured value 841.34 [M +1] + .

[0126]

[0164] Synthesis of compound 25

[0165] To a mixture of compound 3 (13 mg, 1 eq), compound 24 (33 mg, 1.5 eq), and HATU (30 mg, 3 eq) in 2:1 DCM:DMF (3 mL) was added DIPEA (13.7 μL, 3 eq). The mixture was stirred at room temperature for 2 h. 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). 11H NMR (600 MHz, DMSO) δ 10.00 (s, 1H), 8.69 (s, 1H), 8.47 (d, J = 8.9 Hz, 1H), 8.14 (d, J = 7.7 Hz, 1H), 8.07 (t, J = 9.0 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.82 - 7.74 (m, 2H), 7.68 (dd, J = 7.6, 1.0 Hz, 1H), 7.64 - 7.60 (m, 2H), 7.52 - 7.43 (m, 3H), 7.37 (d, J = 8.5 Hz, 2H), 7.35 - 7.27 (m, 2H), 6.54 (s, 1H), 5.99 (t, J = 7.1 Hz, 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 = 14.1 Hz, 1H), 4.38 (dd, J = 13.0, 7.7 Hz, 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, 1.8 Hz, 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] + , measured value 1331.72 [M + 1] + .<0XXXXXX>

[0127]

[0166] Example 9 Synthesis of DBCO-PEG3-GGFG-Exatecan (Compound 29) [ka]

[0128]

[0167] Synthesis of compound 27

[0168] To a mixture of EDCI (273 mg, 1.5 eq) and HOSu (164 mg, 1.5 eq) in dichloromethane (18 mL) was added commercially available Boc-GGFG-OH (compound 26) (415 mg, 1 eq). The mixture was stirred at room temperature for 3.5 hours. The reaction mixture was added dropwise to a mixture of exatecan mesylate (343 mg, 0.83 eq) and triethylamine (0.2 mL, 1.5 eq) in DMF. 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 purified by column chromatography using methanol / dichloromethane to give compound 27 (507 mg, 63% yield) as a pale yellow solid. LC-MS (ESI): m / z [C 44 H 48 FN7O 10 ] calculated value 853.91 [M +1] + , Actual value 854.35 [M +1] + .875.91[M + Na] + , Measured value 875.52 [M + Na] + .

[0129]

[0169] Synthesis of compound 28

[0170] To a solution of compound 27 (507 mg, 1 eq) in dichloromethane (4 mL) was added trifluoroacetic acid (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 as a yellow solid (378 mg, 85% yield). LC-MS (ESI): m / z [C 39 H 40 FN7O8] calculated value 753.79 [M +1] + , Actual value 754.18 [M +1] + .

[0130]

[0171] Synthesis of compound 29

[0172] To a solution of compound 28 (40 mg, 1 eq) in DMF (1 mL) was added DIPEA (0.18 mL, 20 eq). The reaction mixture was stirred in an ice bath for 15 minutes. The reaction mixture was added dropwise to a mixed solution of compound 3 (48 mg, 1.2 eq) and HBTU (30 mg, 1.5 eq) in DMF (1 mL). 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 using 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] + , Actual value 1244.56 [M+1] + . 1266.34[M + Na] + , Measured value 1266.83 [M+ Na] + .

[0131]

[0173] Example 10 Synthesis of DBCO-PEG12-GGFG-Exatecan (Compound 31) [ka]

[0132]

[0174] Synthesis of compound 30

[0175] To a mixture of compound 2 and NH2-PEG12-COOH (1217 mg, 1.0 eq) in dichloromethane / DMF (8 mL / 8 mL) was added DIPEA (0.7 mL, 2 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 using methanol / dichloromethane to give DBCO-PEG12-GGFG-exatecan (compound 31) as a viscous liquid (401 mg, 23% yield). LC-MS (ESI): m / z [C 46 H 68 N2O 16] calculated value 905.05 [M] + , Actual measurement value 905.53 [M] + .

[0133]

[0176] Synthesis of compound 31

[0177] To a mixture of compound 28 (30 mg, 1 eq) in DMF (1 mL) was added DIPEA (0.14 mL, 20 eq). The reaction mixture was stirred in an ice bath for 15 minutes. The reaction mixture was 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 using 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] + , Actual value 1641.07 [M+1] + .

[0134]

[0178] Example 11 Synthesis of DBCO-PEG3-GGFG-DXd2 (Compound 36) [ka]

[0135]

[0179] Synthesis of compound 32

[0180] To a mixture of N-(tert-butoxycarbonyl)-4-aminobutanoic acid (200 mg, 1.0 eq) and EDCI (283 mg, 1.5 eq) in DCM (5 mL) was added HOSu (170 mg, 1.5 eq). The reaction mixture was stirred at room temperature under N2 atmosphere for 2 hours. After the reaction was completed, 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 exatecan mesylate (434 mg, 0.83 eq) and Et3N (0.21 mL, 1.5 eq) in DMF (5 mL). The reaction mixture was stirred at room temperature for 12 hours. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography to give compound 32 as a yellow solid (402 mg, 79% yield). LC-MS (ESI): m / z C 33 H 37 FN4O7[M + H] + Calculated value: 621.26, Measured value: 621.01.

[0136]

[0181] Synthesis of compound 33

[0182] Compound 32 was added to a mixed solution of DCM / TFA = 1 / 1 (9.5 mL / 9.5 mL). The reaction mixture was stirred at room temperature under N2 atmosphere for 2 hours. After the reaction was completed, 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] + : 521.21, Actual value: 521.09. 1HNMR (600 MHz, DMSO) δ 8.53 (d, J = 8.7 Hz, 1H), 7.82(d, J = 10.8 Hz, 1H), 7.32 (s, 3H), 6.56 (s, 1H), 5.65 - 5.53 (m, 1H), 5.43 (s,2H), 5.25 (d, J = 18.7 Hz, 1H), 5.15 (d, J = 18.7 Hz, 1H), 3.17 (t, J = 6.0 Hz,2H), 2.81 (t, J = 7.6 Hz, 2H), 2.42 - 2.37 (m, 3H), 2.26 (t, J = 7.1 Hz, 2H),2.14 (d, J = 5.3 Hz, 2H), 1.99 - 1.70 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0137]

[0183] Synthesis of compound 34

[0184] To a mixture of EDCI (104 mg, 1.5 eq) and HOSu (77 mg, 1.5 eq) in DCM (9 mL) was added compound 26 (252 mg, 1.3 eq). The reaction mixture was stirred at room temperature under N2 atmosphere for 2 hours. 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 mixed solution of compound 33 (231 mg, 1.0 eq) and Et3N (0.1 mL, 1.5 eq). The reaction mixture was stirred at room temperature under N2 atmosphere for 12 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 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, Measured value: 939.68.

[0138]

[0185] Synthesis of compound 35

[0186] Compound 34 was added to a mixed solution of DCM / TFA = 1 / 1 (3 mL / 3 mL). The reaction mixture was stirred at room temperature under N2 atmosphere for 2 hours. After the reaction was completed, 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, Measured value: 839.22.

