Immunoconjugates comprising camptothecin analogs and methods of use thereof

Abstract

To provide an antibody-drug conjugates (ADCs) comprising an antibody and antitumor compound connected by a linker, wherein the linker and the antitumor compound are represented by the formula of -Q-L1-L2-L3-L4-(NH-Exa). The ADC are useful as therapeutics, in particular in the treatment of cancer.

Description

IMMUNOCONJUGATES COMPRISING CAMPTOTHECIN ANALOGS AND METHODS OF USE THEREOF SEQUENCE LISTING

[0001] The content of the following submission of sequence list is incorporated herein by reference in its entirety: ADC SEQLIST.xml, date recorded: 2023.12.25, size: 29 KB) FIELD

[0002] This application pertains to an antibody-drug conjugate (ADC) having an antitumor compound conjugated via a linker with a specific structure, and methods of manufacture and uses thereof, including methods of treating cancer diseases. BACKGROUND

[0003] Antibody Drug Conjugate (ADC) as a novel targeting drug, it generally consists of three moieties, an antibody or antibody-like ligand, one or more cytotoxic drug, and a linker that couples the drug to the antibody.

[0004] ADC allows for targeted delivery of a cytotoxic drug, particularly to cancer cells, which can reduce side-effects commonly associated with chemotherapy. The antibody portion of the ADC, which binds to an antigen expressed on the surface of a cancer cell and is capable of inducing cellular internalization, can deliver the drug selectively to cancer cells and is thus expected to cause accumulation of the drug within cancer cells and to kill the cancer cells. The drug molecule is generally conjugated through a cleavable or non-cleavable linker that attaches to amino acid residues (e.g., cysteine or lysine) on the antibody molecule.

[0005] The drug to antibody ration (DAR) of the ADC is dependent, in part, on the number of moieties within the antibody to which the drug can be linked and the hydrophobicity of the drug. Most ADCs having an average DAR between 1 and 8. Higher DAR allows for more potent ADC for use as therapeutics.

[0006] Topoisomerase I (Topo I) inhibitors represent the most recent breakthrough in ADC payload innovation with the approval of two ADCs containing camptothecin (CPT) analogues: Trastuzumab deruxtecan (DS-8201a—Enhertu®) and sacituzumab govitecan (IMMU-132— Trodelvy®). Despite the breakthrough, DXd and SN-38 ADCs face intrinsic and acquired resistance due to tumor heterogeneity and multidrug resistance (MDR) regulation (Garcia-Alonso S, Ocana A, Pandiella A. Resistance to antibody-drug conjugates. Cancer Res 2018;78:2159–65; Collins DM et al., Acquired resistance to antibody-drug conjugates. Cancers (Basel) 2019;11:394). Exatecan, a camptothecin and the precursor of DXd, has attracted initial attention due to its higherTOP1 inhibition potency (Joto N et al., DX-8951f, a water-soluble camptothecin analog, exhibits potent antitumor activity against a human lung cancer cell line and its SN-38-resistant variant. Int J Cancer 1997;72:680-6; Jo U et al., TOP1-DNA trapping by exatecan and combination therapy with ATR inhibitor. Mol Cancer Ther 2022;21:1090–102.), lower sensitivity to ABCG2 or P-gp (van Hattum AH et al., Induction of breast cancer resistance protein by the camptothecin derivative DX-8951f is associated with minor reduction of antitumor activity. Br J Cancer 2002;87:665-72), and higher permeability and bystander penetration (Khera E, Dong S, Huang H, de Bever L, van Delft FL, Thurber GM. Cellular-resolution imaging of bystander payload tissue penetration from antibody-drug conjugates. Mol Cancer Ther 2022;21:310–21).

[0007] Exatecan is the most potent drug and induced TOP1ccs at a lower concentration than Dxd and SN-38. However, exatecan is too hydrophobic to be conjugated directly to antibodies (Ogitani Y et al., Wide application of a novel topoisomerase I inhibitor-based drug conjugation technology. Bioorg Med Chem Lett 2016; 26:5069-72). Though this active agent presents promising potential but its use as an ADC payload has been limited because of its hydrophobicity and challenging biophysical properties, which appear to be strongly caused by the steric hindrance around the stereo-defined primary amine (Nakada, T et al., Novel Antibody Drug Conjugates Containing Exatecan Derivative-Based Cytotoxic Payloads. Bioorg. Med. Chem. Lett.2016, 26, 1542-1545).

[0008] Most recently, exatecan was also considered as a payload for antibody-drug conjugates and various linker formats were screened, as for example reported by Nakada et al., Bioorg. Med. Chem. Lett. 2016, 26, 1542-1545, incorporated by reference. However, it was observed that exatecan-based ADCs showed extensive aggregation, up to 26%.

[0009] It is essential and urgent to explore the discovery of a more potent antibody-drug conjugate in treating cancer disease.

[0010] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety. BRIEF SUMMARY

[0011] In one aspect, the present application provides an antibody-drug conjugate comprising antibody and an antitumor compound connected by a linker, wherein the linker and the antitumor compound are represented by the formula of -Q-L1-L2-L3-L4-(NH-Exa), wherein，

[0012] Q is an attachment entity that conjugates to the antibody,

[0013] L1 represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-, or a single bond, and n1 is an integer of 2 to 24, optionally, n1 is an integer of 4 to 12,

[0014] L2 represents a peptide residue that is cleavable by cathepsin B,

[0015] L3represents a self-immolative spacer unit that may release the toxin without a separate hydrolysis step,

[0016] L4represents a natural or unnatural amino acid residue, wherein the natural or unnatural amino acid residue is connected to -(NH-Exa) by an amide bond formed between the amine residue of -(NH-Exa) and the carboxylic residue of the natural or unnatural amino acid residue, and

[0017] -(NH-Exa) is a group represented by the following formula: amino group at position 1 is the connecting site.some conjugates to cysteine residues of the antibody and is derived from maleimidoacetic, maleimidopropionic, maleimidobutanoic, maleimidopentanoic, maleimidocaproic, or maleimido-methyl-cyclohexanecarboxylic.

[0021] In some embodiments, wherein Q represents -(Succinimid-3-yl-N)-(CH2)n2-C(═O)-, wherein n2 represents an integer of 2 to 8, and -(Succinimid-3-yl-N)-has a structure represented by the following formula:

[0022]

[0023] which is connected to the antibody at position 3 thereof and is connected to a methylene group on the nitrogen atom at position 1.

[0024] In some embodiments, wherein Q represents -(Succinimid-3-yl-N)-(CH2)2-C(═O)- or - (Succinimid-3-yl-N)-(CH2)5-C(═O)-.

[0025] In some embodiments, wherein L2 is a dipeptide or tripeptide residue. In some preferred embodiments, L2is -Val-Cit-, -Val-Ala-,or -Phe-Lys-.

[0026] In some embodiments, wherein L3is -NH-phenyl-CH2-O-C(═O)- that has the following formula: .

[0027] L4 is a natural α- amino acid residue, unnatural β-amino acid residue, or unnatural D-amino acid residue. In some more preferred embodiments, L4 is a glycine residue, alanine residue, β-alanine residue, or D-alanine residue.

[0028] In some preferred embodiments, the present application provides an antibody-drug conjugate comprising an antibody and an antitumor compound connected by a linker, wherein the linker and the antitumor compound are represented by one of the following formulas:

[0029] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa),

[0030] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)- Val-Ala-NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa),

[0031] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa),

[0032] or

[0033] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)-Val-Ala-NH- phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa),

[0034] Wherein,

[0035] -(Succinimid-3-yl-N)- represents the following formula:

[0036]

[0037] which is connected to the antibody at position 3 thereof and is connected to a methylene group on the nitrogen atom at position 1,

[0038] -(NH-Exa) represents the following formula:

[0039] e amino group at position 1 is the connecting position, and

[0000] - -p eny -C 2-O-C(═O)- represents the following formula: . the antibody targets a tumor cell, optionally the antibodyis selected from an anti-HER2 antibody, an anti-TROP2 antibody or an anti-FRα antibody.

[0045] In some preferred embodiments, wherein the anti-HER2 antibody is selected from Trastuzumab, Disitamab, Pertuzumab, Zanidatamab or Hertuzumab, the anti-TROP2 antibody is selected from Sacituzumab or Datopotamab, or the anti-FRα antibody is selected from Luveltamab, Farletuzumab or Mirvetuximab.

[0046] In another aspect, the present application provides a linker-toxin intermediate compound represented by the following formula: Q’-L1-L2-L3-L4-(NH-Exa), which can be linked to an antibody.

[0047] In the linker toxin intermediate, wherein Q’ represents an attachment entity that can conjugate with amino acid residues on the antibody. Q’ corresponds to Q in that upon conjugation, Q’ is turned into Q.

[0048] L1represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-, or a single bond, and n1 is an integer of 2 to 24, optionally, n1 is an integer of 4 to 12,

[0049] L2represents a peptide residue that is cleavable by cathepsin B,

[0050] L3 represents a self-immolative spacer unit that may release the toxin without a separate hydrolysis step,

[0051] L4 represents a natural or unnatural amino acid residue, wherein the natural or unnatural amino acid residue is connected to -(NH-Exa) by an amide bond formed between the amine residue of -(NH-Exa) and the carboxylic residue of the natural or unnatural amino acid residue, and

[0052] -(NH-Exa) is a group represented by the following formula:e amino group at position 1 is the connecting site.

[0055] In some embodiments, wherein Q’ can conjugate to cysteine residues of the antibody.

[0056] In some preferred embodiments, wherein Q’ comprises (maleimido-N-yl)- group having the following formula:

[0057]

[0058] atom is a connecting position.

[0059] In some preferred embodiments, wherein the Q’ is derived from the one selected from the group consisting of maleimidoacetic, maleimidopropionic, maleimidobutanoic, maleimidopentanoic, maleimidocaproic, and maleimido-methyl-cyclohexanecarboxylic.

[0060] In some more preferred embodiments, wherein Q’ is (maleimido-N-yl)-(CH2)n2-C(═O)-, and n2 is a integer of 2 to 5.

[0061] In an even more preferred embodiment, wherein Q’ is (maleimido-N-yl)-CH2CH2- C(═O)- or (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-.

[0062] In some embodiments, wherein L1represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-, and n1 is an integer of 2 to 24, optionally, n1 is an integer of 4 to 12.

[0063] In some embodiments, wherein L2is a dipeptide or tripeptide residue. In some preferred embodiments, L2 is -Val-Cit-, -Val-Ala-, or -Phe-Lys-.

[0064] In some embodiments, wherein L3is -NH-phenyl-CH2-O-C(═O)- that has the following formula:ments, L4is a natural α- amino acid residue, unnatural β-amino ac d res due, or unna ura -am no acid residue. In some more preferred embodiments, L4is a glycine residue, alanine residue, β-alanine residue, or D-alanine residue.

[0067] In some preferred embodiments, the present application provides a linker-toxin intermediate compound, which has one of the following structures:

[0068] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa),

[0069] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)- Val-Ala-NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa),

[0070] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa), or

[0071] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)-Val-Ala-NH- phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa),

[0072] wherein (maleimido-N-yl)- represents the following formula:

[0073]

[0074] atom is the connecting position,

[0075] -(NH-Exa) represents the following formula:

[0076]

[0078] wherein the nitrogen atom of the amino group at position 1 is the connecting position, and

[0079] -NH-phenyl-CH2-O-C(═O)- represents the following formula: .compositions, kits and articles of manufacture comprising any one of the antibody-drug conjugates described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG.1 shows the in vitro stability results of several linker-toxin compounds in PBS buffer at 37 °C.

[0083] FIG. 2 shows the in vitro stability results of several linker-toxin compounds in human plasma at 37 °C.

[0084] FIG.3A and FIG.3B show the in vitro stability results of several ADCs in human plasma at 37 °C.

[0085] FIG.4 and FIG.5 show the in vitro stability results of several ADCs in human serum and cynomolgus monkey serum, respectively.

[0086] FIG.6A and FIG.6B show the antibody internalization results in JIMT-1 cell line.

[0087] FIG. 7A and 7B show the in vivo results of several ADCs in anti-tumor efficacy test in mice tumor model with NCI-N87 cancer cells. FIG. 7A shows tumor volume changes with time for each group. FIG.7B displays mice body weight changes profiles.

[0088] FIG. 8A and 8B show the in vivo results of several ADCs in anti-tumor efficacy test in mice tumor model with MDA-MB-468 cancer cells. FIG. 8A shows tumor volume changes with time for each group. FIG.8B displays mice body weight changes profiles.

[0089] FIG. 9A and 9B show the in vivo results of several ADCs in anti-tumor efficacy test in mice tumor model with HCC1806 cancer cells. FIG. 9A shows tumor volume changes with time for each group. FIG.9B displays mice body weight changes profiles. DETAILED DESCRIPTION

[0090] The present application provides an antibody-drug conjugate that comprise an antitumor compound or toxin linked to an antibody via a linker moiety, and will be described in detail below.Definitions

[0091] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival. Also encompassed by "treatment" is a reduction of pathological consequence of the disease (such as, for example, tumor volume for cancer). The methods of the application contemplate any one or more of these aspects of treatment.

[0092] The term "antibody" includes full-length antibodies and antigen-binding fragments thereof. A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0093] The antibody of the present invention may be any antibody capable of targeting tumor cells. That is, after a drug having an antitumor activity is linked via a linker, the antibody preferably has one or more of a property capable of recognizing a tumor cell, a property capable of bindingto a tumor cell, a property capable of internalizing into a tumor cell, and a property of damaging a tumor cell.

[0094] The term "antigen-binding fragment" as used herein refers to an antibody fragment including, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragments that bind to an antigen but do not comprise a complete antibody structure. An antigen-binding fragment also includes a fusion protein that comprises the antibody fragment described above. An antigen- binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0095] The term "epitope" as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen. In some embodiments, the epitope of the antibody is preferably localized to the extracellular domain of the target.

[0096] As used herein, a first antibody "competes" for binding to a same target with a second antibody when the first antibody inhibits same target binding of the second antibody by at least about 50% (such as at least about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolar concentration of the first antibody, or vice versa. A high throughput process for "binning" antibodies based upon their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0097] As used herein, the term "specifically binds", "specifically recognizing", or "is specific for" refers to measurable and reproducible interactions, such as binding between a target and an antibody that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically recognizes a target (which can be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or with greater duration than its bindings to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with a binding affinity that is at least about 10 times its binding affinity for other targets.

[0098] An "isolated" antibody as used herein refers to an antibody that (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, (3) is expressed by a cell from a different species, or, (4) does not occur in nature.

[0099] The term "isolated nucleic acid" as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated nucleic acid" (1) is not associated with all or a portion of a polynucleotide in which the "isolated nucleic acid" is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

[0100] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem.252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol.262:732- 745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entireties for use in the present application and for possible inclusion in one or more claims herein. In the present application, CDRs numbering follows the nomenclature of Kabat. TABLE 1: CDR DEFINITIONS Kabat1Chothia2MacCallum3IMGT4AHo5VL CDR2 50-56 50-52 46-55 56-65 58-77 VL CDR3 89-97 91-96 89-96 105-117 109-1373Residue numbering follows the nomenclature of MacCallum et al., supra 4Residue numbering follows the nomenclature of Lefranc et al., supra 5Residue numbering follows the nomenclature of Honegger and Plückthun, supra

[0101] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a biological activity of this application (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0102] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and 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.

[0103] "Single-chain Fv", also abbreviated as "sFv" or "scFv", are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VHand VLdomains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

[0104] The term "diabodies" refers to small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) typically with short linkers (such as about 5 to about 10 residues) between the VHand VLdomains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0105] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can 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 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 also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

[0106] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skilled in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0107] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR of this application is one that binds to an IgG antibody (a γ receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptorsinclude FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review M. in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). The term includes allotypes, such as FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and / or FcγRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)).

[0108] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an α-chain noncovalently bound to β2-microglobulin. The multiple functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays a role in the passive delivery of immunoglobulin IgGs from mother to young and the regulation of serum IgG levels. FcRn can act as a salvage receptor, binding and transporting pinocytosed IgGs in intact form both within and across cells, and rescuing them from a default degradative pathway.

[0109] The "CH1 domain" of a human IgG Fc region usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0110] "Hinge region" is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol.22:161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions.

[0111] The "CH2 domain" of a human IgG Fc region usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol.22:161-206 (1985).

[0112] The "CH3 domain" comprises the stretch of residues of a C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).

[0113] A "functional Fc fragment" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art.

[0114] An antibody with a variant IgG Fc with "altered" FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity (e.g., FcγR or FcRn) and / or ADCC activity compared to a parent polypeptide or to a polypeptide comprising a native sequence Fc region. The variant Fc which "exhibits increased binding" to an FcR binds at least one FcR with higher affinity (e.g., lower apparent Kd or IC50 value) than the parent polypeptide or a native sequence IgG Fc. According to some embodiments, the improvement in binding compared to a parent polypeptide is about 3 fold, such as about any of 5, 10, 25, 50, 60, 100, 150, 200, or up to 500 fold, or about 25% to 1000% improvement in binding. The polypeptide variant which "exhibits decreased binding" to an FcR, binds at least one FcR with lower affinity (e.g., higher apparent Kd or IC50value) than a parent polypeptide. The decrease in binding compared to a parent polypeptide may be about 40% or more decrease in binding.

[0115] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0116] The polypeptide comprising a variant Fc region which "exhibits increased ADCC" or mediates ADCC in the presence of human effector cells more effectively than a polypeptide having wild type IgG Fc or a parent polypeptide is one which in vitro or in vivo is substantially more effective at mediating ADCC, when the amounts of polypeptide with variant Fc region and the polypeptide with wild type Fc region (or the parent polypeptide) in the assay are essentially thesame. Generally, such variants will be identified using any in vitro ADCC assay known in the art, such as assays or methods for determining ADCC activity, e.g., in an animal model, etc. In some embodiments, the variant is from about 5 fold to about 100 fold, e.g. from about 25 to about 50 fold, more effective at mediating ADCC than the wild type Fc (or parent polypeptide).

[0117] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capability are described in US patent No. 6,194,551B1 and WO99 / 51642. The contents of those patent publications are specifically incorporated herein by reference. See also, Idusogie et al. J. Immunol.164: 4178-4184 (2000).

[0118] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0119] The term "operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0120] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0121] An "effective amount" of an antibody, an ADC or composition as disclosed herein, is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and by known methods relating to the stated purpose.

[0122] The term "therapeutically effective amount" refers to an amount of an antibody, an ADC or composition as disclosed herein, effective to "treat" a disease or disorder in an individual. In the case of cancer, the therapeutically effective amount of an antibody, an ADC or composition as disclosed herein can reduce the number of cancer cells; reduce the tumor size or weight; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the antibody, ADC or composition as disclosed herein can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In some embodiments, the therapeutically effective amount is an amount that extends the survival of a patient. In some embodiments, the therapeutically effective amount is an amount that improves progression free survival of a patient.

[0123] As used herein, by "pharmaceutically acceptable" or "pharmacologically compatible" is meant a material that is not biological or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0124] The anti-tumor compounds (also referred to “toxins” herein) disclosed in this application may comprise one or more asymmetric centers, and different diastereomers and / or enantiomers may exist of the compounds. The description of any compound in this application is meant to include all diastereomers, and mixtures thereof, unless stated otherwise. In addition, the description of any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise. When the structure of a compound is depicted as a specific enantiomer, it is to be understood that the application is not limited to that specific enantiomer.

