Improved trop2 conjugated biological molecules, pharmaceutical compositions and applications thereof

Abstract

The present disclosure provides linker-payload conjugates including a bioorthogonal group and a hydrophilic moiety. The improved linker-payloads for antibody conjugation and improved antibody drug conjugates (ADCs) exhibit greater stability in blood circulation and enhanced drug delivery and drug release efficiencies in target cells. The present disclosure also relates to antibodies and antigen-binding fragments thereof targeting TROP2, as well as pharmaceutical compositions including TROP2 ADCs. Also described herein are methods of using TROP2 ADCs for treatment of subjects associated with pathological conditions.

Description

IMPROVED TROP2 CONJUGATED BIOLOGICAL MOLECULES, PHARMACEUTICAL COMPOSITIONS AND APPLICATIONS THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Patent Applications No. 63 / 659,345 (filed on June 13, 2024) and 63 / 696,872 (filed on September 20, 2024). The entirety of the aforementioned application is incorporated herein by reference. FIELD

[0002] The present invention relates to cancer therapies. In particular, the present invention relates to antibody-drug conjugates (ADC) targeted to TROP2 and applications thereof in cancer therapy. BACKGROUND OF THE INVENTION

[0003] A great deal of interest in cancer therapies is focused on the use of monoclonal antibodies (mAbs) for the targeted delivery of cytotoxic agents to cancer cells. The design of antibody-drug conjugates (ADCs), by attaching a drug to an antibody, typically via a linker, involves consideration of a variety of factors. These factors include the types of antibodies and linkers for the conjugation of drugs. Further, if the drug is to be released after antibody internalization, the mechanism of drug release, and the associated structural elements, and the structural modification of the drug after release, if any, also need to be taken into account. For example, the structural elements and mechanisms of drug release must be consistent with the intracellular trafficking of the conjugate.

[0004] The antibodies for ADC construction can be small protein formats (e.g., scFv or Fab fragments, designed ankyrin repeat proteins, affibodies, nanobody, etc.), monoclonal antibodies (mAbs), and bispecific antibodies which have been selected based on their high selectivity and affinity for a given antigen, their long circulating half-lives, and little to no immunogenicity. Thus, mAbs as protein ligands for a carefully selected biological receptor provide an ideal platform for the delivery of drugs to target cells. For example, a monoclonal antibody known to bind selectively with a tumor-associated antigen can be used for delivery of a conjugated cytotoxic agent to the tumor through its target-specific binding to the antigen and subsequent internalization of the cytotoxic agent. The cytotoxic agent may be a small molecule toxin, a protein toxin, an agonist, an antagonist, or in other formats, like oligonucleotides. Hence, the concept of targeted drug delivery to a specific cellular location of choice is a powerful approach to treating a wide range of diseases, with many beneficial aspects versus systemic delivery of the same drug.

[0005] TROP2 (tumor-associated calcium signal transducer 2, encoded by the TACSTD2 gene) is a prevalent tumor surface antigen and mediates cell growth, adhesion, and migration. TROP2 plays an important role in cancer development; however, it may function differently depending on its location in tumor cells. Patients have worse clinical outcomes with membranous TROP2 expression while intracellular TROP2 is associated with a better survival rate and less relapse in breast cancer patients. TROP2 has been studied in the up-regulation of epithelial-mesenchymal transition (EMT) through PI3K / Akt in cervical cancer, gallbladder cancer and gastric cancer. The RAS-Raf-MEK-ERK pathway has been indicated under the regulation of TROP2 to stimulate cell proliferation. TROP2 has been discovered in interaction with other membrane proteins to regulate cellular functions, including IGF-1, MDK, and NRG1. SUMMARY OF THE INVENTION

[0006] The inventors found that cleavable linker-payloads are highly suitable for metal-free click conjugation of drugs to antibodies and that attaching specific hydrophilic moieties to said linker-payloads caused the resulting ADCs to display improved in vitro and in vivo cytotoxicity and antitumor activity. Accordingly, the present disclosure provides improved linker-payloads that may be used in conjugation with antibodies, such as glycan-engineered antibodies, as well as improved antibody conjugates.

[0007] The present disclosure provides an antibody conjugate, such as an antibody-drug conjugate (ADC), prepared by conjugating the linker-payload disclosed herein with an antibody or an antigen-binding fragment thereof. The ADCs disclosed herein exhibit greater stability in blood circulation, enhancing drug delivery and the subsequent drug release efficiencies in target tumor cells, thus improving therapeutic index of the ADCs with lower off-target toxicity. The enhanced stability of the ADCs in the body may be attributed to the hydrophilic moiety (e.g., PEG), which, at an appropriate length and an appropriate distance from the payload, can effectively protect the cleavable linker-payloads.

[0008] In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is an anti-TROP2 antibody selected from hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, R4702, datopotamb, or sacituzumab. In certain preferred embodiment, the antibody is R4702. R4702 is as described in PCT patent publication No. WO2022222992A1, the content of which is incorporated herein by reference in its entirety.

[0009] In certain embodiments, the antibody is a glycan-engineered antibody, including an N- linked glycan at an asparagine residue of the antibody (e.g., the N297 of a human IgG heavy chain constant region) that is modified through deglycosylation and transglycosylation to have a defined glycan structure for drug conjugation. The deglycosylation and transglycosylation may beaccomplished using glycosynthases and variants thereof. In certain embodiments, the glycosynthase variants are EndoSd-D232M and EndoSz-D234M. Exemplary EndoSd-D232M and EndoSz-D234M are as described in PCT patent publication WO2020006176A1.

[0010] In another aspect, the present disclosure provides a method for preparing a glycan- engineered antibody conjugate, including reacting a glycan-engineered antibody with the linker- payload disclosed herein, wherein the glycan-engineered antibody includes a fucosylated or non- fucosylated N-acetylglucosamine (GlcNAc) at an asparagine residue (e.g., the N297 of human IgG heavy chain constant region) coupled to a glycan. The glycan-engineered antibody may be obtained by contacting an antibody with a glycosynthase and a glycan oxazoline to couple the glycan with the fucosylated or non-fucosylated GlcNAc. This method offers a unique glycan- engineered antibody conjugate (e.g., a glycan-engineered ADC) platform that is distinct from conventional ones by high product homogeneity. Therefore, site-specific ADCs with homogeneous DAR (drug-to-antibody ratio) can be prepared readily. These glycan-engineered ADCs have favorable manufacturing, quality control, and in vivo pharmacokinetic profiles.

[0011] In another aspect, the present disclosure provides a pharmaceutical composition, including the ADC described herein and a pharmaceutically acceptable carrier.

[0012] In another aspect, the present disclosure provides a method for inhibiting proliferation of cancer cells, including contacting with the cancer cells with an effective amount of the ADC described herein.

[0013] In another aspect, the present disclosure provides a method for treating a disease such as cancer, infection, nervous system, inflammatory, or autoimmune disorders, including administering to a subject in need thereof an effective amount of the ADC described herein. The effective amount will vary depending on various factors, including but not limited to the type of the ADC, the physiological conditions (e.g., general health or age) of a subject, the type and severity of the cancer, the treatment regimens, and the presence of other diseases. Generally, the effective amount is in the range of 0.01 μg-250 mg per kilogram body weight of a human subject.

[0014] In certain embodiments, the disease is characterized by expressing TROP2. In certain embodiments, the disease is a cancer selected from the group consisting of multiple myeloma, acute myeloid leukemia (AML), sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0015] In another aspect, the present disclosure provides a method for preparing an ADC, including reacting an antibody or an antigen-binding fragment thereof with the linker-payload disclosed herein to obtain the ADC, wherein the payload is the drug moiety.

[0016] The details of one or more embodiments of the invention are set forth in the description below. The features or advantages of the present invention will be apparent from the detailed description of preferred embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A-1E. The chromatograms of R4702 ADCs with varying PEG lengths, analyzed by hydrophobic interaction chromatography (HIC), wherein FIG. 1A shows the chromatogram of R4702-BCN-PEG48-Exatecan ADC (referred to as ADC-1), FIG. 1B shows the chromatogram of R4702-BCN-PEG24-Exatecan ADC (referred to as ADC-2), FIG. 1C shows the chromatogram of R4702-BCN-PEG12-Exatecan ADC (referred to as ADC-3), FIG. 1D shows the chromatogram of R4702-BCN-PEG6-Exatecan ADC (referred to as ADC-4), and FIG. 1E shows the chromatogram of R4702-BCN-Exatecan ADC (referred to as ADC-5).

[0018] Figures 2A-2C. The 3D cytotoxicity assay of R4702 ADCs with varying PEG lengths. FIG. 2A: NCI-N87 shVOID (with high TROP2 expression); FIG. 2B: NCI-N87 shTROP2 (with low TROP2 expression); FIG. 2C: NCI-H1975 (with medium TROP2 expression).

[0019] Figure 3. The payload release efficiency of R4702 ADCs with different PEG lengths.

[0020] Figure 4. Tumor inhibition assay in NCI-H1975 human non-small cell lung cancer xenograft mice treated with R4702 ADCs with varying PEG lengths.

[0021] Figures 5A-5B. The linker-payload assays. FIG. 5A shows the digestion efficiency of exemplary linker-payloads; FIG. 5B shows the exatecan release efficiency of exemplary linker- payloads.

[0022] Figure 6. The cytotoxicity of exemplary linker-payloads in human breast cancer cells SKBR-3.

[0023] Figure 7. The payload release efficiency of R4702 ADCs with different bioorthogonal groups.

[0024] Figure 8. The cytotoxicity of exemplary R4702 ADCs with different bioorthogonal groups in pancreatic adenocarcinoma cells BxPC-3.

[0025] Figures 9A-9D. Tumor inhibition comparison between OBI-902 and Dato-Dxd. FIG. 9A: NCI-N87 human gastric cancer cell-derived xenograft; FIG. 9B: DLD-1 human colorectal cancer cell-derived xenograft; FIG.9C: HPAC human PDAC (pancreatic ductal adenocarcinoma) cell-derived xenograft; FIG. 9D: TFK-1 human Cholangiocarcinoma cell-derived xenograft.

[0026] Figures 10A-10C. The HIC chromatogram and tumor inhibition comparison of sacituzumab_gADC. FIG. 10A shows the chromatogram of sacituzumab-BCN-GGVA-Hydra- PAB-PEG24-Exatecan (sacituzumab_gADC); FIG. 10B: NCI-H2170 human Enhertu^-resistant human non-small cancer (NSCLC) cell lung cell-derived xenograft; FIG. 10C: HPAC human PDAC (pancreatic ductal adenocarcinoma) cell-derived xenograft. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Abbreviations

[0027] ACN: acetonitrile; ADC: Antibody-drug conjugate; ADCC: antibody-dependent cellular cytotoxicity; BCN: Bicyclononyne; bsADC: bispecific ADC; CDR: complementarity- determining region; CE-SDS: capillary electrophoresis-sodium dodecyl sulfate; DAR: drug-to- antibody ratio; DBCO: Dibenzocyclooctyne; DL: linker-payload; DMSO: dimethyl sulfoxide; FA: formic acid; GlcNAc: N-acetylglycosamine; HIC: hydrophobic interaction chromatography; HRMS: high resolution mass spectrometry; mAb: monoclonal antibody; MCCA: 4-(N- Maleimidomethyl)-cyclohexane-1-carboxylate; NaOAc: sodium acetate; NaOH: sodium hydroxide; NSCT: sialylated complex type N-glycan; PAB: Para-aminobenzyl alcohol; PBS: phosphate buffered saline; TFA: trifluoroacetic acid; T785:1-(4-aminobutyl)-2-butylimidazo[4,5- c]quinolin-4-amine; PSar20: polysarcosine 20; PDAC: pancreatic ductal adenocarcinoma; SMCC: succinimidyl-trans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate; MMAE: monomethyl auristatin E; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis. Definitions

[0028] As used herein, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.

[0029] As used herein, the term “glycan” refers to a polysaccharide, oligosaccharide or monosaccharide. Glycans can be monomers or polymers of sugar residues and have a linear or branched structure. A glycan may include natural sugar residues (e.g., glucose, N- acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2’-fluororibose, 2’-deoxyribose, phosphomannose, 6’ sulfo N-acetylglucosamine, etc.).

[0030] As used herein, the terms “fucose”, “core fucose”, and “core fucose residue” are used interchangeably and refer to a fucose in ^-1,6-position linked to the N-acetylglucosamine.

[0031] As used herein, the terms “N-glycan” and “N-linked glycan” are used interchangeably and refer to an N-linked oligosaccharide attached via an N-acetylglucosamine (GlcNAc) to the amide nitrogen of an asparagine residue in a protein or an antibody.

[0032] As used herein, the terms “glycosylation pattern” and “glycosylation profile” are used interchangeably and refer to the characteristic “fingerprint” of the N-glycan species on a glycoprotein or antibody. The glycosylation profile can be obtained by collecting a N-glycan species released from a glycoprotein through enzymatic digestion or chemical hydrolysis, and then analyzing the carbohydrate structure, for example, LC-HPLC, or MALDI-TOF MS, and the like.

[0033] As used herein, the term “antigen” is defined as any substance capable of eliciting an immune response.

[0034] As used herein, the term “epitope” is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.

[0035] As used herein, the term “antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.

[0036] As used herein, the term “specific binding” refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10-6mol / L, about 10-7mol / L, or about 10-8mol / L, or less.

[0037] The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and sometimes may refer to antibody fragments, as described in greater detail herein. An antibody can be chimeric, human, humanized and / or affinity matured. An antibody can be a full- length or a fragment (or a combination of fragments) of an antibody having an antigen-binding portion according to the context. The fragment includes, but are not limited to, Fab, F(ab')2, Fab', F(ab)', Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragment, an CDR, diabodies, triabodies, tetrabodies, nanobodies, single-chain antibody molecules, or multispecific antibodies formed from antibody fragments. Single-chain antibodies produced by encompassed by the present disclosure.

[0038] The antibody disclosed herein may include a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof. The antibody or antigen-binding fragment thereof may be mammalian-derived, including murine and human antibodies.

[0039] The phrase “variable region” or “variable domain” of an antibody refers to the amino- terminal domains of heavy chains or light chain of the antibody. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites. The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence amongantibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).

[0040] The term “Fab” refers to two identical antigen-binding fragments produced by papain digestion of antibodies, each of which with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab’)2fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0041] The term “Fv” refers to the minimum antibody fragment which contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single- chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0042] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0043] The phrase “light chains” of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0044] Depending the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ,respectively. An antibody may be part of a larger fusion molecule, formed by covalent or non- covalent association of the antibody with one or more other proteins or peptides.

[0045] The phrase “antibody fragments” refers to a portion of an intact antibody, wherein the portion retains at least one, or as many as most or all, of the functions normally associated with that portion when present in an intact antibody. In one embodiment, an antibody fragment contains an antigen binding site of an intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment includes a Fc region of an antibody and retains at least one of the biological functions normally associated with the Fc region when present in the intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half-life substantially similar to an intact antibody. For example, such an antibody fragment may contain an antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

[0046] The phrase “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. Such monoclonal antibody typically includes an antibody including a polypeptide sequence that enables the antibody to bind a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target- binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0047] The term “humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and / or capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0048] The term “single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0049] The term “tumor” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

[0050] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, multiple myeloma and leukemia. More particular examples of such cancers include lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer.

[0051] As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct orindirect pathological consequences of the disease, preventing or decreasing inflammation and / or tissue / organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0052] An “individual” or a “subject” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, the vertebrate is a human.

[0053] A “combination therapy” refers to a combination of an amount of an ADC and an amount of other biological or chemical drugs that when administered together (either as co- administration and / or co-formulation), either sequentially or simultaneously, on the same or different days during a treatment cycle, have a synergistic effect that is therapeutically effective and more than therapeutically additive.

[0054] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (also called DM1), anthracycline, pyrrolobenzodiazepine, ^- amanitin, tubulysin, benzodiazepine, erlotinib (TARCEVA®), Genentech / OSI Pharm.), bortezomib (VELCADE®, Millenium Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®), Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (ELOXATIN®, Sanofi), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SARASAR®, SCH 66336), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone), camptothecin (including the synthetic analogue topotecan), bryostatin, callystatin, CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues), cryptophycins (particularly cryptophycin 1 and cryptophycin 8), dolastatin, duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, andranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1), dynemicin, including dynemicin A; aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo- 5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids such as maytansine and ansamitocins; mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine), urethan, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara- C”), cyclophosphamide, thiotepa, taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE®doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil, GEMZAR®gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin; vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, NAVELBINE®vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, capecitabine (XELODA®, Roche), and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0055] The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a cytotoxic agent or an ADC. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2- hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion. Antibody-drug conjugates

[0056] In certain embodiments, the antibody-drug conjugates (ADC) is represented by Formula (I): Ab-(DL)n(I); wherein Ab is an antibody or an antigen-binding fragment thereof capable of binding to one or more of tumor-associated antigens or cell-surface receptors; DL is a linker-payload as described herein where the payload is a drug moiety, and DL forms a covalent linkage with the antibody or the antigen-binding fragment thereof; and n is a drug-to-antibody ratio (DAR) ranging from 2 to 32.

[0057] In some embodiments, the present disclosure provides the linker-payload having the structure of Formula (II):wherein E is a hydrophilic moiety including: polyethylene glycol (PEG), polysarcosine (pSar), poly lactic-co-glycolic acid (PLGA), poly(glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N- dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), saccharides, or any combination thereof; C is a bioorthogonal group for conjugation to an antibody or an antigen-binding fragment thereof;L is a linker unit including a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety; and P is a payload, wherein the payload is a drug moiety.

[0058] In certain embodiments, the hydrophilic moiety has a molecular weight ranging from 60 Da to 13 kDa, from 100 Da to 10 kDa, from 200 Da to 9 kDa, from 250 Da to 8 kDa, from 300 Da to 7 kDa, from 350 Da to 6 kDa, from 400 Da to 5 kDa, from 450 Da to 4 kDa, from 500 Da to 3 kDa, from 600 Da to 3 kDa, from 700 Da to 3 kDa, from 800 Da to 3 kDa, from 900 Da to 3 kDa, or from 1 kDa to 3 kDa, or from 500 Da to 2.5 kDa. In certain embodiments, the hydrophilic moiety includes ethylene glycol or polyethylene glycol (PEG) with or without other chemical moieties at one or two ends of the PEG, and the PEG may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ethylene glycol units, or the ethylene glycol units may range between any two of the numbers listed above.