[0139]

[0187] Synthesis of DBCO-PEG3-GGFG-DXd2 (Compound 36) [ka]

[0140]

[0188] 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) was added compound 35 (20 mg, 1 eq). The reaction mixture was stirred at room temperature under a N2 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, Measured value: 1329.69.

[0141]

[0189] Example 12 Synthesis of DBCO-PEG12-GGFG-DXd2 (Compound 37) [ka]

[0142]

[0190] 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) was added compound 35 (20 mg, 1 eq). The reaction mixture was stirred at room temperature under a N2 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, Measured value: 1726.6.

[0143]

[0191] Example 13 Synthesis of BCN-PEG3-VC-PAB-MMAE (Compound 40) [ka]

[0144]

[0192] Synthesis of compound 39

[0193] 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) was added DIPEA (0.5 mL, 3 eq). The reaction mixture was stirred at room temperature for 18 h. 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 39 (235 mg, 62% yield) as a viscous liquid. LC-MS (ESI): m / z [C 20 H 31 NO7] calculated value 397.47 [M +1] + , Actual value 397.39 [M +1] + .420.47[M + Na] + , Measured value 420.07 [M + Na] + .

[0145]

[0194] Synthesis of compound 40

[0195] 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) was added DIPEA (69 μl, 4 eq). The reaction mixture was stirred at room temperature for 18 h. After the reaction was completed, 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] + , Actual value 1503.13 [M+1] + .1524.90[M + Na] + , Measured value 1525.43 [M+ Na] + .

[0146]

[0196] Example 14 Synthesis of BCN-PEG12-GGFG-Exatecan (Compound 42) [ka]

[0147]

[0197] Synthesis of compound 41

[0198] 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) was added DIPEA (0.5 mL, 3 eq). The reaction mixture was stirred at room temperature for 18 h. 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 41 (598 mg, 79% yield) as a viscous liquid. LC-MS (ESI): m / z [C 38 H 67 NO 16 ] calculated value 793.95 [M +1] + , Actual value 794.25 [M +1] + .

[0148]

[0199] Synthesis of BCN-PEG-GGFG-exatecan (Compound 42)

[0200] 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) was added DIPEA (0.14 mL, 20 eq). The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, 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] + , Actual value 1530.9 [M +1] + .

[0149]

[0201] Example 15 Synthesis of BCN-PEG3-GGFG-Exatecan (Compound 43) [ka]

[0150]

[0202] 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) was added DIPEA (0.14 mL, 20 eq). The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, 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] + , Actual value 1133.63 [M+1] + .

[0151]

[0203] Example 16 Synthesis of BCN-PEG12-GGFG-DXd2 (Compound 44) [ka]

[0152]

[0204] 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) was added DIPEA (0.14 mL, 20 eq). The reaction mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, 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, Measured value: 1615.42.

[0153]

[0205] Example 17 Synthesis of DBCO-PEG3-2(PEG3-VC-PAB-MMAE) (Compound 48) [ka]

[0154]

[0206] Synthesis of compound 45

[0207] To a mixture of EDC (552 mg, 3 eq) and HOSu (333 mg, 3 eq) in dry dichloromethane / dry DMF (2.4 mL / 2.4 mL) was added compound 3 (490 mg, 1 eq). The reaction mixture was stirred at room temperature under a N2 atmosphere for 18 h. 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 viscous liquid compound 45 (856 mg) without further purification.

[0155]

[0208] Synthesis of compound 47

[0209] 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) was added DIPEA (621.7 mg, 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 (DCM / MeOH) to give compound 47 (485 mg, 54% yield) as a viscous liquid. LC-MS(ESI): m / z [C 46 H 65 N3O 16 ] calculated value 916.03 [M] + , Actual measurement value 916.3 [M] + .1H NMR (600 MHz, DMSO) δ 12.17 (s, 2H), 7.77 (t, J =5.6 Hz, 1H), 7.68 (dd, J = 7.7, 1.3 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.48 (m,3H), 7.36 (m, 2H), 7.30 (dd, J = 7.4, 1.4 Hz, 1H), 5.02 (d, J = 14.1 Hz, 1H),3.61 - 3.56 (m, 8H), 3.54 - 3.32 (m, 39H), 3.31 - 3.26 (m, 2H), 3.16 (d, J =4.9 Hz, 1H), 3.13 - 3.03 (m, 3H), 2.63 - 2.54 (m, 4H), 2.43 (td, J = 6.3, 2.7Hz, 4H), 2.23 (dt, J = 15.5, 7.7 Hz, 1H), 1.99 (m, 1H), 1.75 (m, 1H), 1.24 (d,J = 5.9 Hz, 9H).

[0156]

[0210] Synthesis of DBCO-PEG3-2(PEG3-VC-PAB-MMAE) (Compound 48)

[0211] 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) was added DIPEA (22 mg, 3.2 eq). The reaction mixture was stirred at room temperature for 24 h. After the reaction was completed, the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to give compound 48 (41 mg) as a viscous liquid. LC-MS (TOF): m / z [C 162 H 249 N 23 O 38 ] calculated value 3126.9 [M] + , Actual measured value 1042.95 [M] 3+ ,1563.92 [M] 2+

[0157]

[0212] Example 18 Preparation of DCBPR2002-4 (DBCO-vc-MMAE) (Figure 21C)

[0213] Preparation of MES pH 6.5 buffer: 4.881 g of MES free acid (2-morpholinoethanesulfonic acid, CAS 4432-31-9) was suspended in 750 mL of dH2O. The pH was adjusted to 6.5 with 10 N NaOH (aq). Distilled water was then added to the suspension until the volume reached 1 L.

[0158]

[0214] 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 for 18 hours. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-4(DBCO-vc-MMAE). The drug-to-antibody ratio (DAR) of the ADC was determined to be 3.89 by LC-MS.

[0159]

[0215] Example 19 Preparation of DCBPR2002-4 (DBCO-S-DM1) (Figure 21D)

[0216] 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 an argon atmosphere. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-4 (DBCO-S-DM1). The drug-to-antibody ratio (DAR) of the ADC was determined to be approximately 4 by LC-MS.

[0160]

[0217] Example 20 Preparation of DCBPR2002-4 (DBCO-vc-seco DUBA) (Figure 21E)

[0218] 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 for 20 hours. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-4 (DBCO-vc-seco DUBA). The drug-to-antibody ratio (DAR) of the ADC was determined to be approximately 4 by LC-MS.

[0161]

[0219] Example 21 Preparation of DCBPR2002-4 (DBCO-PEG4-vc-PAB-MMAF) (Figure 21F)

[0220] DBCO-PEG4-VC-PAB-MMAF is a commercially available linker-payload.

[0162]

[0221] 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 for 18 hours. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to obtain DCBPR2002-4 (DBCO-PEG4-vc-PAB-MMAF). The drug-to-antibody ratio (DAR) of the ADC was determined to be approximately 4 by LC-MS.