[0125] The compounds or toxins may occur in different tautomeric forms. The compounds according to the application are meant to include all tautomeric forms, unless stated otherwise.When the structure of a compound is depicted as a specific tautomer, it is to be understood that the application of the present application is not limited to that specific tautomer.

[0126] The compounds disclosed in this description and in the claims may further exist as R and S stereoisomers. Unless stated otherwise, the description of any compound in the description and in the claims is meant to include both the individual R and the individual S stereoisomers of a compound, as well as mixtures thereof. When the structure of a compound is depicted as a specific S or R stereoisomer, it is to be understood that the application of the present application is not limited to that specific S or R stereoisomer.

[0127] The compounds disclosed in this description and in the claims may further exist as exo and endo diastereoisomers. Unless stated otherwise, the description of any compound in the description and in the claims is meant to include both the individual exo and the individual endo diastereoisomers of a compound, as well as mixtures thereof. When the structure of a compound is depicted as a specific endo or exo diastereomer, it is to be understood that the application of the present application is not limited to that specific endo or exo diastereomer.

[0128] The compounds according to the application may exist in salt form, which are also covered by the present invention. The salt is typically a pharmaceutically acceptable salt, containing a pharmaceutically acceptable anion. The term “salt thereof’ means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient. For example, in a salt of a compound the compound may be protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0129] The term “half maximal effective concentration (EC50)” corresponds to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after some specified exposure time. It is commonly used as a measure of drug's potency. The EC50 of a graded dose response curve therefore represents the concentration of a compound where 50% of its maximal effect is observed. The EC50 of a quantal dose response curve represents the concentration of a compound where 50% of the population exhibits a response, after specified exposure duration. Concentration measures typically follow a sigmoidal curve, increasing rapidly over a relatively small change in concentration. This can be determined mathematically by derivation of the best-fit line.

[0130] In some embodiments, the EC50, determined in the present invention, characterizes the potency of antibody to bind on the Target exposed on human tumor cells. The EC50 parameter is determined using FACS analysis. The EC50 parameter reflects the antibody concentration for which 50% of the maximal binding on the human Target expressed on tumor cells is obtained. Each EC50 value was calculated as the midpoint of the dose response curve using a four-parameter regression curve fitting program (Prism Software). This parameter has been selected as to be representative of physiological / pathological conditions.

[0131] The term “Half maximal inhibitory concentration (IC50)” is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. toxin) is needed to inhibit, in vitro, a given biological process or biological component by 50%. IC50 values are typically expressed as molar concentration.

[0132] It is understood that embodiments of the application described herein include "consisting" and / or "consisting essentially of" embodiments.

[0133] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0134] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0135] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Antibodies

[0136] The antibody used in the antibody-drug conjugate of the present application refers to a molecule containing an antigen-binding domain that specifically binds to an antigen. The antibody of the present invention may be of any of IgG, IgE, IgM, IgD, IgA and IgY (class), but IgG is preferred. The subclass (subclass) may be any of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, and IgG1 and IgG2 are preferable. Antibodies may be derived from any species, but human, rat, mouse and rabbit may be preferred. When derived from a species other than human, it is preferable to chimeric or humanize it using a known technique. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred.

[0137] The antibody of the present invention may be any antibody capable of targeting tumor cells. That is, after a drug having an antitumor activity (e.g., toxin) is linked to the antibody via a linker, the antibody preferably has one or more of a property capable of recognizing a tumor cell,a property capable of binding to a tumor cell, a property capable of internalizing into a tumor cell, and a property of damaging a tumor cell.

[0138] The present invention comprises antibodies that exhibit high internalization capacity upon their binding to a target. As used herein, an "internalized" or "internalized" antibody (both expressions are similar) is an antibody that is taken up by (meaning "entered into") a mammalian cell upon binding to a target on the cell. This antibody is important as part of the ADC, so it localizes (addresses) the attached cytotoxic agent to the targeted cancer cell. Once internalized, cytotoxic agents trigger cancer cell death.

[0139] It is more preferred but not essential that the antibody itself should have an antitumor effect. For exerting the antitumor effect and also for specifically and selectively damaging tumor cells by the drug, it is important and also preferred that the antibody should have the property of internalizing to migrate into tumor cells. The key to the success of ADC therapy is believed to be the target antigen specificity and internalization of the antigen-antibody complex into the cancer cell. Clearly, internalized antigens deliver cytotoxic agents more efficiently than non-internalized antigens. The internalization process is variable between antigens and depends on a variety of parameters which can be influenced by the antibody.

[0140] The binding activity of the antibody to tumor cells can be confirmed using flow cytometry. The entry of antibodies into tumor cells can be confirmed using the following methods: (1) Inspection of antibodies entering cells using secondary antibodies (fluorescent markers) bound to therapeutic antibodies using fluorescence microscopy (Cell Death and Differentiation (2008) 15, 751-761); (2) Assays that measure the amount of fluorescence that enters a cell using a secondary antibody (fluorescent label) that binds to the therapeutic antibody (Molecular Biology of the Cell vol.15,5268-5282, december 2004); alternatively, (3) Mab-ZAP assays, in which immunotoxins conjugated to therapeutic antibodies are used, which release the toxin after entry into the cell, thereby inhibiting cell proliferation (BioTe chniques 28:162-165, january 2000).

[0141] In a preferred embodiment, the internalization of the antibodies according to the application can be assessed by immunofluorescence or FACS as exemplified herein below, or any method or process known to those skilled in the art, in particular for internalization mechanisms. In a preferred embodiment, an antibody according to the application may induce internalization of at least 30%, preferably 50%, more preferably 80%, upon binding to the target. In some embodiments, the efficiency of antibody-mediated internalization varies significantly depending on the epitope targeted. Selection of effective internalizing antibodies requires various experimental data that not only study target downregulation, but also detect antibody penetration into cells.

[0142] The binding specificity of the antibody disclosed in the present invention or its antigen- binding unit can be determined by in vitro experiments, such as co-immunoprecipitation, radioimmunoassay (RIA), surface plasmon resonance, flow cytometry or enzyme-linked immunosorbent assay (ELISA) to detect.

[0143] In one aspect, the present application provides antibody drug conjugate comprising antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and / or light chain CDRs discussed herein. Contemplated antibodies include, for example, full-length antibodies (e.g., full- length IgG1 or IgG4), scFvs, antibody Fc fusion proteins, multi-specific (such as bispecific) antibodies, immunoconjugates, and the like. In some embodiments, the antibody is a Fab, a Fab', a F(ab)'2, a Fab'-SH, a single-chain Fv (scFv), an Fv fragment, a dAb, a Fd, a nanobody, a diabody, or a linear antibody. In some embodiments, reference to an antibody that specifically binds to a target means that the antibody binds to the target with an affinity that is at least about 10 times (including for example at least about any one of 10, 102, a103, 104, 105, 106, or 107 times) more tightly than its binding affinity for a non-target. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) assay or biolayer interferometry (BLI).

[0144] Although antibodies containing human sequences (e.g., human heavy and light chain variable domain sequences comprising human CDR sequences) are extensively discussed herein, non-human antibodies are also contemplated. In some embodiments, non-human antibodies comprise human CDR sequences from an antibody as described herein and non-human framework sequences. Non-human framework sequences include, in some embodiments, any sequence that can be used for generating synthetic heavy and / or light chain variable domains using one or more human CDR sequences as described herein, including, e.g., mammals, e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), etc. In some embodiments, a non-human antibody includes an antibody generated by grafting one or more human CDR sequences as described herein onto a non- human framework sequence (e.g., a mouse or chicken framework sequence).

[0145] In some embodiments, the antibody cross-reacts with at least one allelic variant of the antigen protein (or fragments thereof). In some embodiments, the allelic variant has up to about 30 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) amino acid substitutions (such as a conservative substitution) when compared to the naturally occurring antigen protein (or fragments thereof).

[0146] In some embodiments, the amino acid substitutions described above are limited to “exemplary substitutions” shown in Table A of this application. In some embodiments, the amino acid substitutions are limited to “preferred substitutions” shown in Table A of this application.

[0147] In some embodiments, competition assays may be used to identify a monoclonal antibody that competes with an antibody described herein for binding to the same target. Competition assays can be used to determine whether two antibodies bind the same epitope by recognizing identical or sterically overlapping epitopes or one antibody competitively inhibits binding of another antibody to the antigen. In certain embodiments, such a competing antibody binds to the same epitope that is bound by an antibody described herein. Exemplary competition assays include, but are not limited to, routine assays such as those provided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). In some embodiments, two antibodies are said to bind to the same epitope if each blocks binding of the other by 50% or more. In some embodiments, the antibody that competes with an antibody described herein is a chimeric, humanized or human antibody.

[0148] In some embodiments, the antibody is selected from the group consisting of an anti- TROP-2 antibody, an anti-HER2 (ErbB2) antibody, an anti-Folate Receptor (FR) α antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, and an anti-EGFR antibody, anti-c-Met antibodies, anti-HER3 (ErbB3) antibodies, anti-HER4 (ErbB4) antibodies, anti-LIV-1 antibodies, anti-ROR1 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD44 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD73 antibodies, anti-CD105 antibodies, anti-CEA antibodies, anti-A33 antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-G250 antibodies, anti-MUCl antibodies, anti-Lewis Y antibodies, anti-VEGFR antibodies, anti-GPNMB antibodies, anti-Integrin antibodies, anti-cancer antibodies. Preferably, the antibody is an anti-cancer antibody. More preferably, the antibody is selected from anti-TROP-2 antibody, an anti-HER2 (ErbB2) antibody and an anti- Folate Receptor (FR) α antibody.

[0149] TROP2

[0150] TROP2 is a member of the TACSTD family expressed in human trophoblasts and is a single-pass transmembrane type 1 cell membrane protein involved in immune resistance, which is common to human trophoblasts and cancer cells.

[0151] As for TROP2 protein to be used in the application, TROP2 protein can be directly purified from the TROP2-expressing cells of a human or a non-human mammal (such as a rat or amouse) and used, or a cell membrane fraction of the above-described cells can be prepared and used. Further, TROP2 can be obtained by in vitro synthesis thereof or production thereof in a host cell through genetic engineering. In the genetic engineering, specifically, after TROP2 cDNA is integrated into a vector capable of expressing TROP2 cDNA, the TROP2 protein can be obtained by synthesizing it in a solution containing an enzyme, a substrate and an energy substance required for transcription and translation, or by expressing TROP2 in another prokaryotic or eucaryotic transformed host cell. Alternatively, the above-described genetically engineered TROP2- expressing cells or a cell line expressing TROP2 may be used as the TROP2 protein.

[0152] The DNA sequence and amino acid sequence of TROP2 are available on a public database and can be referred to, for example, under Accession Nos. NM_002353 and NP_002344 (NCBI).

[0153] Further, a protein which consists of an amino acid sequence wherein one or several amino acids are substituted, deleted and / or added in any of the above-described amino acid sequences of TROP2 and also has a biological activity equivalent to that of the protein is also included in TROP2.

[0154] The human TROP2 protein is constituted by a signal sequence consisting of N-terminal 26 amino acid residues, an extracellular domain consisting of 248 amino acid residues, a transmembrane domain consisting of 23 amino acid residues, and an intracellular domain consisting of 26 amino acid residues.

[0155] In some embodiments, examples of a cell line expressing TROP2 can include human lung cancer lines NCI-H322, PC14, NCIH-H2122, and LCAM1, a human prostate cancer line PC3, human pancreatic cancer lines BxPC-3, Capan-1, and PK-1, a human ovarian cancer line SKOV3, and a human colorectal cancer line COLO205.

[0156] Anti-TROP-2 antibody

[0157] The anti-TROP2 antibody used in the anti-TROP2 antibody-drug conjugate of the present application may be derived from any species, and preferred examples of the species can include humans, rats, mice, and rabbits. In case when derived from other than human species, it is preferably chimerized or humanized using a well-known technique. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody and is preferably a monoclonal antibody.

[0158] The anti-TROP2 antibody is capable of targeting tumor cells, that is, has a property capable of recognizing a tumor cell, a property capable of binding to a tumor cell, a property of internalizing in a tumor cell, or the like, and can be converted into an antibody-drug conjugate by conjugation to a compound having antitumor activity via a linker.

[0159] In some embodiments, the anti-TROP2 antibody described herein have the following properties: (a) specifically binding to TROP2, and having an activity of internalizing in TROP2- expressign cells by binding to TROP2.

[0160] The anti-TROP2 antibody can be obtained using a method usually carried out in the art, which involves immunizing animals with an antigenic polypeptide and collecting and purifying antibodies produced in vivo. The origin of the antigen is not limited to humans, and the animals may be immunized with an antigen derived from a non-human animal such as a mouse, a rat and the like. In this case, the cross-reactivity of antibodies binding to the obtained heterologous antigen with human antigens can be tested to screen for an antibody applicable to a human disease.

[0161] Alternatively, antibody-producing cells which produce antibodies against the antigen are fused with myeloma cells according to a method known in the art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; and Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y. (1980)) to establish hybridomas, from which monoclonal antibodies can in turn be obtained.

[0162] The antigen can be obtained by genetically engineering host cells to produce a gene encoding the antigenic protein. Specifically, vectors that permit expression of the antigen gene are prepared and transferred to host cells so that the gene is expressed. The antigen thus expressed can be purified. The antibody can be also obtained using a method of immunizing animals with the above-described genetically engineered antigen-expressing cells or a cell line expressing the antigen.

[0163] The anti-TROP2 antibody that can be used in the present invention is not particularly limited, and, for example, those specified by the amino acid sequences shown in the Sequence Listing of the present application can be preferably used.

[0164] In some embodiments, the antibody used in the present invention specifically binds to TROP2. In a specific embodiment, the anti-TROP2 antibody binds to the same epitope on TROP2 as Sacituzumab or Datopotamab. In a more specific embodiment, the anti-TROP2 antibody has the same variable region CDRs as Sacituzumab or Datopotamab. In yet a more specific embodiment, the anti-TROP2 antibody has the same variable regions (i.e., VH and VL) as Sacituzumab or Datopotamab.

[0165] In a preferred embodiment of the application, the anti-TROP2 antibody in the antibody- drug conjugate of the application is selected from Sacituzumab or Datopotamab.

[0166] In a preferred embodiment of the application, the anti-TROP2 antibody in the antibody- drug conjugate of the application comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VLcomprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0167] In a preferred embodiment of the application, the anti-TROP2 antibody in the antibody- drug conjugate of the application comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0168] In a more preferred embodiments of the application, the anti-TROP2 antibody in the antibody-drug conjugate of the application comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0169] HER2(Human epidermal growth factor receptor 2)

[0170] HER2, also known as ErbB2, pi 85 and CD340, is a receptor tyrosine kinase that is involved in the regulation of various cellular functions. Amplification of the gene encoding HER2 with consequent overexpression of the receptor was observed in breast and ovarian cancers and correlates with a poor prognosis (Slamon et ah, 1987, Science 235(4785): 177-82; Slamon et ak, 1989, Science 244:707-12; Anbazhagan et ah, 1991, Annals Oncology 2(1):47-53; Andrulis et ak, 1998, J Clinical Oncology 16(4): 1340-9). Overexpression of HER2 (frequently but not necessarily due to gene amplification) has also been observed in other tumor types including gastric, endometrial, non-small cell lung cancer, colon, pancreatic, bladder, kidney, prostate and cervical (Scholl et ak, 2001, Annals Oncology 12 (Suppk 1): S81-7; Menard et ak, 2001, Ann Oncol 12(Suppl l):S15-9; Martin et ak, 2014, Future Oncology 10: 1469-86).

[0171] As used herein, the term “HER2” refers to a transmembrane tyrosine kinase receptor that belongs to the EGFR family. The wild type human HER2 protein is described, for example, in Semba et ah, 1985, PNAS 82:6497-6501 and Yamamoto et ah, 1986, Nature 319:230-4 and Genbank Accession Number X03363. The term “HER2” includes variants, isoforms, homologs, orthologs and paralogs.

[0172] Anti-HER2 antibody

[0173] In some aspects of the application, antibodies and antibody-drug conjugates cross-react with HER2 from species other than human, such as HER2 of mouse, rat, or primate, as well as different forms of HER2 (e.g., glycosylated HER2). In other aspects, the antibodies and antibody- drug conjugates may be completely specific for human HER2 and may not exhibit species or othertypes of cross-reactivity. As used herein the term HER2 refers to naturally occurring human HER2 unless contextually dictated otherwise. Therefore, a “HER2 antibody”, “anti-HER2 antibody”, or other similar designation, means an antibody that associates, binds, or reacts with the HER2 type ligand or isoform, or fragment or derivative thereof. The anti-HER2 antibody is capable of targeting tumor cells expressing HER2, that is, has a property capable of recognizing a tumor cell, a property capable of binding to a tumor cell, a property of internalizing in a tumor cell, or the like, and can be converted into an antibody-drug conjugate by conjugation to a compound having antitumor activity via a linker. In some embodiments, the anti-HER2 antibody described herein have the following properties: (a) specifically binding to HER2, and having an activity of internalizing in HER2-expressign cells by binding to HER2. Further, a “HER2 antibody-drug conjugate”, “anti-HER2 antibody-drug conjugate” refers to an antibody-drug conjugate or ADC (as defined herein) that comprises an anti-HER2 antibody as defined herein.

[0174] In some embodiments, the antibody used in the present invention specifically binds to HER2. In a specific embodiment, the HER2 antibody binds to the same epitope on HER2 as Trastuzumab, Disitamab, Pertuzumab, Zanidatamab or Hertuzumab. In a more specific embodiment, the HER2 antibody has the same variable region CDRs as trastuzumab, Disitamab, Pertuzumab, Zanidatamab or Hertuzumab. In yet a more specific embodiment, the HER2 antibody has the same variable regions (i.e., VH and VL) as Trastuzumab, Disitamab, Pertuzumab, Zanidatamab or Hertuzumab.

[0175] In a preferred embodiment of the application, the anti-HER2 antibody in the antibody- drug conjugate of the application is selected from Trastuzumab, Disitamab, Pertuzumab, Zanidatamab or Hertuzumab.

[0176] In a preferred embodiment of the application, the anti-HER2 antibody in the antibody- drug conjugate of the application comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0177] In a preferred embodiment of the application, the anti-HER2 antibody in the antibody- drug conjugate of the application comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 17; and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a variantthereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0178] In a more preferred embodiments of the application, the anti-HER2 antibody in the antibody-drug conjugate of the application comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:19, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:20.

[0179] HER2-Expressing Cancers

[0180] Cancers that may be treated with the dosing regimen or method provided by the present disclosure include HER2 expressing (“HER2 positive” or “HER2+”) solid tumors. HER2- expressing cancers can express HER2 at ahigh, moderate, or low level. Methods for identifying levels of expression and / or amplification of the HER2 gene are known in the art, such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH or ISH). In some embodiments, the cancers to be treated are breast cancers that are hormone receptor (HR) positive (+). The term “hormone receptor positive” or ‘HR+” means the tumor is estrogen receptor (ER) positive, progesterone receptor (PR) positive, or both ER positive and PR positive. In some particular embodiments, patients with breast cancer are HR+ (including documentation of estrogen receptor (ER) positive and / or progesterone receptor positive tumor ((>1% positive stained cells) based on most recent tumor biopsy utilizing an assay consistent with local standards) and HER2 IHC+ / ISH negative (-) or equivocal. In some other embodiments, the cancer to be treated is resistant to, refractory to and / or relapsed from treatment with trastuzumab and / or trastuzumab emtansine (T-DM1) either of which alone or in combination with a taxane. Examples of cancers to be treated include breast cancer, ovarian cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, urothelial cancer, pancreatic cancer, salivary gland cancer and brain cancer or metastases of the aforementioned cancers. In a more specific embodiment, the breast cancer is hormone receptor positive breast cancer, estrogen receptor and progesterone receptor negative breast cancer, or triple negative breast cancer (TNBC). In another embodiment, the lung cancer is non-small cell lung cancer (NSCLC).