[0059] The term “bioorthogonal group (C)” refers to a non-native chemical group that can be used to conjugate the linker-payload with an antibody or an antigen-binding fragment thereof under the conditions of living systems without affecting the activity of the antibody or antigen- binding fragment thereof. In certain embodiments, the bioorthogonal group is selected from a dibenzocyclooctyne (DBCO) group, a bicyclononyne (BCN) group, a alkyne group, a maleimide group, a ^,^-unsaturated carbonyl group, a sulfonyl pyrimidine group, a 4-dibenzocyclooctynol (DIBO) group, a aza-dibenzocyclooctynes (DIBAC) group, a tetrazine group, a tetrazole group, a norbornene group, a cyclooctyne group, a methylcyclopropene group, an aminooxy group, a hydrazine group, an isocyanide group, an isocyanopropanoate group, a phosphine-containing thioester group, a phosphine phenolic ester group, or an alpha-halo carbonyl group. In certain embodiments, the bioorthogonal group is an alkyne group, which can react with an azide introduced at a specific site on an antibody through metal-free azide-alkyne cycloaddition, resulting in antibody conjugation via a triazole linkage. In other embodiments, the bioorthogonal group is a cyclooctyne, DBCO, or BCN group, which can react with an azide on an antibody (e.g., an azide on an N-glycan at an asparagine residue in an antibody heavy chain) without a catalyst via strain-promoted azide-alkyne cycloaddition to achieve antibody conjugation via a triazoline linkage. In other embodiments, the bioorthogonal group is a tetrazine group, which can react with a trans-cyclooctene (TCO) on an antibody to form a dihydropyridazine linkage, resulting in antibody conjugation.

[0060] The term “linker unit (L)” refers to an element connecting the bioorthogonal group with the payload and including a cleavable moiety. In certain embodiments, the linker unit is a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety or a hydrolysable moiety, each of which being directly linked with the bioorthogonal group and / or directly linked with the payload. In certain embodiments, the linker unit includes any one or more of the listed cleavable moieties and an intervening spacer (usually with a molecular weight of between 15 Da and 1800 Da) for connecting with the bioorthogonal group or the payload. The spacer may be a di-functional or tri-functional chemical moiety that is capable of covalently linking together the cleavable moiety of the linker unit and any one or two of the bioorthogonal group, the payload, and the hydrophilic moiety. Examples of the spacer include but not limited to amino acids and aminobenzyl alcohols.

[0061] The protease-cleavable peptide moiety may be a peptide composed of two or more natural or non-natural amino acids that can be cleaved by a peptidase. Preferably, the protease- cleavable peptide moiety may be a dipeptide selected from valine-alanine (VA), valine-cysteine (VC), phenylalanine-glycine (FG), phenylalanine-lysine (FK), alanine-alanine (AA), glycine- valine (GV), or glycine-cysteine (GC); alternatively, a peptide may be composed of said dipeptide and one to eight additional amino acids, for example, a tetrapeptide where two additional glycine or alanine residues are attached to the N-terminal of said dipeptide.

[0062] The glycosidase-cleavable sugar moiety may a sugar residue linked via an oxygen glycosidic bond to a self-immolative group, for example, a glucuronic acid linked via an oxygen glycosidic bond to a p-aminobenzyl alcohol linker, which can be cleaved by β-glucuronidase.

[0063] The pH sensitive moiety may be an acid-liable moiety that can be hydrolyzed at acidic pH and optionally an intracellularly-cleavable moiety cleavable by the low pH environment of endosomal and lysosomal vesicles. The acidic pH condition is beneficial to release payload from the ADC. Examples of the pH sensitive moiety may be one or more amino acids spacers between the drug and the linker.

[0064] The hydrolysable moiety may be a chemical moiety that can be cleaved by hydrolase. Preferably, the hydrolase is an esterase.

[0065] The term “payload” as used herein refers to a molecule to be carried and delivered by an antibody or an antigen-binding fragment thereof. The term “drug moiety” refers to a drug molecule (e.g., a cytotoxic agent), an inhibitor of an enzyme, a ligand of a receptor, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, or a prodrug thereof. In certain embodiments, the payload is selected from a chemotherapeutic agent, a toxin, a cytokine, a growth inhibitory agent, a protein degrader, a peptide, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, or an immunoregulatory agent.

[0066] In certain embodiments, the toxin is selected from pyrrolobenzodiazepine compounds or derivatives thereof (e.g., PBD), auristatin compounds or derivatives thereof (e.g., MMAE, MMAF), maytansinoid compounds or derivatives thereof (e.g., maytansine, DM1, DM4, DM21), duocarmycin or derivatives thereof, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors or derivatives thereof, tubulysin compounds or derivatives thereof, enediyne compounds or derivatives thereof (e.g., calicheamicin), anthracycline compounds or derivatives thereof (e.g., doxorubicin), pyrrole-based kinesin spindle protein (KSP) inhibitors or derivatives thereof, cryptophycin compounds or derivatives thereof (e.g., cryptophycin 52), drug efflux pump inhibitors or derivatives thereof, sandramycin or derivatives thereof, amanitin compounds or derivatives thereof, or camptothecin compounds or derivatives thereof (e.g., SN-38, belotecan, exatecan, deruxtecan).

[0067] In certain embodiments, the linker unit (L) of Formula (II) has the structure of LP-QCL- QSP, and the linker-payload is further represented by Formula (III):wherein: (a) C is a bioorthogonal group for conjugation to an antibody or an antigen-binding fragment thereof; (b) E is a hydrophilic moiety as defined in Formula (II); (c) QSPis a spacer unit including an aromatic group or amino methylene; (d) QCLis a cleavable unit including a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety; (e) LPis a connector unit that covalently connects QCL, C and E, wherein the connector unit includes one or more amino acids; and (f) P is a payload, wherein the payload is a drug moiety.

[0068] In certain embodiments, the hydrophilic moiety E of Formula (III) includes PEG and may have the formula of:, wherein the wavy line indicates the site of covalent attachment to LP, R1 is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or, R2is selected from H, SO3H, PO3H2, a sugar derivative, C1-C10(hetero) alkyl group, C3-C10(hetero) cycloalkyl group, C2-C10alkyl-NH2, C1-C10alkyl-COOH, C2-C10alkyl-NH(C1-C3alkyl), C2-C10alkyl-N (C1-C3 alkyl)2, or sarcosines, and the subscript n is an integer ranging from 2 to 72.

[0069] In certain embodiments, the linker unit (L) of Formula (II) has the structure of LB-QCL- BP, and the linker-payload is further represented by Formula (IV):wherein: (a) C is a bioorthogonal group for conjugation to an antibody or an antigen-binding fragment thereof; (b) E is a hydrophilic moiety as defined in Formula (II); (c) BPis a branch unit that covalently connects QCL, E and P, wherein the branch unit includes a functional group defined as, wherein A is an aromatic group; R3 is linked to E and is selected from -C(O)-, -C(O)O-, -C(O)NH-, alkyl-O-, alkyl-NH-, alkyl- C(O)-, alkyl-C(O)-O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-SO2-, alkyl-S-, alkyl-O-P(O)-O2-, alkyl-O-C(O)-NH-, or triazole; and the bond extending directly from one benzene carbon of the functional group is covalently linked to QCL; (d) QCLis a cleavable unit including a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety; (e) LBis a bridge unit that covalently connects C and QCL; and (f) P is a payload, wherein the payload is a drug moiety.

[0070] In certain embodiments, the hydrophilic moiety E of Formula (IV) includes PEG and may have the formula of:, wherein the wavy line indicates the site of covalent attachment to BP, R1 is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or, R2is selected from H, SO3H, PO3H2, a sugar derivative, C1-C10(hetero) alkyl group, C3-C10(hetero) cycloalkyl group, C2-C10alkyl-NH2, C1-C10alkyl-COOH, C2-C10alkyl-NH(C1-C3alkyl), C2-C10alkyl-N (C1-C3 alkyl)2, or sarcosines, and the subscript n is an integer ranging from 2 to 72.

[0071] In certain embodiments, the linker-payload of Formula (IV) includes (i) the drug moiety selected from camptothecin compounds or derivatives thereof (such as exatecan) and (ii) the hydrophilic moiety including PEG. Said linker-payload is further represented by Formula (V):wherein EPEG refers to the hydrophilic moiety including PEG, and PCAM refers to the drug moiety selected from camptothecin compounds or derivatives thereof. The EPEG may have the formula of:, wherein the wavy line indicates the site of covalent attachment to BP, R1 is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or, R2 is selected from H, SO3H, PO3H2, a sugar derivative, C1-C10 (hetero) alkyl group, C3-C10 (hetero) cycloalkyl group, C2-C10alkyl-NH2, C1-C10alkyl-COOH, C2-C10alkyl-NH(C1-C3alkyl), C2-C10alkyl-N (C1-C3 alkyl)2, or sarcosines, and the subscript n is an integer ranging from 2 to 72.

[0072] In certain embodiments, the linker-payload has the structure of the following formula:, wherein z is an integer ranging from 6 to 48. In one embodiment, z is 24, and the linker-payload has the structure of Formula (VI) (referred to as MCCA-PEG24-VA-PAB-Exatecan or DL-1):(VI). The compound of Formula (VI) is an example of the compound of Formula (III), where QCLincludes a protease-cleavable dipeptide of valine and alanine. The dipeptide is connected to PAB, an example of QSPthat further connects exatecan. The dipeptide is also connected to LPthat includes a glutamic acid residue via the sidechain of the said residue. The LPfurther connects an MCCA group (an example of the maleimide group) at N-terminal end of the glutamic acid residue and also connects PEG at C-terminal end of the said residue via an amide linkage.

[0073] In certain embodiments, the linker-payload has the structure of the following formula:,wherein z is an integer ranging from 6 to 48. In one embodiment, z is 24, and the linker-payload has the structure of Formula (VII) (referred to as DBCO-PEG24-VA-PAB-Exatecan or DL-2):

[0074] In certain embodiments, the linker-payload has the structure of the following formula:, wherein z is an integer ranging from 6 to 48.

[0075] In certain embodiments, the linker-payload has the structure of the following formula:, wherein z is an integer ranging from 6 to 48.

[0076] In one embodiment, the linker-payload has the structure of Formula (VIII) (referred to as BCN-GGVA-Hydra-PAB-PEG24-Exatecan or DL-8):

[0077] In another aspect, the present disclosure provides a glycosite-specific ADC as formula (IX):(IX); wherein Ab is a glycoengineered antibody that binds to TROP2; G represents a glycan moiety connected to an Neu5Ac and the glycoengineered antibody, wherein the glycan moiety is a linear or branched chain of saccharides selected from the group consisting of galactose, N-acetyl-glucosamine, glucose, mannose, fucose, and derivatives thereof; Y is a connector moiety independently comprising polyethylene glycol (PEG), polysarcosine (pSar), poly lactic-co-glycolic acid (PLGA), poly(glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N- dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), or any combinations thereof; Z is independently selected from a triazole or imine linkage; L is a linker connecting D and Z; D is a drug moiety; m is a number of Neu5Ac units branching from G, and is an integer from 1 to 4; and p is a number of Y-Z-L-D units attached to each of the Neu5Ac units, and is an integer from 1 to 4.

[0078] In certain embodiments, the glycosite-specific ADC is represented as formula (X):wherein Ab is a glycoengineered antibody capable of binding to TROP2; L is a linker connecting exatecan and the glycoengineered antibody; and a, b are independently 0 or 1. The glycosite- specific ADC represented by formula (X) includes a glycoengineered antibody carrying two glycan moieties, whereby the two glycan moieties are conjugated with two units of linker-exatecan.

[0079] In certain embodiments, the antibody or antigen-binding fragment thereof of the ADC is monospecific or multispecific. In certain embodiments, the antibody or antigen-binding fragment thereof is capable of specifically binding to TROP2. Preparation of antibody conjugates

[0080] The present disclosure further provides a method for preparing an antibody-drug conjugate (ADC), including reacting an antibody with the linker-payload disclosed herein to obtain the ADC, wherein the payload is a drug moiety. The ADC prepared accordingly has enhanced tumor suppression efficacy and / or improved circulation stability when administered to a subject in need.

[0081] In some embodiments, the ADC preparation method further includes the step of adding a co-solvent into a reaction mixture including the linker-payload and the antibody. In some embodiments, the linker-payload includes a hydrophilic moiety that includes PEG, and the co- solvent is propylene glycol. In some embodiments, the hydrophilic moiety of the linker-payload includes PEG having 2 to 48 ethylene glycol units, 6 to 48 ethylene glycol units, 12 to 48 ethylene glycol units, or 24 to 48 ethylene glycol units. Preferably, the hydrophilic moiety of the linker- payload includes PEG having 12 to 48 ethylene glycol units. In some embodiments, the propylene glycol has a volume ratio ranging from 0-50% to the total volume of the reaction mixture.

[0082] The present disclosure further provides a method for preparing a glycan-engineered antibody conjugate, including reacting a glycan-engineered antibody with the linker-payload disclosed herein to obtain the glycan-engineered antibody conjugate, wherein the glycan- engineered antibody includes a fucosylated or non-fucosylated N-acetylglucosamine (GlcNAc) at an asparagine residue coupled to a glycan.

[0083] In some embodiments, the glycan-engineered antibody is obtained by contacting an antibody with a glycosynthase and a glycan oxazoline to couple the glycan with the fucosylated or non-fucosylated GlcNAc. In some embodiments, the glycosynthase includes EndoSd-D232M and EndoSz-D234M.Therapeutic Applications

[0084] The present disclosure further provides a method for killing or inhibiting the proliferation of tumor cells or cancer cells, including contacting the cells with an effective amount of the ADC disclosed herein.

[0085] The present disclosure also provides a method for treating a disease, including administering to a subject in need thereof an effective amount of the ADC disclosed herein or a pharmaceutical composition that includes one or more of the ADCs described herein. By varying the characteristics of the ADC (such as the type of the antibody or the payload), the disease to be treated may be cancer, infection (including viral and bacterial infections), nervous system disorders, inflammatory disorders, or autoimmune disorders.

[0086] In some embodiments, the subject (e.g., a human patient) in need of the treatment is diagnosed with, suspected of having, or at risk for cancer. Examples of the cancer include, but are not limited to, multiple myeloma, acute myeloid leukemia (AML), sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0087] In some embodiments, the treatment results in reduction of tumor size, elimination of malignant cells, prevention of metastasis, prevention of relapse, reduction or killing of disseminated cancer, prolongation of survival and / or prolongation of time to tumor cancer progression.

[0088] In some embodiments, the method for treatment further includes administering an additional therapy to said subject prior to, during or subsequent to said administering of the ADCs. In some embodiments, the additional therapy is treatment with a chemotherapeutic agent. In some embodiments, the additional therapy is radiation therapy, photodynamic therapy, chemotherapy, immunotherapy, targeted therapy, or hormone therapy.

[0089] The methods for treating cancers described herein are particularly advantageous in treating and preventing early stage tumors, thereby preventing progression to the more advanced stages resulting in a reduction in the morbidity and mortality associated with advanced cancer. The methods are also advantageous in preventing the recurrence of a tumor or the regrowth of a tumor, for example, a dormant tumor that persists after removal of the primary tumor, or in reducing or preventing the occurrence of a tumor.

[0090] The subject to be treated by the treatment methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, or rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having cancer, which include, but are not limited to, lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, or oral cancer. A subject having cancer can be identified by medical examination.

[0091] The phrase “an effective amount” refers to the amount of each active agent or a pharmaceutical composition required to achieve the desired therapeutic result (“therapeutically effective amount”) or the desired prophylactic result (“prophylactically effective amount”), either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0092] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a disease such as cancer, a symptom of cancer, or a predisposition toward cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect cancer, the symptom of cancer, or the predisposition toward cancer, or to delay the development or progression of cancer. “Development” or “progression” of cancer means initial manifestations and / or ensuing progression of cancer. Development of cancer can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein, “onset” or “occurrence” of cancer includes initial onset and / or recurrence. Administration of ADC, pharmaceutical compositions, and pharmaceutical formulations

[0093] The present disclosure also provides a pharmaceutical composition including the ADC described herein and a pharmaceutically acceptable carrier. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer an ADC or a pharmaceutical composition including an ADC to the subject, depending upon the type of disease to be treated or the site of the disease. A pharmaceutical composition can be administered via various routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0094] The pharmaceutical compositions of ADC may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables. Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble ADCs can be administered by the drip method, whereby a pharmaceutical formulation containing the ADCs and a physiologically acceptable excipients is infused. Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitanmonooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

[0095] The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL / hour can occur. Subcutaneous (bolus) administration may be effected with about 1.5 ml or less of total volume and a concentration of about 100 mg ADC per ml. For ADC that require frequent and chronic administration, the subcutaneous route may be employed, such as by pre-filled syringe or autoinjector device technology.

[0096] As a general proposition, the initial effective amount of ADC administered per dose will be in the range of about 0.01-100 mg / kg, namely about 0.1 to 20 mg / kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg / kg / day. The dose may be escalated to the maximally tolerated dose (MTD). The dosing schedule may be about every 3 weeks, but according to diagnosed condition or response, the schedule may be more or less frequent. The dose may be further adjusted during the course of treatment to be at or below MTD which can be safely administered for multiple cycles, such as about 4 or more.

[0097] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. EXAMPLES Example 1. Preparation of NSCT derivates 1-1. Preparation of NSCT-2 (Oxazoline-NSCT-N3)

[0098] NSCT-1 (235 mg, 0.097 mmol; purchased from Glytech, Inc. Catalog No. GT-25261; HPLC purity > 90%) and triethylamine (605 ^L, 0.44 mmol) were dissolved in water (10 mL) and cooled to 0oC. 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride aqueous solution (1 M, 1.44 mL) was added slowly and the resulting mixture was stirred at 0 to 5oC for 4 hours. NaOH solution was added (0.01 M, 1 mL) and the resulting mixture was concentrated under reduced pressure. After most of the triethylamine was evaporated, the residual mixture was purified by Sephadex®g-15 column. Using 0.01 M NaOH as eluent to stabilize NSCT-2. Fractions with desired product were combined and freeze-dried to afford NSCT-2 (170 mg) as a white solid. 1HNMR (D2O): δ 6.10 (d, J = 7.26 Hz, 1H, H1 of oxazoline), 5.24 (s, 1H, H1 of GluNAc), 4.97 (s, 1H, H1 of GluNAc), 4.76 (s, 1H, H1 of Man), 4.64-4.60 (m, 2H, H1 of two Gal), 4.46 (s, 1H, H1 of Neu5Ac), 4.45 (s, 1H, H1 of Neu5Ac), 4.40 (s, 1H, H3 of β-form Man), 4.18 (d, J = 21 Hz, 4H, H2 of two Man and two GluNAc), 3.99-3.48 (m), 2.71 (dd, J1 = 12.9 Hz, J2 = 4.2 Hz, 2H, H3eq of two Neu5Ac), 2.15-2.02 (m, 15H), 1.58 (dd, J1 = J2 = 12.2 Hz, 2H, H3ax of two Neu5Ac).1-2. Preparation of NSCT-5

[0099] NSCT (50.0 mg, 0.0247 mmol; purchased from Glytech Inc.) dissolved in DMF (2.0 mL) with DBCO-NH2(41.0 mg, 0.148 mmol), PyBOP (257.0 mg, 0.494 mmol) and DIPEA (63.8 mg, 0.494 mmol) was added. This mixture was stirred under room temperature for 20 hours. After the reaction was completed, the reaction mixture was extracted with DCM / H2O. The H2O layer was collected and lyophilized to afford crude product. The crude product was purified by reverse phase chromatography (eluent: ACN / Water). The pure fractions were combined and lyophilized to obtain NSCT-5 (41.5 mg, 66.2% yield). MS calculate for [C112H153N9O57 + 2Na+] / 2: 1290.9573, found: 1290.9553.1H NMR (D2O): δ 7.69 (t, 2H), 7.52-7.59 (m, 12H), 7.48 (t, 2H), 7.43 (t, 2H), 7.37 (t, 2H), 5.12 (t, 4H), 4.59 (t, 4H), 4.43 (dd, 2H), 4.28 (s, 1H), 4.21 (s, 1H), 4.13 (s, 1H), 4.05- 3.47(m), 3.38-3.46 (m, 2H), 3.35-3.23 (m, 2H), 3.21-3.12 (m, 2H) 2.58-2.42 (m, 3H), 2.09-2.00 (m), 1.76 (td, 1H).1-3. Preparation of NSCT-6 (Oxazoline-NSCT-DBCO)

[0100] A method similar to that described in NSCT-2 preparation was applied to obtain the oxazoline product NSCT-6 from NSCT-5.Example 2. Deglycosylation, transglycosylation and purification of monoclonal antibodies 2-1. Deglycosylation of mAbs by EndoSz-D234M to generate mAb-GlcNAc(Fuc)

[0101] One monoclonal antibody was used to illustrate the process to obtain glycan-engineered antibodies. R4702 is an anti-TROP2 monoclonal antibody. R4702 mAb was deglycosylated with EndoSz-D234M in 50 mM Tris pH 7.2 at 37 °C for 24-49 hours. Only the antibodies with high mannose N-glycan modification were further supplied with Endo H and incubated at 25 °C overnight to remove glycans completely and produced mAb-GlcNAc(Fuc). The complete cleavage of Fc N-glycans were analyzed by SDS-PAGE and CE-SDS.