[0163]

[0222] Example 22 Preparation of DCBPR2002-4 (DBCO-DTPA) (Figure 21G)

[0223] 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 for 18 hours. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to obtain DCBPR2002-4(DBCO-DTPA). The drug-to-antibody ratio (DAR) of the ADC was determined to be approximately 4 by LC-MS.

[0164]

[0224] Example 23 Preparation of DCBPR2002-4 (DBCO-PEG3-vc-exatecan) (Figure 21H)

[0225] 0.04 mL of DBCO-PEG3-VC-exatecan (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 for 18 hours. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-4 (DBCO-PEG3-VC-exatecan). The drug-to-antibody ratio (DAR) of the ADC was determined to be 3.77 by LC-MS.

[0165]

[0226] Example 24 Preparation of DCBPR2002-4 (DBCO-PEG3-GGFG-Exatecan) (Figure 21I)

[0227] 0.02 mL of DBCO-PEG3-GGFG-exatecan (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 for 18 hours. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-4 (DBCO-PEG3-GGFG-exatecan). The drug-to-antibody ratio (DAR) of the ADC was determined to be 3.21 by LC-MS.

[0166]

[0228] Example 25 Preparation of DCBPR2002-4 (DBCO-PEG12-GGFG-Exatecan) (Figure 21J)

[0229] 0.02 mL of DBCO-PEG12-GGFG-exatecan (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 for 18 hours. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-4 (DBCO-PEG12-GGFG-exatecan). The drug-to-antibody ratio (DAR) of the ADC was determined to be 3.91 by LC-MS.

[0167]

[0230] Example 26 Preparation of DCBPR2002-4 (DBCO-PEG3-GGFG-DXd2) (Figure 21K)

[0231] 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 for 18 hours under argon atmosphere. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to obtain DCBPR2002-4 (DBCO-branched PEG3-GGFG-exatecan). The drug-to-antibody ratio (DAR) of the ADC was determined to be 3.12 by LC-MS.

[0168]

[0232] Example 27 Preparation of DCBPR2002-4 (DBCO-PEG12-GGFG-DXd2) (Figure 21L)

[0233] 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 for 18 hours under argon atmosphere. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to obtain DCBPR2002-4 (DBCO-PEG12-GGFG-DX8951). The drug-to-antibody ratio (DAR) of the ADC was determined to be 3.52 by LC-MS.

[0169]

[0234] Example 28 Preparation of DCBPR2002-4 (BCN-PEG-VC-PAB-MMAE) (Figure 21M)

[0235] 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 for 18 hours under an argon atmosphere. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to obtain DCBPR2002-4 (BCN-PEG3-VC-PAB-MMAE). The drug-to-antibody ratio (DAR) of the ADC was determined to be 2.67 by LC-MS.

[0170]

[0236] Example 29 Preparation of DCBPR2002-4 (BCN-PEG12-GGFG-Exatecan) (Figure 21N)

[0237] 0.02 mL of BCN-PEG12-GGFG-exatecan (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 for 42 hours. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-4 (BCN-PEG12-GGFG-exatecan). The drug-to-antibody ratio (DAR) of the ADC was determined to be 3.51 by LC-MS.

[0171]

[0238] Example 30 Preparation of DCBPR2002-4 (BCN-PEG3-GGFG-Exatecan) (Figure 21O)

[0239] 0.01 mL of BCN-PEG3-GGFG-exatecan (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 for 18 hours. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-4 (BCN-PEG3-GGFG-exatecan). The drug-to-antibody ratio (DAR) of the ADC was determined to be 3.73 by LC-MS.

[0172]

[0240] Example 31 Preparation of DCBPR2002-4 (BCN-PEG-GGFG-DXd) (Figure 21P)

[0241] 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 for 18 hours under argon atmosphere. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to obtain DCBPR2002-4 (DBCO-PEG12-GGFG-DXd2). The drug-to-antibody ratio (DAR) of the ADC was determined to be 3.51 by LC-MS.

[0173]

[0242] Example 32 Preparation of DCBPR2002-4 (DBCO-PEG-VC-PAB-MMAE) (Figure 21Q)

[0243] 0.02 mL of DBCO-branched-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 for 18 h under argon atmosphere. The antibody preparation was desalted and concentrated in MES pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to obtain DCBPR2002-4 (DBCO-PEG3-2(PEG3-VC-PAB-MMAE)). The drug-to-antibody ratio (DAR) of the ADC was determined to be 5.68 by LC-MS.

[0174]

[0244] Example 33 Preparation of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco DUBA) (Figure 21R)

[0175]

[0245] Synthesis of DCBPR2002-2 (DBCO-vc-MMAE)

[0246] 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 for 20 hours. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-trimannosyl-2(DBCO-vc-MMAE). The drug-to-antibody ratio (DAR) of the ADC was determined to be approximately 2 by LC-MS.

[0176]

[0247] General synthesis of DCBPR2002-2(linker-payload)-2Az

[0248] Five mg of DCBPR2002-2 (linker-payload) and UDP-GlcNAz (2.5 mg) in 1000 μl of 1× buffer SP (25 mM MES, 10 mM MnCl, pH 6.5) were incubated in the presence of rat MGAT-2 (0.05 mg) at 37°C for 16 hours. After the reaction, the antibody product was purified using an Amicon Ultra-15 centrifugal filter device to obtain DCBPR2002-2 (linker-payload) with two active GlcNAz groups bound to the remaining terminal mannose of the heavy chain. This product was subjected to reduced mass chromatographic analysis.

[0177]

[0249] Synthesis of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco DUBA).

[0250] 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 for 18 hours under an argon atmosphere. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco DUBA). The drug-to-antibody ratio (DAR) of the ADC was determined to be approximately 4 by LC-MS.

[0178]

[0251] Example 34 Preparation of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-S-DM1) (Figure 21S)

[0179]

[0252] Synthesis of DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-S-DM1).

[0253] 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 for 18 hours. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-2(DBCO-vc-MMEA)-2(DBCO-S-DM1). The drug-to-antibody ratio (DAR) of the ADC was determined to be approximately 4 by LC-MS.

[0180]

[0254] Example 35 Preparation of DCBPR2002-2 (DBCO-vc-seco DUBA)-2 (DBCO-S-DM1) (Figure 21T)

[0181]

[0255] Synthesis of DCBPR2002-2 (DBCO-vc-seco DUBA)

[0256] 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 for 20 hours. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-2 (DBCO-vc-seco DUBA). The drug-to-antibody ratio (DAR) of the ADC was determined to be approximately 2 by LC-MS.