[0181] FRα (Folate receptor α)

[0182] Folate, an essential vitamin required for DNA synthesis and repair, cell division processes, is transported by endocytosis by binding to folate receptors on the cell surface, where folate receptors are internalized and then recycled back to the cell membrane. The folate receptor is a transmembrane single-chain glycoprotein linked to glycosylated phosphatidylinositol and has a high affinity for folate. Folate receptors include three subtypes, namely folate receptor α (Folate Receptorα, FOLR1, FRα), folate receptor β (Folate Receptorβ, FOLR2) and folate receptor γ (Folate Receptorγ, FOLR3). The expression of folate receptors is highly restricted in normal cells,but significantly high in tumor cells. Among them, folate receptor α has been found to be overexpressed in various malignant tumors, so it has become one of the popular targets of anticancer drugs.

[0183] Folate receptor-α (FRα or FOLR1) is a glycosylphosphatidylinositol-linked cell- surface glycoprotein that has high affinity for folates. Its physiologic role in normal and cancerous tissues has not yet been fully elucidated. Most normal tissues do not express FRα, and transport of physiologic folates into most cells is thought to be mediated by several other proteins, most notably, reduced folate carrier. High levels of FRα have been found in serous and endometrioid epithelial ovarian cancer, endometrial adenocarcinoma, and non-small cell lung cancer of the adenocarcinoma subtype. Importantly, FRα expression is maintained in metastatic foci and recurrent carcinomas in ovarian cancer patients, and after chemotherapy in epithelial ovarian and endometrial cancers. These properties, together with the highly restricted expression of FRα on normal tissues, make FRα a highly promising target for targeted therapies such as ADCs.

[0184] As used herein, the term "tumor overexpressing folate receptor alpha" refers to a tumor (including benign tumors and cancers) overexpressing folate receptor alpha. In some embodiments, folate receptor alpha expression in a tumor sample above background levels in immune tissue (eg, as determined by immunohistochemical staining) indicates that the tumor is a folate receptor alpha overexpressing tumor. Methods for detecting folate receptor alpha expression in tumors are known in the art, such as immunohistochemical assays. In some embodiments, "FRα negative cells" are cells in a sample of cells that lack folate receptor alpha above background (eg, as determined by immunohistochemical techniques).

[0185] anti-FRα antibody

[0186] The antibody provided by the application is an anti-folate receptor α antibody. In one or more embodiments, an antibody of the application is capable of specifically binding human folate receptor alpha. In one or more embodiments, the application provides a humanized anti-FRα antibody. Herein, these antibodies are collectively referred to as anti-FRα antibodies. The anti- FRα antibody of the present invention has characteristics including but not limited to binding to FRα (such as human FRα) in vitro, binding to cells expressing FRα, high affinity, and strong internalization ability.

[0187] In one or more embodiments, the anti-FRα antibody of the present invention can specifically bind to folate receptor α (FOLR1), but not to folate receptor β (FOLR2) or folate receptor γ (FOLR3).

[0188] In some embodiments, the antibody used in the present invention specifically binds to FRα. In a specific embodiment, the anti-FRα antibody binds to the same epitope on FRα as Luveltamab, Farletuzumab or Mirvetuximab. In a more specific embodiment, the anti- FRαantibody has the same variable region CDRs as Luveltamab, Farletuzumab or Mirvetuximab. In yet a more specific embodiment, the anti- FRα antibody has the same variable regions (i.e., VH and VL) as Luveltamab, Farletuzumab or Mirvetuximab.

[0189] In a preferred embodiment of the application, the anti- FRα antibody in the antibody-drug conjugate of the application is selected from Luveltamab, Farletuzumab or Mirvetuximab.

[0190] In a preferred embodiment of the application, the anti- FRα antibody in the antibody-drug conjugate of the application comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0191] In a preferred embodiment of the application, the anti- FRα antibody in the antibody-drug conjugate of the application comprises a VH comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and a VL comprising the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28.

[0192] In a more preferred embodiments of the application, the anti-FRα antibody in the antibody-drug conjugate of the application comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:29, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:30.

[0193] Exemplary anti-HER2 antibody, anti-TROP2 antibody, and anti-FRα antibody sequences are shown in Tables 2-4. Those skilled in the art will recognize that many algorithms are known for prediction of CDR positions and for delimitation of antibody heavy chain and light chain variable regions. Full-length antibody

[0194] The antibody in some embodiments is a full-length antibody. In some embodiments, the full-length antibody is an IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length antibody comprises IgG constant domains, such as constant domains of any of IgG1, IgG2, IgG3, and IgG4 including variants thereof. In some embodiments, the full-length antibody comprises a lambda light chain constant region. In some embodiments, the full-length antibody comprises a kappa light chain constant region. In some embodiments, the full-length antibody is a full-lengthhuman antibody. In some embodiments, the full-length antibody comprises an Fc sequence of a mouse immunoglobulin. In some embodiments, the full-length antibody comprises an Fc sequence that has been altered or otherwise changed so that it has enhanced antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) effector function.

[0195] In some embodiments, according to any of the antibodies described herein, the antibody comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the antibody comprises an IgG1 heavy chain constant region. In some embodiments, the antibody comprises an IgG2 heavy chain constant region. In some embodiments, the antibody comprises an IgG3 heavy chain constant region. In some embodiments, the antibody comprises an IgG4 heavy chain constant region. Binding affinity

[0196] Binding affinity can be indicated by Kd, Koff, Kon, or Ka. The term "Koff", as used herein, is intended to refer to the off-rate constant for dissociation of an antibody from the antibody / antigen complex, as determined from a kinetic selection set up. The term "Kon", as used herein, is intended to refer to the on-rate constant for association of an antibody to the antigen to form the antibody / antigen complex. The term dissociation constant "Kd", as used herein, refers to the dissociation constant of a particular antibody-antigen interaction, and describes the concentration of antigen required to occupy one half of all of the antibody-binding domains present in a solution of antibody molecules at equilibrium, and is equal to Koff / Kon. The measurement of Kd presupposes that all binding agents are in solution. In the case where the antibody is tethered to a cell wall, e.g., in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC50, which gives a good approximation of Kd. The affinity constant, Ka, is the inverse of the dissociation constant, Kd.

[0197] The dissociation constant (Kd) is used as an indicator showing affinity of antibody moieties to antigens. For example, easy analysis is possible by the Scatchard method using antibodies marked with a variety of marker agents, as well as by using Biacore (made by Amersham Biosciences), analysis of biomolecular interactions by surface plasmon resonance, according to the user's manual and attached kit. The Kd value that can be derived using these methods is expressed in units of M. An antibody that specifically binds to a target may have a Kd of, for example, ≤ 10-7M, ≤ 10-8M, ≤ 10-9M, ≤ 10-10M, ≤ 10-11M, ≤ 10-12M, or ≤ 10-13M.

[0198] Binding specificity of the antibody can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore-tests and peptide scans.

[0199] In some embodiments, the antibody specifically binds to a target with a Kd of about 10-7M to about 10-13M (such as about 10-7M to about 10-13M, about 10-8M to about 10-13M, about 10-9M to about 10-13M, or about 10-10M to about 10-12M). Thus in some embodiments, the Kd of the binding between the antibody and the target, is about 10-7M to about 10-13M, about 1×10-7M to about 5×10-13M, about 10-7M to about 10-12M, about 10-7M to about 10-11M, about 10-7M to about 10-10M, about 10-7M to about 10-9M, about 10-8M to about 10-13M, about 1×10-8M to about 5×10-13M, about 10-8M to about 10-12M, about 10-8M to about 10-11M, about 10-8M to about 10-10M, about 10-8M to about 10-9M, about 5×10-9M to about 1×10-13M, about 5×10-9M to about 1×10-12M, about 5×10-9M to about 1×10-11M, about 5×10-9M to about 1×10-10M, about 10-9M to about 10-13M, about 10-9M to about 10-12M, about 10-9M to about 10-11M, about 10-9M to about 10-10M, about 5×10-10M to about 1×10-13M, about 5×10-10M to about 1×10-12M, about 5×10-10M to about 1×10-11M, about 10-10M to about10-13M, about 1×10-10M to about 5×10-13M, about 1×10-10M to about 1×10-12M, about 1×10-10M to about 5×10-12M, about 1×10-10M to about 1×10-11M, about 10-11M to about 10-13M, about 1×10-11M to about 5×10-13M, about 10-11M to about 10-12M, or about 10-12M to about 10-13M. In some embodiments, the Kd of the binding between the antibody and the target is about 10-7M to about 10-13M. Nucleic Acids

[0200] Nucleic acid molecules encoding the antibodies are also contemplated. In some embodiments, there is provided a nucleic acid (or a set of nucleic acids) encoding a full-length antibody. In some embodiments, the nucleic acid (or a set of nucleic acids) encoding the antibody described herein may further comprises a nucleic acid sequence encoding a peptide tag (such as protein purification tag, e.g., His-tag, HA tag).

[0201] Also contemplated here are isolated host cells comprising an antibody, an isolated nucleic acid encoding the polypeptide components of the antibody, or a vector comprising a nucleic acid encoding the polypeptide components of the antibody described herein.

[0202] The present application also includes variants to these nucleic acid sequences. For example, the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the antibodies of the present application under at least moderately stringent hybridization conditions.

[0203] The present application also provides vectors in which a nucleic acid of the present application is inserted.

[0204] In brief summary, the expression of an antibody (e.g., full-length antibody) by a natural or synthetic nucleic acid encoding the antibody can be achieved by inserting the nucleic acid intoan appropriate expression vector, such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including for example a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR). The vectors can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0205] The nucleic acids of the present application may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In some embodiments, the application provides a gene therapy vector.

[0206] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0207] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No.6,326,193).

[0208] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0209] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

[0210] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the application should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the application. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0211] In some embodiments, the expression of the antibody is inducible. In some embodiments, a nucleic acid sequence encoding the antibody is operably linked to an inducible promoter, including any inducible promoter described herein. Inducible promoters

[0212] The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Exemplary inducible promoter systems for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see, e.g., Spencer, D. M. et al. (1993) Science 262: 1019- 1024) and ionizing radiation-regulated elements (e.g., see Manome, Y. et al. (1993) Biochemistry 32: 10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1014- 10153). Furtherexemplary inducible promoter systems for use in in vitro or in vivo mammalian systems are reviewed in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system for use to express the antibody is the Tet system. In some embodiments, the inducible promoter system for use to express the antibody is the lac repressor system from E. coli.

[0213] An exemplary inducible promoter system for use in the present application is the Tet system. Such systems are based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, a polynucleotide of interest is under the control of a promoter that comprises one or more Tet operator (TetO) sites. In the inactive state, Tet repressor (TetR) will bind to the TetO sites and repress transcription from the promoter. In the active state, e.g., in the presence of an inducing agent such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox), or an active analog thereof, the inducing agent causes release of TetR from TetO, thereby allowing transcription to take place. Doxycycline is a member of the tetracycline family of antibiotics having the chemical name of 1-dimethylamino-2,4a,5,7,12-pentahydroxy-11-methyl-4,6-dioxo- 1,4a,11,11a,12,12a-hexahydrotetracene-3-carboxamide.

[0214] In one embodiment, a TetR is codon-optimized for expression in mammalian cells, e.g., murine or human cells. Most amino acids are encoded by more than one codon due to the degeneracy of the genetic code, allowing for substantial variations in the nucleotide sequence of a given nucleic acid without any alteration in the amino acid sequence encoded by the nucleic acid. However, many organisms display differences in codon usage, also known as "codon bias" (i.e., bias for use of a particular codon(s) for a given amino acid). Codon bias often correlates with the presence of a predominant species of tRNA for a particular codon, which in turn increases efficiency of mRNA translation. Accordingly, a coding sequence derived from a particular organism (e.g., a prokaryote) may be tailored for improved expression in a different organism (e.g., a eukaryote) through codon optimization.

[0215] Other specific variations of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-Off system, transcription is inactive in the presence of Tc or Dox. In that system, a tetracycline-controlled transactivator protein (tTA), which is composed of TetR fused to the strong transactivating domain of VP16 from Herpes simplex virus, regulates expression of a target nucleic acid that is under transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE is made up of TetO sequence concatamers fused to a promoter (commonly the minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter). In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive.

[0216] Conversely, in the Tet-On system, transcription is active in the presence of Tc or Dox. The Tet-On system is based on a reverse tetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain. However, a four amino acid change in the TetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of Dox. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.

[0217] Another inducible promoter system is the lac repressor system from E. coli (See Brown et al., Cell 49:603-612 (1987)). The lac repressor system functions by regulating transcription of a polynucleotide of interest operably linked to a promoter comprising the lac operator (lacO). The lac repressor (lacR) binds to LacO, thus preventing transcription of the polynucleotide of interest. Expression of the polynucleotide of interest is induced by a suitable inducing agent, e.g., isopropyl- β-D-thiogalactopyranoside (IPTG).

[0218] In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic- resistance genes, such as neo and the like.

[0219] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui- Tel et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0220] In some embodiments, there is provided nucleic acid encoding a full-length antibody described herein. In some embodiments, the nucleic acid comprises one or more nucleic acidsequences encoding the heavy and light chains of the full-length antibody. In some embodiments, each of the one or more nucleic acid sequences are contained in separate vectors. In some embodiments, at least some of the nucleic acid sequences are contained in the same vector. In some embodiments, all of the nucleic acid sequences are contained in the same vector. Vectors may be selected, for example, from the group consisting of mammalian expression vectors and viral vectors (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses).

[0221] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0222] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well- known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, the introduction of a polynucleotide into a host cell is carried out by calcium phosphate transfection.

[0223] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus 1, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

[0224] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0225] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with amicelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0226] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present application, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the application. Preparation of antibodies

[0227] In some embodiments, the antibody is a monoclonal antibody or derived from a monoclonal antibody. In some embodiments, the antibody comprises VH and VL domains, or variants thereof, from the monoclonal antibody. In some embodiments, the antibody further comprises CH1 and CLdomains, or variants thereof, from the monoclonal antibody. Monoclonal antibodies can be prepared, e.g., using known methods in the art, including hybridoma methods, phage display methods, or using recombinant DNA methods.

[0228] In a hybridoma method, a hamster, mouse, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro. The immunizing agent can include a polypeptide or a fusion protein of the protein of interest. To obtain epitope specific antibody, the immunizing agents can be a polypeptide essentially comprising or consisting of the epitope, or an antigen fragment or domain essentially comprising or consisting of the epitope and over-expression cell line (Greenfield EA. Standard Immunization of Mice, Rats, and Hamsters. Cold Spring Harb Protoc. 2020 Mar 2;2020(3):100297; Holzlöhner P, Hanack K. Generation of Murine Monoclonal Antibodies by Hybridoma Technology. J Vis Exp.2017 Jan 2;(119):54832). The epitope specific antibody can be identified via the methods well-known in the field, including but not limited inantigen domain swapping, alanine scanning and antigen-Fab complex crystal-structural study (Toride King M, Brooks CL. Epitope Mapping of Antibody-Antigen Interactions with X-Ray Crystallography. Methods Mol Biol. 2018;1785:13-27; Morrison KL, Weiss GA. Combinatorial alanine-scanning. Curr Opin Chem Biol. 2001 Jun;5(3):302-7). Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevents the growth of HGPRT-deficient cells.

[0229] In some embodiments, the immortalized cell lines fuse efficiently, support stable high- level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. In some embodiments, the immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies.

[0230] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide. The binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme- linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0231] After the desired hybridoma cells are identified, the clones can be sub-cloned by limiting dilution procedures and grown by standard methods. Goding, supra. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0232] The monoclonal antibodies secreted by the sub-clones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures suchas, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0233] In some embodiments, according to any of the antibodies described herein, the antibody comprises sequences from a clone selected from an antibody library (such as a phage library presenting scFv or Fab fragments). The clone may be identified by screening combinatorial libraries for antibody fragments with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0234] In certain phage display methods, repertoires of VHand VLgenes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433- 455 (1994). Phage typically display antibody fragments, either as scFv fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0235] The antibodies can be prepared using phage display to screen. The library can be a human scFv phage display library having a diversity of at least 1 × 109(such as at least about any of 1 × 109, 2.5 × 109, 5 × 109, 7.5 × 109, 1 × 1010, 2.5 × 1010, 5 × 1010, 7.5 × 1010, or 1 × 1011) unique human antibody fragments. In some embodiments, the library is a naïve human library constructed from DNA extracted from human PMBCs and spleens from healthy donors, encompassing allhuman heavy and light chain subfamilies. In some embodiments, the library is a naïve human library constructed from DNA extracted from PBMCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library, wherein heavy chain CDR3 is completely randomized, with all amino acids (with the exception of cysteine) equally likely to be present at any given position (see, e.g., Hoet, R.M. et al., Nat. Biotechnol.23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 of the semi-synthetic human library has a length from about 5 to about 24 (such as about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) amino acids. In some embodiments, the library is a fully-synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0236] Phage clones that bind to the target with high affinity can be selected by iterative binding of phage to the target, which is bound to a solid support (such as, for example, beads for solution panning or mammalian cells for cell panning), followed by removal of non-bound phage and by elution of specifically bound phage. The bound phage clones are then eluted and used to infect an appropriate host cell, such as E. coli XL1-Blue, for expression and purification. The panning can be performed for multiple (such as about any of 2, 3, 4, 5, 6 or more) rounds with solution panning, cell panning, or a combination of both, to enrich for phage clones binding specifically to the target. Enriched phage clones can be tested for specific binding to the target by any methods known in the art, including for example ELISA and FACS.

[0237] An alternative method for screening antibody libraries is to display the protein on the surface of yeast cells. Wittrup et al. (US Patent Nos. 6,699,658 and 6,696,251) have developed a method for a yeast cell display library. In this yeast display system, a component involves the yeast agglutinin protein (Aga1), which is anchored to the yeast cell wall. Another component involves a second subunit of the agglutinin protein Aga2, which can display on the surface yeast cells through disulfide bonds to Aga1 protein. The protein Aga1 is expressed from a yeast chromosome after the Aga1 gene integration. A library of single chain variable fragments (scFv) is fused genetically to Aga2 sequence in the yeast display plasmid, which, after transformation, is maintained in yeast episomally with a nutritional marker. Both of the Aga1 and Aga2 proteins were expressed under the control of the galactose-inducible promoter.

[0238] Human antibody V gene repertoire (VHand VKfragments) are obtained by PCR method using a pool of degenerate primers (Sblattero, D. & Bradbury, A. Immunotechnology 3, 271-278 1998). The PCR templates are from the commercially available RNAs or cDNAs, including PBMC, spleen, lymph nodes, bone marrow and tonsils. Separate VH and VK PCR libraries were combined, then assembled together in the scFv format by overlap extension PCR (Sheets, M.D. et al., Proc. Natl. Acad. Sci. USA 95, 6157-6162 1998.). To construct the yeast scFv display library,the resultant scFv PCR products are cloned into the yeast display plasmid in the yeasts by homologous recombination. (Chao, G, et al., Nat Protoc. 2006;1(2):755-68. Miller KD, et al., Current Protocols in Cytometry 4.7.1-4.7.30, 2008).