[0102] The EndoSz-D234M was applied to other mAbs for further deglycosylation and tranglycosylation investigation. For the deglycosylation studies, the mAbs were incubated with EndoSz-D234M at a weight ratio of 1:30 (EndoSz-D234M: mAbs). All the mAb-GlcNAc(Fuc) from different mAbs reached to >90% by EndoSz-D234M cleavage. For the tranglycosylation investigation with NSCT-2, 20 or 38 equivalents of NSCT-2 was added to mAb-GlcNAc(Fuc) for incubation with EndoSz-D234M at 37 °C for 1.5 to 2 hours. Table 1 showed that the yield of R4702-(NSCT-di-N3)2was 95.61%, analyzed by CE-SDS. This data indicate that EndoSz-D234M can be applied to various mAbs.

[0103] Table 1. Deglycosylation and transglycosylation of R4702 mAb2-2. Transglycosylation of mAb-GlcNAc(Fuc) with Oxazoline-NSCT-N3 (NSCT-2) to generate mAb-(NSCT-di-N3)2

[0104] In general, mAb-GlcNAc(Fuc) were incubated with 20-38 equivalents of NSCT-2 at 37 °C for 1.5-2 hours to generate mAb-(NSCT-di-N3)2. The transglycosylation efficiency was monitored by SDS-PAGE and CE-SDS. 2-3. Purification of mAb-(NSCT-di-N3)2

[0105] Sodium chloride was added into the transglycosylation mixture to reach a final concentration of 3M and then applied to PBS and 3M NaCl pre-equilibrated HiTrap Phenyl HP (Cytiva). The non-bound contaminations were washed by 5CV of equilibration buffer (PBS and 3M NaCl). mAb-(NSCT-di-N3)2was eluted with a 30-100% elution buffer (Sodium phosphate 20 mM and 20% IPA pH 7.2) in 20CV linear gradient. The eluted fractions were applied to a prepacked column, HiTrap Protein A HP (Cytiva). The impurities were washed by two steps pH gradient, 100 mM Sodium citrate pH 6.0 and pH 5.5, with 5CV in each step.50 mM Sodium citrate pH 3.5 was employed to elute bound antibody. The eluted fractions were immediately neutralized with 1 M Tris-HCl pH 9.0 to natural pH and change buffer to 20 mM Sodium Acetate pH 5.0 with Amicon centrifugation membrane (30 kDa cutoff, Millipore). The purified mAb-(NSCT-di-N3)2were stored at -80°C.Example 3. Synthesis of linker-payload 3-1. Preparation of MCCA-PEG24-VA-PAB-Exatecan (also termed DL-1 / N-PM-0017)

[0106] Step 1: To a suspension of exatecan mesylate in DMF, N-PM-0015 and DIPEA were added at room temperature. The suspension became a clear brown solution within 5 minutes. This mixture was stirred at room temperature for 20 hours. After the reaction was completed, the reaction mixture was added to a stirring TBME to get precipitate. After stirring for 30 minutes, the solids were collected by filtration and followed with high vacuum drying to obtain crude N-PM-0016. This crude product was used in the next step without further purification.

[0107] Step 2: A stirring suspension of N-PM-0016 in DCM was cooled to -20 ⁰C. A -10 ⁰C pre-cooled TFA liquid was added to N-PM-0016 solution over 60 minutes. This mixture was stirred at -20 ⁰C for 10 hours. After the reaction was completed, the reaction mixture was added to a stirring TBME to get precipitate. After stirring for 30 minutes, the solids were collected by filtration and followed with high vacuum drying to obtain crude N-PM-0018. This crude product was used in the next step without further purification.

[0108] Step 3: A solution of N-DT-0013 in DMF, HATU and NMM was added. This mixture was stirred at room temperature for 2 hours. A solution of N-PM-0018 and NMM in DMF was added to the N-DT- 0013 solution at room temperature over 30 minutes. This mixture was stirred at room temperature for further 2 hours. After the reaction was completed, the reaction mixture was added to a stirring TBME to get precipitate. After stirring for 30 minutes, the solids were collected by filtration and purified by reverse phase chromatography (eluent: ACN / Water). The pure fractions were combined and extracted with 10% MeOH / DCM to obtain N-PM-0017.3-2. Preparation of DBCO-PEG24-VA-PAB-Exatecan (also termed DL-2)

[0109] Step 1: N-(9-Fmoc)-L-glutamic acid g-tert-butyl ester monohydrate (152.2 mg, 0.35 mmol), m-PEG24- amine (380.9 mg, 0.35 mmol) and HATU (159.7 mg, 0.42 mmol) were dissolved in DMF / CH2Cl2= 1 / 1 (3.5 mL) under room temperature. NMM (115.8 µL, 1.05 mmol) was added. After the addition, the resulting mixture was stirred at room temperature for 18 hours. The reaction solution was concentrated in vacuo at 30-35oC, and the residue was purified by a flash silica gel column (CH2Cl2 / MeOH = 15 / 1 to 12 / 1) to obtain 502.5 mg of Glu-1 with 96.0% yield.

[0110] Step 2: To a solution of Glu-1 (413.8 mg, 0.27 mmol) in CH2Cl2 / MeOH = 1 / 1 (13.8 ml), Et2NH (1.38 ml) was added. The mixture was stirred at room temperature for 24 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC and then azeotroped with toluene (5 mL x 3) to remove excess Et2NH. The resulting material was dried in high vacuum to obtain crude Glu-2. The crude product was used in the next step without further purification.

[0111] Step 3: To a solution of crude Glu-2 (352.3 mg, 0.27 mmol) and DBCO-acid (101.4 mg, 0.33 mmol) in DMF / CH2Cl2= 1 / 1 (5.5 ml), HATU (157.8 mg, 0.45 mmol) and NMM (91.6 µL, 0.83 mmol) were added separately. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was concentrated in vacuo at 30-35oC, and the residue was purified with a flash silica gel column (CH2Cl2 / MeOH = 15 / 1) to obtain 367.9 mg of DBCO-1 with 85.2% yield from Glu-1.

[0112] Step 4: A solution of DBCO-1 (350.0 mg, 0.22 mmol) in CH2Cl2(6.9 mL) was cooled to 0oC. TFA (1.8 mL) was added dropwise. The reaction mixture was stirred at 0-4oC for 4-6 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC, and the residue was purified with a flash silica gel column (CH2Cl2 / MeOH = 12 / 1) to obtain 175.4 mg of DBCO-2 with 52.0% yield.

[0113] Step 5: N-PM-0018 (26.5 mg, 0.031 mmol), DBCO-2 (45.9 mg, 0.031 mmol) and HATU (13.9 mg, 0.037 mmol) were dissolved in DMF (0.61 mL). NMM (10.1 µL, 0.092 mmol) was added. The reaction mixture was stirred at room temperature for 20 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 48.9 mg of DL-2 with 71.5% yield.1H NMR (600 MHz, d-MeOH) δ 7.70-7.50 (m, 6H), 7.46-7.36 (m, 5H), 7.33-7.10 (m, 3H), 5.56 (d, 1H, J = 16.0 Hz), 5.36 (dd, 1H, J = 16.0, 6.2 Hz), 5.33-5.29 (m, 1H), 5.27 (d, 1H, J = 20.2 Hz), 5.21-5.13 (m, 2H), 5.13-5.05 (m, 2H), 4.47-4.41 (m, 1H), 4.23-4.12 (m, 2H), 3.73-3.52 (m, 102H), 3.50-3.46 (m, 1H), 3.43-3.38 (m, 1H), 3.35 (s, 3H), 3.27-3.17 (m, 1H), 3.15-3.07 (m, 1H), 2.81- 2.70 (m, 1H), 2.37 (s, 3H), 2.36-2.24 (m, 4H), 2.45-2.40 (m, 1H), 2.24-2.00 (m, 5H), 2.00-1.78 (m, 4H), 1.43 (d, 1H, J = 7.1 Hz), 1.03-0.94 (m, 9H); HRMS (ESI) m / z found [(M+2H) / 2]+, 1121.0605 C113H164FN9O362+, required 1121.0553.

[0114] Table 2. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-3. Preparation of MCCA-linear PEG12-VA-PAB-Exatecan (also termed DL-3)

[0115] Mal-2 (14.0 mg, 0.0162 mmol) in DMF (0.6 mL) was cooled to 0-4 ⁰C. N-PM-0018 (16.9 mg, 0.0194 mmol), NMM (4.1 mg, 0.0405 mmol) and HOAt (5.5 mg, 0.0405 mmol) were added sequentially under 0-4 ⁰C. This mixture was stirred at 0-4 ⁰C for 16 hours. After the reaction was completed, the reaction mixture was purified by reverse phase chromatography (eluent: ACN / Water). The pure fractions were combined and extracted with 10% MeOH / DCM to obtain DL-3 (13.6 mg, 55.8% yield). MS calculate for [C74H101FN8O24+ 2H+] / 2: 753.3529, found: 753.3534.3-4. Preparation of DBCO-VA-Hydra-PAB-PEG24-Exatecan (also termed DL-5)

[0116] Step 1:(2R)-hydroxy(4-nitrophenyl)acetic acid (150.0 mg, 0.76 mmol), m-PEG24-amine (909.4 mg, 0.84 mmol) and HATU (346.4 mg, 0.91 mmol) were dissolved in anhydrous DMF (7.0 mL) under room temperature. NMM (0.25 mL, 2.30 mmol) was added. The resulting mixture was stirred at room temperature for 6-8 hours.7% NaHCO3 (aq)(7.0 mL) was then added, and the mixture solution was extracted with CH2Cl2(10 mL x 3). The combined organic layers were dried with MgSO4and then concentrated in vacuo at 30-35oC. The crude H-PAB-1 was dried in high vacuum and then used in the next step without further purification.

[0117] Step 2: To a solution of crude H-PAB-1 in EA / MeOH (25 mL, 95 / 5), 10% Pd / C (165.0 mg, 5 wt%) was added. The reaction was stirred at room temperature with H2balloon for 24 hours. After the reaction was completed, the reaction mixture was passed through celite (3.3 g) and the celite was washed with MeOH (10 mL x 3). The desired fraction was concentrated in vacuo at 30-35oC. The crude H-PAB-2 was dried in high vacuum and then used in the next step without further purification. HRMS (ESI) m / z found [M+H]+, 1237.7278 C57H109N2O26+, required 1237.7269.

[0118] Step 3: To a solution of crude H-PAB-2 in anhydrous CH2Cl2(25 mL), Boc-VA-OH (792.8 mg, 2.75 mmol) and EEDQ (741.8 mg, 3.00 mmol) were added. The mixture was stirred at room temperature for 18-24 hours. After the reaction was completed, 1 M HCl (aq) (15 mL) was added and then the mixture solution was extracted with CH2Cl2(15 mL x 3). The combined organic layers were washed with H2O (20 mL) and then concentrated in vacuo at 30-35oC. The resulting mixture was purified with preparative HPLC to obtain 440.0 mg of H-PAB-3 with 38.4% yield for three steps. HRMS (ESI) m / z found [(M+2Na) / 2]+, 776.4278 C70H130N4O30Na22+, required 776.4282.

[0119] Table 3. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0120] Steps 4 and 5: H-PAB-3 (440.0 mg, 0.29 mmol) and Bis(4-nitrophenyl) carbonate (177.5 mg, 0.58 mmol) in anhydrous CH2Cl2(5.8 mL) was cooled to 0-4oC. 2,6-lutidine (51.0 µL, 0.43 mmol) and DIPEA (50.9 µL, 0.29 mmol) were added sequentially at 0-4oC. The resulting mixture was stirred at 0-4oC and monitored by HPLC. After H-PAB-3 was consumed, Et2NH (6.0 µL x 5) was added separately at 0-4oC to quench excess Bis(4-nitrophenyl) carbonate. Anhydrous DMF (11.6 ml), exatecan mesylate (278.4 mg, 0.52 mmol), and DIPEA (188.0 µL, 1.07 mmol) were added into the reaction solution at 0-4oC. The reaction mixture was stirred at 0-4oC until H-PAB-4 was consumed by checking with HPLC. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 229.8 mg of H-PAB-5 with 40.0% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 985.0144 C95H152FN7O352+, required 985.0156.

[0121] Table 4. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0122] Step 6: A solution of H-PAB-5 (45.0 mg, 0.02 mmol) in CH2Cl2 (0.9 mL) was cooled to 0oC. TFA (225.0 0 µL) was added dropwise. The reaction mixture was stirred at 0oC for 4-6 hours. After the reaction was completed, the reaction mixture was added dropwise into a stirring ether (11.3 mL) to get precipitate. The solids were collected by filtration and washed with ether (5 mL x 3), and followed by dried in high vacuum to obtain crude H-PAB-6. The crude product was used in the next step without further purification.

[0123] Step 7:To a solution of crude H-PAB-6 (0.023 mmol) in anhydrous DMF (0.45 mL), HATU (10.4 mg, 0.027 mmol) and DBCO-acid (6.9 mg, 0.023) were added and the reaction mixture was cooled to 0-4oC. After 5 minutes, Et3N (9.62 µL, 0.069 mmol) was added slowly. The reaction mixture was stirred at 0-4oC for 4 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 27.6 mg of DL-5 with 56.2% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1078.5447 C109H157FN8O352+, required 1078.5367.

[0124] Table 5. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-5. Preparation of BCN-VA-Hydra-PAB-PEG24-Exatecan (also termed DL-6)

[0125] A solution of H-PAB-5 (36.8 mg, 0.0187 mmol) in CH2Cl2(0.74 mL) was cooled to 0oC. TFA (184.0 µL) was added dropwise. The reaction mixture was stirred at 0oC for 4 hours. After the reaction was completed, the reaction mixture was diluted with MeOH (5 mL) and then concentrated in vacuo at 30-35oC and then azeotroped with toluene (5 mL x 3) to remove excess TFA. The resulting material was dried in high vacuum to obtain crude deprotected intermediate. A solution of the crude intermediate (0.0187 mmol) in anhydrous DMF (0.37 mL), BCN-Osu (5.4 mg, 0.0187 mmol) and Et3N (7.8 µL, 0.0561 mmol) were added sequentially. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 23.8 mg of DL-6 with 62.2% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1023.0380 C101H154FN7O352+, required 1023.0235.

[0126] Table 6. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-6. Preparation of DBCO-GGVA-Hydra-PAB-PEG24-Exatecan (also termed DL-7)

[0127] Step 1:DBCO-acid (265.7 mg, 0.87 mmol), tert-butyl 2-(2-aminoacetamido)acetate (163.8 mg, 0.87 mmol) and HATU (363.9 mg, 0.96 mmol) were dissolved in CH2Cl2 (8.7 mL) under room temperature. NMM (287.0 µL, 2.61 mmol) was added. After the addition, the resulting mixture was stirred at room temperature for 16-18 hours. The reaction solution was concentrated in vacuo at 30-35oC, and the residue was purified by a flash silica gel column (CH2Cl2 / EA = 1 / 2 to 1 / 4) to obtain 342.2 mg of DBCO-4 with 82.7% yield.

[0128] Step 2: A solution of DBCO-4 (340.0 mg, 0.71 mmol) in CH2Cl2 (6.8 mL) was cooled to 0oC. TFA (1.7 mL) was added dropwise. The reaction mixture was warmed to room temperature (rt) slowly and then stirred for 1-2 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC and then azeotroped with toluene (5 mL x 3) to remove excess TFA. The resulting mixture was purified with preparative HPLC to obtain 168.4 mg of DBCO-5 with 56.1% yield.

[0129] Table 7. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0130] Step 3:DBCO-5 (3.98 mg, 0.0095 mmol), H-PAB-6 (17.8 mg, 0.0095 mmol) and HATU (3.97 mg, 0.0104 mmol) were dissolved in anhydrous DMF (0.32 mL). Et3N (4.0 µL, 0.0285 mmol) was added. The reaction mixture was stirred at room temperature for 3-4 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 10.7 mg of DL-7 with 49.6% yield.

[0131] Table 8. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-7. Preparation of BCN-GGVA-Hydra-PAB-PEG24-Exatecan (also termed DL-8)

[0132] Step 1: A solution of H-PAB-5 (57.0 mg, 0.0289 mmol) in CH2Cl2 (1.2 mL) was cooled to 0oC. TFA (285.0 µL) was added dropwise. The reaction mixture was stirred at 0-4oC for 3-4 hours. After the reaction was completed, the reaction mixture was diluted with MeOH (2 mL). The solution was concentrated in vacuo at 30-35oC and then azeotroped with toluene (5 mL x 3) to removeexcess TFA. The residue was dried in high vacuum to generate crude deprotected intermediate. The prepared crude intermediate, Boc-Gly-OH (5.1 mg, 0.0289 mmol) and HATU (13.2 mg, 0.0347 mmol) were dissolved in anhydrous DMF (0.58 mL). Et3N (12.1 µL, 0.0868 mmol) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 40.4 mg of H- PAB-7 with 68.9% yield.