[0182]

[0257] Synthesis of DCBPR2002-2(DBCO-vc-seco DUBA)-2(DBCO-S-DM1)

[0258] 2.4 mL of DBCO-S-DM1 (10 mM in DMA) was slowly added to a solution of DCBPR2002-2(DBCO-vc-seco DUBA)-2Az (6 mL, 5 mg / mL) in buffer (MES pH 6.5). The reaction mixture was stirred at 37°C for 18 hours under an argon atmosphere. The antibody preparation was desalted and concentrated in sodium citrate pH 6.5 buffer using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL to yield DCBPR2002-2(DBCO-vc-seco DUBA)-2(DBCO-S-DM1). The drug-to-antibody ratio (DAR) of the ADC was determined to be approximately 4 by LC-MS.

[0183]

[0259] Example 36 SDS-PAGE

[0260] The ADCs of the invention can be analyzed by techniques known in the art, such as SDS-PAGE and HPLC. For example, solutions of anti-MSLN mAbs and anti-MSLN ADCs can be analyzed using 4-12% non-reducing and reducing SDS-PAGE gels followed by Coomassie brilliant blue staining.

[0184]

[0261] Example 37 Payload Coupling Assay

[0262] Assessment of the drug-to-antibody ratio (DAR) is important for monitoring the payload conjugation efficiency of target antibodies. The drug-to-antibody ratio can affect the therapeutic efficacy of anti-MSLN ADC products. Liquid chromatography-mass spectrometry (LC-MS) is the method of choice for determining the drug-to-antibody ratio (DAR) and drug loading distribution of lysine-linked antibody-drug conjugates (ADCs). Peak area percentages represent the relative distribution of specific drug-loaded ADC species. The weighted average DAR is then calculated using the percentage peak area information and drug loading number.

[0185]

[0263] Figure 7 shows an example of a mass spectrometry analysis of an ADC of the invention (DCBPR2002-4(DBCO-vc-MMAE)), which shows the distribution of various numbers of drugs conjugated to the antibody, with the most abundant species having four drugs conjugated to the antibody. The average drug-to-antibody ratio (DAR) for this sample is 4.07.

[0186]

[0264] Example 38 ELISA Binding Affinity

[0265] Mesothelin (100 μL in coating buffer at a concentration of 1 μg / mL) was added to each well of the plate for coating. The plate was sealed and incubated overnight at 4°C. The wells were aspirated and washed three times with 300 μL of PBST (0.05% Tween 20). 200 μL of PBS-5% skim milk was added to block the wells and incubated at 37°C for 1 hour. The wells were aspirated and washed three times with 300 μL / well of PBST (0.05% Tween 20). 100 μL of 400 ng ADC sample diluted in PBS was added to each well, and the plate was incubated at 37°C for 1 hour. The wells were aspirated and washed three times with 300 μL of PBST (0.05% Tween 20). 50 μL of anti-human kappa light chain (1:5000) was added to each well, and the plate was incubated at 37°C for 1 hour. The wells were aspirated and washed three times with 300 μL / well of PBST (0.05% Tween 20). 100 μL of TMB was added to each well, and the plate was incubated at room temperature for 15 minutes. Color development was stopped by adding 100 μL of 1N HCl. The plate was measured at an absorbance of 450-650 nm using an ELISA reader. The data are shown in Figure 5.

[0187]

[0266] 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-DU BA)Kd=1.580e-010; and DCBPR2002-2(DBCO-vc-seco-DUBA)-2(DBCO-s-DM1)Kd=7.315e-011.

[0188]

[0267] Example 39 Binding Kinetics of Anti-Mesothelin ADCs

[0268] The rate constant for the interaction of anti-mesothelin ADCs with mesothelin was determined by surface plasma resonance (Biacore® T100, Biacore, Inc., Piscataway, NJ). The flow cell of a CM5 wafer was immobilized with approximately 10,000 response units (RU) of anti-human IgG-Fc (Biacore®) in 10 mM glycine, pH 5.0, at 10 μL / min for 600 s. Anti-mesothelin antibodies and anti-mesothelin ADCs diluted in TBS at 10 μg / mL were captured onto the CM5 chip at 10 μL / min. Recordings were performed in PBS containing 1 mM CaCl2 for 3 min upon binding with four concentrations (3.7–100 nM) of human mesothelin recombinant protein and zero concentration (flow buffer) at 100 μL / min. Complex dissociation was measured for 10 min. The water surface was regenerated by injecting 3 M MgCl2 and 3 mM EGTA at 10 μl / min for 60 seconds. Using Biacore® T100 evaluation software (Biacore®), the curves obtained after subtracting the reference and buffer signals were fitted to a 1:1 Langmuir binding model. The K, Kd, ​​and KD are shown in Table 4. Kinetic analysis indicated that the anti-mesothelin antibody and anti-mesothelin ADC had similar K(on) and Kd(off) rates.

[0189]

[0269] Table 4 [Table 4]

[0190]

[0270] Example 40 In vitro cytotoxicity test (KLM-1 and OVCAR-3)

[0271] The pancreatic cancer cell line KLM-1 was grown in RPMI 1640 Medium (ATCC modified) supplemented with 10% fetal bovine serum. The ovarian cancer cell line OVCAR-3 was grown in RPMI 1640 Medium (ATCC modified) supplemented with 20% fetal bovine serum. KLM-1 and OVCAR-3 cell lines were maintained in a humidified 37°C incubator with a 5% CO2 atmosphere. The day before treatment, cells were harvested and seeded into 96-well plates (4,000 cells per well). On day 2, cells were treated with 3-fold serial dilutions of toxic payload and ADC. Each treatment was performed in triplicate with eight data points. After 72 hours of treatment, cell viability was assessed using the CellTiter-Glo® kit (Promega) according to the manufacturer's instructions. At the end of the incubation period, luminescence was measured using a SpectraMax i3x MultiMode Detection Platform (Molecular Devices). The cytotoxicity of the compounds was assessed relative to cells treated with 0.05% PBS (ADC) or 0.05% DMSO (toxic payload). IC 50 Values ​​were calculated by fitting the survival data with a four-parameter logistic equation using GraphPad Prism 5.0 software. The results are shown in Table 5.

[0191]

[0272] Table 5: Toxic payloads and IC of ADCs 50 value [Table 5]

[0192]

[0273] Example 41 Internalization Assay

[0274] KLM-1 or OVCAR3 cells were trypsinized, harvested, and resuspended in FACS 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, then washed with 1 mL of FACS buffer. The supernatant was discarded. Test group: KLM-1 or OVCAR3 cells were preincubated with 10 μg / mL trimannosyl anti-mesothelin ADC in FACS buffer for 60 minutes on ice, 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 (BD LSRFortessa), and the results are shown in Figure 6.

[0193]

[0275] Example 42 In vivo PK

[0276] In this study, we performed pharmacokinetic analysis of DCBPR2002-4 (DBCO-vc-MMAE) in BALB / c mouse and rat samples using the Meso Scale Discovery (MSD) electrochemiluminescent (ECL) method. The MSD assay can measure both conjugated and unconjugated antibodies. As shown in this example, or total antibody assay, the plate is coated with goat anti-human IgG, which can capture all humanized antibodies (conjugated and unconjugated). For conjugated antibody assays, the plate is coated with an antibody against the payload (drug), such as an anti-MMAE antibody.