[0239] The antibodies can be discovered using mammalian cell display systems in which antibody moieties are displayed on the cell surface and those specific to the target are isolated by the antigen-guided screening method, as described in U.S. patent No. 7,732,195B2. A Chinese hamster ovary (CHO) cell library representing a large set of human IgG antibody genes can be established and used to discover the clones expressing high-affinity antibody genes. Another display system has been developed to enable simultaneous high-level cell surface display and secretion of the same protein through alternate splicing, where the displayed protein phenotype remains linked to genotype, allowing soluble secreted antibody to be simultaneously characterized in biophysical and cell-based functional assays. This approach overcomes many limitations of previous mammalian cell display, enabling direct selection and maturation of antibodies in the form of full-length, glycosylated IgGs (Peter M. Bowers, et al., Methods 2014,65:44-56). Transient expression systems are suitable for a single round of antigen selection before recovery of the antibody genes and therefore most useful for the selection of antibodies from smaller libraries. Stable episomal vectors offer an attractive alternative. Episomal vectors can be transfected at high efficiency and stably maintained at low copy number, permitting multiple rounds of panning and the resolution of more complex antibody libraries.

[0240] The IgG library is based on germline sequence V-gene segments joined to rearranged (D)J regions isolated from a panel of human donors. RNA collected from 2000 human blood samples was reverse-transcribed into cDNA, and the VH and VK fragments were amplified using VH- and VK-specific primers and purified by gel extraction. IgG libraries were generated by sub-cloning the VH and VK fragments into the display vectors containing IgG1 or K constant regions respectively and then electroporating into or transducing 293T cells. To generate the scFv antibody display library, scFvs were generated by linking VH and VK, and then sub-cloned into the display vector, which were then electroporated into or transduce 293T cells. As we known, the IgG library is based on germline sequence V-gene segments joined to rearranged (D)J regions isolated from a panel of donors, the donor can be a mouse, rat, rabbit, or monkey.

[0241] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the application can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells as described above or target-specific phage clones of the application can serve as a source of such DNA. Once isolated, the DNA can be placedinto expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains and / or framework regions in place of the homologous non-human sequences (U.S. Patent No.4,816,567; Morrison et al., supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the application, or can be substituted for the variable domains of one antigen-combining site of an antibody of the application to create a chimeric bivalent antibody.

[0242] The antibodies can be monovalent antibodies. Methods for preparing monovalent antibodies are known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

[0243] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using any method known in the art.

[0244] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant-domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. Human and Humanized Antibodies

[0245] The antibodies (e.g., full-length antibodies) can be humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibody moieties are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding subsequences of antibodies) that typically contain minimal sequence derived from non-human immunoglobulin. Humanized antibody moieties include humanimmunoglobulins, immunoglobulin chains, or fragments thereof (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibody moieties can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody can comprise substantially at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.

[0246] Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. According to some embodiments, humanization can be essentially performed following the method of Winter and co- workers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibody moieties are antibody moieties (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibody moieties are typically human antibody moieties in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0247] As an alternative to humanization, human antibody moieties can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852. Alternatively, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed that closelyresembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

[0248] Human antibodies may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147(1): 86-95 (1991). Antibody variants

[0249] In some embodiments, amino acid sequences of the antibody variants (e.g., full-length antibody) provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence of an antibody variant may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

[0250] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., improved bioactivity, retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0251] Conservative substitutions are shown in Table A below. TABLE A: CONSERVATIVE SUBSTITUTIONS Original Exemplary PreferredArg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln hainproperties: a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that influence chain orientation: Gly, Pro; f. aromatic: Trp, Tyr, Phe.

[0253] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0254] An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibody moieties displayed on phage and screened for a particular biological activity (e.g., bioactivity based on binding affinity or ligand blocking assay). Alterations (e.g., substitutions) may be made in HVRs, e.g., to improvebioactivity based on binding affinity or ligand blocking assay. Such alterations may be made in HVR "hotspots", i.g., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or specificity determining residues (SDRs), with the resulting variant VH and VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0255] In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0256] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR "hotspots" or SDRs. In some embodiments of the variant VHand VLsequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0257] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or glu) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations to demonstrate functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be determined to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0258] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. Fc Region Variants

[0259] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody (e.g., a full-length antibody or antibody Fc fusion protein) provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC effector function, often related to binding to Fc receptors (FcRs). In some embodiments, the Fc region variant has decreased ADCC effector function. There are many examples of changes or mutations to Fc sequences that can alter effector function. For example, WO 00 / 42072 and Shields et al., J Biol. Chem.9(2): 6591-6604 (2001) describe antibody variants with improved or diminished binding to FcRs. The contents of those publications are specifically incorporated herein by reference.

[0260] Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is a mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell (e.g., a cancer cell), whose membrane-surface antigens have been bound by specific antibodies (e.g., an anti-TROP2 antibody). The typical ADCC involves activation of NK cells by antibodies. An NK cell expresses CD16 which is an Fc receptor. This receptor recognizes, and binds to, the Fc portion of an antibody bound to the surface of a target cell. The most common Fc receptor on the surface of an NK cell is called CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of an antibody results in NK cell activation, release of cytolytic granules and consequent target cell apoptosis. The contribution of ADCC to tumor cell killing can be measured with a specific test that uses NK-92 cells that have been transfected with a high-affinity FcR. Results are compared to wild-type NK- 92 cells that do not express the FcR.

[0261] In some embodiments, the application contemplates an antibody variant (such as a full- length antibody variant) comprising an Fc region that possesses some but not all effector functions, which makes it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcRexpression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No.5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CYTOTOX 96™ non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol.18(12):1759-1769 (2006)).

[0262] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

[0263] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No.6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem.9(2): 6591- 6604 (2001).)

[0264] In some embodiments, there is provided an antibody (such as a full-length antibody) variant comprising a variant Fc region comprising one or more amino acid substitutions which improve ADCC. In some embodiments, the variant Fc region comprises one or more amino acid substitutions which improve ADCC, wherein the substitutions are at positions 298, 333, and / or 334 of the variant Fc region (EU numbering of residues). In some embodiments, the antibody (e.g., full-length antibody) variant comprises the following amino acid substitution in its variant Fc region: S298A, E333A, and K334A.

[0265] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol.164: 4178-4184 (2000).

[0266] In some embodiments, there is provided an antibody (such as a full-length antibody) variant comprising a variant Fc region comprising one or more amino acid substitutions which increase half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-lives and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 concerning other examples of Fc region variants.

[0267] Antibodies (such as full-length antibodies) comprising any of the Fc variants described herein, or combinations thereof, are contemplated. Glycosylation Variants

[0268] In some embodiments, an antibody (such as a full-length antibody) provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence of the antibody or polypeptide portion thereof such that one or more glycosylation sites are created or removed.

[0269] Wherein the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the application may be made in order to create an antibody variants with certain improved properties.

[0270] The N-glycans attached to the CH2 domain of Fc is heterogeneous. Antibodies or Fc fusion proteins generated in CHO cells are fucosylated by fucosyltransferase activity. See Shoji- Hosaka et al., J. Biochem.2006, 140:777- 83. Normally, a small percentage of naturally occurringafucosylated IgGs may be detected in human serum. N-glycosylation of the Fc is important for binding to FcγR; and afucosylation of the N-glycan increases Fc's binding capacity to FcγRIIIa. Increased FcγRIIIa binding can enhance ADCC, which can be advantageous in certain antibody therapeutic applications in which cytotoxicity is desirable.

[0271] In some embodiments, an enhanced effector function can be detrimental when Fc- mediated cytotoxicity is undesirable. In some embodiments, the Fc fragment or CH2 domain is not glycosylated. In some embodiments, the N-glycosylation site in the CH2 domain is mutated to prevent from glycosylation.

[0272] In some embodiments, antibody (such as a full-length antibody) variants are provided comprising an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. Specifically, antibodies are contemplated herein that have reduced fucose relative to the amount of fucose on the same antibody produced in a wild-type CHO cell. That is, they are characterized by having a lower amount of fucose than they would otherwise have if produced by native CHO cells (e.g., a CHO cell that produce a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene). In some embodiments, the antibody is one wherein less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon comprise fucose. For example, the amount of fucose in such an antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. In some embodiments, the antibody is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the antibody is completely without fucose, or has no fucose or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such asα-1,6- fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0273] Antibody (such as a full-length antibody) variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody (such as a full-length antibody) variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No.6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5): 851-861 (2006). Antibody (such as full-length antibody) variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0274] In some embodiments, the antibody (such as a full-length antibody) variants comprising an Fc region are capable of binding to an FcγRIII. In some embodiments, the antibody (such as a full-length antibody) variants comprising an Fc region have ADCC activity in the presence of human effector cells (e.g., T cell) or have increased ADCC activity in the presence of human effector cells compared to the otherwise same antibody (such as a full-length antibody) comprising a human wild-type IgG1Fc region. Cysteine Engineered Variants

[0275] In some embodiments, it may be desirable to create cysteine engineered antibodies (such as a full-length antibody) in which one or more amino acid residues are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an antibody immunoconjugate, as described further herein. Cysteine engineered antibodies (e.g., full-length antibodies) may be generated as described, e.g., in U.S. Pat. No.7,521,541. Derivatives

[0276] In some embodiments, an antibody (such as a full-length antibody) provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are notlimited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc. Toxin (also referred as anti-tumor compounds, cytotoxic drugs or payloads)

[0277] Toxin linked to the antibody-drug conjugates of the application are described. The toxin is not particularly limited as long as it is a compound having an antitumor effect, a compound having a substituent capable of being linked to a linker structure, or a partial structure. In the case of a toxin, a part or all of the linker is cleaved in tumor cells to release the toxin moiety, thereby exhibiting antitumor effects.

[0278] There are several classes of small molecules with cytotoxicity for antibody drug conjugates, examples of the antitumor compound include doxorubicin (doxorubicin), daunorubicin (daunorubicin), mitomycin C (mitomycin C), bleomycin (bleomycin), amitabine (cyclidine), vincristine (vinblastine), vinblastine (vinblastine), methotrexate (methotrerate), a platinum-based antitumor agent (cisplatin or a derivative thereof), paclitaxel (taxol) or a derivative thereof, camptothecin or a derivative thereof (an antitumor agent described in Japanese patent application laid-open No. 6-87746), and the like. One class of these is camptothecin derivatives, which have an antitumor effect by inhibiting topoisomerase I.

[0279] TOPOISOMERASE I INHIBITORS

[0280] The toxin of the antibody-drug conjugate of the application is a topoisomerase I inhibitor. A topoisomerase I inhibitor is a compound, which is capable of forming a ternary complex with topoisomerase I and DNA, thereby preventing DNA re-ligation and introducing DNA strand breaks in the cellular genome. The topoisomerase I inhibitor may be e.g. selected from camptothecin or analogs thereof, indenoisoquinolines and indolocarbazoles.

[0281] In a particular embodiment, the topoisomerase- I-inhibitor is camptothecin or an analog thereof, i.e., a compound comprising the pentacyclic basic structure of camptothecin and modified substituents optionally resulting in the presence of a further ring. Specific examples are camptothecin, topotecan, irinotecan, SN-38, belotecan, exatecan including derivatives thereof such as deruxtecan, lurtotecan or atiratecan.

[0282] Exatecan

[0283] Exatecan, chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4- methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10,13(9H,15H)-dione) is a water soluble derivative of camptothecin, representing the formula below: topoisomerase I inhibitor is exatecan. In some embodiments,exatecan is typically conjugated to an antibody via its NH2group. The present inventors have been able to prepare an ADC with exatecan payloads that were found to display significant in vitro and in vivo efficacy. Linker

[0285] With regard to the antibody-drug conjugate of the application, the linker structure for conjugating an antitumor compound (e.g., exatecan) to the antibody is explained. The linker is represented by the following formula: -Q-L1-L2-L3-L4-.

[0286] The antibody is connected to the terminal of Q (opposite terminal to which L1is connected), and the anti-tumor compound (e.g., exatecan) is connected at its amino group to the carbonyl group or the C-terminal of L4.

[0287] 1. Q

[0288] Q comprises an attachment entity that conjugates to the antibody. In a further embodiment, the attachment entity may for example connect the antibody and the linker, where the attachment entity is the reaction product between an antibody amino acid side chain and a reactive attachment group (e.g., Q’ of the present application) in the linker precursor. Examples of an attachment entity comprising or consisting of maleimide moiety, N-hydroxysuccinimide,reactive attachment groups directed to modified or unmodified protein-bound carbohydrate, peptide sequences that are required for enzymatic reactions, azides or alkynes or being derived from these by reaction with the antibody or a chemically or enzymatically generated derivative thereof. In some embodiments, attachment entity is conjugated to cysteine using a maleimide group, which is commonly used to link a thiol group and a linker, because the nucleophilic reactivity of the thiol group of a cysteine residue to a maleimide group is about 1000 times greater than the amine group or the N-terminal amine group of other amino acid residues such as lysine residues.

[0289] In some embodiments, the attachment entity is derived from maleimidoacetic, maleimidopropionic, maleimidobutanoic, maleimidopentanoic, maleimidocaproic, maleimido- methyl-cyclohexanecarboxylic.

[0290] Preferably, Q represents the following formula: -(Succinimid-3-yl-N)-(CH2)n2-C(=O)-.

[0291] In the above formula, n2 is an integer of 2 to 8, preferably 2 to 5.

[0292] In the above formula, “-(Succinimid-3-yl-N)-” has a structure represented by the following formula:

[0293]

[0294] The structure above is connected to the antibody at position 3 thereof and is connected to a methylene group on the nitrogen atom at position 1. The connection to the antibody at position 3 is characterized by forming a thioether bond. The nitrogen atom at position 1 of structure moiety is connected to the carbon atom of methylene which is present within the linker including the structure.

[0295] Specifically, Ab-(Succinimid-3-yl-N)-(CH2)n2-C(=O)-L1- is a structure represented by the following formula:

[0296]

[0297] In e ormu a, n body-S- s der ved rom an an body.

[0298] In the formula, n2 is an integer of 2 to 8, and preferably 2 to 5.

[0299] Specific examples of Q include, but not limited to, the followings:

[0300] -(Succinimid-3-yl-N)-CH2-CH2-C(=O)- (abbreviated as “-Mal-” in the examples),

[0301] -(Succinimid-3-yl-N)-CH2-CH2-CH2-C(=O)-,

[0302] -(Succinimid-3-yl-N)-CH2-CH2-CH2-CH2-C(=O)-, or,

[0303] -(Succinimid-3-yl-N)-CH2-CH2-CH2-CH2-CH2-C(=O)- (abbreviated as “-MC-” in the examples).

[0304] 2. L1

[0305] L1 is a PEG derived linker moiety or a single bond.

[0306] Preferably, L1 represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(=O)- (abbreviated as “PEG(n1)” in the examples), and n1 is an integer of 1 to 24, optionally, n1 is an integer of 4 to 12.

[0307] L1may not be present. And in such a case, L1is a single bond.

[0308] L1is connected to Q with its terminal amino group and is connected to L2with the carbonyl group of the opposite terminal. More specifically, L1 is connected to the carbonyl group of Q with its terminal amino group and connected to the amino group or N-terminal of L2 with its carbonyl group.

[0309] Specific examples of L1include, but not limited to, the following:

[0310] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-,

[0311] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-,

[0312] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)- (abbreviated as “PEG(4)” in the examples), or,

[0313] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-.

[0314] 3. L2

[0315] L2 represents a peptide residue that is cleavable by lysosomal protease, allowing for intracellular release of the anti-tumor compound inside the target cells. In some embodiments, L2 is stable to serum proteases, yet is cleaved by lysosomal enzymes in the target cell. In a preferred embodiment, L2 represents a peptide residue that is cleavable by cathepsin B.

[0316] Specifically, L2 is connected to L1 (or Q in the case of L1 is absent) at its amino group or N-terminal and connected to L3at its carbonyl group or C-terminal. More specifically, L2is connected to the carbonyl group of L1 (or Q in the case of L1 is absent) at its amino group or N- terminal and connected to the amino group of L3at its carbonyl group or C-terminal.

[0317] Preferably, the peptide residue is a dipeptide (n = 2) or tripeptide (n = 3) residue, most preferably the peptide residue is a dipeptide residue. Although any peptide may be used, preferably the peptide residue is selected from -Val-Cit-, -Val-Ala-, -Val-Lys-, -Val-Arg-, -AcLys-Val-Cit-, -AcLys-Val-Ala-, -Glu-Val-Ala-, -Asp-Val-Ala-, -Phe-Cit-, -Phe-Ala-, -Phe-Lys-, -Phe-Arg-, - Ala-Lys-, -Leu-Cit-, -lle-Cit-, -Trp-Cit-, -Ala-Ala-Asn-, -Ala-Asn-Lys-, more preferably -Val-Cit-, -Val-Ala-, -Glu-Val-Ala-, -Val-Lys-, -Phe-Cit-, -Phe-Ala-, -Phe-Lys-, -Ala-Ala-Asn-, more preferably -Val-Cit-, -Val-Ala-, -Ala-Ala-Asn-, most preferably -Val-Cit- (abbreviated as “VC” in the examples) or -Val-Ala- (abbreviated as “VA” in the examples).

[0318] In a more preferred embodiment of the present application, -Val-Cit- has the structure of .

[0320] L3 represents a self-immolative spacer unit that may release the antitumor compound without a separate hydrolysis step.

[0321] L3 is connected to L2 at its amino group and to the L4 at its carbonyl group. More specifically, L3is connected to the carbonyl group or C-terminal of L2at its amino group, and to the amino group or N-terminal of L4at its carbonyl group.

[0322] In some embodiments, L3 is derived from p-aminobenzoic acid (PAB), para-aminobenzyl carbamate (PABC). In a particular embodiment, L3 is derived from PABC.

[0323] In a preferred embodiment, L3represents the following formula (also indicated as “-NH- phenyl-CH2-O-C(=O)-” and abbreviated as “-PABC-” or “P” in the examples):clude, but are not limited to, aromatic compounds that are electrically (electronically) equivalent to the PAB group, such as the residue of a 2- aminoimidazole-5-methanol derivative and the residue of an ortho-or para-aminobenzyl acetal.

[0326] 5. L4

[0327] L4represents a natural or unnatural amino acid residue, wherein the natural or unnatural amino acid residue is connected to -(NH-Exa) by an amide bond formed between the amine residue of -(NH-Exa) and the carboxylic residue of the natural or unnatural amino acid residue.

[0328] In some preferred embodiments, L4 is a natural α- amino acid residue, unnatural β-amino acid residue, or unnatural D-amino acid residue. In some more preferred embodiments, L4 is a glycine residue, alanine residue, β-alanine residue, or D-alanine residue.

[0329] With regard to the linker structure of the application, in some embodiments, the linker may have one of the following structures:

[0330] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2- C(═O)-,

[0331] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)- NH-CH2-C(═O)-,

[0332] - (Succinimid-3-yl-N)-CH2CH2-C(═O)-Val-Ala-NH-phenyl-CH2-O-C(=O)-NH-CH2- C(═O)-,

[0333] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-Val-Ala-NH-phenyl-CH2-O-C(=O)- NH-CH2-C(═O)-.

[0334] With regard to the linker structure of the application, in some embodiments, the linker may have one of the following structures:

[0335] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-, wherein n is an integer between 2 to 24, optionally, n is an integer of 4 to 12.

[0336] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-, wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12.

[0337] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-Val-Ala- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-, wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12, or,

[0338] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2- C(═O)-Val-Ala-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-, wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12.