[0133] Table 9. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0134] Step 2: A solution of H-PAB-7 (38.5 mg, 0.0190 mmol) in CH2Cl2 (0.77 mL) was cooled to 0oC. TFA (192.5 µL) was added dropwise. The reaction mixture was stirred at 0-4oC for 3-4 hours. After the reaction was completed, the reaction mixture was diluted with MeOH (2 mL). The solution was concentrated in vacuo at 30-35oC and then azeotroped with toluene (5 mL x 3) to removeexcess TFA. The residue was dried in high vacuum to generate crude deprotected intermediate. The prepared crude intermediate, Boc-Gly-OH (3.4 mg, 0.0190 mmol) and HATU (8.7 mg, 0.0228 mmol) were dissolved in anhydrous DMF (0.38 mL). Et3N (7.9 µL, 0.0570 mmol) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 28.7 mg of H-PAB-8 with 72.6% yield.

[0135] Table 10. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0136] Step 3: A solution of H-PAB-8 (25.0 mg, 0.0120 mmol) in CH2Cl2 (0.50 mL) was cooled to 0oC. TFA (125.0 µL) was added dropwise. The reaction mixture was stirred at 0oC for 4 hours. After the reaction was completed, the reaction mixture was diluted with MeOH (5 mL) and then concentrated in vacuo at 30-35oC and then azeotroped with toluene (5 mL x 3) to remove excess TFA. The resulting material was dried in high vacuum to obtain crude deprotected intermediate. A solution of the crude intermediate (0.0120 mmol) in anhydrous DMF (0.24 mL), BCN-OSu (3.5 mg, 0.0120 mmol) and Et3N (5.1 µL, 0.0360 mmol) were added sequentially. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 15.7 mg of DL-8 with 60.9% yield.

[0137] Table 11. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-8. Preparation of BCN-GGVA-Hydra-PAB-MMAE (also termed DL-14) and BCN-GGVA- Hydra-PAB-T785 (also termed DL-15)

[0138] Step 1: To a solution of N-DT-0024 (50.0 mg, 0.065 mmol) and payload (MMAE or T785, 1.1 eq) in anhydrous DMF (0.65 mL), DIPEA (33.4 μL, 0.192 mmol) was added slowly. The resulting mixture was stirred at room temperature until N-DT-0024 was consumed. After the reaction was completed, the reaction mixture was added dropwise into a stirring TBME (6.5 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude HL-1 (X=MMAE with 75.6% yield) or HL-2 (X=T785 with 79.1% yield). This crude product was used in the next step without further purification.

[0139] Step 2: A stirring suspension of HL-1 or HL-2 (83.4 mg) in DCM (1.46 mL) was cooled to 0 ⁰C. TFA (0.63 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4 ⁰C for 20-24 hours. After the reaction was completed, the reaction mixture was added dropwise into a stirring TBME (20.9 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude intermediate. This crude product was used in the next step without further purification.

[0140] The intermediate and BCN-OSu (1 eq) were dissolved in anhydrous DMF (0.05 M). Et3N (3 eq) was added into the reaction solution at room temperature. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring TBME (10 times amount of intermediate) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude BCN intermediate. This crude product was used in the next step without further purification.

[0141] The BCN intermediate (1 eq), NH2-PEG24-Ome (2 eq) and HATU (1.2 eq) were dissolved in anhydrous DMF (0.05 M). NMM (5 eq) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain DL-14 or DL-15. DL-14 was obtained with 26.0% yield.MS (ESI) m / z found [(M+H+NH4 / 2]+, 1229.810 C120H210N12O402+, required 1229.738. DL-15 was obtained with 27.7% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1018.0743 C99H165N11O332+, required 1018.0785.

[0142] Table 12. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-9. Preparation of BCN-GGVA-Hydra-PAB-Eribulin (also termed DL-16)

[0143] Step 1: Fmoc-VA-PAB(COOtBu)-OH (202.8 mg, 0.329 mmol) in anhydrous CH2Cl2 (3.8 mL) was cooled to 0oC. After five minutes, TFA (1.27 mL) was added slowly. The reaction mixture was stirred at0-4 ⁰C overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring TBME (50.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain a crude intermediate (75.71 mg). The previous intermediate (75.71 mg, 0.135 mmol), NH2-PEG24-OMe (161.9 mg, 0.149 mmol) and HATU (61.7 mg, 0.162 mmol) were dissolved in anhydrous DMF (1.35 mL). NMM (44.8 μL, 0.406 mmol) was added and the reaction mixture was stirred at room temperature for 6-8 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC, and the residue was purified by a flash silica gel column (CH2Cl2 / MeOH = 12 / 1 to 9 / 1) to obtain 212.7 mg of HL-3 with 39.6% yield.

[0144] Step 2: HL-3 (22.57 mg, 0.014 mmol) and Bis(4-nitrophenyl) carbonate (8.42 mg, 0.028 mmol) in anhydrous CH2Cl2 (0.28 mL) was cooled to 0-4oC.2,6-lutidine (2.4 µL, 0.021 mmol) and DIPEA (2.4 µL, 0.014 mmol) were added sequentially at 0-4oC. The resulting mixture was stirred at 0-4oC for 20-24 hours. Et2NH (1.4 µL, 0.014 mmol) was added slowly at 0-4oC to quench excess Bis(4-nitrophenyl) carbonate. Anhydrous DMF (0.55 mL), Eribulin (15.2 mg, 0.021 mmol), DIPEA (7.2 µL, 0.042 mmol) were added into previous reaction solution at 0-4oC. The reaction mixture was stirred at 0-4oC overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 7.54 mg of HL-4 with 22.8% yield.

[0145] Table 13. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0146] Step 3: HL-4 (7.54 mg, 0.003 mmol) was dissolved in CH2Cl2 / MeOH (226.0 µL, 1 / 1) and then cooled to 0oC. Et2NH (45.2 µL) was added and the reaction mixture was stirred at 0-4oC overnight. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC and azeotroped with toluene (1 mL x 3) to remove excess Et2NH. The crude compound was dried in high vacuum to obtain a crude intermediate (8.2 mg). The intermediate (8.2 mg), Fmoc-GG-OH (1.61 mg, 0.004 mmol) and HATU (2.16 mg, 0.005 mmol) were dissolved in anhydrous DMF (0.2 mL). NMM (1.25 µL, 0.011 mmol) was added at room temperature. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture wasconcentrated in vacuo at 30-35oC, and the residue was purified by a flash silica gel column (CH2Cl2 / MeOH = 100 / 0 to 87 / 13) to obtain Fmoc-GG-intermediate. Fmoc-GG-intermediate was dissolved in CH2Cl2 / MeOH (0.6 mL, 1 / 1) and then cooled to 0oC. Et2NH (0.13 mL) was added and the reaction mixture was stirred at 0-4oC for 20-24 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC and azeotroped with toluene (2 mL x 3) to remove excess Et2NH. The crude compound was dried in high vacuum to obtain a crude intermediate. The intermediate (19.3 mg), BCN-OSu (2.51 mg, 0.008 mmol) were dissolved in anhydrous DMF 0.17 mL. Et3N (3.61 µL, 0.026 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 2.85 mg of DL-16 with 37.4% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1227.1713 C121H199N7O442+, required 1227.1697.

[0147] Table 14. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-10. Preparation of BCN-Glu(PEG24)-VA-Hydra-PAB-Exatecan (also termed DL-17)

[0148] Step 1: H-PAB-6 (57.3 mg, 0.029 mmol), Fmoc-Glu(PEG24) (43.7 mg, 0.030 mmol) and HATU (22.7 mg, 0.060 mmol) were dissolved in anhydrous DMF (0.29 mL). NMM (9.5 μL, 0.087 mmol) was added and the reaction mixture was stirred at room temperature for 1-2 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 72.6 mg of HL-5 with 76.1% yield. HRMS (ESI) m / z found [(M+4H) / 4]+, 823.1910 C159H262FN9O614+, required 823.1915.

[0149] Table 15. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0150] Step 2: HL-5 (70. 4 mg, 0.021 mmol) was dissolved in CH2Cl2 / MeOH (1.69 mL, 1 / 1) and then cooled to 0oC. Et2NH (0.42 mL) was added and the reaction mixture was stirred at 0-4oC for 8-10 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC and azeotroped with toluene (5 mL x 3) to remove excess Et2NH. This crude product HL-6 was used in the next step without further purification.

[0151] Step 3: HL-6 (38.6 mg, 0.013 mmol) and BCN-OSu (3.7 mg, 0.013 mmol) were dissolved in anhydrous DMF 0.26 mL. Et3N (5.3 µL, 0.038 mmol) was added. The resulting mixture was stirred at room temperature for 1-2 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 27.8 mg of DL-17 with 65.9% yield. HRMS (ESI) m / z found [(M+3Na) / 3]+, 1103.9109 C155H160FN9O61Na33+, required 1103.9065.

[0152] Table 16. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-11. Preparation of DBCO-Glu(PEG24)-VA-Hydra-PAB-Exatecan (also termed DL-18)

[0153] HL-6 (15.2 mg, 0.0050 mmol), DBCO-acid (1.5 mg, 0.0050 mmol) and HATU (2.83 mg, 0.0074 mmol) were dissolved in anhydrous DMF (0.10 mL). Et3N (2.1 µL, 0.0149 mmol) was added. The reaction mixture was stirred at room temperature for 1-2 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 10.2 mg of DL-18 with 61.6% yield. HRMS (ESI) m / z found [(M+3H) / 3]+, 1118.9224 C163H264FN10O613+, required 1118.9282.

[0154] Table 17. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-12. Preparation of 2SP-Glu(PEG24)-VA-Hydra-PAB-Exatecan (also termed DL-19)

[0155] To a solution of HL-6 (25.7 mg, 0.0084 mmol), 6-(2-(Methylsulfonyl)pyrimidin-5- yl)hex-5-ynoic acid (2.3 mg, 0.0084 mmol) and HATU (4.8 mg, 0.0126 mmol) in anhydrous DMF (0.17 mL), Et3N (3.5 µL, 0.0252 mmol) was added. The reaction mixture was stirred at room temperature for 1-2 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 16.5 mg of DL-19 with 59.4% yield. HRMS (ESI) m / z found [(M+3H) / 3]+, 1106.5778 C155H261FN11O62S3+, required 1106.5771

[0156] Table 18. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-13. Preparation of MCCa-Glu(PEG24)-VA-Hydra-PAB-Exatecan (also termed DL-20)

[0157] To a solution of HL-6 (19.9 mg, 0.0065 mmol) and SMCC (2.6 mg, 0.0078 mmol) in anhydrous DMF (0.13 mL), Et3N (2.7 µL, 0.0195 mmol) was added. The reaction mixture was stirred at room temperature for 1-2 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 10.9 mg of DL-20 with 51.4% yield. HRMS (ESI) m / z found [(M+3H) / 3]+, 1096.2614 C156H264FN10O623+, required 1096.2599.

[0158] Table 19. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-14. Preparation of BCN-GGVA-PAB(PSar20)-Exatecan (also termed DL-21)

[0159] N-PM-0025 (12.5 mg, 0.0115 mmol), polysarcosine (26.4 mg, 0.0172 mmol) and HATU (5.2 mg, 0.0138 mmol) were dissolved in anhydrous DMF (0.23 mL). NMM (3.8 µL, 0.0344 mmol) was added. The reaction mixture was stirred at 50oC for 5-6 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 10.4 mg of DL-21 with 34.8% yield. HRMS (ESI) m / z found [(M+3Na) / 3]+, 878.0733 C120H170FN29O33Na33+, required 878.2725.

[0160] Table 20. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-15. Preparation of BCN-Glu(PEG24)-VA-PAB(PSar20)-Exatecan (also termed DL-22)

[0161] Step1: Fmoc-VA-OH (703.5 mg, 1.713 mmol) and PAB(COOtBu)-OH (318.8 mg, 1.428 mmol) were dissolved in anhydrous CH2Cl2 (6.39 mL) and anhydrous MeOH (6.38 mL). EEDQ (529.7 mg, 2.142 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC. The crude product was purified by a flash silica gel column (EA / CH2Cl2= 99 / 1 to 97 / 3, then CH2Cl2 / MeOH = 9 / 1) to obtain 687.6 mg of Fmoc-VA-PAB(COOtBu) with 78.2% yield.

[0162] Fmoc-VA-PAB(COOtBu) (674.5 mg, 1.095 mmol) and Bis(4-nitrophenyl) carbonate (866.4 mg, 2.848 mmol) were dissolved in DMF (3.3 mL). DIPEA (0.76 mL, 4.382 mmol) was added into the reaction solution. The reaction mixture was stirred at room temperature for 4-6 hours. After the reaction was completed, the reaction solvent was concentrated in vacuo at 30-35oC. The crude product was purified by flash silica gel column (CH2Cl2 / MeOH = 15 / 1 to 9 / 1) to obtain 496.1 mg of Fmoc-VA-PAB(COOtBu)-PNP with 58.0% yield.

[0163] To a suspension of exatecan mesylate (295.0 mg, 0.555 mmol), Fmoc-VA- PAB(COOtBu)-PNP (481.5 mg, 0.617 mmol) in anhydrous DMF (6.1 mL), DIPEA (0.32 mL, 1.849 mmol) was added at room temperature. The suspension became a clear brown solution within five minutes. This mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring TBME (65.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtaincrude Fmoc-VA-PAB(COOtBu)-Exatecan (398.5 mg, 60.0% yield). This crude product was used in the next step without further purification.

[0164] Fmoc-VA-PAB(COOtBu)-Exatecan (152.9 mg, 0.142 mmol) was dissolved in CH2Cl2 / MeOH (3.4 mL, 1 / 1) and then cooled to 0oC. Et2NH (0.85 mL) was added and the reaction mixture was stirred at 0-4oC for 4-5 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC to reduce the 2 / 3 solvent amount, and then added dropwise into a stirring TBME (30.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude HL-7 (86.7 mg, 71.5% yield). This crude product was used in the next step without further purification.

[0165] Step 2: HL-7 (81.3 mg, 0.095 mmol), Fmoc-Glu(PEG24) (145.1 mg, 0.100 mmol) and HATU (43.3 mg, 0.114 mmol) were dissolved in anhydrous DMF (0.95 mL). NMM (31.4 μL, 0.285 mmol) was added and the reaction mixture was stirred at room temperature for 1-2 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 48.4 mg of Fmoc-Glu(PEG24)-VA-PAB(COOtBu)-Exatecan with 22.4% yield. HRMS (ESI) m / z found [(M+H)]+, 2276.1406 C114H168FN8O38+, required 2276.1444.

[0166] Table 21. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0167] Fmoc-Glu(PEG24)-VA-PAB(COOtBu)-Exatecan (42.6 mg, 0.018 mmol) was dissolved in CH2Cl2 / MeOH (1.0 mL, 1 / 1) and then cooled to 0oC. Et2NH (0.26 mL) was added and the reaction mixture was stirred at 0-4oC for 4-5 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC and azeotroped with toluene (5 mL x 3) to remove excess Et2NH. This crude product was then dissolved in anhydrous CH2Cl2(0.71 mL) and cooled to 0oC. TFA (0.35 mL) was added dropwise. The reaction mixture was stirred at 0oC overnight. After the reaction was completed, the reaction mixture was added dropwise into astirring ether (11.0 mL) to get precipitate. The solids were collected by filtration and washed with ether (5 mL x 3), and followed by dried with high vacuum to obtain crude HL-8. The crude product was purified with preparative HPLC to obtain 11.8 mg of HL-8 with 31.6% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 999.5108 C114H168FN8O38+, required 999.5107.

[0168] Table 22. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0169] Step 3: HL-8 (10.1 mg, 0.0051 mmol) and BCN-OSu (2.2 mg, 0.0076 mmol) were dissolved in anhydrous DMF (0.10 mL). Et3N (2.2 µL, 0.0153 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC. The crude intermediate was dissolved in anhydrous DMF 0.10 mL, followed by addition of PSar20(15.2 mg, 0.0102 mmol) and HATU (2.9 mg, 0.0076 mmol). NMM (1.7 µL ,0.0153 mmol) was added. The reaction mixture was stirred at room temperature for 4-6 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 8.3 mg of DL-22 with 45.0% yield. HRMS (ESI) m / z found [(M+4H) / 4]+, 913.2349 C170H274FN29O574+, required 913.2354.

[0170] Table 23. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-16. Preparation of BCN-Glu(PSar20)-VA-PAB(PSar20)-Exatecan (also termed DL-23)

[0171] Step 1: HL-7 (71.5 mg, 0.083 mmol), Fmoc-Glu(PSar20) (162.1 mg, 0.088 mmol) and HATU (38.2 mg, 0.100 mmol) were dissolved in anhydrous DMF (1.6 mL). NMM (27.5 μL, 0.249 mmol) was added and the reaction mixture was stirred at room temperature for 1-2 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 89.0 mg of Fmoc- Glu(PSar20)-VA-PAB(COOtBu)-Exatecan with 39.7% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1341.6494 C129H179FN28O322+, required 1341.6561.

[0172] Table 24. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0173] Fmoc-Glu(PSar20)-VA-PAB(COOtBu)-Exatecan (40.8 mg, 0.015 mmol) was dissolved in CH2Cl2 / MeOH (0.99 mL, 1 / 1) and then cooled to 0oC. Et2NH (0.24 mL) was added and the reaction mixture was stirred at 0-4oC for 4-5 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC, and azeotroped with toluene (5 mL x 3) to remove excess Et2NH. This crude product was then dissolved in anhydrous CH2Cl2 (0.68 mL) and cooled to 0oC. TFA (0.33 mL) was added dropwise. The reaction mixture was stirred at 0oC overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring ether (15.0 mL) to get precipitate. The solids were collected by filtration and washed with ether (5 mL x 3), and followed by dried with high vacuum to obtain crude HL-9. The crude product was purified with preparative HPLC to obtain 10.8 mg of HL-9 with 29.6% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1202.5877 C110H161FN28O322+, required 1202.5908.

[0174] Table 25. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0175] Step 2: HL-9 (9.5 mg, 0.0040 mmol) and BCN-OSu (1.5 mg, 0.0051 mmol) were dissolved in anhydrous DMF (0.10 mL). Et3N (1.7 µL, 0.0120 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC. The crude intermediate was dissolved in anhydrous DMF 0.10 mL, followed by addition of PSar20 (12.0 mg, 0.0080 mmol) and HATU (1.8 mg, 0.0048 mmol). NMM (1.4 µL ,0.0120 mmol) was added. The reaction mixture was stirred at room temperature for 4-6 hours. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 3.9 mg of DL-23 with 24.9% yield.