[0194]

[0277] Mice were administered a dose of 3 mg / kg via the tail vein. Blood samples were then collected at different time points to measure the concentration of DCBPR2002-4 (DBCO-vc-MMAE) in the mice using the MESO QuickPlex SQ 120 method. The pharmacokinetic parameters of DCBPR2002-4 (DBCO-vc-MMAE) were analyzed by non-compartmental analysis using the Phoenix™ WinNonlin program, version 6.3.

[0195]

[0278] Table 6 summarizes the results of the PK studies. Whole Antibody MSD Assay: Measures both conjugated and unconjugated antibodies. Conjugated Antibody MSD Assay: Measures only conjugated antibodies. The in vivo half-life of DCBPR2002-4 (DBCO-vc-MMAE) was approximately 87.2 hours, which may be attributed to the enzyme carboxylase 1C, for which the valine-citrulline linker on our ADC is a substrate, as higher proteolysis of the linker was observed in mice than in other species.

[0196]

[0279] An in vivo pharmacokinetic study was designed to compare the linker-payload stability of trimannosyl-conjugated and cysteine-conjugated synthetic ADCs (Adcetris). Rats were administered DCBPR2002-4 (DBCO-vc-MMAE) and Adcetris at a dose of 5 mg / kg via the tail vein. Serum samples were then obtained at different time points, and the concentrations of DCBPR2002-4 (DBCO-vc-MMAE) and Adcetris in the rats were determined using the MESO QuickPlex SQ 120 method. The pharmacokinetic parameters of DCBPR2002-4 (DBCO-vc-MMAE) and Adcetris were analyzed by noncompartmental analysis using the Phoenix™ WinNonlin program, version 6.3.

[0197]

[0280] Table 7 summarizes the results of the PK studies. Whole Antibody MSD Assay: Measures both conjugated and unconjugated antibody. Conjugated Antibody MSD Assay: Measures only conjugated antibody. The in vivo half-life of DCBPR2002-4 (DBCO-vc-mMAE) is 194 ± 35.0 hours for whole antibody; the half-life of the conjugated antibody is 148 ± 8.14 hours for DCBPR2002-4 (DBCO-vc-MMAE) and 182 ± 10.9 hours for ADCETRIS. (Figure 8)

[0198]

[0281] Table 6 [Table 6]

[0199]

[0282] Table 7 [Table 7]

[0200]

[0283] In a comparison of the pharmacokinetic profiles of DCBPR2002-4 (DBCO-vc-MMAE) and ADCETRIS between whole and conjugated antibodies, the difference between the whole and conjugated antibody curves is closer for DCBPR2002-4 (DBCO-vc-MMAE) than for ADCETRIS. In vivo results demonstrated that the proposed trimannosyl conjugation has a difference in the stability of the conjugated linker payload compared to the cysteine ​​conjugation (ADCETRIS).

[0201]

[0284] Example 43: Xenograft model of anti-MSLN ADC (pancreatic cancer)

[0285] The objective of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002-lysine-DBCO-vc-MMAE (DBCO-vc-MMAE linked to an antibody polypeptide via an azide-activated lysine) and DCBPR2002-4 (DBCO-vc-MMAE) in the KLM-1 human pancreatic cancer xenograft model in male NOD SCID mice.

[0202]

[0286] Formulations containing test article DCBPR2002-lysine-DBCO-vc-MMAE, test article DCBPR2002-4 (DBCO-vc-MMAE), and the corresponding vehicle were formulated by diluting the stock with 25 mM sodium citrate buffer (pH 6.5). Each formulation was administered intravenously (IV) to mice once a week for 3 weeks.

[0203]

[0287] KLM-1 cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0204]

[0288] Male NOD-SCID mice, 6-7 weeks old, were purchased from BioLasco Taiwan Co., Ltd. and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility at 19-25°C with a 12-hour light / 12-hour dark cycle. The animals had free access to rodent chow and water.

[0205]

[0289] KLM-1 cells were implanted subcutaneously (SC) into the right flank of male NOD SCID mice (4 × 10 in a 1:1 PBS / Matrigel mixture). 6 (0.1 mL of cells were transplanted per mouse). The average tumor volume was approximately 200 mm 3 When the mice reached 100 mg / kg, they were randomly divided into three groups (N = 6 per group) and intravenously administered vehicle, DCBPR2002-lysine-DBCO-vc-MMAE (15 mg / kg), or DCBPR2002-4 (DBCO-vc-MMAE) (15 mg / kg) once a week for 3 weeks.

[0206]

[0290] Tumor volume, body weight, mortality, and signs of overt toxicity were monitored and recorded three times a week for 28 days. 3 ) was measured three times a week using a caliper and calculated according to the following formula: tumor volume = (w 2 × l) / 2, where w = width and l = length of tumor diameter (mm). Tumor growth inhibition (TGI) was calculated using the following formula: %TGI = [1-(T / C)] × 100%, where T and C represent the mean tumor volumes of the treatment and control groups, respectively. A TGI (%) value of ≥ 58% was considered significant antitumor activity. One-way analysis of variance followed by Dunnett's test was applied to compare vehicle and test article-treated groups. *Differences were considered significant at P<0.05. Animals were weighed three times a week until the completion of the study.

[0207]

[0291] Figure 9 shows tumor growth curves in male NOD SCID mice implanted with KLM-1 tumors. The test products DCBPR2002-lysine-DBCO-vc-MMAE (15 mg / kg) and DCBPR2002-4 (DBCO-vc-MMAE) (15 mg / kg) were intravenously administered once weekly for 3 weeks. A tumor growth inhibition rate (TGI) of ≥ 58% was considered significant (#) compared with the vehicle group. Comparisons between vehicle and test product-treated groups were performed using one-way analysis of variance followed by Dunnett's test. * Differences were considered significant at P<0.05. DCBPR2002-4 (DBCO-vc-MMAE) at 15 mg / kg significantly reduced KLM-1 tumor growth from days 7 to 28. DCBPR2002-lysine-DBCO-vc-MMAE at 15 mg / kg did not exhibit significant antitumor activity.

[0208]

[0292] Figure 10 shows the weight change in male NOD SCID mice implanted with KLM-1. Test articles DCBPR2002-lysine-DBCO-vc-MMAE (15 mg / kg) and DCBPR2002-4 (DBCO-vc-MMAE) (15 mg / kg) were intravenously administered once a week for three weeks. No weight loss was observed throughout the experiment.

[0209]

[0293] Example 44: Xenograft model of anti-MSLN ADC (pancreatic cancer)

[0294] The objective of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002 and DCBPR2002-4 (DBCO-vc-MMAE) in the KLM-1 human pancreatic cancer xenograft model in male NOD SCID mice.