[0339] With regard to the linker-structure of the application, in some embodiments, the linker may have one of the following structures:

[0340] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-,

[0341] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-,

[0342] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-,

[0343] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-,

[0344] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-,

[0345] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-,

[0346] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-, or,

[0347] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-.

[0348] In the linker structure mentioned above, wherein -(Succinimid-3-yl-N)- is a group represented by the following formula:

[0349]

[0350] In the linker structure mentioned above, wherein “-NH-phenyl-CH2-O-C(=O)-” is a group represented by the following formula:the linker-toxin compound which can conjugate to the antibody is explained. The linker-toxin compound is represented by the following formula: Q’- L1-L2-L3-L4-(NH-Exa). In some embodiments, the linker-toxin compound of the application is a production intermediate used in the production of the antibody-drug conjugate of the application.

[0353] In some embodiments, following known chemical procedures, synthesis of the linker- toxin compound was conducted.

[0354] The antibody reacts with the terminal of Q’ (opposite terminal to which L1is connected) resulting in a chemical bond. Upon reaction, Q’ is transformed into Q.

[0355] 1. Q’

[0356] Q’ corresponds to Q, comprising an attachment entity that can react and conjugate to the antibody.

[0357] In some embodiments, the attachment entity is derived from maleimidoacetic, maleimidopropionic, maleimidobutanoic, maleimidopentanoic, maleimidocaproic, or maleimido- methyl-cyclohexanecarboxylic.

[0358] Preferably, Q’ represents the following formula: (maleimido-N-yl)-(CH2)n2-C(=O)-.

[0359] In the above formula, n2 is an integer of 2 to 8, preferably 2 to 5.

[0360] In the above formula, “(maleimido-N-yl)-” has a structure represented by the following formula:above is connected to a methylene group on the nitrogen atom at position 1 and could react with Cys residue in an antibody resulting in a thioether bond at position 3. Thenitrogen atom at position 1 of structure moiety is connected to the carbon atom of methylene which is present within the linker including the structure.

[0363] Specifically, (maleimido-N-yl)-(CH2)n2-C(=O)-L1- (one of the examples of Q’-L1-) can react with Cys residue in an antibody resulting in Ab-(Succinimid-3-yl-N)-(CH2)n2-C(=O)-L1- (one of the examples of Ab-Q-L1-), which is a structure represented by the following formula:

[0364]

[0365]

[0366] In the formula, n2 is an integer of 2 to 8, and preferably 2 to 5.

[0367] Specific examples of Q’ include, but not limited to, the followings:

[0368] (maleimido-N-yl)-CH2-CH2-C(=O)- (abbreviated as “Mal-” in the examples),

[0369] (maleimido-N-yl)-CH2-CH2-CH2-C(=O)-,

[0370] (maleimido-N-yl)-CH2-CH2-CH2-CH2-C(=O)-, or,

[0371] (maleimido-N-yl)-CH2-CH2-CH2-CH2-CH2-C(=O)- (abbreviated as “MC-” in the examples).

[0372] 2. L1

[0373] L1 is a PEG derived linker moiety or a single bond.

[0374] Preferably, L1 represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(=O)- (abbreviated as “PEG(n1)” in the examples), and n1 is an integer of 2 to 24, optionally, n1 is an integer of 4 to 12.

[0375] L1 may not be present. And in such a case, L1 is a single bond.

[0376] L1 is connected to Q’ at its terminal amino group and is connected to L2 at the carbonyl group of the opposite terminal. More specifically, L1is connected to the carbonyl group of Q’ at its terminal amino group and connected to the amino group or N-terminal of L2 at its carbonyl group.

[0377] Specific examples of L1 include, but not limited to, the following:

[0378] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-,

[0379] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-,

[0380] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)- (abbreviated as “PEG(4)” in the examples), or,

[0381] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-.

[0382] 3. L2

[0383] L2 represents a peptide residue that is cleavable by lysosomal protease, allowing for intracellular release of the anti-tumor compound inside the target cells. In some embodiments L2is stable to serum proteases, yet is cleaved by lysosomal enzymes in the target cell. In a preferred embodiment, L2represents a peptide residue that is cleavable by cathepsin B.

[0384] Specifically, L2is connected to L1 (or Q’ in the case of L1is absent) at its amino group or N-terminal and connected to L3 at its carbonyl group or C-terminal. More specifically, L2 is connected to the carbonyl group of L1 (or Q’ in the case of L1is absent) at its amino group or N- terminal and connected to the amino group of L3 at its carbonyl group or C-terminal.

[0385] Preferably, the peptide residue is a dipeptide (n = 2) or tripeptide (n = 3) residue, most preferably the peptide residue is a dipeptide residue. Although any peptide may be used, preferably the peptide residue is selected from -Val-Cit-, -Val-Ala-, -Val-Lys-, -Val-Arg-, -AcLys-Val-Cit-, -AcLys-Val-Ala-, -Glu-Val-Ala-, -Asp-Val-Ala-, -Phe-Cit-, -Phe-Ala-, -Phe-Lys-, -Phe-Arg-, - Ala-Lys-, -Leu-Cit-, -lle-Cit-, -Trp-Cit-, -Ala-Ala-Asn-, -Ala-Asn- Lys-, more preferably -Val- Cit-, -Val-Ala-, -Glu-Val-Ala-, -Val-Lys-, -Phe-Cit-, -Phe-Ala-, -Phe-Lys-, -Ala-Ala-Asn-, more preferably -Val-Cit-, -Val-Ala-, -Ala-Ala-Asn-, most preferably -Val-Cit- (abbreviated as “VC” in the examples) or -Val-Ala- (abbreviated as “VA” in the examples).

[0386] In a more preferred embodiment of the present application, -Val-Cit- has the structure of .

[0388] L3 represents a self-immolative spacer unit may release the antitumor compound without a separate hydrolysis step.

[0389] L3is connected to L2at its amino group and to the L4at its carbonyl group. More specifically, L3 is connected to the carbonyl group or C-terminal of L2 at its amino group, and to the amino group or N-terminal of L4at its carbonyl group.

[0390] In some embodiments, L3is derived from p-aminobenzoic acid (PAB), para-aminobenzyl carbamate (PABC). In a particular embodiment, L3 is derived from PABC.

[0391] In a preferred embodiment, L3 represents the following formula (also indicated as “-NH- phenyl-CH2-O-C(=O)-” and abbreviated as “-PABC-” or “P” in the examples): but are not limited to, aromatic compounds that areto the PAB group, such as the residue of a 2- aminoimidazole-5-methanol derivative and the residue of an ortho-or para-aminobenzyl acetal.

[0394] 5. L4

[0395] L4 represents a natural or unnatural amino acid residue, wherein the natural or unnatural amino acid residue is connected to -(NH-Exa) by an amide bond formed between the amine residue of -(NH-Exa) and the carboxylic residue of the natural or unnatural amino acid residue.

[0396] In some preferred embodiments, L4 is a natural α- amino acid residue, unnatural β-amino acid residue, or unnatural D-amino acid residue. In some more preferred embodiments, L4 is a glycine residue, alanine residue, β-alanine residue, or D-alanine residue.

[0397]

[0398] 6. -(NH-Exa)

[0399] -(NH-Exa) represents the antitumor compound exatecan in the drug-antibody conjugate, which is a group represented by the following formula:group of the antitumor compound is connected to the carbonyl group or C-terminal of L4 (for example, Gly residue).

[0402] As for -Gly-(NH-Exa), it is a group represented by the following formula (also abbreviated as “Gly-Exa” in examples):up of the formula is connected to L3. More specifically, the amino group of the formula is connected to the carbonyl group of L3.

[0405] With regard to the linker-toxin compound of the present invention, in some embodiments, the linker-toxin compound may be represented by one of the following formulas:

[0406] (maleimido-N-yl)-CH2CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)- (NH-Exa),

[0407] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)- NH-CH2-C(═O)-(NH-Exa),

[0408] (maleimido-N-yl)-CH2CH2-C(═O)-Val-Ala-NH-phenyl-CH2-O-C(=O)-NH-CH2- C(═O)-(NH-Exa),

[0409] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-Val-Ala-NH-phenyl-CH2-O-C(=O)- NH-CH2-C(═O)-(NH-Exa).

[0410] With regard to the linker-toxins of the application, in some embodiments, the linker-toxin compound may be represented by one of the following formulas:

[0411] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12.

[0412] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12.

[0413] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-Val-Ala-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12, or,

[0414] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)- Val-Ala-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12.

[0415] With regard to the application, in some embodiments, the linker-toxin may be represented by one of the following formulas:

[0416] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0417] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0418] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0419] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0420] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0421] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0422] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), or,

[0423] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa).

[0424] In the linker-toxin compound mentioned above, wherein (maleimido-N-yl)- is a group represented by the following formula:toxin compound mentioned above, wherein “-NH-phenyl-CH2-O-C(=O)-” is a group represented by the following formula:

[0428] The linker-toxin compound mentioned above, wherein -(NH-Exa) is a group represented by the following formula, which has the nitrogen atom of the amino group at position 1 as a binding site.antibody conjugate of the application, the antibody-drug conjugate comprises an antibody and an antitumor compound connected by a linker, wherein the linker and the antitumor compound are represented by the formula: -Q-L1-L2-L3-L4-(NH-Exa).

[0430] 1. Q

[0431] Q comprises an attachment entity that conjugates to the antibody. In a further embodiment, the attachment entity may for example connect the antibody and the linker, where the attachment entity is the reaction product between an antibody amino acid side chain and a reactive attachment group in the linker precursor. Examples of an attachment entity comprising or consisting of maleimide moiety, N-hydroxysuccinimide, reactive attachment groups directed to modified or unmodified protein-bound carbohydrate, peptide sequences that are required for enzymatic reactions, azides or alkynes or being derived from these by reaction with the antibody or a chemically or enzymatically generated derivative thereof. In some embodiments, attachment entity is conjugated to cysteine using a maleimide group, which is commonly used to link a thiol group and a linker, because the nucleophilic reactivity of the thiol group of a cysteine residue to a maleimide group is about 1000 times greater than the amine group or the N-terminal amine group of other amino acid residues such as lysine residues.

[0432] In some embodiments, the attachment entity is derived from maleimidoacetic, maleimidopropionic, maleimidobutanoic, maleimidopentanoic, maleimidocaproic, maleimido- methyl-cyclohexanecarboxylic.

[0433] Preferably, Q is represented by the following formula: -(Succinimid-3-yl-N)-(CH2)n2- C(=O)-.

[0434] In the above formula, n2 is an integer of 2 to 8, preferably 2 to 5.

[0435] In the above formula, “-(Succinimid-3-yl-N)-” has a structure represented by the following formula:

[0436]

[0437] connected to the antibody at position 3 thereof and is connected to a methylene group on the nitrogen atom at position 1. The connection to the antibody at position 3 is characterized by forming a thioether bond. The nitrogen atom at position 1 of structure moiety is connected to the carbon atom of methylene which is present within the linker including the structure.

[0438] Specifically, Ab-(Succinimid-3-yl-N)-(CH2)n2-C(=O)-L1- is a structure represented by the following formula:

[0439]

[0440] In the formula, “Antibody-S-” is derived from an antibody.

[0441] In the formula, n2 is an integer of 2 to 8, and preferably 2 to 5.

[0442] Specific examples of Q include, but not limited to, the followings:

[0443] -(Succinimid-3-yl-N)-CH2-CH2-C(=O)- (abbreviated as “-Mal-” in the examples),

[0444] -(Succinimid-3-yl-N)-CH2-CH2-CH2-C(=O)-,

[0445] -(Succinimid-3-yl-N)-CH2-CH2-CH2-CH2-C(=O)-, or,

[0446] -(Succinimid-3-yl-N)-CH2-CH2-CH2-CH2-CH2-C(=O)- (abbreviated as “-MC-” in the examples).

[0447] 2. L1

[0448] L1is a PEG derived linker moiety or a single bond.

[0449] Preferably, L1represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(=O)- (abbreviated as “PEG(n1)” in the examples), and n1 is an integer of 2 to 24, optionally, n1 is an integer of 4 to 12.

[0450] L1may not be present. And in such a case, L1is a single bond.

[0451] L1is connected to Q at its terminal amino group and is connected to L2at the carbonyl group of the opposite terminal. More specifically, L1 is connected to the carbonyl group of Q at its terminal amino group and connected to the amino group or N-terminal of L2at its carbonyl group.

[0452] Specific examples of L1include, but not limited to, the following:

[0453] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-,

[0454] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-,

[0455] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)- (abbreviated as “PEG(4)” in the examples), or,

[0456] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-.

[0457] 3. L2

[0458] L2represents a peptide residue that is cleavable by lysosomal protease, allowing for intracellular release of the anti-tumor compound inside the target cells. In some embodiments L2 is stable to serum proteases, yet is cleaved by lysosomal enzymes in the target cell. In a preferred embodiment, L2 represents a peptide that is cleavable by cathepsin B.

[0459] Specifically, L2 is connected to L1 (or Q in the case of L1 is absent) at its amino group or N-terminal and connected to L3at its carbonyl group or C-terminal. More specifically, L2is connected to the carbonyl group of L1 (or Q in the case of L1 is absent) at its amino group or N- terminal and connected to the amino group of L3at its carbonyl group or C-terminal.

[0460] Preferably, the peptide residue is a dipeptide (n = 2) or tripeptide (n = 3) residue, most preferably is a dipeptide residue. Although any peptide may be used, preferably the peptide residue is selected from -Val-Cit-, -Val-Ala-, -Val-Lys-, -Val-Arg-, -AcLys-Val-Cit-, -AcLys-Val-Ala-, - Glu-Val-Ala-, -Asp-Val-Ala-, -Phe-Cit-, -Phe-Ala-, -Phe-Lys-, -Phe-Arg-, -Ala-Lys-, -Leu-Cit-, - lle-Cit-, -Trp-Cit-, -Ala-Ala-Asn-, -Ala-Asn-Lys-, more preferably -Val-Cit-, -Val-Ala-, -Glu-Val- Ala-, -Val-Lys-, -Phe-Cit-, -Phe-Ala-, -Phe-Lys-, -Ala-Ala-Asn-, more preferably -Val-Cit-, -Val- Ala-, -Ala-Ala-Asn-, most preferably -Val-Cit- (abbreviated as “VC” in the examples) or -Val- Ala- (abbreviated as “VA” in the examples).

[0461] In a more preferred embodiment of the present application, -Val-Cit- has the structure of .

[0463] L3 represents a self-immolative spacer unit may release the antitumor compound without a separate hydrolysis step.

[0464] L3is connected to L2at its amino group and to the L4at its carbonyl group. More specifically, L3 is connected to the carbonyl group or C-terminal of L2 at its amino group, and to the amino group or N-terminal of L4 at its carbonyl group.

[0465] In some embodiments, L3is derived from p-aminobenzoic acid (PAB), para-aminobenzyl carbamate (PABC). In a particular embodiment, L3is derived from PABC.

[0466] In a preferred embodiment, L3 represents the following formula (also indicated as “-NH- phenyl-CH2-O-C(=O)-” and abbreviated as “-PABC-” or “P” in the examples):but are not limited to, aromatic compounds that are electrically (electronically) equivalent to the PAB group, such as the residue of a 2- aminoimidazole-5-methanol derivative and the residue of an ortho-or para-aminobenzyl acetal.

[0469] 5. L4

[0470] L4 represents a natural or unnatural amino acid residue, wherein the natural or unnatural amino acid residue is connected to -(NH-Exa) by an amide bond formed between the amine residue of -(NH-Exa) and the carboxylic residue of the natural or unnatural amino acid residue.

[0471] In some preferred embodiments, L4is a natural α- amino acid residue, unnatural β-amino acid residue, or unnatural D-amino acid residue. In some more preferred embodiments, L4is a glycine residue, alanine residue, β-alanine residue, or D-alanine residue.

[0472] 6. -(NH-Exa)

[0473] -(NH-Exa) represents the antitumor compound exatecan in the drug-antibody conjugate, which has the following structure:o group of the antitumor compound is connected to the carbonyl group or C-terminal of L4(for example, Gly residue).

[0476] As for -Gly-(NH-Exa), it is a group represented by the following formula (also abbreviated as “Gly-Exa” in examples):of the formula is connected to L3. More specifically, the amino group of the formula is connected to the carbonyl group of L3.

[0479] With regard to the drug-antibody conjugate of the application, in some embodiments, the antibody-drug conjugate comprises an antibody and an antitumor compound connected by a linker, wherein the linker and the antitumor compound is represented by one of the following formulas:

[0480] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2- C(═O)-(NH-Exa),

[0481] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)- NH-CH2-C(═O)-(NH-Exa),

[0482] - (Succinimid-3-yl-N)-CH2CH2-C(═O)-Val-Ala-NH-phenyl-CH2-O-C(=O)-NH-CH2- C(═O)-(NH-Exa),

[0483] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-Val-Ala-NH-phenyl-CH2-O-C(=O)- NH-CH2-C(═O)-(NH-Exa).

[0484] With regard to the drug-antibody conjugate of the application, in some embodiments, the linker drug-antibody conjugate comprises an antibody and an antitumor compound connected bya linker, wherein the linker and the antitumor compound is represented by one of the following formulas:

[0485] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12.

[0486] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12.

[0487] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-Val-Ala- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12, or,

[0488] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2- C(═O)-Val-Ala-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12.

[0489] With regard to the drug-antibody conjugate of the application, in some embodiments, the drug-antibody conjugate comprises an antibody and an antitumor compound connected by a linker, wherein the linker and the antitumor compound is represented by one of the following formulas:

[0490] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0491] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0492] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0493] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0494] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0495] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0496] -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), or,

[0497] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa).

[0498] In the antibody-drug conjugate mentioned above, wherein -(Succinimid-3-yl-N)- is a group represented by the following formula:

[0499]

[0500] mentioned above, wherein “-NH-phenyl-CH2-O-C(=O)-” is a group represented by the following formula:

[0502] In the antibody-drug conjugate mentioned above, wherein “-(NH-Exa)” is a group represented by the following formula:in the antibody-drug conjugate mentioned above, the antibody is selected from humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and / or light chain CDRs discussed herein. Contemplated antibodies include, for example, full-length antibodies (e.g., full-length IgG1 or IgG4), scFvs, antibody Fc fusion proteins, multi-specific (such as bispecific) antibodies, immunoconjugates, and the like. In some embodiments, the antibody is a Fab, a Fab', a F(ab)'2, aFab'-SH, a single-chain Fv (scFv), an Fv fragment, a dAb, a Fd, a nanobody, a diabody, or a linear antibody.

[0505] In some embodiments of the application, in the antibody-drug conjugate mentioned above, the antibody is selected from the group consisting of an anti-TROP-2 antibody, an anti- HER2 (ErbB2) antibody, an anti-Folate Receptor (FR) α antibody, an anti-EGFR antibody, an anti- B7-H3 antibody, and an anti-EGFR antibody, anti-c-Met antibodies, anti-HER3 (ErbB3) antibodies, anti-HER4 (ErbB4) antibodies, anti-LIV-1 antibodies, anti-ROR1 antibodies, anti- CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD44 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD73 antibodies, anti-CD105 antibodies, anti-CEA antibodies, anti-A33 antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-G250 antibodies, anti-MUCl antibodies, anti-Lewis Y antibodies, anti-VEGFR antibodies, anti-GPNMB antibodies, anti-Integrin antibodies, anti-cancer antibodies. Preferably, the antibody is an anti-cancer antibody. More preferably, the antibody is selected from anti-TROP- 2 antibody, an anti-HER2 (ErbB2) antibody and an anti- Folate Receptor (FR) α antibody.