[0176] Table 26. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-17. Preparation of BCN-VC-Hydra-PAB-Exatecan (also termed DL-24)

[0177] Step 1: Fmoc-VC-OH (489.3 mg, 0.985 mmol) and PAB(COOtBu)-OH (200.0 mg, 0.895 mmol) were dissolved in anhydrous CH2Cl2(2.0 mL) and anhydrous MeOH (2.0 mL). EEDQ (310.1 mg, 1.254 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (50.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude Fmoc-VC-PAB(COOtBu)-OH (445.1 mg, 71.0% yield). This crude product was used in the next step without further purification.

[0178] Fmoc-VC-PAB(COOtBu) (445.0 mg, 0.634 mmol) and Bis(4-nitrophenyl) carbonate (578.7 mg, 1.902 mmol) were dissolved in DMF (4.5 mL). DIPEA (0.33 mL, 1.902 mmol) was added into the reaction solution. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (50.0 mL) to get precipitate. The solids were collected by filtration and followed with highvacuum drying to obtain crude Fmoc-VC-PAB(COOtBu)-PNP (300.0 mg, 81.0% yield). This crude product was used in the next step without further purification.

[0179] To a suspension of exatecan mesylate (183.9 mg, 0.346 mmol) and Fmoc-VC- PAB(COOtBu)-PNP (300.0 mg, 0.346 mmol) in anhydrous DMF (1.73 mL), DIPEA (0.18 mL, 1.038 mmol) was added at room temperature. The suspension became a clear brown solution within 5 minutes. This mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (20.0 mL) to get precipitate. The solids were collected by filtration and followed with vacuum drying to obtain a crude product. The crude product was purified by a flash silica gel column (CH2Cl2 / MeOH = 12 / 1 to 7 / 1) to obtain 250.0 mg of HL-10 with 62.0% yield.

[0180] Step 2: A stirring suspension of HL-10 (100.0 mg, 0.115 mmol) in DCM (4.0 mL) was cooled to 0 ⁰C. TFA (1.98 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4 ⁰C overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (60 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude Fmoc-VC-PAB(COOH)-Exatecan (64.5 mg, 68% yield). This crude product was used in the next step without further purification.

[0181] Fmoc-VC-PAB(COOH)-Exatecan (64.5 mg, 0.058 mmol), NH2-PEG24-OMe (75.7 mg, 0.070 mmol) and HATU (33.1 mg, 0.087 mmol) were dissolved in anhydrous DMF (0.65 mL). NMM (12.8 µL, 0.116 mmol) was added and the reaction mixture was stirred at room temperature for 1-2 hours. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (10 mL) to get precipitate. The solids were collected by filtration and followed with vacuum drying to obtain a crude product. The crude product was purified with preparative HPLC to obtain 47.8 mg of Fmoc-VC-Hydra-PAB-Exatecan with 37.0% yield.

[0182] Table 27. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0183] Fmoc-VC-Hydra-PAB-Exatecan (47.8 mg, 0.022 mmol) was dissolved in CH2Cl2 / MeOH (1.4 mL, 1 / 1). Et2NH (0.16 mL) was added and the reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (15 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude NH2-VC-Hydra-PAB-Exatecan. This crude product was used in the next step without further purification. The crude product and BCN-OSu (6.4 mg, 0.022 mmol) were dissolved in anhydrous DMF (0.4 mL). DIPEA (11.5 µL, 0.066 mmol) was added. The resulting mixture was stirred at room temperature for 0.5-1 hour. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 21.3 mg of DL-24 with 40.0% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1066.0572 C104H162FN9O362+, required 1066.0553.

[0184] Table 28. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-18. Preparation of BCN-GGVC-Hydra-PAB-Exatecan (also termed DL-25)

[0185] HL-10 (150.0 mg, 0.129 mmol) was dissolved in CH2Cl2 / MeOH (4.0 mL, 1 / 1) and the reaction solution was cooled to 0-4oC. Et2NH (0.45 mL) was added and the reaction mixture was stirred at 0-4oC for three days. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (45 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude NH2-VC-PAB(COOtBu)- Exatecan (82.5 mg) without purification. The crude NH2-VC-PAB(COOtBu)-Exatecan (82.5 mg, 0.088 mmol), Boc-GG-OH (20.4 mg, 0.088 mmol) and HATU (40.1 mg, 0.106 mmol) were dissolved in anhydrous DMF (1.76 mL). NMM (29.1 µL, 0.264 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 66.7 mg of Boc-GGVC-PAB(COOtBu)- Exatecan with 65.9% yield.

[0186] Table 29. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)

[0187] A stirring suspension of Boc-GGVC-PAB(COOtBu)-Exatecan (65.0 mg, 0.056 mmol) in CH2Cl2 (1.1 mL) was cooled to 0 ⁰C. TFA (0.54 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4 ⁰C overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (20 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude NH2-GGVC-PAB(COOH)- Exatecan without further purification. The crude NH2-GGVC-PAB(COOH)-Exatecan and BCN- OSu (16.3 mg, 0.056 mmol) were dissolved in anhydrous DMF (1.1 mL). DIPEA (29.3 µL, 0.168 mmol) was added. The resulting mixture was stirred at room temperature for 0.5-1 hour. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (45 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude BCN-GGVC-PAB(COOH)-Exatecan 18.4 mg without purification. Crude BCN-GGVC-PAB(COOH)-Exatecan (18.4 mg, 0.016 mmol), NH2-PEG24-OMe (34.8 mg, 0.032 mmol) and HATU (7.3 mg, 0.019 mmol) were dissolved in anhydrous DMF (0.32 mL). NMM (5.3 µL, 0.048 mmol) was added and the reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was purified with preparativeHPLC to obtain 17.8 mg of DL-25 with 50.6% yield. HRMS (ESI) m / z found [(M+3H) / 3]+, 749.0508 C108H169FN11O383+, required 749.0459.

[0188] Table 30. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)3-19. Preparation of BCN-GGFG-Hydra-PAB-Exatecan (also termed DL-26)

[0189] Step 1: Boc-GGFG-OH (498.0 mg, 1.141 mmol) and PAB(COOtBu)-OH (212.3 mg, 0.951 mmol) were dissolved in anhydrous CH2Cl2 (4.2 mL) and anhydrous MeOH (4.2 mL). EEDQ (352.7 mg, 1.426 mmol) was added and the resulting mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC. The crude product was purified by a flash silica gel column (CH2Cl2 / MeOH = 12 / 1 to 9 / 1) to obtain 603.5 mg of Boc-GGFG-PAB(COOtBu) with 98.9% yield.

[0190] Boc-GGFG-PAB(COOtBu) (603.5 mg, 0.940 mmol) and Bis(4-nitrophenyl) carbonate (1.43 g, 4.703 mmol) were dissolved in anhydrous CH2Cl2 / DMF (9.4 mL, 4 / 1). DIPEA (0.82 mL, 4.703 mmol) was added into the reaction solution. The reaction mixture was stirred at room temperature for 5-6 hours. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC. The crude product was purified by a flash silica gel column (CH2Cl2 / EA = 3 / 1 to CH2Cl2 / MeOH = 15 / 1) to obtain 350.5 mg of Boc-GGFG-PAB(COOtBu)-PNP with 46.2% yield.

[0191] To a suspension of exatecan mesylate (254.0 mg, 0.478 mmol) and Boc-GGFG- PAB(COOtBu)-PNP (350.5 mg, 0.434 mmol) in anhydrous CH2Cl2 / DMF (4.3 mL, 1 / 1), DIPEA (227.0 µL, 1.303 mmol) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC. The crude product was purified by a flash silica gel column (CH2Cl2 / EA = 1 / 1 to CH2Cl2 / MeOH = 15 / 1) to obtain 221.7 mg of HL-11 with 46.3% yield.

[0192] Step 2: A stirring suspension of HL-11 (100.0 mg, 0.091 mmol) in CH2Cl2(1.7 mL) was cooled to 0oC. TFA (0.83 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4oC overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (25 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude NH2-GGFG-PAB(COOH)-Exatecan without further purification. The crude NH2-GGFG-PAB(COOH)-Exatecan and BCN-OSu (26.5 mg, 0.091 mmol) were dissolved in anhydrous DMF (1.8 mL). DIPEA (47.6 µL, 0.273 mmol) was added. The resulting mixture was stirred at room temperature for 0.5-1 hour. After the reaction was completed, the reaction mixture was added dropwise into a stirring ethyl ether (30 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude BCN- GGFG-PAB(COOH)-Exatecan (65.1 mg) without purification. Crude BCN-GGFG-PAB(COOH)- Exatecan (65.1 mg, 0.058 mmol), NH2-PEG24-OMe (126.1 mg, 0.116 mmol) and HATU (26.4 mg, 0.069 mmol) were dissolved in anhydrous DMF (1.2 mL). NMM (19.2 µL, 0.174 mmol) was added and the reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was purified with preparative HPLC to obtain 52.8 mg of DL-26 with 41.6% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1097.0405 C108H180FN9O372+, required 1097.0449.

[0193] Table 31. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µm, 12 nm)Example 4. Synthesis of linker-payloads with different PEG lengths 4-1. Preparation of BCN-GGVA-Hydra-PAB-PEG24-Exatecan (also termed DL-8), BCN-GGVA- Hydra-PAB-PEG6-Exatecan (also termed DL-10), BCN-GGVA-Hydra-PAB-PEG12-Exatecan (also termed DL-11), and BCN-GGVA-Hydra-PAB-PEG48-Exatecan (also termed DL-12)

[0194] Step 1:A solution of (2R)-hydroxy(4-nitrophenyl)acetic acid (1.0 g, 5.07 mmol) in anhydrous CH2Cl2(25 mL), AcCl (0.54 mL, 7.61 mmol) and pyridine (1.23 mL, 15.22 mmol) were added dropwise sequentially. The resulting mixture was stirred at room temperature for 2-3 hours.0.1N HCl(aq) (50 mL) was added, and the mixture solution was extracted with CH2Cl2(50 ml x 3). The combined organic layers were washed with saturated brine and then dried with MgSO4.The organic solvent was filtered and concentrated in vacuo at 30-35oC. The crude PNB(COOH)-OAc was dried in high vacuum and then used in the next step without further purification.

[0195] Step 2: Anhydrous DCM (11 mL) and anhydrous MgSO4 (1.9 g, 15.74 mmol) were mixed in a reactor and stirred, then H2SO4(0.21 mL, 3.78 mmol) was added. The reaction mixture was stirred at room temperature for 15 minutes. PNB(COOH)-OAc (753.0 mg, 3.14 mmol) and tBuOH (1.49 mL, 17.741 mmol) in anhydrous CH2Cl2 (3 mL) was added into the previous solution at room temperature and then the mixture solution was stirred overnight. After the reaction was completed, it was quenched by adding saturated NaHCO3(aq)(15 mL) slowly. The mixture solution was extracted with CH2Cl2 (15 mL x 3) and the combined organic layers were washed with saturated brine and then dried with MgSO4. The organic solvent was filtered and concentrated in vacuo at 30-35oC. The crude product was purified by a flash silica gel column (Hexanes / EA = 5 / 1) to obtain 790.2 mg of PNB(COOtBu)-OAc with 85% yield.1H NMR (600 MHz, CDCl3) δ 8.23 (dd, 2H, J = 6.6, 1.8 Hz), 7.65 (q, 2H, J = 6.6, 1.8 Hz), 5.89 (s, 1H), 2.21 (s, 3H), 1.38 (s, 9H).

[0196] Step 3:A solution of PNB(COOtBu)-OAc (366.6 mg, 1.24 mmol) in MeOH (24.8 mL) was cooled to 0-4oC. After 5 min, 5.4 M NaOMe in MeOH (22.9 L, 0.12 mmol) was added slowly. The reaction mixture was stirred at 0-4oC for 30-40 minutes. After the reaction was completed, saturated NH4Cl(aq)(30 mL) was added to quench the reaction, and then the mixture solution was extracted with CH2Cl2(20 ml x 3). The combined organic layers were dried with MgSO4, filtered, and then concentrated in vacuo at 30-35oC. The crude product was purified by a flash silica gel column (Hexanes / EA / CH2Cl2= 7 / 1 / 1 to 5 / 1 / 1) to obtain 273.1 mg of PNB(COOtBu)-OH with 87% yield.

[0197] Step 4: To a solution of PNB(COOtBu)-OH (273.1 mg, 1.08 mmol) in MeOH (10.8 mL), 10% Pd / C (41.0 mg, 15 wt%) was added. The reaction was stirred at room temperature with H2balloon overnight. After the reaction was completed, the reaction mixture was passed through celite (3.0 g) and the celite was washed with MeOH (10 mL x 3). The desired fraction was concentrated in vacuo at 30- 35oC. The crude PAB(COOtBu)-OH was dried in high vacuum and then used in the next step without further purification.

[0198] Step 5: PAB(COOtBu)-OH (99.9 mg, 0.45 mmol) and Boc-GGVA-OH (216.2 mg, 0.54 mmol) were dissolved in anhydrous CH2Cl2(2.0 ml) and IPA (2.0 mL). EEDQ (166.1 mg, 0.67 mmol) was added. The resulting mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC. The crude product waspurified by a flash silica gel column (EA / CH2Cl2= 99 / 1 to 97 / 3, then CH2Cl2 / MeOH = 9 / 1) to obtain 198.5 mg of N-DT-0025 with 73% yield.

[0199] Step 6: N-DT-0025 (316.9 mg, 0.52 mmol) and Bis(4-nitrophenyl) carbonate (396.6 mg, 1.30 mmol) were dissolved in anhydrous DMF / CH2Cl2(6.3 mL, v / v = 1 / 9). DIPEA (0.36 mL, 2.09 mmol) was added into the reaction solution. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC. The crude product was purified by a flash silica gel column (EA / CHCl3= 99 / 1, then CH2Cl2 / MeOH = 15 / 1) to obtain 275.3 mg of N-DT-0024 with 68.3% yield.1H NMR (600 MHz, CDCl3) δ 8.34 (d, 2H, J = 9.0 Hz), 7.71 (d, 2H, J = 8.5 Hz), 7.51 (d, 2H, J = 9.0 Hz), 7.45 (d, 2H, J = 8.5 Hz), 5.83 (s, 1H), 4.47 (q, 1H, J = 7.1 Hz), 4.19 (d, 1H, J = 6.6 Hz), 3.97 (d, 1H, J = 16.5 Hz), 3.89 (d, 1H, J = 16.5 Hz), 3.72 (d, 2H, J = 5.3 Hz), 2.19-2.13 (m, 1H), 1.47 (d, 3H, J = 7.2 Hz), 1.44 (s, 9H), 1.42 (s,N-DT-0024 Chemical Formula: C36H48N6O13Molecular Weight: 772.8090

[0200] Step 7: To a suspension of exatecan mesylate (34.0 mg, 0.06 mmol) and N-DT-0024 (49.4 mg, 0.06 mmol) in anhydrous DMF (0.64 mL), DIPEA (33.4 µL, 0.19 mmol) was added at room temperature. The suspension became a clear brown solution within 5 minutes. This mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring TBME (6.4 mL) to get precipitate. The solids were collected by filtration and followed by high-vacuum drying to obtain crude N-PM-0023 (65.4 mg, 95.7% yield). This crude product was used in the next step without further purification.

[0201] Step 8: A stirring suspension of N-PM-0023 (83.4 mg, 0.08 mmol) in DCM (1.40 mL) was cooled to 0 ⁰C. TFA (6.88 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4 ⁰C for 20-24 hours. After the reaction was completed, the reaction mixture was added dropwise into a stirring TBME (20.9 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude N-PM-0024 (73.4 mg, 91.7% yield). This crude product was used in the next step without further purification.

[0202] Step 9: N-PM-0024 (56.9 mg, 0.05 mmol) and BCN-OSu (16.2 mg, 0.05 mmol) were dissolved in anhydrous DMF (1.1 mL). Et3N (23.2 µL, 0.16 mmol) was added into the reaction solution at room temperature. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was added dropwise into a stirring TBME (11 mL) to get precipitate. The solids were collected by filtration and followed by high-vacuum drying to obtain crude N-PM-0025 (60.2 mg, 99.7% yield). This crude product was used in the next step without further purification.

[0203] Step 10: N-PM-0025 (1 eq) and m-PEG-amine with various PEG length (2 eq) and HATU (1.2 eq) were dissolved in anhydrous DMF (0.05 M). NMM (5 eq) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain DL-8 (with PEG24), DL-10 (with PEG6), DL-11 (with PEG12), and DL-12 (with PEG48). DL-8 (BCN-GGVA-Hydra-PAB-PEG24-Exatecan) was obtained with 31.5% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1080.0534 C105H161FN9O372+, required 1079.5488. DL-10 (BCN-GGVA-Hydra-PAB-PEG6-Exatecan) was obtained with 35.2% yield. HRMS (ESI) m / z found [(M+2Na) / 2]+, 705.8000 C69H88FN9O19Na22+, required 705.7988. DL-11 (BCN-GGVA-Hydra-PAB-PEG12-Exatecan) was obtained with 42.7% yield. HRMS (ESI) m / z found [(M+2Na) / 2]+, 837.8782 C81H112FN9O25Na22+, required 837.8774. DL-12 (BCN- GGVA-Hydra-PAB-PEG48-Exatecan) was obtained with 47.1% yield. HRMS (ESI) m / z found [(M+4H) / 4]+, 804.6855 C153H257FN9O614+, required 804.6870.Molecular Weight: 1630.8234 DL-8 (n=24): Chemical Formula: C105H160FN9O37Molecular Weight: 2159.4594 DL-12 (n=48): Chemical Formula:C153H256FN9O61Molecular Weight: 3216.7314

[0204] Table 32. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm)4-2. Preparation of BCN-GGVA-Hydra-PAB-Exatecan (also termed DL-13)

[0205] Steps 1 and 2: N-PM-0018 (41.2 mg, 0.047 mmol), Fmoc-GG-OH (16.8 mg, 0.047 mmol) and HATU (21.6 mg, 0.057 mmol) were dissolved in anhydrous DMF (0.95 mL). NMM (15.7 µL, 0.142 mmol) was added. The reaction was stirred at room temperature for 4-5 hours. After the reaction was completed, CH2Cl2 / MeOH (1.56 mL, v / v = 1 / 1) was added. Et2NH (155.1 µL) was added at room temperature. Then the reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 21.7 mg of GGVA-PAB-Exa with 52.6% yield.Exact Mass: 868.3556 Molecular Weight: 868.9204

[0206] Table 33. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm)

[0207] Step 3: GGVA-PAB-Exa (21.6 mg, 0.025 mmol) and BCN-Osu (7.3 mg, 0.025 mmol) were dissolved in anhydrous DMF (0.5 mL). Et3N (10.4 µL, 0.075 mmol) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 11.7 mg of DL-13 (BCN-GGVA-Hydra-PAB-Exatecan) with 45.0% yield.Molecular Weight: 868.9204Exact Mass: 1044.4393 Molecular Weight: 1045.1354

[0208] Table 34. HPLC condition (Column: YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm)Example 5. ADC preparation and in vitro 3D cytotoxicity assay of ADCs with different PEG lengths 5-1. Preparation of ADCs with different PEG lengths (ADC-1 to ADC-5)

[0209] BCN linker-payload (8-16 eq) was dissolved in propylene glycol / DMSO (1 mg linker- payload / 50 µL propylene glycol / DMSO; 0-50% (v / v) propylene glycol in DMSO) and then added slowly to R4702-(NSCT-di-N3)2 solution (antibody concentration 5 mg / mL in 20 mM NaOAc, pH 5.0), and the mixture was shaken at 25 ^C for 6-24 hours. After the conjugation was completed, the crude ADC was further purified by using Spectrum®Hollow Fiber Filter Modules (buffer: 20 mM NaOAc, pH 5.0) to afford R4702-BCN-ADC. ADC was adjusted to around 10 mg / mL and sterilized by passing through ProMaxTMSyringe Filter (PVDF, 0.22 µm). The DAR value of the ADC is determined by hydrophobic interaction chromatography (HIC).