[0210]

[0295] Formulations containing test article DCBPR2002, test article DCBPR2002-4 (DBCO-vc-MMAE), and the corresponding vehicle were prepared by diluting the stock with 25 mM sodium citrate buffer (pH 6.5). Each formulation was administered intravenously (IV) to mice once a week for 3 weeks.

[0211]

[0296] KLM-1 cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0212]

[0297] Male NOD-SCID mice, 6-7 weeks old, were purchased from BioLasco Taiwan Co., Ltd. and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility at 19-25°C with a 12-hour light / 12-hour dark cycle. The animals had free access to rodent chow and water.

[0213]

[0298] KLM-1 cells were implanted subcutaneously (SC) into the right flank of male NOD SCID mice (4 × 10 in a 1:1 PBS / Matrigel mixture). 6 (0.1 mL of cells were transplanted per mouse). The average tumor volume was 200 mm 3 When the mice reached 100 mg / kg, they were randomly divided into four groups (N=6 per group). Vehicle, DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg), and naked antibody (DCBPR2002, 30 mg / kg) were intravenously administered once a week for 3 weeks.

[0214]

[0299] Tumor volume, body weight, mortality, and signs of overt toxicity were monitored and recorded three times a week for 28 days. 3 ) was measured three times a week using a caliper and calculated according to the following formula: tumor volume = (w 2× l) / 2, where w = width and l = length of tumor diameter (mm). Tumor growth inhibition (TGI) was calculated using the following formula: %TGI = [1-(T / C)] × 100%, where T and C represent the mean tumor volumes of the treatment and control groups, respectively. A TGI (%) value of ≥ 58% was considered significant antitumor activity. One-way analysis of variance followed by Dunnett's test was applied to compare vehicle and test article-treated groups. * Differences were considered significant at P<0.05. Animals were weighed three times a week until the completion of the study.

[0215]

[0300] Figure 11 shows tumor growth curves in male NOD SCID mice implanted with KLM-1. Test articles DCBPR2002 (30 mg / kg) and DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg), respectively, were intravenously administered once weekly for 3 weeks. A tumor growth inhibition rate (TGI) of ≥ 58% was considered significant (#) compared with the vehicle group. Comparisons between vehicle and test article-treated groups were performed using one-way analysis of variance followed by Dunnett's test. * Differences were considered significant at P<0.05. DCBPR2002-4 (DBCO-vc-MMAE) at 15 and 30 mg / kg significantly reduced KLM-1 tumor growth from days 7 to 28. DCBPR2002 at 30 mg / kg did not exhibit significant antitumor activity.

[0216]

[0301] Figure 12 shows the weight change in male NOD SCID mice implanted with KLM-1. Test products DCBPR2002 (30 mg / kg) and DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg), respectively, were intravenously administered once a week for 3 weeks. No weight loss was observed throughout the experiment.

[0217]

[0302] Example 45: Xenograft model of anti-MSLN ADC (pancreatic cancer)

[0303] The objective 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 the KLM-1 human pancreatic cancer xenograft model in male NOD SCID mice.

[0218]

[0304] Formulations containing test article DCBPR2002-4(DBCO-vc-MMAE) (15 mg / kg), test article DCBPR2002-4(DBCO-vc-seco-DUBA) (15 mg / kg), test article DCBPR2002-4(DBCO-s-DM1) (15 mg / kg), test article DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco-DUBA) (15 mg / kg), test article DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-s-DM1) (15 mg / kg), test article DCBPR2002-2(DBCO-vc-seco-DUBA)-2(DBCO-s-DM1) (15 mg / kg), and the corresponding vehicle were formulated by diluting the stock with 25 mM sodium citrate buffer (pH 6.5). Each formulation was administered intravenously (IV) once weekly for three weeks.

[0219]

[0305] KLM-1 cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0220]

[0306] Male NOD-SCID mice, 6-7 weeks old, were purchased from BioLasco Taiwan Co., Ltd. and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility at 19-25°C with a 12-hour light / 12-hour dark cycle. The animals had free access to rodent chow and water.

[0221]

[0307] KLM-1 cells were implanted subcutaneously (SC) into the right flank of male NOD SCID mice (4 × 10 in a 1:1 PBS / Matrigel mixture). 6 (0.1 mL of cells were transplanted per mouse). The average tumor volume was 300 mm 3 Upon reaching 10 days, mice were randomly assigned to seven groups (N = 6 per group) and administered vehicle, 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), or DCBPR2002-2(DBCO-vc-seco-DUBA)-2(DBCO-s-DM1) intravenously at 15 mg / kg once weekly for 3 weeks.

[0222]

[0308] Tumor volume, body weight, mortality, and overt signs of toxicity were monitored and recorded three times a week for 28 days. Tumor volume was measured three times a week using calipers and calculated according to the following formula: tumor volume = (w 2 × l) / 2, where w = width and l = length of tumor diameter (mm). Tumor growth inhibition (TGI) was calculated using the following formula: %TGI = [1-(T / C)] × 100%, where T and C represent the mean tumor volumes of the treatment and control groups, respectively. A TGI (%) value of ≥ 58% was considered significant antitumor activity. One-way analysis of variance followed by Dunnett's test was applied to compare vehicle and test article-treated groups. * Differences were considered significant at P<0.05. Animals were weighed three times a week until the completion of the study.

[0223]

[0309] Figure 13 shows tumor growth curves in male NOD SCID mice implanted with KLM-1. Test articles 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) were intravenously administered at 15 mg / kg once weekly for 3 weeks. A tumor growth inhibition rate (TGI) of ≥ 58% was considered significant antitumor activity (#) compared to the vehicle group. One-way analysis of variance followed by Dunnett's test was applied to compare vehicle and test article treatment groups. * Differences were considered significant at P<0.05. All anti-MSLN ADCs demonstrated significant antitumor activity. The rank order of efficacy was DCBPR2002-4(DBCO-vc-seco-DUBA) = DCBPR2002-2(DBCO-vc-MMAE)-2(DBCO-vc-seco-DUBA) = DCBPR2002-2(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).

[0224]

[0310] Figure 14 shows the weight change in male NOD SCID mice implanted with KLM-1. Test articles 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) were each administered intravenously at 15 mg / kg once weekly for 3 weeks. No weight loss was observed in any of the treatment groups.

[0225]

[0311] Example 46 Xenograft model of anti-MSLN ADC (ovarian cancer)

[0312] The objective 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 the OVCAR-3 human ovarian cancer xenograft model in female NOD SCID mice.

[0226]

[0313] Formulations containing test article DCBPR2002-4 (DBCO-vc-MMAE), test article DCBPR2002-4 (DBCO-vc-seco-DUBA), and the corresponding vehicle were prepared by diluting the stock with 25 mM sodium citrate buffer (pH 6.5). Each formulation was administered intravenously (IV) once weekly for 3 weeks.

[0227]

[0314] OVCAR-3 cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 20% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0228]

[0315] Six- to seven-week-old female NOD-SCID mice were purchased from BioLasco Taiwan Co., Ltd. and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility at 19–25°C with a 12-hour light / 12-hour dark cycle. The animals had free access to rodent chow and water.