[0506] In a preferred embodiment of the application, in the antibody-drug conjugate mentioned above, the antibody is selected from Sacituzumab or Datopotamab.

[0507] In a preferred embodiment of the application, in the antibody-drug conjugate of mentioned above, the antibody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0508] In a preferred embodiment of the application, in the antibody-drug conjugate of mentioned above, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0509] In a more preferred embodiment of the application, in the antibody-drug conjugate mentioned above, the antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0510] In a preferred embodiment of the application, in the antibody-drug conjugate mentioned above, the antibody is selected from Trastuzumab, Disitamab, Pertuzumab, Zanidatamab or Hertuzumab.

[0511] In a preferred embodiment of the application, in the antibody-drug conjugate of mentioned above, the antibody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0512] In a preferred embodiment of the application, in the antibody-drug conjugate of mentioned above, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 17; and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0513] In a more preferred embodiment of the application, in the antibody-drug conjugate mentioned above, the antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:19, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:20.

[0514] In a preferred embodiment of the application, in the antibody-drug conjugate mentioned above, the antibody is selected from Luveltamab, Farletuzumab or Mirvetuximab.

[0515] In a preferred embodiment of the application, in the antibody-drug conjugate of mentioned above, the antibody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0516] In a preferred embodiment of the application, in the antibody-drug conjugate of mentioned above, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 27; and a VL comprising the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 28.

[0517] In a more preferred embodiment of the application, in the antibody-drug conjugate mentioned above, the antibody comprises: a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 29, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0518] Another antibody-drug conjugate is also disclosed in the present application, which comprises an anti-TROP2 antibody and an antitumor compound connected by a linker, wherein the linker and the antitumor compound is represented by the following formula:

[0519] -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-GGFG-NH-CH2-O-CH2-C(=O)- (NH-Exa),

[0520] wherein -(Succinimid-3-yl-N)- is a group represented by the following formula:

[0521]

[0522] -tetrapeptide residue of “-Gly-Gly-Phe-Gly-”, of which the structure ,the following formula: .antibody-drug conjugate of mentioned above, the anti- TROP2 antibody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL comprising a LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LC-CDR2 comprising the aminoacid sequence of SEQ ID NO: 5, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0526] In a preferred embodiment, in the antibody-drug conjugate of mentioned above, the anti- TROP2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0527] In a more preferred embodiment, in the antibody-drug conjugate mentioned above, the anti-TROP2 antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0528] The antibody-drug conjugates of the application can be prepared by any method known to those skilled in the art. In a preferred embodiment according to the present application, the antibody-drug conjugates of the application can be prepared by the conjugation of a cysteine present on the antibody to an electrophilic group of the linker-toxin moiety, preferably of a cysteine present on the antibody to a maleimide moiety present on the linker-toxin moiety.

[0529] The maleimide-cysteine coupling may be performed by methods well known to those skilled in the art.

[0530] Typically, antibodies do not contain many, if any, free and reactive cysteine thiols that can be attached to the drug-linker moiety. Most cysteine thiol residues in antibodies exist as disulfide bridges and must be reduced with a reducing agent such as Dithiothreitol (DTT) or TCEP under partially or fully reducing conditions. The loading (drug / antibody ratio) of the ADC can be controlled in a number of different ways, including: (i) limiting the molar excess of linker-toxin intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reduction conditions for cysteine thiol modification.

[0531] The disulfide bond structure of human IgG is now well defined (reviewed in Liu and May, mAbs 4(2012): 17-23). In fact, there are many similarities and some differences in disulfide bond structures for the 4 human IgG subclasses (i.e., IgG1, IgG2, IgG3, and IgG 4). All IgG subclasses always contain 12 intrachain disulfide bridges, and differences exist in the interchain disulfide bonds they form between heavy and light chains. Each intra-chain disulfide bond is associated with a single IgG domain, namely the variable (VL and VH) and constant (CL, CH1, CH2 and CH3) domains. The 2 heavy chains are connected at their hinge region by a variable number of disulfide bridges: there were 2 for IgG1 and IgG4, 4 for IgG2, and 11 for IgG 3. The heavy and light chains of IgG1 are linked by a disulfide bond between the last cysteine residue of the light chain and the fifth residue of the heavy chain, whereas for the other subclasses IgG2, IgG3 andIgG4, the light chain is linked to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the third cysteine residue of the heavy chain, which is located at the interface of the VH and CH1 domains. Disulfide bond structures of IgG2 and IgG4 in addition to these classical structures have been described (reviewed in Liu and May, mAbs 4(2012): 17-23). Interchain disulfide bonds are highly solvent exposed and therefore more active than intrachain disulfide bonds, which are buried in antiparallel beta-sheet structures within each domain and which are not solvent exposed. For these reasons, coupling will occur on interchain exposed cysteine residues after mild reduction, regardless of antibody isotype. Thus, each interchain disulfide bridge can theoretically form two conjugation sites.

[0532] Additional nucleophilic groups can be introduced into the antibody by reaction of lysine with 2-iminothiolane (Traut's) reagent, converting the amine to a sulfhydryl group. Reactive thiol groups can also be introduced into an antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., making a mutant antibody comprising one or more non-native cysteine amino acid residues). US 7521541 teaches the engineering of antibodies by the introduction of reactive cysteine amino acids.

[0533] Cysteine amino acids may be engineered at the reactive site of an antibody and they do not form intra-or intermolecular disulfide bridges (Junutula, et al, 2008b Nature Biotech.,26(8): 925-. The engineered cysteine thiol may be reacted with a linker reagent or linker-toxin reagent of the application having a thiol-reactive electrophilic group, such as maleimide or an alpha- haloamide, to form an ADC having a cysteine-engineered antibody and a drug moiety. Thus, the location of the drug moiety can be designed, controlled and known. Drug loading can be controlled because the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or linker-toxin reagents in high yields. IgG antibodies were engineered to introduce cysteine amino acids by substitution at a single site on either the heavy or light chain, creating two new cysteines on the symmetric antibody. Drug loading close to 2 can be achieved with the nearly homogeneous conjugation product ADC (homogeneity).

[0534] When more than one nucleophilic or electrophilic group of an antibody is reacted with a linker-toxin intermediate, or with a linker reagent and then with a toxin moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of toxin moieties attached to the antibody, e.g., 1,2,3, etc. Liquid chromatography, such as polymeric reversed phase (PLRP) and Hydrophobic Interaction (HIC), can separate compounds in a mixture by drug loading value. Formulations of ADCs with a single drug loading value (p) can be isolated, however, these single loading values of ADCs are still likely to be a heterogeneous mixture, as the toxin moiety can be attached to different sites of the antibody via a linker.

[0535] For some antibody-drug conjugates, the drug ratio may be limited by the number of attachment sites on the antibody. High drug loadings (e.g., drug ratios >5) can result in aggregation, insolubilization, toxicity or loss of cell permeability of certain antibody-drug conjugates. Typically, a drug moiety smaller than the theoretical maximum is conjugated to the antibody during the conjugation reaction.

[0536] Drug loading, also known as the drug-antibody ratio (DAR), is the average amount of drug per antibody.

[0537] In the case of antibodies IgG1 and IgG4 isotypes, where the drug is bound to cysteine after partial antibody reduction, the drug loading may be 1 to 8 drugs per antibody, i.e., where 1,2,3, 4,5, 6, 7, and 8 drug moieties are covalently linked to the antibody.

[0538] In the case of the antibody IgG2 isotype, where the drug is bound to cysteine after partial antibody reduction, the drug loading may be 1 to 12 drugs per antibody, i.e., where 1,2,3, 4,5, 6, 7, 8, 9, 10, 11, and 12 drug moieties are covalently linked to the antibody.

[0539] The compositions of ADCs comprise a collection of cell binding agents (e.g., antibodies) conjugated with 1 to 8 or 1 to 12 drugs.

[0540] The average number of drugs per antibody in the ADC preparation from the conjugation reaction can be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectrometry, ELISA assays and electrophoresis.

[0541] In some embodiments, the antibody-drug conjugate comprises any antibody, any liker or any linker toxin compounds described herein.

[0542] In a preferred embodiment, the present application provides a conjugation of toxin to an antibody via a special linker moiety, the antibody-drug conjugates reported herein ultimately provides (i) a homogeneous conjugation, (ii) an improved hydrophilic profile of the resulting ADC, (iii) an exquisite linker-toxin plasma stability, (iv) a potent in vivo activity against breast and gastric cancer models, and (v) a highly potent in vitro strong cytotoxicity against cancer cells. Production Method

[0543] Next, explanations are given for the representative method for producing the antibody- drug conjugate of the present invention or a production intermediate thereof. Meanwhile, the compounds are hereinbelow described with the number or letter shown in each reaction scheme. Specifically, they are referred to as “compound of the formula (a)”, “compound (a)”, or the like.

[0544] In a representative example, the antibody-drug conjugate of the present application can be produced by the processes as shown in Scheme 1.

[0545] Scheme 1:

[0546]

[0547] Process 1: Synthesis of H-L4-(NH-Exa) (Compound 1);

[0548] Process 2: Preparation of compounds represented by the formula of Q’-L1-L2-L3-PNP (Compound 2);

[0549] Process 3: Contacting Compound 1 with Compound 2, resulting in the linker-toxins represented by the formula of Q’-L1-L2-L3-L4-(NH-Exa) (Compound 3);

[0550] Process 4: Contacting compound 3 (also referred to as linker-toxin) obtained in Process 3 with antibody, resulting in the antibody-drug conjugate.

[0551] Process 1:

[0552] In the process 1, H-L4-(NH-Exa) (Compound 1) can be prepared by using exatecan as starting material.

[0553] L4 represents a natural or unnatural amino acid residue, wherein the natural or unnatural amino acid residue is connected to -(NH-Exa) by an amide bond formed between the amine residue of -(NH-Exa) and the carboxylic residue of the natural or unnatural amino acid residue.

[0554] In some preferred embodiments, L4is a natural α- amino acid residue, unnatural β-amino acid residue, or unnatural D-amino acid residue. In some more preferred embodiments, L4is a glycine residue, alanine residue, β-alanine residue, or D-alanine residue.

[0555] The reaction of the process is well-known in the art. Taking L4being Gly residue as an example, the exemplary reaction is shown in Scheme 2, which was also disclosed previously in publications (see, e.g., Nakada T et al, Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads, Bioorganic & Medicinal Chemistry Letters, 2016, 26(6):1542-1545, the content of which is incorporated herein by its entirety). The compound 1 can be used as starting materials to obtain the antibody-drug conjugate of the present application.

[0556] Scheme 2:

[0557]

[0558] In case .

[0559] Process 2

[0560] In the process, the intermediate compounds represented by the formula of Q’-L1-L2-L3- PNP (compound 2), which is used as one of starting material to prepare the linker-toxins of the present application, are synthesized.

[0561] In a preferred embodiment of the present application, the exemplary reactions of the process 2 are shown in Scheme 3, where there are 4 steps included.

[0562] Scheme 3:

[0563]

[0564] Wherein,

[0565] 1. Q’

[0566] Q’ comprises an attachment entity that can react and conjugate to the antibody. After conjugation, Q’ is transformed into Q.

[0567] In some embodiments, the attachment entity is derived from maleimidoacetic, maleimidopropionic, maleimidobutanoic, maleimidopentanoic, maleimidocaproic, or maleimido- methyl-cyclohexanecarboxylic.

[0568] Preferably, Q’ represents the following formula: (maleimido-N-yl)-(CH2)n2-C(=O)-.

[0569] In the above formula, n2 is an integer of 2 to 8, preferably 2 to 5.

[0570] In the above formula, “(maleimido-N-yl)-” has a structure represented by the following formula:above is connected to a methylene group on the nitrogen atom at position 1 and could react with Cys residue in an antibody resulting in a thioether bond at position 3. The nitrogen atom at position 1 of the structure moiety is connected to the carbon atom of methylene which is present within the linker including the structure.

[0573] Specifically, (maleimido-N-yl)-(CH2)n2-C(=O)-L1- (one of the examples of Q’-L1-) can react with Cys residue in an antibody resulting in Ab-(Succinimid-3-yl-N)-(CH2)n2-C(=O)- L1- (one of the examples of Ab-Q-L1-), which is a structure represented by the following formula:

[0574]

[0575] In the formula, “Antibody-S-” is derived from an antibody.

[0576] In the formula, n2 is an integer of 2 to 8, and preferably 2 to 5.

[0577] Specific examples of Q’ include, but not limited to, the followings:

[0578] (maleimido-N-yl)-CH2-CH2-C(=O)- (abbreviated as “Mal-” in the examples),

[0579] (maleimido-N-yl)-CH2-CH2-CH2-C(=O)-,

[0580] (maleimido-N-yl)-CH2-CH2-CH2-CH2-C(=O)-, or,

[0581] (maleimido-N-yl)-CH2-CH2-CH2-CH2-CH2-C(=O)- (abbreviated as “MC-” in the examples).

[0582] 2. L1

[0583] L1 is a PEG derived linker moiety or a single bond.

[0584] Preferably, L1 represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(=O)- (abbreviated as “PEG(n1)” in the examples), and n1 is an integer of 2 to 24, optionally, n1 is an integer of 4 to 12.

[0585] L1 may not be present. And in such a case, L1 is a single bond.

[0586] L1 is connected to Q’ at its terminal amino group and is connected to L2 at the carbonyl group of the opposite terminal. More specifically, L1 is connected to the carbonyl group of Q’ at its terminal amino group and connected to the amino group or N-terminal of L2at its carbonyl group.

[0587] Specific examples of L1 include, but not limited to, the following:

[0588] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-,

[0589] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-,

[0590] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)- (abbreviated as “PEG(4)” in the examples), or,

[0591] -NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)-.

[0592] 3. L2

[0593] L2represents a peptide residue that is cleavable by lysosomal protease, allowing for intracellular release of the anti-tumor compound inside the target cells. In some embodiments L2 is stable to serum proteases, yet is cleaved by lysosomal enzymes in the target cell. In a preferred embodiment, L2 represents a peptide residue that is cleavable by cathepsin B.

[0594] Specifically, L2 is connected to L1 (or Q’ in the case of L1 is absent) at its amino group or N-terminal and connected to L3 at its carbonyl group or C-terminal. More specifically, L2 is connected to the carbonyl group of L1(or Q’ in the case of L1is absent) at its amino group or N- terminal and connected to the amino group of L3 at its carbonyl group or C-terminal.

[0595] Preferably, the peptide residue is a dipeptide (n = 2) or tripeptide (n = 3) residue, most preferably the peptide residue is a dipeptide residue. Although any peptide may be used, preferably the peptide residue is selected from -Val-Cit-, -Val-Ala-, -Val-Lys-, -Val-Arg-, -AcLys-Val-Cit-,-AcLys-Val-Ala-, -Glu-Val-Ala-, -Asp-Val-Ala-, -Phe-Cit-, -Phe-Ala-, -Phe-Lys-, -Phe-Arg-, - Ala-Lys-, -Leu-Cit-, -lle-Cit-, -Trp-Cit-, -Ala-Ala-Asn-, -Ala-Asn-Lys-, more preferably -Val-Cit-, -Val-Ala-, -Glu-Val-Ala-, -Val-Lys-, -Phe-Cit-, -Phe-Ala-, -Phe-Lys-, -Ala-Ala-Asn-, more preferably -Val-Cit-, -Val-Ala-, -Ala-Ala-Asn-, most preferably -Val-Cit- (abbreviated as “VC” in the examples) or -Val-Ala- (abbreviated as “VA” in the examples).

[0596] In a more preferred embodiment of the present application, -Val-Cit- has the structure of .

[0598] L3represents a self-immolative spacer unit may release the antitumor compound without a separate hydrolysis step.

[0599] L3 is connected to L2 at its amino group and to the L4 at its carbonyl group. More specifically, L3 is connected to the carbonyl group or C-terminal of L2 at its amino group, and to the amino group or N-terminal of L4at its carbonyl group.

[0600] In some embodiments, L3is derived from p-aminobenzoic acid (PAB), para-aminobenzyl carbamate (PABC). In a particular embodiment, L3 is derived from PABC.

[0601] In a preferred embodiment, L3 represents the following formula (also indicated as “-NH- phenyl-CH2-O-C(=O)-” and abbreviated as “-PABC-” or “P” in the examples):but are not limited to, aromatic compounds that are electrically (electronically) equivalent to the PAB group, such as the residue of a 2- aminoimidazole-5-methanol derivative and the residue of an ortho-or para-aminobenzyl acetal.

[0604] In the compounds mentioned above, wherein (maleimido-N-yl)- is a group represented by the following formula:[00605 [00606 pounds mentioned above, wherein “-NH-phenyl-CH2-O-C(=O)-” is a group represented by the following formula:above, wherein PNP is a group represented by the following formula of “-O- .

[0609] Step (1):

[0610] In step (1), compound (a) is allowed to react with compound (b), which are commercially available, resulting the production of compound (c).

[0611] Specifically, in a preferred embodiment of the present application, compound (a) is dissolved in N,N-dimethylformamide (DMF) and mixed with compound (b). Then, triethylamine (TEA) is then added. The reaction is allowed to proceed at room temperature for several hours and compound (c) is obtained and purified by e.g., flash chromatography.

[0612] In some preferred embodiments, compound (a) may have the following structure represented by:

[0613] NH2-(CH2-CH2-O)n1-CH2-CH2-C(═O)-O-C(CH3)3 ),

[0614] wherein n1 is an integer of 2 to 24.

[0615] Specific examples of compound (a) include, but not limited to, the following:

[0616] NH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(═O)-O-C(CH3)3,

[0617] NH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(═O)-O-C(CH3)3,

[0618] NH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(═O)-O-C(CH3)3 or,

[0619] NH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(═O)- O-C(CH3)3.

[0620] In some preferred embodiments, compound (b) is selected from 6-maleimidohexanoic acid N-hydroxysuccinimide or 3-Maleimidopropionic acid N-succinimidyl, whose chemical , respectively.

[0621] one of the following structures:

[0622] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-O-C(CH3)3, wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12; or

[0623] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2- C(═O)-O-C(CH3)3(or, also ),

[0624] wherein n1 is an integerto 12.

[0625] In more preferred embodiments, compound (c) may have one of the following structures:

[0626] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-O-C(CH3)3,

[0627] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)- O-C(CH3)3,

[0628] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)-O-C(CH3)3,

[0629] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)- O-C(CH3)3,

[0630] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)-O-C(CH3)3,

[0631] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)- O-C(CH3)3,

[0632] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)-O-C(CH3)3, or,

[0633] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)- O-C(CH3)3.

[0634] Step (2):

[0635] In step (2), t-Butyl alcohol is removed from compound (c), resulting in compound (d).

[0636] Specifically, in a preferred embodiment of the present application, the reaction is allowed in the presence of trifluoroacetic acid (TFA) in dichloromethane (DCM).

[0637] In some preferred embodiments, compound (d) may have one of the following structures:

[0638] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-OH, wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12, or

[0639] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2- C(═O)-OH (or, also referred ),

[0640] wherein n1 is an of 4 to 12.

[0641] In more preferred embodiments, compound (d) may have one of the following structures:

[0642] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-OH,

[0643] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)- OH,

[0644] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)-OH,

[0645] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)- OH,

[0646] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)-OH,

[0647] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)- OH,

[0648] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)-OH, or,

[0649] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)- OH.

[0650] Step (3):

[0651] In step (3), compound (d) is allowed to react with compound (e) under the suitable conditions, resulting in compound (f).

[0652] Specifically, in some embodiments, the purified compound (d) is reacted with compound (e) in the presence of HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) and HOAt (1-Hydroxy-7- azabenzotriazole) in DMF at room temperature.