[0210] FIGs.1A-1E indicated the HIC results of five R4702 (anti-TROP2) ADCs with different PEG lengths. FIG. 1A indicated the DAR distribution of R4702-BCN-PEG48-Exatecan ADC (ADC-1) was 0.09% for DAR0, 5.56% for DAR2, and 92.63% for DAR4. The average DAR was 3.8. FIG. 1B indicated the DAR distribution of R4702-BCN-PEG24-Exatecan ADC (ADC-2) was 0.35% for DAR0, 7.93% for DAR2, and 91.73% for DAR4. The average DAR was 3.8. FIG. 1C indicated the DAR distribution of R4702-BCN-PEG12-Exatecan ADC (ADC-3) was 0.15% for DAR0, 6.54% for DAR2, and 93.31% for DAR4. The average DAR was 3.9. FIG. 1D indicated the DAR distribution of R4702-BCN-PEG6-Exatecan ADC (ADC-4) was 0.25% for DAR0, 8.16%for DAR2, and 91.60% for DAR4. The average DAR was 3.8. FIG. 1E indicated the DAR distribution of R4702-BCN-Exatecan ADC (ADC-5) was 0.24% for DAR0, 5.47% for DAR2, and 94.29% for DAR4. The average DAR was 3.9. 5-2. In vitro 3D cytotoxicity assay of ADCs with different PEG lengths

[0211] Tumor cells (NCI-N87 shVOID: high TROP2 expressing gastric carcinoma; NCI-N87 shTROP2: low TROP2 expressing gastric carcinoma; NCI-H1975: medium TROP2 expressing non-small cell lung carcinoma, NSCLC) were purchased from ATCC and further infected with adenovirus. The cells were seeded at a density of 1-2 × 10² cells per well in 50 μL of complete medium supplemented with 1% Matrigel (Corning, New York, USA) in ultralow attachment 96- well plates (Thermo Scientific, Waltham, Massachusetts, USA). The cells were allowed to form compact 3D aggregates. After six days, the spheroid size was assessed by measuring the perimeter using ImageJ software (NIH, Bethesda, Maryland, USA), with sizes ranging between 200-300 μm².

[0212] Spheroids were then treated with various ADCs at the indicated concentrations. Following an additional six days of incubation, cell viability was measured using the CellTiter- Glo®3D cell viability assay (Promega Corporation, Madison, Wisconsin, USA), and the luminescent signal was recorded according to the manufacturer’s instructions.

[0213] The in vitro therapeutic efficacy of R4702 (anti-TROP2) ADCs were evaluated by 3D cytotoxicity assay (FIGs. 2A-2C). The EC50values observed in NCI-N87 shVOID (high TROP2) were ranging from 14.11 to 23.16 nM (FIG. 2A). The EC50 values in NCI-N87 shTROP2 (low TROP2) were ranging from 104.7 to 193.7 nM (FIG.2B). The EC50 values in NCI-H1975 (medium TROP2) were ranging from 119.4 to 237.6 nM (FIG.2C). These data indicated that the EC50values for the ADCs with different PEG length were similar. Table 35. The EC50 values for the ADCs with different PEG lengths5-3. The payload release efficiency of ADCs with different PEG lengths

[0214] This study was conducted to evaluate how the length of PEG in the linker-payload would affect drug release from ADC. The study was evaluated by digesting ADCs with different PEG lengths (PEG0 to PEG48) using cathepsin B and qualifying the released exatecan (as an example of drug moiety) by LC-UV. 25 µg of ADCs were treated with 10 µg / mL of activated cathepsin B in digestion buffer for exatecan cleavage at 37 °C. The reaction mixture was sampled at 2, 4, 6, 824, 30 and 50 hours after adding the activated cathepsin B into the ADCs. A C18 column (Symmetry 5 ^m, 2.1^150 mm Waters) was applied for this analysis. Samples were eluted with ACN / water gradient at a flow rate of 0.5 mL / minute, and the chromatogram was monitored at 263 nm.

[0215] FIG. 3 indicated the results of exatecan release efficiency of ADCs with different PEG lengths after cathepsin B cleavage. The %released exatecan was derived by the ratio of the released exatecan to theoretical exatecan on DAR4 ADC. As shown in the figure, ADC-5 (no PEG), was completely digested by cathepsin B and released most of exatecan after two hours of digestion (98%). Other ADCs had not reached the highest exatecan release until fifty hours of digestion. The exatecan release efficiency of ADCs, from the highest to the lowest, was 88% for ADC-4 (PEG6), 83% for ADC-3 (PEG12), 78% for ADC-2 (PEG24), and 76% for ADC-1 (PEG48). ADCs with PEG24or PEG48linker-payloads had similar payload release efficiency. The results demonstrated that the length of PEG may affect its ability to protect linker-payloads from destruction. Since cathepsin B cleavage might mimic the in-vivo circumstance, the results suggested that ADCs with longer PEG moieties would enhance stability of ADCs in blood circulation of a subject. Example 6. In-vivo efficacy comparison of ADCs with different PEG lengths

[0216] 6.1 Test substances and dosing patterns (a) ADC-5: PEG0(9.8 mg / mL) (b) ADC-4: PEG6 (8.33 mg / mL) (c) ADC-3: PEG12 (10.0 mg / mL) (d) ADC-2: PEG24(9.9 mg / mL) (e) ADC-1: PEG48(9.9 mg / mL) Table 36. Study Design and sampling

[0217] 6.2 Cell line: NCI-H1975 cells (TROP2-expressing human non-small cell lung cancer cells) (ATCC, CRL-5908)

[0218] 6.3 Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at the start of the study: 15-25 g (g) Animal grouping: The mice were divided into six groups and each group contained five mice. A total of thirty-six mice were involved in the study.

[0219] 6.4 Equipment and Materials (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0220] 6.5 Methods (a) Establishment of a xenograft mouse model Subcutaneous inoculation of tumor cells: 5x106NCI-H1975 cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test articles: The first dosing day was denoted as Day 1 when average tumor volume reaches 150-200 mm3. All test articles (test item 1 to 5) or reference items (sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage of 3 mg / kg, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group, respectively, at the end of the study (Day 23), whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group, respectively, at the beginning of test item administration (Day 1).(d) Statistical analysis Results were presented as mean and standard error of the mean (mean±SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0221] 6.6 Results

[0222] FIG. 4 indicated the tumor volume of the vehicle group was 1457^210.36 mm3on Day 23. Until Day 34, the tumor volume of the ADC-5 (PEG0) group was 1167.48^188.75 mm3. The tumor volume of the ADC-4 (PEG6) group was 1121.61^61.41 mm3. The tumor volume of the ADC-3 (PEG12) group was 794.48^35.46 mm3. The tumor volume of the ADC-2 (PEG24) group was 543.43^173.22 mm3. The tumor volume of the ADC-1 (PEG48) group was 698.70^69.64 mm3. The results demonstrated that PEG could enhance tumor suppression efficacy of ADCs. Furthermore, the maximum tumor inhibition efficacy was observed in the ADC with PEG24. Example 7. The digestion efficiency, payload release efficiency and in-vitro cytotoxicity assay of different linker-payloads

[0223] This study was conducted to evaluate how the DBCO and BCN in the linker-payloads would affect drug release. The study also included the linker-payloads with the two-amino acid spacer “GG” in the digestion comparison. The study was evaluated by digesting the linker- payloads using cathepsin B and quantifying the released exatecan by LC-UV. To perform exatecan release test by cathepsin B cleavage, 5 µg of the linker-payloads were treated with 10 µg / mL of activated cathepsin B in digestion buffer for payload cleavage at 37 °C. The reaction mixture was sampled every two hours for 24 hours, and the amounts of released exatecan and residual linker- payload were analyzed by LC-UV. A C18 column, Symmetry 5um, 2.1^150 mm Waters, was applied for this analysis. Samples were eluted with ACN / water gradient at a flow rate of 0.5 mL / min, and the chromatogram was monitored at 263 nm.

[0224] FIG.5A indicated the digestion efficiency of different linker-payloads. The calculated percentage of linker-payloads existed (% linker-payload) after cathepsin B cleavage reaction were 75% for DBCO-VA-Hydra-PAB-PEG24-Exatecan (DL-5), 66% for DBCO-GGVA-Hydra-PAB- PEG24-Exatecan (DL-7), 14% for BCN-VA-Hydra-PAB-PEG24-Exatecan (DL-6), and 4% for BCN-GGVA-Hydra-PAB-PEG24-Exatecan (DL-8). The results demonstrated that the linker- payload with BCN had better digestion efficiency. Furthermore, FIG. 5B indicated the exatecan release efficiency of different linker-payloads. The uppermost amount of released exatecan (71.1%) was observed in BCN-VA-Hydra-PAB-PEG24-Exatecan (DL-6), followed by 52.6% for BCN- GGVA-Hydra-PAB-PEG24-Exatecan (DL-8), 15.9% for DBCO-GGVA-Hydra-PAB-PEG24- Exatecan (DL-7), and 10.8% for DBCO-GGVA-Hydra-PAB-PEG24-Exatecan (DL-5). Bothresults demonstrated the BCN group in linker-payloads has better payload release efficiency than the DBCO group.

[0225] Cytotoxicity was determined using CellTiter-Glo™ luminescent assay reagent (Promega). Human SKBR-3 breast cancer cells (ATCC, HTB-30) were used in this study. The cells were plated in 96-well plates with 8E+03 cells per well and incubated at 37 °C overnight. Different linker-payloads (DL-5 to DL-8) were prepared from 3000 to 0.5 nM with three-fold serial dilution in cell culture medium (McCoy’s 5A medium contain 10% FBS) and then added to the plates. After incubated at 37 °C for six days, the CellTiter-Glo luciferase assay reagent was added to each well. The plate was placed on an orbital shaker for two minutes to induce cell lysis and then incubated at room temperature for ten minutes before recording the luminescent signals by Luminometer. Luminescence was determined using a microplate luminometer SpectraMax L (Molecular Devices, Sunnyvale, CA). The percentage of the cytotoxicity was calculated by dividing the non-treated cell luminescence minus experimental cell luminescence by the non- treated cell luminescence and multiplying by 100. IC50was determined by plotting x (concentration in nM) – y (drug cytotoxicity in %) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software. FIG. 6 indicated the IC50 of DL-5 (DBCO-VA-Hydra-PAB-PEG24- Exatecan) was 243.5 nM, the IC50of DL-6 (BCN-VA-Hydra-PAB-PEG24-Exatecan) was 221.4 nM, the IC50of DL-8 (BCN-GGVA-Hydra-PAB-PEG24-Exatecan) was 137.7 nM, and the IC50of DL-7 (DBCO-GGVA-Hydra-PAB-PEG24-Exatecan) was 96.7 nM. The IC50 data for the different linker-payloads were similar. Example 8. Synthesis of single-arm saccharide derivatives and oxazoline-hexasaccharide-N3 8-1. Scheme of tetra-saccharide formation

[0226] A mixture of donor G-C1-0039 (639 mg, 0.967 mmol, 1.2 equiv., manufactured by OBI Pharma, Inc.), acceptor compound 1 (669 mg, 0.820 mmol, 1.0 equiv.), and activated 4 Å molecular sieves (1.93 g) in anhydrous CH2Cl2(32 mL) was stirred at room temperature under an N2 atmosphere for 30 minutes. It was then cooled to -78 °C, and NIS (256 mg, 1.178 mmol, 1.44 equiv.) was added. After stirring at -78 °C for 10 minutes, TfOH (16 μL, 0.164 mmol, 0.2 equiv.) was added, and the reaction mixture was gradually warmed up to -50 °C. After stirring for four hours, the mixture was quenched with triethylamine (50 μL, 0.68 mmol, 0.8 equiv.). The mixture was filtered and concentrated in vacuo. The residue was diluted with CH2Cl2(50 mL) and washed with saturated NaHCO3(aq.)(30 mL), finally saturated NaS2O3(aq.)(30 mL). Organic layer was dried over anhydrous MgSO4 and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound 2 (951 mg, 86%) as white solid. TLC (EtOAc / hexanes= 30%, v / v,CAM, anisaldehyde and UV) Rf= 0.5; HRMS (ESI) m / z calculated for C81H81N3NaO16[M+Na]+1374.5509; found 1374.5454.

[0227] Compound 2 (429 mg, 0.317 mmol, 1.0 equiv.) was dissolved in a solution MeOH / CH2Cl2(12 / 6, 2 / 1 = v / v) in a round bottom flask followed by addition of NaOMe (137 mg, 2.536 mmol, 8.0 equiv.). The mixture was stirred at room temperature for eighteen hours. The reaction mixture was diluted with CH2Cl2 (50 mL) and washed with 1N HCl(aq.) (40 mL) twice. Organic layer was dried over anhydrous MgSO4, filtered, and concentrated by evaporation. The concentrated crude was purified by flash column chromatography (EtOAc / hexanes = 10% →20% →30%, v / v) to afford compound 3 (369 mg, 93%) as white solid. TLC (EtOAc / hexanes = 30%, v / v, CAM, anisaldehyde and UV) Rf = 0.3.

[0228] A mixture of donor G-C1-0040 (436 mg, 0.596 mmol, 1.2 equiv., manufactured by OBI Pharma, Inc.), acceptor compound 1 (620 mg, 0.496 mmol, 1.0 equiv.), and activated 4 Å molecular sieves (1.2 g) in anhydrous CH2Cl2 (20 mL) was stirred at room temperature under an N2 atmosphere for 30 minutes. It was then cooled to -78 °C, and NIS (161 mg, 0.715 mmol, 1.44 equiv.) was added. After stirring at -78 °C for 10 minutes, TfOH (8.8 μL, 0.099 mmol, 0.2 equiv.) was added, and the reaction mixture was gradually warmed up to -50 °C. After stirring for three hours, the mixture was quenched with triethylamine (20 μL, 0.15 mmol, 0.3 equiv.). The mixture was filtered and concentrated in vacuo. The residue was diluted with CH2Cl2(40 mL) and washed with saturated NaHCO3(aq.)(20 mL), and then saturated NaS2O3(aq.)(20 mL). Organic layer wasdried over anhydrous MgSO4, filtered, and concentrated by evaporation. The concentrated crude was purified by MPLC to afford compound 4 (813 mg, 88%) as white solid. TLC (EtOAc / hexanes= 30%, v / v, CAM, anisaldehyde and UV) Rf = 0.5; HRMS (ESI) m / z calculated for C104H107Cl3N4Na2O21[M+2Na]+2949.3139; found 949.3201.

[0229] Compound 4 (669 mg, 0.360 mmol, 1.0 equiv.) was dissolved in a solution AcO2 (682 μL, 7.214 mmol, 20 equiv.), AcOH (682 μL), and THF (7 mL) in a round bottom flask followed by addition of Zn powder (1340 mg). The mixture was stirred at room temperature for twenty hours. Zn powder was removed by celite and concentrated by evaporation. The residue was purified by flash column chromatography (EtOAc / hexanes= 40% →45% →50%, v / v) to afford compound 5 (479 mg, 76%) as white solid. TLC (EtOAc / hexanes= 50%, v / v, CAM, anisaldehyde and UV) Rf= 0.3; HRMS (ESI) m / z calculated for C105H112N2Na2O21[M+2Na]+2891.3771; found 891.3785.

[0230] Compound 5 (218 mg, 0.125 mmol) was dissolved in a solution MeOH / H2O (12 mL, 2 / 1= v / v) in a round bottom flask followed by addition of Pd(OH)2 / C (400 mg). The flask was sealed and stirred under a H2 atmosphere at room temperature for twenty hours. Pd(OH)2 / C was removed by celite and concentrated by evaporation. The residue was purified by G-10 column chromatography to afford compound 6 (90 mg, 96%) as white solid. TLC (Butanol / Acetic acid / H2O = 5 / 3 / 2, v / v, anisaldehyde and CAM) Rf= 0.3; HRMS (ESI) m / z calculated for C28H49N2O21 [M+H]+749.2822; found 749.2840.

[0231] To a solution of Compound 6 (13 mg, 17.4 µmol) in water were successively added Et3N (109 µL, 783 µmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 87 µL of a 1M solution) at 0oC. The mixture was stirred at 0oC for two hours. After this time HPLC-UV analysis indicated the completion of reaction. The mixture was then subjected to gel filtration chromatography on a Sephadex G-10 column. The product was eluted with an aqueous solution of NaOH (0.01 M). Pure fractions containing the product were combined and lyophilized to afford compound 7 as a white foam (9 mg, 71%).1H NMR (D2O, 600 MHz) δ 6.09 (d, 1H, J= 7.3 Hz), 5.12 (s, 1H), 4.73 (s, 1H), 4.55 (d, 1H, J= 8.4 Hz), 4.39-4.36 (m, 1H), 4.21-4.17 (m, 2H), 4.14 (d, 1H, J= 2.9 Hz), 3.97-3.88 (m, 4H), 3.80-3.59 (m, 10H), 3.57-3.48 (m, 2H), 3.46-3.38 (m, 4H), 2.07 (d, 3H, J= 1.1 Hz), 2.05 (s, 3H).8-2. Scheme of hexa-saccharide formation

[0232] To a 1 mL solution containing Compound 6 (1.5 mg, 2 µmol), ATP (11 µg, 0.01 µmol), UTP (27 µg, 0.025 µmol), galactose (0.72 mg, 2 µmol), phosphoenolpyruvate (1.82 mg, 4.4 µmol, monopotassium salt), and MgCl2(final 10 mM) were added the enzymes galactokinase (GalK, 0.02 U), UDP-sugar pyrophosphorylase (AtUSP, 0.06 U), α1,4-galactosyltransferase (Sigma- Aldrich, 0.75 U), pyruvate kinase (PK, 0.08 U), and inorganic pyrophosphatase (PPA, 0.08 U). The final volume of the mixture was adjusted to 1 mL, and the reaction was carried out at room temperature over the course of twenty-four hours with the pH controlled at 8.6. The reaction was monitored by thin layer chromatography (5:3:2 butanol / acetate / water). To the corresponding penta-saccharide mixture (11 mg, 15 µmol), Neu5Ac (0.42 mg, 15 µmol), ATP (0.82 µg, 0.17 µmol), CTP (0.04 µg, 1.7 µmol), phosphoenolpyruvate (PEP, 0.31 mg, 10 µmol, monopotassium salt), cytidine monophosphate kinase (CMK, 0.167 U), CMP-sialic acid synthetases (CSS, 0.4 U) and α-2,6-sialyltransferase (SHIZ-145, 0.5 U) were added. The reaction was carried out at 37 ^Cfor six hours with the pH controlled at pH 9.0 solution of Tris buffer. The glycosylation reaction was monitored by TLC until completion. The reaction was stopped by the treatment of MeOH, and precipitated protein was removed by centrifugation at 20,000g for five minutes. The supernatant was concentrated and purified by silica gel column chromatography (25% H2O in ACN) to give Compound 8 as a white solid (14.3 mg, 81% over two steps).1H NMR (D2O, 600 MHz) δ 5.21 (d, 0.6H, J= 3.2 Hz), 5.15 (s, 1H), 4.79 (1H, overlapped with solvent signal), 4.72 (d, 0.4H, J= 7.7 Hz), 4.61 (d, 1H, J= 7.6 Hz), 4.45 (s, 1H, J= 7.9 Hz), 4.25 (dd, 1H, J= 7.1, 3.2 Hz), 4.22-4.19 (m, 1H), 4.04-3.45 (m, 35H), 2.67 (dd, 1H, J= 12.4, 4.7 Hz), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.72 (pt, 1H, J= 12.4 Hz). HRMS calculated for [C45H75N3O34+Na]+1224.4130, found 1224.4096.