[0229]

[0316] OVCAR-3 cells were implanted subcutaneously (SC) into the right flank of female NOD SCID mice (1 × 10 in a 1:1 PBS / Matrigel mixture). 7 (0.2 mL of cells were transplanted per mouse). The average tumor volume was 300 mm 3 When the mice reached 100 mg / kg, they were randomly divided into four groups (N = 6 per group) and intravenously administered vehicle, DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg), or DCBPR2002-4 (DBCO-vc-seco-DUBA) (15 mg / kg) once weekly for 3 weeks.

[0230]

[0317] Tumor volume was measured three times a week using calipers and estimated using the following formula: tumor volume = (w 2 × l) / 2, where w = width and l = length of tumor diameter (mm). Tumor growth inhibition (TGI) was calculated using the following formula: %TGI = [1-(T / C)] × 100%, where T and C represent the mean tumor volumes of the treatment and control groups, respectively. A TGI (%) value of ≥ 58% was considered significant antitumor activity. One-way analysis of variance followed by Dunnett's test was applied to compare vehicle and test article-treated groups. * Differences were considered significant at P<0.05. Animals were weighed three times a week until the completion of the study.

[0231]

[0318] Figure 15 shows tumor growth curves in female NOD SCID mice implanted with OVCAR-3. Test articles DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg) and DCBPR2002-4 (DBCO-vc-seco-DUBA) (15 mg / kg) were intravenously administered once weekly for 3 weeks. A tumor growth inhibition rate (TGI) of ≥ 58% was considered significant (#) compared to the vehicle group. Comparisons between vehicle and test article-treated groups were performed using one-way analysis of variance followed by Dunnett's test. * Differences were considered significant at P < 0.05. 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 ​​of >90%, respectively.

[0232]

[0319] Figure 16 shows the weight change in female NOD SCID mice implanted with OVCAR-3. Test articles DCBPR2002-4 (DBCO-vc-MMAE) (15 and 30 mg / kg) and DCBPR2002-4 (DBCO-vc-seco-DUBA) (15 mg / kg) were intravenously administered once weekly for three weeks. No weight loss was observed in the treatment groups.

[0233]

[0320] Example 47 Xenograft model of anti-MSLN ADC (ovarian cancer)

[0321] The objective of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002-4 (DBCO-vc-MMAE) and DCBPR2002-TM in the OVCAR-3 human ovarian cancer xenograft model in female NOD SCID mice.

[0234]

[0322] Formulations containing test article DCBPR2002-4 (DBCO-vc-MMAE), test article DCBPR2002-TM, and the corresponding vehicle were prepared by diluting the stock with 25 mM sodium citrate buffer (pH 6.5). Each formulation was administered intravenously (IV) once weekly for 3 weeks.

[0235]

[0323] OVCAR-3 cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 20% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0236]

[0324] Six- to seven-week-old female NOD-SCID mice were purchased from BioLasco Taiwan Co., Ltd. and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility at 19–25°C with a 12-hour light / 12-hour dark cycle. The animals had free access to rodent chow and water.

[0237]

[0325] OVCAR-3 cells were implanted subcutaneously (SC) into the right flank of female NOD SCID mice (1 × 10 in a 1:1 PBS / Matrigel mixture). 7 (0.2 mL of cells were transplanted per mouse). The average tumor volume was 300 mm 3 When the mice reached 100 mg / kg, they were randomly divided into four groups (N=5 per group). Vehicle, DCBPR2002-4 (DBCO-vc-MMAE) (5 and 15 mg / kg), and DCBPR2002-TM (15 mg / kg) were intravenously administered once weekly for 3 weeks.

[0238]

[0326] Tumor volume was measured three times a week using calipers and estimated using the following formula: tumor volume = (w 2× l) / 2, where w = width and l = length of tumor diameter (mm). Tumor growth inhibition (TGI) was calculated using the following formula: %TGI = [1-(T / C)] × 100%, where T and C represent the mean tumor volumes of the treatment and control groups, respectively. A TGI (%) value of ≥ 58% was considered significant antitumor activity. One-way analysis of variance followed by Dunnett's test was applied to compare vehicle and test article-treated groups. * Differences were considered significant at P<0.05. Animals were weighed three times a week until the completion of the study.

[0239]

[0327] Figure 17 shows tumor growth curves in female NOD SCID mice implanted with OVCAR-3. Test articles DCBPR2002-4 (DBCO-vc-MMAE) (5 and 15 mg / kg) and DCBPR2002-TM (15 mg / kg) were intravenously administered once weekly for 3 weeks. A tumor growth inhibition rate (TGI) of ≥ 58% was considered significant (#) compared with the vehicle group. Comparisons between vehicle and test article-treated groups were performed using one-way analysis of variance followed by Dunnett's test. * Differences were considered significant at P<0.05. DCBPR2002-4 (DBCO-vc-MMAE) (5 and 15 mg / kg) significantly reduced OVCAR-3 tumor growth. DCBPR2002-TM at 15 mg / kg did not exhibit antitumor activity.

[0240]

[0328] Figure 18 shows the weight change in female NOD SCID mice implanted with OVCAR-3. Test articles DCBPR2002-4 (DBCO-vc-MMAE) (5 and 15 mg / kg) and DCBPR2002-TM (15 mg / kg) were intravenously administered once weekly for three weeks. No weight loss was observed in the treatment groups.

[0241]

[0329] Example 48 Xenograft model of anti-MSLN ADC (ovarian cancer)

[0330] The objective of this study was to evaluate the in vivo antitumor efficacy of DCBPR2002-4 (DBCO-vc-MMAE) in the OVCAR-3 human ovarian cancer xenograft model in female NOD SCID mice.

[0242]

[0331] Formulations containing the test article DCBPR2002-4 (DBCO-vc-MMAE) and the corresponding vehicle were prepared by diluting the stock with 25 mM sodium citrate buffer (pH 6.5). Each formulation was administered intravenously (IV) once weekly for 3 weeks.

[0243]

[0332] OVCAR-3 cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 20% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0244]

[0333] Six- to seven-week-old female NOD-SCID mice were purchased from BioLasco Taiwan Co., Ltd. and quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility at 19–25°C with a 12-hour light / 12-hour dark cycle. The animals had free access to rodent chow and water.

[0245]

[0334] OVCAR-3 cells were implanted subcutaneously (SC) into the right flank of female NOD SCID mice (1 × 10 in a 1:1 PBS / Matrigel mixture). 7 (0.2 mL of cells were transplanted per mouse). The average tumor volume was 300 mm 3 When the mice reached 100 mg / kg, they were randomly divided into four groups (N = 5 per group). Vehicle and DCBPR2002-4 (DBCO-vc-MMAE) (5, 10, and 15 mg / kg) were intravenously administered once a week for 3 weeks.