[0653] Compound (e) is commercially available or can be synthesized by well-known methods which are previously published (see, e.g., Dubowchik et al (Bioconjugate Chem. 2002, 13:855- 869), the context of which is incorporated herein by its entirety).

[0654] In a preferred embodiment, compound (e) may have the following structure of Val-Cit-NH-phenyl-CH2-OH, or more .

[0655] In some preferred of the following structures:

[0656] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-OH, wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12, or

[0657] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-OH (more preferred, ).integer of 4 to 12.

[0659] In some more preferred embodiments, compound (f) may have one of the following structures:

[0660] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-OH,

[0661] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-OH,

[0662] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-OH,

[0663] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-OH,

[0664] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-OH,

[0665] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-OH,

[0666] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-OH, or,

[0667] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-OH.

[0668] Step (4)

[0669] In step (4), compound 2 is synthesized from compound (f) under suitable conditions.

[0670] Specifically, in some embodiments, compound (f) is reacted with 4-nitrophenylchloroformate ( ) in DMF in the presence of DIEA (N,N-resulting in the production of compound 2. The product may be subject to further purification and confirmation.

[0671] In some preferred embodiments, compound 2 may have one of the following structures:

[0672] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-O-phenyl-NO2, wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12;

[0673] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-O-phenyl-NO2(more preferred, )

[0675] In more preferred embodiments, compound 2 may have one of the following structures:

[0676] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-O-phenyl-NO2,

[0677] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-O-phenyl-NO2,

[0678] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-O-phenyl-NO2,

[0679] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-O-phenyl-NO2,

[0680] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-O-phenyl-NO2,

[0681] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-O-phenyl-NO2,

[0682] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-O-phenyl-NO2, or,

[0683] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-O-phenyl-NO2.

[0684] Process 3

[0685] In the process, the linker-toxins (i.e., Compound 3) of the present application are produced and can be used in the production of antibody-drug conjugate.

[0686] In a representative example, compound 2 is allowed to contact with compound 1 in suitable condition in order to produce compound 3. Specifically, in a preferred embodiment, to a mixture of Compound 1, Compound 2, HOBt (Hydroxybenzotriazole), EDC-HCl (1-Ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride) in DMF, DIEA was added. The reaction mixture was stirred at room temperature for 4-5 hours. Then, the mixture was quenched with water and extracted with ethyl acetate followed by flashy chromatography to obtain the purified compound 3.

[0687] With regard to the compound 3 of the present invention, in some embodiments, the compound may have one of the following structures:

[0688] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), wherein n1 is an integer between 2 to 24, optionally, n1 is an integer of 4 to 12, or

[0689] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), or further preferred:

[0692] With regard to the application, in some embodiments, the compound 3 may have one of the following structures:

[0693] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0694] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0695] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0696] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)6-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0697] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0698] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)8-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa),

[0699] (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa), or,

[0700] (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)12-CH2-CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa).

[0701] In the compound 3 mentioned above, wherein (maleimido-N-yl)- is a group represented by the following formula:[00702 [00703 mpound 3 mentioned above, wherein “-NH-phenyl-CH2-O-C(=O)-” is represented by the following formula: above, wherein -(NH-Exa) is a group represented by thefollowing formula, which has the nitrogen atom of the amino group at position 1 as a binding site.

[0708] In the process, the antibody-drug conjugate can be produced by reacting the compound 3, which is obtained in the process 3, with antibody having a sulfhydryl group.

[0709] In some embodiments, the antibody is selected from humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and / or light chain CDRs discussed herein. Contemplated antibodies include, for example, full-length antibodies (e.g., full-length IgG1 or IgG4), scFvs, antibody Fc fusion proteins, multi-specific (such as bispecific) antibodies, immunoconjugates, and the like. In some embodiments, the antibody is a Fab, a Fab', a F(ab)'2, a Fab'-SH, a single-chain Fv (scFv), an Fv fragment, a dAb, a Fd, a nanobody, a diabody, or a linear antibody.

[0710] In some embodiments, the antibody is selected from the group consisting of an anti- TROP-2 antibody, an anti-HER2 (ErbB2) antibody, an anti-Folate Receptor (FR) α antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, and an anti-EGFR antibody, anti-c-Met antibodies,anti-HER3 (ErbB3) antibodies, anti-HER4 (ErbB4) antibodies, anti-LIV-1 antibodies, anti-ROR1 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD44 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD73 antibodies, anti-CD105 antibodies, anti-CEA antibodies, anti-A33 antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-G250 antibodies, anti-MUCl antibodies, anti-Lewis Y antibodies, anti-VEGFR antibodies, anti-GPNMB antibodies, anti-Integrin antibodies, anti-cancer antibodies. Preferably, the antibody is an anti-cancer antibody. More preferably, the antibody is selected from anti-TROP-2 antibody, an anti-HER2 (ErbB2) antibody and an anti- Folate Receptor (FR) α antibody.

[0711] In a preferred embodiment, the antibody is selected from Sacituzumab or Datopotamab.

[0712] In a preferred embodiment, the antibody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0713] In a preferred embodiment, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0714] In a more preferred embodiment, the antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0715] In a preferred embodiment, the antibody is selected from Trastuzumab, Disitamab, Pertuzumab, Zanidatamab or Hertuzumab.

[0716] In a preferred embodiment, the antibody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0717] In a preferred embodiment, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 17; and a VL comprising the amino acid sequence ofSEQ ID NO: 18, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0718] In a more preferred embodiment, the antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:19, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:20.

[0719] In a preferred embodimen, the antibody is selected from Luveltamab, Farletuzumab or Mirvetuximab.

[0720] In a preferred embodiment, the antibody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0721] In a preferred embodiment, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 27; and a VL comprising the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 28.

[0722] In a more preferred embodiment, the antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 29, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0723] The antibody-drug conjugates of the application can be prepared by any method known to those skilled in the art (see, e.g., Ting Shao et al., Signal Transduction and Targeted Therapy (2020)5:132; U.S. Pat. NO.10,195,288, the content of which are incorporated herein by its entirety). In a preferred embodiment according to the present application, the antibody-drug conjugates of the application can be prepared by the conjugation of a cysteine present on the antibody to an electrophilic group of the linker-toxin moiety, preferably of a cysteine present on the antibody to a maleimide moiety present on the linker-toxin moiety.

[0724] The antibody having a sulfhydryl group can be obtained by a method well known in the art (Hermanson, G.T, Bioconjugate , pp.56-136, pp.456-493, Academic Press (1996)). Examples include: Traut's reagent is reacted with the amino group of the antibody; N-succinim-idyl S- acetylthioalkanoates are reacted with the amino group of the antibody followed by reaction with hydroxylamine; after reacting with N-succinimidyl 3-(pyridyldithio)propi-onate, it is reacted with a reducing agent; the antibody is reacted with a reducing agent such as dithiothreitol, 2-mer-captoethanol, and tris(2-carboxyethyl)phosphine hydrochlo-ride (TCEP) to reduce the disulfide bond at a hinge part in the antibody to form a sulfhydryl group, but it is not limited thereto.

[0725] In a preferred embodiment, specifically, using 0.3 to 3 molar equivalents of TCEP as a reducing agent per disulfide bonds at hinge part in the antibody and reacting with the antibody in a buffer solution containing a chelating agent, the antibody which the disulfide bonds at hinge part in the antibody is partially or completely reduced can be obtained. Examples of the chelating agent include ethylenediamine tetraacetic acid (EDTA) and diethylenetriamine pentaacetic acid (DTPA). It can be used at concentration of 1 mM to 20 mM. Examples of the buffer solution which may be used include a solution of sodium phosphate, sodium borate, or sodium acetate. Specifically, by reacting the antibody with TCEP at 4 °C to 37 °C for 1 to 4 hours, the antibody having partially or completely reduced sulfhydryl groups can be obtained.

[0726] By performing a reaction of a sulfhydryl group to the compound 3, the linker-toxins can be conjugated to the antibody by a thioether bond, the methods of which are well-known in the art (see, e.g., Dubowchik et al, Bioconjugate Chem.2002, 13:855-869).

[0727] In an exemplary embodiment, using 2 to 20 molar equivalents of the compound 3 per the antibody having a sulfhydryl group, the antibody-drug conjugate in which 2 to 8 drug molecules are conjugated per antibody can be produced. Specifically, it is sufficient that the solution containing the compound 3 dissolved therein is added to a buffer solution containing the antibody having a sulfhydryl group for the reaction. Herein, examples of the buffer solution which may be used include sodium acetate solution, sodium phosphate, and sodium borate, pH for the reaction is 5 to 9, and more preferably the reaction is performed near pH7. Examples of the solvent for dissolving the compound 3 include an organic solvent such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP).

[0728] The reaction may be carried out by adding the organic solvent solution containing the compound 3 dissolved therein at 1 to 20% v / v to a buffer solution containing the antibody having a sulfhydryl group. The reaction temperature is 0 to 37 °C, more preferably 10 to 25 °C, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by deactivating the reactivity of unreacted compound 3 with a thiol-containing reagent. Examples of the thiol-containing reagent include cysteine and N-acetyl-L-cysteine (NAC). More specifically, by adding 1 to 2 molar equivalents of NAC to the compound 3 used and, by incubating at room temperature for 10 to 30 minutes, the reaction can be terminated.

[0729] The produced antibody-drug conjugate can be subjected to, after concentration, buffer exchange, purification, and measurement of antibody concentration and average number ofconjugated drug molecules pre antibody molecule, all of which procedures are well-known in the art. Pharmaceutical Compositions

[0730] A further aspect of the present invention is a pharmaceutical composition comprising the antibody-drug conjugate according to the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0731] Examples of suitable carriers and excipients for formulating antibody drug conjugates include saline and aqueous buffer solutions and are well known in the art. Typically, the pharmaceutical composition is adapted for parenteral administration, e.g., for subcutaneous, intramuscular or intravenous injection or by infusion.

[0732] The ADC according to the application may be in the form of a pharmaceutically acceptable salt.

[0733] In the present invention, "pharmaceutically acceptable" means that it can be used in the preparation of pharmaceutical compositions, is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and is acceptable for veterinary as well as human pharmaceutical use.

[0734] Substances used in a pharmaceutical composition containing antibody-drug conjugate of the present invention can be suitably selected and applied from formulation additives or the like that are generally used in the art, in view of the dosage or administration concentration.

[0735] The antibody-drug conjugate of the present invention can be administered as a pharmaceutical composition containing at least one pharmaceutically suitable ingredient.

[0736] For example, the pharmaceutical composition above typically contains at least one pharmaceutical carrier (for example, sterilized liquid). for example, water and oil (petroleum oil and oil of animal origin, plant origin, or synthetic origin (the oil may be, for example, peanut oil, soybean oil, mineral oil, sesame oil or the like)). Water is a more typical carrier when the pharmaceutical composition above is intravenously administered. Saline solution, an aqueous dextrose solution, and an aqueous glycerol solution can be also used as a liquid carrier, in particular, for an injection solution. A suitable pharmaceutical vehicle is known in the art. If desired, the composition above may also contain a trace amount of a moisturizing agent, an emulsifying agent, or a pH buffering agent. Examples of suitable pharmaceutical carrier are disclosed in “Remington's Pharmaceutical Sciences” by E. W. Martin. The formulations correspond to an administration mode.

[0737] Various delivery systems are known and they can be used for administering the antibody- drug conjugate of the present invention. Examples of the administration route include intradermal,intramuscular, intraperitoneal, intravenous, and subcutaneous routes, but not limited thereto. The administration can be made by injection or bolus injection, for example. According to a specific preferred embodiment, the administration of the antibody-drug conjugate is performed by injection. Parenteral administration is a preferred administration route.

[0738] According to a representative embodiment, the pharmaceutical composition is prescribed, as a pharmaceutical composition suitable for intravenous administration to human, according to the conventional procedures. The composition for intravenous administration is typically a solution in a sterile and isotonic aqueous buffer solution. If necessary, the drug may contain a solubilizing agent and local anesthetics to alleviate pain at injection site (for example, lignocaine). Generally, the ingredient above is provided individually as any one of lyophilized powder or an anhydrous concentrate contained in a container which is obtained by sealing in an ampoule or a sachet having an amount of the active agent or as a mixture in a unit dosage form. When the drug is to be administered by injection, it may be administered from an injection bottle containing water or saline of sterile pharmaceutical grade. When the drug is administered by injection, an ampoule of sterile water or saline for injection may be provided such that the aforementioned ingredients are admixed with each other before administration.

[0739] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the antibody-drug conjugate of the present invention or a pharmaceutical composition containing the antibody-drug conjugate and at least one cancer treating agent other than the conjugate. The antibody-drug conjugate of the present invention can be administered with other cancer treating agent. The anti-cancer effect may be enhanced accordingly. Another anti-cancer agent used for such purpose may be administered to an individual simultaneously with, separately from, or subsequently to the antibody-drug conjugate, and it may be administered while varying the administration interval for each. Examples of the cancer treating agent include abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinorelbine, drugs described in International Publication No. WO 2003 / 038043, LH-RH analogues (leuprorelin, goserelin, or the like), estramustine phosphate, estrogen antagonist (tamoxifen, raloxifene, or the like), and an aromatase inhibitor (anastrozole, letrozole, exemestane, or the like), but it is not limited as long as it is a drug having an antitumor activity.

[0740] The pharmaceutical composition can be formulated into a lyophilization formulation or a liquid formulation as a formulation having desired composition and required purity. When formulated as a lyophilization formulation, it may be a formulation containing suitable formulation additives that are used in the art. Also for a liquid formulation, it can be formulated as a liquid formulation containing various formulation additives that are used in the art.

[0741] Composition and concentration of the pharmaceutical composition may vary depending on administration method. However, the antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exhibit the pharmaceutical effect even at a small dosage when the antibody-drug conjugate has higher affinity for an antigen, that is, higher affinity (=lower Kd value) in terms of the dissociation constant (that is, Kd value) for the antigen. Thus, for determining dosage of the antibody-drug conjugate, the dosage can be determined in view of a situation relating to the affinity between the antibody-drug conjugate and antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg can be administered once or administered several times with an interval of one time for 1 to 180 days

[0742] By "pharmaceutically acceptable salt" of a compound is meant to a pharmaceutically acceptable salt, as defined herein, and which possesses the desired pharmacological activity of the parent compound.

[0743] The pharmaceutically acceptable salts comprise, inter alia:

[0744] (1) pharmaceutically acceptable acid addition salts with pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and the like; or a pharmaceutically acceptable acid addition salt with a pharmaceutically acceptable organic acid such as acetic acid, trifluoroacetic acid, propionic acid, succinic acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, methanesulfonic acid, stearic acid, lactic acid and the like; and

[0745] (2) pharmaceutically acceptable base addition salts formed when the acid proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, alkaline earth metal ion, or aluminum ion; or a pharmaceutically acceptable base addition salt formed when coordinated to a pharmaceutically acceptable organic base, such as lysine, arginine, and the like; or a pharmaceutically acceptable base addition salt formed when coordinated to a pharmaceutically acceptable inorganic base, such as sodium hydroxide, potassium hydroxide (potash), calcium hydroxide, and the like.

[0746] These salts can be prepared from the compounds of the application containing a base or acid functional group with the corresponding acid or base using conventional chemical methods. Medical Application

[0747] The antibody-drug conjugates according to the application are especially suitable in the treatment of cancer. The application thus further concerns the use of the antibody-drug conjugate according to the application in medicine. In a further aspect, the application also concerns a methodof treating a subject in need thereof, comprising administering the antibody-drug conjugate according to the application to the subject. The method according to this aspect can also be worded as the antibody-drug conjugate according to the application for use in treatment, in particular for use in the treatment of a subject in need thereof. The method according to this aspect can also be worded as use of the antibody-drug conjugate according to the application for the manufacture of a medicament. Herein, administration typically occurs with a therapeutically effective amount of the antibody-drug conjugate according to the application.

[0748] The application further concerns a method for the treatment of a specific disease in a subject in need thereof, comprising the administration of the antibody-drug conjugate according to the application as defined above. The specific disease may be selected from cancer and an autoimmune disease, preferably the disease is cancer. The subject in need thereof is typically a cancer patient. The use of antibody-drug conjugates is well-known in such treatments, especially in the field of cancer treatment, and the antibody-drug conjugates according to the application are especially suited in this respect. In the method according to this aspect, the antibody-drug conjugate is typically administered in a therapeutically effective amount. The present aspect of the application can also be worded as an antibody-drug conjugate according to the application for use in the treatment of a specific disease in a subject in need thereof, preferably for the treatment of cancer. In other words, this aspect concerns the use of an antibody-drug conjugate according to the application for the preparation of a medicament or pharmaceutical composition for use in the treatment of a specific disease in a subject in need thereof, preferably for use in the treatment of cancer. The antibody-drug conjugate of the present application can be preferably administered to a mammal, but it is more preferably administered to a human.

[0749] The cancer to be prevented and / or treated may be any kind of cancer, wherein the term "cancer or tumor" is used herein to refer to proliferative diseases.

[0750] The cancer may preferably be selected from target-associated cancers comprising all or part of the tumor cells expressing or overexpressing the target on their surface.

[0751] In one embodiment, the cancer is a HER2-positive cancer, such as HER2-positive breast cancer, HER2-positive stomach cancer, HER2-positive colon cancer, HER2-positive lung cancer, HER2-positive pancreatic cancer, HER2-positive urothelial cancer, HER2-positive brain cancer, HER2-positive ovarian cancer. Thus, in one embodiment, the patient is HER2-positive.

[0752] In one embodiment, the cancer is a TROP2-positive cancer, such as breast, ovarian, cervical, endometrial, lung, prostate, colorectal, stomach, esophageal, bladder, renal, pancreatic, thyroid, and head-and-neck cancer. The ADC may be of particular use for treatment of cancersthat are resistant to one or more standard anti-cancer therapies, such as colorectal cancer, pancreatic ductal cancer, triple-negative breast cancer or small-cell lung cancer.

[0753] In one embodiment, the cancer is a FRα-positive cancer. Overexpression of FRα was found in 90% epithelial ovarian cancers, as well as numerous other cancers including endometrial cancer, kidney cancer, lung cancer, mesothelioma, breast cancer, brain cancer, and myeloid leukemia, whereas most normal tissues express low to negligible levels (Coney et al., 1991, Cancer Res. 51:6125-6132). In certain embodiments, the cancer cell is a folate receptor alpha (FRα)- positive lymphoma or folate receptor alpha (FRα)-positive leukemia cell. In certain embodiments, the cancer is ovarian cancer, lung cancer, uterine cancer, testicular choriocarcinoma, ependymoma, mesothelioma, breast cancer, colon cancer, or renal cell carcinoma.

[0754] Depending on the stage and the severity of the disorder, the pharmaceutical composition or the antibody-drug conjugate of the application may be administered once or several times in a therapeutically effective dose to a subject in need thereof, particularly to a human subject. For example, it may be administered once or several times daily, each second day, two times weekly or weekly for a suitable period, e.g., of at least one week, or at least one month.

[0755] The antibody-drug conjugate may be administered alone or together with a further active agent, which may be selected from chemotherapeutic agents, e.g., anti-metabolites, alkylating agents, intercalating agents, or anti-mitotic agents), inhibitors of specific kinases e.g. tyrosine kinase inhibitors, serine / threonine kinase inhibitors or phosphoinositide kinase inhibitors, immunotherapeutic compounds, e.g., immune checkpoint inhibitors, CAR-T cells, therapeutic vaccines or oncolytic viruses. Kits

[0756] The application therefore also relates to a kit comprising at least i) an antibody-drug- conjugate according to the application and / or a pharmaceutical composition according to the application and ii) a syringe or vial or ampoule in which the antibody-drug-conjugate and / or the pharmaceutical composition is contained.