[0233] Following the same procedure as described for the preparation of Compound 7, Compound 9 was obtained as a white foam (9.8 mg, 98%) from Compound 8.1H NMR (D2O, 600 MHz) δ 6.09 (d, 1H, J= 7.3 Hz), 5.14 (s, 1H), 4.74 (s, 1H), 4.60 (d, 1H, J= 8.0 Hz), 4.44 (d, 1H, J= 7.9 Hz), 4.38 (dd, 1H, J= 3.0, 1.7 Hz), 4.22-4.17 (m, 2H), 4.15 (d, 1H, J= 3.1 Hz), 4.03-3.49 (m, 31H), 3.46-3.39 (m, 2H), 2.67 (dd, 1H, J= 12.4, 4.7 Hz), 2.07 (s, 6H), 2.03 (s, 3H), 1.72 (pt, 1H, J= 12.4 Hz).8-3. Preparation of oxazoline-hexasaccharide-N3

[0234] Step 1. Synthesis of hexasaccharide-N3 (Compound 9)

[0235] To a solution of Compound 8 (23.6 mg, 19.6 µmol) and H2N-PEG3-N3(12.9 mg, 58.9 µmol) in DMSO (270 µL) were successively added DIPEA (20.6 µL, 118 µmol) and benzotriazol- 1-yl-oxy-tripyrrolidino-phosphonium hexafluorophosphate (PyBOP, 61.3 mg, 118 µmol). The resulting mixture was stirred at room temperature for 4 hours and quenched with water. The residue was washed by CH2Cl2, concentrated, and purified by reverse phase silica gel column chromatography (15% MeOH in water, C18 silica gel) to afford hexasaccharide-N3 (Compound 9) as a white fluffy solid (20.6 mg, 75%).1H NMR (D2O, 600 MHz) δ 5.21 (d, 0.6H, J = 3.1 Hz), 5.15 (s, 1H), 4.79 (1H, overlapped with solvent signal), 4.72 (m, 0.4H), 4.60 (d, 1H, J = 7.3 Hz), 4.45 (d, 1H, J = 7.9 Hz), 4.25 (dd, 1H, J = 7.1, 3.2 Hz), 4.22 – 4.19 (m, 1H), 4.02 – 3.45 (m, 51H), 2.71 (dd, 1H, J = 12.8, 4.3 Hz), 2.07 (s, 3H), 2.04 (s, 6H), 1.85 (pt, 1H, J = 12.3 Hz). HRMS calcd for [C53H91N7O36+2Na]2+723.7645, found 723.7669.

[0236] Step 2. Synthesis of oxazoline-hexasaccharide-N3 (Compound 10)

[0237] To a solution of Compound 9 (12.5 mg, 8.9 µmol) in water (60 µL) were successively added Et3N (56 µL, 402 µmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 44.5 µL of a 1M solution, 44.5 µmol) at 0oC. The mixture was stirred at 0oC for 4 hours. After this time HPLC-UV analysis indicated the completion of reaction. The mixture was thensubjected to gel filtration chromatography on a Sephadex G-10 column. The product was eluted with an aqueous solution of NaOH (0.01 M). Pure fractions containing the product were combined and lyophilized to afford oxazoline-hexasaccharide-N3 (Compound 10) as a white fluffy solid (12.2 mg, quantitative yield).1H NMR (D2O, 600 MHz) δ 6.09 (d, 1H, J = 7.3 Hz), 5.13 (s, 1H), 4.73 (s, 1H), 4.59 (d, 1H, J = 8.0 Hz), 4.44 (d, 1H, J = 7.9 Hz), 4.38 – 4.35 (m, 1H), 4.21 – 4.17 (m, 2H), 4.14 (d, 1H, J = 2.9 Hz), 4.00 – 3.38 (m, 49H), 2.70 (dd, 1H, J = 12.9, 4.6 Hz), 2.07 (d, 3H, J = 1.7 Hz), 2.06 (s, 3H), 2.04 (s, 3H), 1.83 (pt, 1H, J = 12.9 Hz).Example 9. DAR2 ADC (R4702-hexasaccharide-N3 conjugated with BCN-GGVA-Hydra- PAB-PEG24-Exatecan) preparation

[0238] 9-1. Generation of R4702-(hexasaccharide-N3)2by glycan remodeling

[0239] R4702 (Anti-TROP2 mAb) firstly underwent deglycosylation by treated with EndoSz- D234M (enzyme / mAb ratio of 1 / 60, w / w) and Endo H (enzyme / mAb ratio of 1 / 5000, w / w) in 100 mM MOPS buffer (pH 7.0) at 37 °C for 72 hours. The resulting R4702-GlcNAc(Fuc) was incubated with ten equivalents of Compound 10 at 25oC for 2.5 hours. Additional ten equivalents of oxazoline were added, and the mixture was further incubated at 15oC for 2.5 hours. The residue was applied to a protein A column, followed by incubation with activated carbon to give pure R4702-(hexasaccharide-N3)2.

[0240] 9-2. Bioconjugation of R4702-(hexasaccharide-N3)2 with BCN-GGVA-Hydra-PAB- PEG24-Exatecan

[0241] To a solution of R4702-(hexasaccharide-N3)2in 20 mM NaOAc buffer (pH 5.0), four equivalents of BCN-GGVA-Hydra-PAB-PEG24-Exatecan (DL-1) dissolved in DMSO were added at 25 ^C. This mixture was stirred under 25 ^C for 16 hours. After the reaction was completed, the crude DAR2 ADC was purified by UF / DF with a 30 kDa cut-off cassette. The concentration of final R4702-DAR2-ADC was adjusted to 10 mg / mL in 20 mM NaOAc buffer (pH 5.0), and sterilized by passing through Millex-GP Syringe Filter (PES, 0.22 um). The drug-to-antibody ratio (DAR) value of final ADC is determined by hydrophobic interaction chromatography (HIC). Example 10. The payload release efficiency and in-vitro cytotoxicity assay of ADCs with different bioorthogonal groups

[0242] This study was to evaluate the effect of different bioorthogonal groups (BCN or DBCO groups) of DAR4 ADCs on payload release. The study was conducted by digesting R4702-BCN- GGVA-Hydra-PAB-PEG24-Exatecan (also termed OBI-902) or R4702-DBCO-GGVA-Hydra- PAB-PEG24-Exatecan using cathepsin B and monitoring the released exatecan (an example of drug moiety) by LC-UV. 25 µg of ADCs were treated with 10 µg / mL of activated cathepsin B in digestion buffer for exatecan cleavage at 37 °C. The reaction mixture was sampled at 2, 4, 6, 8, 18, 24, 48 and 57 hours after adding the activated cathepsin B into ADCs. A Cortecs C18 column (2.7 ^m, 3.0^150 mm) was used for this analysis. Samples were eluted with ACN / water gradient at a flow rate of 0.3 mL / min, and the chromatogram was monitored at 263 nm.

[0243] FIG. 7 indicated the results of exatecan release efficiency of ADCs after cathepsin B cleavage. The %released exatecan was derived by the ratio of the released exatecan to theoretical exatecan on ADCs (BCN: 87% and DBCO: 68%). The results demonstrated the bioorthogonal group of ADCs would affect the payload digestion efficiency and releasing.

[0244] Cytotoxicity was determined using CellTiter-Glo™ Luminescent assay reagent (Promega). Human BxPC-3 pancreatic adenocarcinoma cancer cells (ATCC, CRL-1687) were used in this study. The cells were plated in 96-well plates with 8E+03 cells per well and incubated at 37 °C overnight. Different bioorthogonal groups of ADCs were prepared from 30 to 0.005 nM with three-fold serial dilution in cell culture medium (McCoy’s 5A medium contain 10% FBS) and then added to the plates. After incubated at 37 °C for six days, the CellTiter-Glo luciferase assay reagent was added to each well. The plate was placed on an orbital shaker for two minutes to induce cell lysis and then incubated at room temperature for ten minutes before recording the luminescent signals by Luminometer. Luminescence was determined using a microplate luminometer SpectraMax L (Molecular Devices, Sunnyvale, CA). The percentage of the cytotoxicity was calculated by dividing the non-treated cell luminescence minus experimental cell luminescence by the non-treated cell luminescence and multiplying by 100. IC50was determinedby plotting x (concentration in nM) – y (drug cytotoxicity in %) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software. FIG. 8 indicated the IC50 of R4702-DBCO- GGVA-Hydra-PAB-PEG24-Exatecan was 0.23 nM, and the IC50 of OBI-902 (R4702-BCN- GGVA-Hydra-PAB-PEG24-Exatecan) was 0.28 nM. The IC50data for the ADCs with different bioorthogonal groups were similar. Example 11. In-vivo anti-tumor efficacy comparison between OBI-902 (R4702-BCN-GGVA- Hydra-PAB-PEG24-Exatecan) and datopotamab deruxtecan (Dato-Dxd)

[0245] 11-1. Measurement of anti-tumor activity in NCI-N87 human gastric cancer cell-derived xenograft in BLAB / c nude mice

[0246] 11-1-1. Test substances and dosing pattern (a) OBI-902: 3 mg / mL (b) Dato-Dxd: 3 mg / mL Table 37. Study Design and sampling

[0247] 11-1-2. Cell line: NCI-N87 (High TROP2 expressed cancer cell)

[0248] 11-1-3. Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal grouping: The mice were divided into 3 groups and each group contained 5 mice. A total of 15 mice were involved in study of NCI-N87 CDX model.

[0249] 11-1-4. Equipment and material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0250] 11-1-5. Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 2.5x106NCI-N87 cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, Cat. No.354248). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article The first dosing day was denoted as Day 1 when average tumor volume reaches 200-250 mm3in NCI-N87 CDX model. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 3 mg / kg for NCI-N87 CDX model, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 57). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (d) Statistical analysis Results were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t- test. p < 0.05 is considered significance.

[0251] 11-1-6. Result FIG. 9A indicated the in-vivo efficacy result in NCI-N87 CDX model. The mean^SEM of tumor volume to vehicle group was 1292.48^315.89 mm3on Day 26. Mean^SEM of tumor volume to treated groups were 240.48^164.37 to OBI-902 and 944.0^482.01 to Dato-Dxd on Day 57. It demonstrated OBI-902 had better inhibition efficacy than Dato-DxD in NCI-N87 gastric cancer.

[0252] 11-2. Measurement of anti-tumor activity in DLD-1 human colorectal cancer cell- derived xenograft in BLAB / c nude mice

[0253] 11-2-1. Test substances and dosing pattern (a) OBI-902: 10 mg / mL (b) Dato-Dxd: 10 mg / mL Table 38. Study Design and sampling

[0254] 11-2-2. Cell line: DLD-1 (Middle TROP2 expressed cancer cell)

[0255] 11-2-3. Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal grouping: The mice were divided into 3 groups and each group contained 5 mice. A total of 15 mice were involved in study of DLD-1 CDX model.

[0256] 11-2-4. Equipment and material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0257] 11-2-5. Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 5x106DLD-1 cells were mixed with the equal volume of matrigel (volume ratio 1:1). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article The first dosing day was denoted as Day 1 when average tumor volume reaches 250-300 mm3in DLD-1 CDX model. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 10 mg / kg for DLD-1 CDX model, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 56). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (d) Statistical analysisResults were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t- test. p < 0.05 is considered significance.

[0258] 11-2-6. Result

[0259] FIG. 9B indicated the in-vivo efficacy result in DLD-1 CRC CDX model. The mean^SEM of tumor volume to vehicle group was 1038.67^309.98 mm3on Day 11. Mean^SEM of tumor volume to treated groups were 329.35^302.27 to OBI-902 on Day 56 and 980.90^605.56 to Dato-Dxd on Day 18. It demonstrated OBI-902 had better inhibition efficacy than Dato-DxD in DLD-1 colorectal cancer.

[0260] 11-3. Measurement of anti-tumor activity in HPAC human PDAC (pancreatic ductal adenocarcinoma) cell-derived xenograft in BLAB / c nude mice

[0261] 11-3-1. Test substances and dosing pattern (a) OBI-902: 5 mg / mL (b) Dato-Dxd: 5 mg / mL Table 39. Study Design and sampling

[0262] 11-3-2. Cell line: HPAC (Middle TROP2 expressed cancer cell)

[0263] 11-3-3. Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal grouping: The mice were divided into 3 groups and each group contained 5 mice. A total of 15 mice were involved in study of HPAC CDX model.

[0264] 11-3-4. Equipment and material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line)(d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0265] 11-3-5. Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 3x106HPAC cells were mixed with the equal volume of matrigel (volume ratio 1:1). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article The first dosing day was denoted as Day 1 when average tumor volume reaches 150-200 mm3in HPAC CDX model. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 5 mg / kg for HPAC CDX model, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 43). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (d) Statistical analysis Results were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t- test. p < 0.05 is considered significance.

[0266] 11-3-6. Result

[0267] FIG. 9C indicated the in-vivo efficacy result in HPAC PDAC CDX model. The mean^SEM of tumor volume to vehicle group was 1085.60^586.09 mm3on Day 24. Mean^SEM of tumor volume to treated groups were 426.13^336.02 to OBI-902 and 1064.30^504.94 to Dato- Dxd on Day 43. It demonstrated OBI-902 had better inhibition efficacy than Dato-DxD in HPAC PDAC cancer.

[0268] 11-4. Measurement of anti-tumor activity in TFK-1 human cholangiocarcinoma cell- derived xenograft in BLAB / c nude mice

[0269] 11-4-1. Test substances and dosing pattern (a) OBI-902: 6 mg / mL (b) Dato-Dxd: 6 mg / mL Table 40. Study Design and sampling

[0270] 11-4-2. Cell line: TFK-1 (High TROP2 expressed cancer cell)

[0271] 11-4-3. Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal grouping: The mice were divided into 3 groups and each group contained 5 mice. A total of 15 mice were involved in study of TFK-1 CDX model.

[0272] 11-4-4. Equipment and material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0273] 11-4-5. Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 1x107TFK-1 cells were mixed with the equal volume of matrigel (volume ratio 1:1). Subcutaneous injection volume was 200 ^L / mouse. (b) Route and administration of test article The first dosing day was denoted as Day 1 when average tumor volume reaches 150-200 mm3in TFK-1 CDX model. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 6 mg / kg for TFK-1 CDX model, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 62). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).(d) Statistical analysis Results were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t- test. p < 0.05 is considered significance.

[0274] 11-4-6. Result

[0275] FIG.9D indicated the in-vivo efficacy result in TFK-1 cholangiocarcinoma CDX model. The mean^SEM of tumor volume to vehicle group was 577.37^232.58 mm3on Day 44. Mean^SEM of tumor volume to treated groups were complete regression (0 mm3) to OBI-902 and 254.01^317.99 to Dato-Dxd on Day 62. It demonstrated OBI-902 had better inhibition efficacy than Dato-DxD in TFK-1 cholangiocarcinoma cancer. Example 12. Formulation study OBI-902 (R4702-BCN-GGVA-Hydra-PAB-PEG24-Exatecan)

[0276] 12-1. pH value and buffer screening

[0277] 12-1-1. Material and method

[0278] We tried to evaluate the stability indicators by stress conditions and get the suitable pH and buffer for OBI-902. Six buffers were tested in this study. It included 20 mM sodium acetate with pH 4.0 and 5.0; 20 mM sodium citrate pH 5.0 and 6.0; 20 mM Histidine buffer with pH 5.5 and 20 mM sodium succinate with pH 5.0. The target OBI-902 concentration was 10 mg / mL. Two stress conditions were conducted in this study: thermal stress at 50 ±3°C for five days, and mechanical stress condition with agitation (uu mode, 50 rpm) for four hours. OBI-902 stored at 5±3°C was used as the control samples in this study. Took 1 mL of OBI-902 DS and exchanged the buffer by 30 kDa Amicon Ultra-0.5 into the desired formulation for three times. Adjusted OBI- 902 concentration and filtrated. Aliquoted 0.5mL into each 1.5 mL eppendorf and placed at each specified condition. (a) Buffer condition 1. 20 mM sodium acetate pH 4.0+10 mg / mL OBI-902 2. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-902 3. 20 mM sodium citrate pH 5.0+10 mg / mL OBI-902 4. 20 mM sodium citrate pH 6.0+10 mg OBI-902 5. 20 mM Histidine pH 5.5+10 mg / mL OBI-902 6. 20 mM sodium succinate pH 5.0+10 mg / mL OBI-902 (b) Stress condition 1. Control: 5±3 °C 2. Thermal stress condition: 50±3 °C for five days 3. Agitation: uu mode, 50 rpm, four hours(c) Test item 1. Protein concentration by NanoDrop. (A280 nm=1.59, EC=1.59 mg-1mLcm-1) 2. Drug load distribution and DAR by HIC-UV 3. Aggregation by SEC-UV 4. Free drugs by HPLC-FLD

[0279] 12-1-2. Study result

[0280] The results for this study were shown in Table 41. There was no significant difference for OBI-902 in six buffer conditions at 5±3 °C. The appearance of all the samples at stress conditions was still clear. OBI-902 after agitation for four hours were still unchanged except that in sodium citrate buffer. Protein concentration of OBI-902 in sodium citrate with pH 5.0 decreased from 10 mg / mL to 4.0 mg / mL and %HMWS increased to 3.54% for sodium citrate with pH 6.0 after agitation.