[0246]

[0335] Tumor volume was measured three times a week using calipers and estimated using the following formula: tumor volume = (w2 × l) / 2, where w = width and l = length of tumor diameter (mm). Tumor growth inhibition (TGI) was calculated using the following formula: %TGI = [1-(T / C)] × 100%, where T and C represent the mean tumor volumes of the treatment and control groups, respectively. A TGI (%) value of ≥ 58% was considered significant antitumor activity. One-way analysis of variance followed by Dunnett's test was applied to compare vehicle and test article-treated groups. * Differences were considered significant at P<0.05. Animals were weighed three times a week until the completion of the study.

[0247]

[0336] Figure 19 shows tumor growth curves in female NOD SCID mice implanted with OVCAR-3. Test article DCBPR2002-4 (DBCO-vc-MMAE) (5, 10, and 15 mg / kg) was intravenously administered once weekly for 3 weeks. A tumor growth inhibition rate (TGI) of ≥ 58% was considered significant (#) compared with the vehicle group. Comparisons between vehicle and test article-treated groups were performed using one-way analysis of variance followed by Dunnett's test. * Differences were considered significant at P<0.05. DCBPR2002-4 (DBCO-vc-MMAE) (5, 10, 15 mg / kg) significantly reduced OVCAR-3 tumor growth in a dose-dependent manner.

[0248]

[0337] Figure 20 shows the weight change in female NOD SCID mice implanted with OVCAR-3. Test article DCBPR2002-4 (DBCO-vc-MMAE) (5, 10, and 15 mg / kg) was intravenously administered once a week for three weeks. No weight loss was observed in the treatment groups.

[0249]

[0338] The above examples clearly demonstrate various methods for obtaining and characterizing the ADCs of the present invention, as well as the effectiveness of the ADCs of the present invention in treating cancer. Although embodiments of the present invention have been illustrated by a limited number of examples, those skilled in the art will appreciate that other variations and modifications are possible without departing from the scope of the present invention. Therefore, the scope of protection of the present invention should be limited only by the appended claims.

Claims

1. An antibody comprising an antigen-binding fragment that specifically binds to an epitope in mesothelin, an N-glycan-binding domain, and an N-glycan having the structure of formula (1): 【Chemistry 1】 (In the formula, “ ＊ " represents a bond or a protecting group); " ＊ " represents a bond, " in the N-glycan ＊ a linker connecting each of The same or different payloads A and B, independently conjugated to a linker. An immunoconjugate comprising: An immunoconjugate, wherein 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.

2. The antibody may be a monoclonal antibody, a humanized antibody, an antibody Fab fragment, or an F(ab') ２ , an Fv fragment from a truncated antibody, an scFv-Fc fragment, a minibody, a diabody, or an scFv.

3. The immunoconjugate of claim 1 , wherein the antibody comprises a heavy chain constant region and the N-glycan binding domain is located in the heavy chain constant region.

4. The immunoconjugate of claim 1 , wherein the antibody comprises two N-glycans.

5. the linker is selected from the group consisting of a linear or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamine, arylamine group, ether, ester, amide, carbamate, carbonate, Formula (3) through Formula (7), a disulfide-containing linker, an acid-labile linker, a photolabile linker, a peptidase-labile linker, and an esterase-labile linker, or a combination thereof, having 2 to 20 carbon atoms; 【Chemistry 2】 In formula (3) and formula (4): R １ are independently hydrogen, halogen, -OR ５ , -NO ２ , -CN, -S(O) ２ R ５ , C １ ~C ２４ Alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ (hetero)arylalkyl groups, The alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted; Two substituents R １ may be linked together to form a fused cycloalkyl or fused (hetero)arene substituent; R ５ are independently hydrogen, halogen, C １ ~C ２４ Alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups, and C ７ ~C ２４ (hetero)arylalkyl groups; X is C(R １ ) ２ , O, S or NR ２ and R ２ is 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; L is selected from the group consisting of linear or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamine, arylamine groups, ethers, esters, amides, carbamates, carbonates, disulfide-containing linkers, acid labile linkers, photolabile linkers, peptidase labile linkers, and esterase labile linkers having 2 to 20 carbon atoms, or combinations thereof; 【Transformation 3】 In formula (5): R １ are independently hydrogen, halogen, -OR ５ , -NO ２ , -CN, -S(O) ２ R ５ , C １ ~C ２４ Alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ (hetero)arylalkyl groups, The alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted; Two substituents R １ may be linked together to form a fused cycloalkyl or fused (hetero)arene substituent; R ５ are independently hydrogen, halogen, C １ ~C ２４ Alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ (hetero)arylalkyl groups; L is selected from the group consisting of linear or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamine, arylamine groups, ethers, esters, amides, carbamates, carbonates, disulfide-containing linkers, acid labile linkers, photolabile linkers, peptidase labile linkers, and esterase labile linkers having 2 to 20 carbon atoms, or combinations thereof; R ３ are independently hydrogen, halogen, C １ ~C ２４ Alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ (hetero)arylalkyl groups; R ４ is hydrogen, halogen, C １ ~C ２４ Alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl(hetero)aryl groups, C ７ ~C ２４ (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N and S, and the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are independently optionally substituted; 【Chemistry 4】 In formula (6) and formula (7): L is selected from the group consisting of linear or branched alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, alkoxy, acyl, alkylamine, arylamine groups, ethers, esters, amides, carbamates, carbonates, disulfide-containing linkers, acid-labile linkers, photolabile linkers, peptidase-labile linkers, and esterase-labile linkers having 2 to 20 carbon atoms, or combinations thereof; The immunoconjugate of claim 1.

6. The immunoconjugate of claim 1 , wherein payload A and payload B are independently a therapeutic agent or a label.

7. The immunoconjugate of claim 6, wherein the therapeutic agent is an antimetabolite, an alkylating agent, an alkylating-like agent, a DNA minor groove alkylating agent, an anthracycline, an antibiotic, a calicheamicin, a mitotic inhibitor, a topoisomerase inhibitor, a proteasome inhibitor, a radioisotope, or an isotope chelator.

8. 7. The immunoconjugate of claim 6, wherein the therapeutic agent is monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), maytansinoid, duocarmycin-hydroxybenzamidoazaindole (DUBA), diethylenetriamine-N,N,N',N'',N''-pentaacetate (DTPA), exatecan, or Dxd2.

9. The immunoconjugate of claim 6 , wherein the label is a fluorescent label, a chromophore label, an electron-dense label, a chemiluminescent label, a radioactive label, an enzyme label, or a positron emitter.

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

11. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising a therapeutically effective amount of the immunoconjugate of any one of claims 1 to 10.

13. The pharmaceutical composition of claim 12, wherein the cancer is a mesothelin-expressing cancer.

14. The pharmaceutical composition according to claim 12, wherein the cancer is ovarian cancer, mesothelioma, pancreatic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, biliary tract cancer, colon cancer, endometrial cancer, or breast cancer.

15. The pharmaceutical composition of claim 12, wherein the cancer is ovarian cancer.

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