[0281] OBI-902 changed significantly after thermal stress incubation. The aggregation formation was observed after thermal stress especially in sodium acetate pH 4.0 and sodium succinate pH 5.0. The percentage %HMWS of OBI-902 in 20 mM sodium acetate pH 4.0 exceed 45% after incubation at 50 °C for five days. The drug load distribution (DLD) of OBI-902 also changed and unexpected peaks were observed behind the DAR4 peak. Only sodium acetate pH 5.0, sodium citrate pH 5.0 and histidine buffer pH 5.5 could maintain the DLD profile after thermal incubation and make average DAR keep at 3.86. The worst condition was sodium acetate with pH 4.0.

[0282] Free drug (exatecan) increased after thermal incubation for all the buffer system. It indicated OBI-902 in 20 mM sodium acetate pH 5.0 had the lowest free drug (exatecan) amount, 67.8 ng / mL. No significant different on Free drug amount of linker-payload related substance in OBI-902 after thermal incubation. Table 41. Summary table of pH and buffer screening

[0283] 12-1-3. Summary

[0284] In the test results, the most suitable buffer for OBI-902 was 20 mM sodium acetate with pH 5.0. It prevented serious aggregation and free drug releasing after thermal stress. Low pH buffer condition with pH ≤ 4.0 should be avoided.

[0285] 12-2. Buffer concentration screening

[0286] 12-2-1. Material and method

[0287] After sodium acetate was selected for OBI-902 formulation, the buffer concentration, pH range and OBI-902 concentration were further investigated. We designed four different formulations, including variance of buffer concentration from 20 mM to 100 mM, pH range from 5.0 to 5.5 and protein concentration from 10 to 20 mg / mL to evaluate buffer concentration. Two stress conditions were conducted in this study including freeze-thaw (-80±5 °C to 25±3 °C) for ten cycles and thermal stress condition (40 ±5 °C for four weeks). OBI-902 stored at 5±3 °C was used as the control samples in this study. (a) Buffer condition 1. 50 mM sodium acetate pH 5.0+10 mg / mL OBI-902 2. 100 mM sodium acetate pH 5.0+10 mg / mL OBI-902 3. 20 mM sodium acetate pH 5.0+20 mg / mL OBI-902 4. 20 mM sodium acetate pH 5.5+10 mg / mL OBI-902 (b) Stress condition 1. Control: 5±3 °C 2. Thermal stress condition: 40±3 °C for two weeks 3. Freeze thaw ten cycles (c) Test item 1. Protein concentration by NanoDrop. (A280 nm=1.59, EC=1.59 mg-1mLcm-1) 2. Drug load distribution and DAR by HIC-UV 3. Aggregation by SEC-UV 4. Free drugs by HPLC-FLD

[0288] 12-2-2. Study result

[0289] The results for this study were shown in Table 42. There was no obvious change of OBI- 902 observed after freeze-thaw ten cycles. OBI-902 showed slightly change in SEC-UV and free drug after thermal degradation at 40 °C for four weeks. The increase of aggregation was observed after thermal stress. In the groups with 10 mg / mL of OBI-902, 20 mM sodium acetate with pH 5.5 presented the lowest %HMWS, 1.05%. OBI-902 in 100 mM sodium acetate with pH 5.0 has thehighest %HMWS, 2.11%. Furthermore, 20 mg / mL of OBI-902 has higher %HWMS (1.45%) than that of 10 mg / mL OBI-902. Low molecular weight species (%LHWS) were also detected after thermal incubation after two weeks. Based on SEC-UV results, 10 mg / mL of OBI-902 in 20 mM sodium acetate with pH 5.5 is the best condition for OBI-902.

[0290] Free exatecan increased after thermal stress for all four samples.20 mM sodium acetate pH5.5, OBI-902 has lowest free drug (exatecan) amount, 174.8 ng / mL. No significant different on free drug of linker-payload related substances in OBI-902 after thermal stress. Table 42. Summary table of buffer concentration screening F

[0291] 12-2-3. Summary

[0292] Based on the test results, the protein concentration of OBI-902 should be within 10 mg / mL to reduce the risk of aggregation. Furthermore, high concentration of acetate buffer led to higher %HMWS formation. Therefore, 20 mM sodium acetate was suitable as the buffer concentration. Combining the results from pH and buffer screening study, the pH range of 20 mM sodium acetate could be within pH 5.0 to pH 5.5.

[0293] 12-3. Excipient screening

[0294] 12-3-1. Material and method

[0295] In this study, different excipients with the function of protein stabilizer and cryoprotectant were studied to enhance stability OBI-902 in liquid or frozen solution. Sucrose, sodium chloride and polysorbate 80 were tested. Two stress conditions were conducted to this study: one is thermal stress (40 ±5°C for four weeks), and another is Freeze-thaw (-80±5°C to 25±3°C) for ten cycles. OBI-902 stored at 5±3°C was used as the control samples in this study. (a) Buffer and excipient condition 1. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-9022. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-902+50 mM NaCl 3. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-902+100 mM NaCl 4. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-902+100 mM Sucrose 5. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-902+200 mM Sucrose 6. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-902+0.02% (w / v) Polysorbate 80 7. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-902+0.05% (w / v) Polysorbate 80 8. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-902+50 mM NaCl+100 mM Sucrouse+0.02% (w / v) Polysorbate 80 9.20 mM sodium acetate pH 5.0+10 mg / mL OBI-902+50 mM NaCl+0.02% (w / v) Polysorbate 80 10. 20 mM sodium acetate pH 5.0+10 mg / mL OBI-902+100 mM Sucrouse+0.02% (w / v) Polysorbate 80 (b) Stress condition 1. Control: 5±3 °C 2. Thermal stress condition: 40±3 °C for four weeks 3. Freeze thaw ten cycles (c) Test item 1. Protein concentration by NanoDrop. (A280 nm=1.59, EC=1.59 mg-1mLcm-1) 2. Drug load distribution and DAR by HIC-UV 3. Aggregation by SEC-UV 4. Free drugs by HPLC-FLD

[0296] 12-3-2. Study result

[0297] The results for this study were shown in Table 43. There was no obvious change of OBI- 902 after freeze-thaw ten cycles. OBI-902 was stable in all ten formulations and could stand freeze -thaw cycle up to ten. OBI-902 in thermal stress showed slightly changed on DLD by HIC-UV and aggregation and fragments by SEC. The %DAR0 ratios of Sample 1, 2, 3, 6, 7, and 9 increased to around 1% after two weeks of treatment, and some over 1% after four weeks. %DAR0 ratio of Sample 4, 5, 8 and 10 were kept at around 0.7% after treatment. Samples 4, 5, 8 and 10 are formulated in sucrose contained sodium acetate buffer. Sample 2, 3, and 9 had a %HMWS greater than 1% after 2 weeks incubation. After four weeks, the %HMWS of Sample 3 even exceed 1.5 and %HMWS of Sample 8 exceed 1%. The increase of %LMWS was also observed after thermal treatment. %LMWS of samples 1, 3, 6, and 9 were exceed 1% after two weeks incubation. After four weeks of thermal treatment, almost all the samples had a %LMWS of 5% or even higher, except for Sample 5, whose %LMWS remained around 2%. OBI-902 formulated in buffer containing sodium chloride had the increase of %HMWS and %LMWS after thermal treatment.Comparing the results of Samples 1, 6, and 7, Polysorbate 80 did not appear to be effective in preventing aggregation or fragmentation of OBI-902. Sample 5, OBI-902 formulated in 20 mM sodium acetate pH 5.0 with 200 mM sucrose showed the best test results than others. Table 43. Summary table of excipient screening

[0298] 12-3-3. Summary

[0299] Sodium chloride and polysorbate 80 also did not contribute benefit to the stability of OBI-902. Especially, sodium chloride has negative effect, particularly on aggregation (increase of %HMWS). Regarding to the thermal stress testing results, sucrose contained formulations could alleviate the increase of %DAR0, %HMWS and %LMWS, helping to stabilize OBI-902. It showed that sucrose was an importance excipient for OBI-902 and required in OBI-902 formulation. Furthermore, the higher the sucrose concentration, the better the outcome occurred. The results of the 200 mM sucrose formulation were better than those of the 100 mM sucrose formulation. Example 13. In-vivo anti-tumor efficacy comparison between sacituzumab_gADC (sacituzumab-BCN-GGVA-Hydra-PAB-PEG24-Exatecan) and datopotamab deruxtecan (Dato-Dxd)

[0300] 13-1. Sacituzumab_gADC preparation

[0301] BCN linker-payload (8-16 eq) was dissolved in propylene glycol / DMSO (1 mg linker- payload / 50 µL propylene glycol / DMSO; 0-50% (v / v) propylene glycol in DMSO) and then added slowly to sacituzumab-(NSCT-di-N3)2 solution (antibody concentration 5 mg / mL in 20 mM NaOAc, pH 5.0), and the mixture was shaken at 25 ^C for 6-24 hours. After the conjugation was completed, the crude ADC was further purified by using Spectrum®Hollow Fiber Filter Modules (buffer: 20 mM NaOAc, pH 5.0) to afford sacituzumab_gADC. ADC was adjusted to around 10 mg / mL and sterilized by passing through ProMaxTMSyringe Filter (PVDF, 0.22 µm). The DAR value of the ADC is determined by hydrophobic interaction chromatography (HIC). FIG. 10A indicated the HIC result of sacituzumab_gADC. It indicated the DAR distribution was 0.51% for DAR0, 4.65% for DAR2, and 94.84% for DAR4. The average DAR was 3.89.

[0302] 13-2. Measurement of anti-tumor activity in NCI-H2170-Enhertu^-resistant human non-small cell lung cancer (NSCLC) cell-derived xenograft in BLAB / c nude mice

[0303] 13-2-1. Test substances and dosing pattern (a) Sacituzumab_gADC: 3 mg / mL (b) Dato-Dxd: 3 mg / mL Table 44. Study Design and sampling

[0304] 13-2-2. Cell line: NCI-H2170-Enhertu^-resistant cells (Middle TROP2 expressed)

[0305] 13-2-3. Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal grouping: The mice were divided into 4 groups and each group contained 6 mice. A total of 24 mice were involved in the study.

[0306] 13-2-4. Equipment (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V)

[0307] 13-2-5. Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 5x106NCI-H2170-Enhertu^-resistant cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, Cat. No.354248). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article The first dosing day was denoted as Day 1 when average tumor volume reaches 200-250 mm3. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 3 mg / kg and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 28). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (d) Statistical analysisResults were presented as mean and standard error of the mean (mean±SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0308] 13-2-6. Result FIG. 10B indicated the in-vivo efficacy result in NCI-H2170-Enhertu^-resistant CDX model. The mean^SEM of tumor volume to vehicle group was 1724.51^465.60 mm3on Day 24. Mean^SEM of tumor volume to treated groups were 322.91^292.98 mm3to sacituzumab_gADC on Day 28 and 1590.14^562.62 mm3to Dato-Dxd on Day 24. It demonstrated sacituzumab_gADC also had better inhibition efficacy than Dato-DxD.

[0309] 13-3. Measurement of anti-tumor activity in HPAC human pancreatic adenocarcinoma cell-derived xenograft in BLAB / c nude mice

[0310] 13-3-1. Test substances and dosing pattern (a) Sacituzumab_gADC: 5 mg / mL (b) Dato-Dxd: 5 mg / mL Table 45. Study Design and sampling

[0311] 13-3-2. Cell line: HPAC cells (Middle TROP2 expressed)

[0312] 13-3-3. Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal grouping: The mice were divided into 4 groups and each group contained 6 mice. A total of 24 mice were involved in the study.

[0313] 13-3-4. Equipment (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002)(c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V)

[0314] 13-3-5. Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 3x106HPAC cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, Cat. No.354248). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article The first dosing day was denoted as Day 1 when average tumor volume reaches 200-250 mm3. All test articles (test item a and b) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 5 mg / kg and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 43). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (d) Statistical analysis Results were presented as mean and standard error of the mean (mean±SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0315] 13-3-6. Result FIG. 10C indicated the in-vivo efficacy result in HPAC CDX model. The mean^SEM of tumor volume to vehicle group was 1085.60^586.09 mm3on Day 24. Mean^SEM of tumor volume to treated groups were 331.79^424.25 mm3to sacituzumab_gADC and 861.47^490.82 mm3to Dato- Dxd on Day 43. It demonstrated sacituzumab_gADC also had better inhibition efficacy than Dato- DxD.

[0316] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

[0317] All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

Claims

Claims 1. An antibody-drug conjugate (ADC) having a structure of Formula (I): Ab-(DL)n (I); wherein: (a) Ab is an antibody or an antigen-binding fragment thereof capable of binding to TROP2; (b) DL is a linker-payload of Formula (III) or (IV) that forms a covalent linkage with the antibody or the antigen-binding fragment thereof:wherein: C is a bioorthogonal group for conjugation to the antibody or the antigen-binding fragment thereof; QCLis a cleavable unit, wherein the cleavable unit comprises a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety; QSPis a spacer unit including an aromatic group or amino methylene; LPis a connector unit that covalently connects QCL, C and E, wherein the connector unit includes one or more amino acids; BP is a branch unit comprising a functional group defined as, whereinA is an aromatic group; R3is linked to E and is selected from -C(O)-, -C(O)O-, -C(O)NH-, alkyl-O-, alkyl-NH-, alkyl-C(O)-, alkyl-C(O)-O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-SO2-, alkyl-S-, alkyl-O-P(O)-O2-, alkyl-O- C(O)-NH-, or triazole; E is a hydrophilic moiety comprising polyethylene glycol (PEG), polysarcosine (pSar), poly lactic- co-glycolic acid (PLGA), poly(glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2- hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), saccharides, or any combination thereof; P is a payload, wherein the payload is a drug moiety; and LBis a bridge unit; and (c) n is a drug-to-antibody ratio (DAR) ranging from 2 to 32.The ADC of claim 1, wherein the hydrophilic moiety E has a formula of:, wherein the wavy line indicates the site of covalent attachment to the branch unit BPor the connector unit LP, R1 is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or, R2is H, SO3H, PO3H2, a sugar derivative, C1-C10(hetero) alkyl group, C3-C10(hetero) cycloalkyl group, C2-C10 alkyl-NH2, C1-C10 alkyl-COOH, C2-C10 alkyl-NH(C1-C3 alkyl), C2-C10 alkyl-N (C1- C3 alkyl)2, or sarcosines; and n is an integer ranging from 2 to 72. The ADC of claim 1, wherein the bioorthogonal group C is selected from a dibenzocyclooctyne (DBCO) group, a bicyclononyne (BCN) group, a alkyne group, a maleimide group, a ^,^- unsaturated carbonyl group, a sulfonyl pyrimidine group, a 4-dibenzocyclooctynol (DIBO) group, a aza-dibenzocyclooctynes (DIBAC) group, a tetrazine group, a tetrazole group, a norbornene group, a cyclooctyne group, a methylcyclopropene group, an aminooxy group, a hydrazine group, an isocyanide group, an isocyanopropanoate group, a phosphine-containing thioester group, a phosphine phenolic ester group, or an alpha-halo carbonyl group. The ADC of claim 1, wherein the payload P is selected from a chemotherapeutic agent, a toxin, a cytokine, a growth inhibitory agent, a protein degrader, a peptide, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, or an immunoregulatory agent. The ADC of claim 4, wherein the toxin is selected from pyrrolobenzodiazepine compounds or derivatives thereof, auristatin compounds or derivatives thereof, maytansinoid compounds or derivatives thereof, duocarmycin or derivatives thereof, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors or derivatives thereof, tubulysin compounds or derivatives thereof, enediyne compounds or derivatives thereof, anthracycline compounds or derivatives thereof, pyrrole-based kinesin spindle protein (KSP) inhibitors or derivatives thereof, cryptophycin compounds or derivatives thereof, drug efflux pump inhibitors or derivatives thereof, sandramycin or derivatives thereof, amanitin compounds or derivatives thereof, or camptothecin compounds or derivatives thereof.The ADC of claim 1, wherein the linker-payload DL has a structure of the following formula:, wherein z is an integer ranging from 6 to 48. The ADC of claim 1, wherein the linker-payload DL has a structure of the following formula:wherein z is an integer ranging from 6 to 48.^The ADC of claim 1, wherein the linker-payload DL has a structure of any one of the following formulas:

9. An antibody-drug conjugate (ADC) having a structure of Formula (IX):(IX); wherein: Ab is a glycoengineered antibody or an antigen-binding fragment thereof capable of binding to TROP2; G represents a glycan moiety connected to an Neu5Ac and the glycoengineered antibody or the antigen-binding fragment thereof, wherein the glycan moiety is a linear or branched chain of saccharides selected from the group consisting of galactose, N-acetyl-glucosamine, glucose, mannose, fucose, and derivatives thereof; Y is a connector moiety independently comprising polyethylene glycol (PEG), polysarcosine (pSar), poly lactic-co-glycolic acid (PLGA), poly(glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N- dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), or any combinations thereof; Z is independently selected from a triazole or imine linkage; L is a linker connecting D and Z; D is a drug moiety; m is a number of Neu5Ac units branching from G, and is an integer from 1 to 4; and p is a number of Y-Z-L-D units attached to each of the Neu5Ac units, and is an integer from 1 to 4.

10. The ADC of claim 9, wherein the glycoengineered antibody is selected from hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, R4702, datopotamb, or sacituzumab.

11. The ADC of claim 9, wherein the drug moiety is selected from MMAE, T785, camptothecin, SN- 38, belotecan, exatecan, or deruxtecan.

12. An antibody-drug conjugate (ADC) having a structure of Formula (X):(X); wherein Ab is a glycoengineered antibody capable of binding to TROP2; L is a linker connecting exatecan and the glycoengineered antibody; and a, b are independently 0 or 1.

13. A pharmaceutical composition comprising the ADC according to any one of claims 1-12 and a pharmaceutically acceptable carrier.

14. A method for treating a cancer, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 13.

15. The method of claim 14, wherein the cancer is a TROP2 expressing cancer.

16. The method of claim 14, wherein the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

17. A liquid formulation comprising the ADC according to any one of claims 1-12.

18. The liquid formulation of claim 17, further comprising a buffer solution, wherein the buffer solution comprises a buffer and / or an excipient.

19. The liquid formulation of claim 18, wherein the buffer is selected from histidine, sodium citrate buffer, sodium acetate buffer or sodium succinate buffer.

20. The liquid formulation of claim 18, wherein the excipient is selected from sucrose, sodium chloride, or polysorbate 80.

21. The liquid formulation of claim 19, wherein the buffer has a pH value range from pH 4.0 to 6.

0.

22. The liquid formulation of claim 19, wherein the buffer has a concentration range from 10 mM to 500 mM.

23. The liquid formulation of claim 20, wherein the sucrose has a concentration range from 10 mM to 500 mM.

24. The liquid formulation of claim 20, wherein the sodium chloride has a concentration range from 10 mM to 500 mM.

25. The liquid formulation of claim 20, wherein the polysorbate 80 has a concentration range from 0.01% (w / v) to 0.1% (w / v).

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