[0757] Their mode of administration, dosage and optimal pharmaceutical form can be determined according to criteria usually considered when establishing a treatment suitable for a patient, such as the age or weight of the patient, the severity of his / her overall condition, tolerance to the treatment and noted side effects.

[0758] Exemplary kits may contain at least one anti-Trop-2 ADC, anti-HER2 ADC, or anti-FRα ADC as described herein. If the composition containing components for administration is not formulated for delivery via the alimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included. One type of device, for applications such as parenteral delivery, is a syringe that is used to inject the composition into the body of a subject. Inhalation devices may also be used. In certain embodiments, an anti-Trop-2 ADC, anti-HER2 ADC, or anti-FRα ADC may be provided in the form of a prefilled syringe or autoinj ection pen containing a sterile, liquid formulation or lyophilized preparation of antibody (e.g., Kivitz et al., Clin. Ther.2006, 28:1619-29).

[0759] The kit components may be packaged together or separated into two or more containers. In some embodiments, the containers may be vials that contain sterile, lyophilized formulations of a composition that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents. Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers. Another component that can be included is instructions for use of the kit. EXAMPLES

[0760] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the application as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within. Example 1: Production of antibodies

[0761] Anti-Trop2 antibody (the same sequence as Sacituzumab, also referred to as hRS7)

[0762] hRS7 antibody was produced as described in U.S. Pat. NO. 7,238,785, the content of which was incorporated herein in its entirety by reference.

[0763] hRS7 antibody is a humanized IgG1κ antibody targeted against Trop2 (trophoblast antigen 2). The amino acid sequences of hRS7 antibody were shown in Table 2. Table 2 SEQ Description SequenceSEQ Description Sequence ID NO Q A L YI Q A L V S D L S D L L YI A

[0764] Anti-HER2 antibody (Trastuzumab)

[0765] Trastuzumab is a humanized IgG1κ antibody targeted against the extracellular domain of the human epidermal growth factor receptor 2 (HER2) and is indicated for the treatment of HER2-positive early or metastatic breast cancer. Trastuzumab (Herceptin®, Lot 9848160) waspurchased commercially and used without further modification. The amino acid sequences of trastuzumab were shown in Table 3. Table 3 SEQ Description Sequence ID NO P Q G T S I C V F QSEQ Description Sequence ID NO L

[0767] The anti-folate receptor (Anti-FRα) targeting antibody used in the examples is a humanized IgG1κ antibody. It exerts its activity against FRα positive cancer cells via multiple modes of action. The antibody was produced in accordance with a method known in the art. The amino acid sequences of the antibody are shown in Table 4. Table 4 SEQ ID NO Description Sequence A F S G I A F S W C F G KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS G I D E L

[0768] All ADC preparation involves three distinctive steps that have been previously reported elsewhere (see, for example, Ting Shao et al., Signal Transduction and Targeted Therapy (2020)5:132; U.S. Pat. NO. 10,195,288) with modifications that are specifically adapted to this particular linker-toxin.

[0769] 1) Reduction of antibody: the antibody solution was buffer exchanged with 10 mM pH5.0 acetate buffer. Protein concentration of the buffer exchanged antibody solution was determined by NanoDrop One Spectrophotometer at 280 nm using an appropriate extinction coefficient to make sure that the protein concentration was in the range of 1-10 mg / mL. A 10 mM Tris (2- carboxyethyl) phosphine hydrochloride (TCEP) in aqueous solution was prepared separately. The calculated amount of TCEP at 4-10:1 (TCEP to antibody) molar ratio was added to the antibody solution. The solution was agitated and then placed in a 37 °C water bath for exactly 2 hours.

[0770] 2) Conjugation between the linker-toxin and antibody: All the linker-toxin (i.e., linker- Exa toxin) were either purchased commercially or custom synthesized according to the known art in the field. If it was customarily synthesized, its purity is generally above 90% based on HPLC analysis. Following known chemical procedure, synthesis of the linker-toxin was conducted. The initial step is to synthesize exatecan amino acid amide. There are many ways to form an amide bond between the amino group of exatecan to the carboxylic group of an amino acid (e.g., Glycine). One of the methods is reported by Nakada T et al (Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads, Bioorganic & Medicinal Chemistry Letters, 2016, 26(6):1542-1545). Briefly, commercially available N-(tert- Butoxycarbonyl) glycine (BOC-Glycine) or other BOC protected amino acid was dissolved in dichloromethane. N-Hydroxysuccinimide (HO-Su) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (WSCI) were added. To this mixture, exatecan was added followed by addition of triethylamine solution in N,N’-dimethylformamide (DMF). The Boc- Glycine-exatecan was deprotected by trifluoroacetic acid (TFA). Other catalyst such as 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) may also be used to synthesize the exatecan amino acid amide. The purified exatecan amino acid amide can then react with commercially available maleimide Val-Cit linker such as Mal-Amide-PEG4-Val-Cit-PAB-PNP (BroadPharm, CA, USA, Cat # BP-40404) in the presence of an organic base, N,N-Diisopropylethylamine (DIPEA) in N,N’-dimethylformamide (DMF). The reaction can be done at room temperature for a couple of hours followed by purification by flash chromatography. MC-PEG4-Val-Cit-PABC-PNP is custom synthesized according to scheme described by Dubowchik et al (Bioconjugate Chem. 2002, 13:855-869).

[0771] The linker-toxins used in the examples represent the following formulas:

[0772] MC-VCP-Exa (also referred to as MC-VC-PABC-Exa): (maleimido-N-yl)- CH2CH2CH2CH2CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-(NH-Exa).

[0773] MC-PEG4-VCP-Gly-Exa (also referred to as MC-PEG4-VC-PABC-Gly-Exa): (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa).

[0774] Mal-PEG4-VCP-Gly-Exa (also referred to as Mal-PEG4-VC-PABC-Gly-Exa): (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH-phenyl- CH2-O-C(=O)-NH-CH2-C(═O)-(NH-Exa).

[0775] Each linker-toxin was weighed to a plastic tube, a suitable amount of dimethylformamide (DMF) was added so that the final linker-toxin concentration was around 10 mM. To the reduced antibody solution, the linker-toxin solution was then added at linker-toxin to antibody molar ratio 8-15:1. Conjugation reaction progression was checked by RP-HPLC.

[0776] 3) Purification: the ADC was purified by either buffer exchange or through a protein A affinity column. When buffer exchange was utilized, a suitable protein concentrator was used and generally 5 cycles of buffer exchange was needed with ~10 times volume reduction in each cycle. If a protein A column was used, the column was conditioned with 1×PBS. The ADC sample was then loaded and followed by 1×PBS wash and then 1×PBS with 0.5M sodium chloride solution wash. The ADC was then eluted off using a 10 mM citric acid (pH3.0) solution. The eluted solution was further buffer exchanged with 10 mM pH5.0 acetate buffer.

[0777] Drug-to-Antibody Ratio (DAR) Determination: DAR was determined using a RP-HPLC method reported by Jun Ouyang (Antibody-Drug Conjugates, Methods in Molecular Biology, Vol 1045, Laurent Ducry (ed.), pp 275-283). Briefly, 50 µL purified ADC at 1-5 mg / mL protein concentration was pipetted into a tube. Dithiothreitol (DTT) solution (5 µL, 0.5 M) and Tris buffer (1 µL, 1M pH8.0) were then added. The tube was incubated in a 37 °C water bath for 25 minutes. The solution was then injected onto a HPLC column for analysis. The weighted average DAR was obtained by calculating percentage of peak area for each assigned peak.

[0778] The RP-HPLC analysis was performed using the following conditions for conjugation reaction progress check as well as DAR analysis. HPLC System: Agilent 1100 with DAD detector Detection: 280 nm and 380 nm Column: Agilent Zorbax 300SB CN, 3.5 µ, 3.0 × 150 mm or Zorbax 300SB C8, 3.5 µ, 3.0 × 150 mm Column temperature: 65 °C Mobile phase A: water with 0.1% trifluoracetic acid (TFA) Mobile phase B: acetonitrile with 0.1% trifluoracetic acid (TFA) Flow rate: 0.8 mL / min Gradient program: 20-30% B (0-2 min), 30-60% B (2-20 min), 60-80% B (20-22 min), 80-80% B (22-24 min), 80-20% B (24-25 min), 20-20% B (25-26 min) Sample injection volume: 5-10 µL

[0779] Production of Trastuzumab ADC (1)

[0780] Trastuzumab-Maleimide propionic-PEG4-Val-Cit-PABC-Gly-Exa (Tras-Mal-PEG4- VCP-Gly-Exa) ADC Preparation, the formula of which is “Tras-(Succinimid-3-yl-N)-CH2CH2- C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2- C(═O)-(NH-Exa)”.

[0781] 100 µL Trastuzumab at protein concentration of 23.3 mg / mL was buffer exchanged using a VivaSpin500 MWCO 50,000 concentrator. After buffer exchange, the final volume was adjusted to 0.5 mL with the pH5.0, 10 mM acetate buffer. Protein concentration was measured to be 4.55 mg / mL by NanoDrop One and an extinction coefficient 1.48 mLmg-1cm-1. To that tube, 10 mM TCEP 12.3 µL at molar ratio 8:1 (TCEP: Trastuzumab) was added and the tube was vortexed and then placed in a 37 °C water bath for exactly 2 hours. The tube was cooled on ice and 27.4 µL linker-toxin (Mal-PEG4-VCP-Gly-Exa) DMF solution (5.6 mM) at 10:1 linker-toxin to Trastuzumab molar ratio was added. The tube was kept in a 4 °C refrigerator for 1 hour. HPLC analysis of the 1-hour sample showed there were no free light and heavy chain peaks. The sample was then purified by buffer exchange using the same VivaSpin500 concentrator.

[0782] Physicochemical characterization: Final obtained ADC protein concentration measured by NanoDrop One spectrophotometer at 280 nm was 4.29 mg / mL for a total of 0.5 mL. DAR was measured by RP-HPLC to be 7.92, and the yield was 94%.

[0783] Production of Trastuzumab ADC (2)

[0784] Trastuzumab-Maleimide Caproic-PEG4-Val-Cit-PABC-Gly-Exatecan (Tras-MC- PEG4-VCP-Gly-Exa) ADC Preparation, the formula of which is “Tras-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O- C(=O)-NH-CH2-C(═O)-(NH-Exa)”

[0785] The same procedure was used except the concentrator was Spin-X UF500 MWCO 30,000. After buffer exchange, Trastuzumab concentration was measured to be 4.36 mg / mL and therefore, 11.8 µL TCEP (10 mM) was added at molar ratio 8:1 (TCEP: Trastuzumab). Reduction was done in a 37 °C water bath for exactly 2 hours. The tube was cooled to room temperature followed by the addition of 18.4 µL of the linker-toxin (MC-PEG4-VCP-Gly-Exa) DMF solution (8.0 mM) at 10:1 linker-toxin to Trastuzumab molar ratio. The tube was kept at room temperature for 2.5 hours. The sample was then purified by buffer exchange using the same Spin-X UF500 concentrator.

[0786] Physicochemical characterization: Final obtained ADC protein concentration measured by NanoDrop One Spectrophotometer at 280 nm was 5.16 mg / mL for a total of 0.4 mL. DAR was measured by RP-HPLC to be 5.75, and the yield was 95%.

[0787] Production of Trastuzumab ADC (3)

[0788] Trastuzumab-Maleimide Caproic-Val-Cit-PABC-Exatecan (Tras-MC-VCP-Exa) ADC Preparation, the formula of which is “Tras-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(=O)-(NH-Exa)”

[0789] The same procedure was used as described above. After buffer exchange, trastuzumab concentration was measured to be 4.38 mg / mL and therefore, 11.8 µL TCEP (10 mM) was added at molar ratio 8:1 (TCEP: Trastuzumab). Reduction was done in a 37 °C water bath for exactly 2 hours. After reduction, the tube was cooled to room temperature. MC-VCP-Exa was purchased from MedChemExpress (Cat No. HY-145929, Lot 151333, Monmouth, NJ) and was dissolved in DMF. Its concentration was measured by RP-HPLC to be 7.42 mM. 19.9 µL of this linker-toxin solution was added into the reduced protein tube at 10:1 linker-toxin to antibody molar ratio. The tube was kept at room temperature for 2.5 hours. The sample was then purified by buffer exchange using the same Spin-X UF500 MWCO 30,000 concentrator.

[0790] Physicochemical characterization: Final obtained ADC protein concentration measured by NanoDrop One spectrophotometer at 280 nm was 2.39 mg / mL for a total of 0.5 mL. DAR was measured by RP-HPLC to be 7.73, and the yield was 55%.

[0791] Reference ADC: Enhertu®

[0792] Enhertu® (Fam-trastuzumab deruxtecan-nxki) is an ADC of the anti-Her2 antibody Trastuzumab with deruxtecan. Deruxtecan is an ADC linker-toxin conjugate composed of an DX- 8951 derivative (DXd) and a maleimide-GGFG peptide linker. This ADC is described by Ogitani et al., “DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1”, Clinical CancerResearch (22)20, October 15, 2016, pp. 5097- 5108 (DOI: 10.1158 / 1078-0432.CCR-15-2822). Enhertu® used in experiments was purchased commercially (Daiichi-Sankyo, Lot 9848160).

[0793] Production of hRS7 ADC (1)

[0794] hRS7-Maleimide Caproic-PEG4-Val-Cit-PABC-Gly-Exatecan (hRS7-MC-PEG4-VCP- Gly-Exa) ADC Preparation, the formula of which is “hRS7-(Succinimid-3-yl-N)- CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O- C(=O)-NH-CH2-C(═O)-(NH-Exa)”

[0795] To prepare this ADC, the general ADC preparation procedures described above were followed. After buffer exchange with 10 mM pH5.0 acetate buffer using a Spin-X UF (20 mL, 30k MWCO), the final hRS7 concentration was measured to be 8.55 mg / mL for a total of 4.0 mL. To 3.0 mL of the antibody solution, 138.6 µL TCEP (10 mM) was added at a molar ratio 8:1 (TCEP: hRS7). Reduction was done in a 37 °C water bath for exactly 2 hours. After reduction, the tube was cooled to room temperature and 1.4 mL of the reduced antibody solution was transferred to another tube. MC-PEG4-VCP-Gly-Exa linker-toxin solution in DMF (94.2 µL, 7.73mM) at 9:1 linker-toxin to hRS7 antibody molar ratio was added. The tube was kept at room temperature for 2 hours. The sample was then purified by buffer exchange using the same Spin-X UF 20 mL concentrator.

[0796] Physicochemical characterization: Final obtained ADC protein concentration measured by NanoDrop One spectrophotometer at 280 nm was 8.75 mg / mL for a total of 1.2 mL. DAR was measured by RP-HPLC to be 7.79, and the yield was 88%.

[0797] Production of hRS7 ADC (2)

[0798] hRS7-Maleimide propionic-PEG4-Val-Cit-PABC-Gly-Exatecan (hRS7-Mal-PEG4- VCP-Gly-Exa) ADC Preparation, the formula of which is “hRS7-(Succinimid-3-yl-N)-CH2CH2- C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH-CH2- C(═O)-(NH-Exa)”

[0799] To prepare this hRS7 ADC, the same procedure was used as described above. To 0.25 mL of the buffer exchanged hRS7 solution with protein concentration 2.55 mg / mL, 3.5 µL TCEP (10 mM) was added at molar ratio 8:1 (TCEP: hRS7). Reduction was carried out in a 37 °C water bath for exactly 2 hours. After reduction, the tube was cooled on ice. Mal-PEG4-VCP-Gly-Exa DMF solution (7.1 µL, 6.10 mM) was added into the reduced antibody solution at 10:1 linker- toxin to hRS7 antibody molar ratio. The tube was kept in a refrigerator overnight for a total of 17 hours. The sample was then purified by buffer exchange using the Spin-X UF500 with MWCO 30k concentrator.

[0800] Physicochemical characterization: Final obtained ADC protein concentration measured by NanoDrop One Spectrophotometer at 280 nm was 1.10 mg / mL for a total of 0.3 mL. DAR was measured by RP-HPLC to be 7.73, and the yield was 52%

[0801] Production of hRS7 ADC (3)

[0802] hRS7-Deruxtecan ADC (hRS7-MC-GGFG-NH-CH2-Dxd) Preparation

[0803] 0.25 mL reduced hRS7 antibody obtained from the above experiment was used to prepare hRS7-Deruxtecan. Deruxtecan was purchased from BroadPharm (Cat No BP-29531, Lot 20230119J, San Diego, CA).2.1 mg of the linker-toxin was dissolved in 0.20 mL DMF to make a 10 mM solution. Concentration measured by RP-HPLC was 8.27 mM. To 0.25 mL of the reduced hRS7 solution, 20.1 µL of this linker-toxin solution was added at 10:1 linker-toxin to hRS7 antibody molar ratio. The tube was kept in a refrigerator overnight for a total of 17 hours. The sample was then purified by buffer exchange using VivaSpin500 with MWCO 50k concentrator.

[0804] Physicochemical characterization: Final obtained ADC protein concentration measured by NanoDrop One Spectrophotometer at 280 nm was 5.06 mg / mL for a total of 0.3 mL. DAR was measured by RP-HPLC to be 7.53, and the yield was 62%.

[0805] Reference ADC: Trodelvy® (hRS7-CL2A-SN-38)

[0806] Trodelvy® (sacituzumab govitecan-hziy) that consists of a three-part combination of the hRS7 antibody and the cytotoxin SN-38 via the linker CL2A. Trodelvy® used in experiments was purchased commercially (Gilead, Lot 7530298).

[0807] Production of Anti-FRα ADC (1)

[0808] Anti-FRα-Maleimide Caproic-PEG4-Val-Cit-PABC-Gly-Exatecan (Anti-FRα-MC- PEG4-VCP-Gly-Exa) ADC Preparation, the formula of which is “Anti-FRα-(Succinimid-3-yl-N)- CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O- C(=O)-NH-CH2-C(═O)-(NH-Exa)”

[0809] To prepare this ADC, the same procedure was used as described above. After buffer exchange with 10 mM pH5.0 acetate buffer using a Spin-X UF500 MWCO 30,000 concentrator, the final anti-FRα antibody concentration was measured to be 2.74 mg / mL for a total of 0.5 mL. 7.4 µL TCEP (10 mM) was added at a molar ratio 8:1 (TCEP: Anti-FRα). Reduction was done in a 37 °C water bath for exactly 2 hours. After reduction, the tube was cooled on ice followed by addition of the linker-toxin (MC-PEG4-VCP-Gly-Exa) DMF solution (13.9 µL, 10 mM) at 15:1 linker-toxin to anti-FRα antibody molar ratio. The tube was kept at a refrigerated temperature for 2 hours. The sample was then purified by buffer exchange using the same Spin-X UF500 concentrator.

[0810] Physicochemical characterization: Final obtained ADC protein concentration measured by NanoDrop at 280 nm was 2.10 mg / mL for a total of 0.5 mL. DAR was measured by RP-HPLC to be 7.33, and the yield was 77%.

[0811] Production of Anti-FRα ADC (2)

[0812] Anti-FRα-Maleimide propionic -PEG4-Val-Cit-PABC-Gly-Exatecan (Anti-FRα-Mal- PEG4-VCP-Gly-Exa) ADC Preparation, the formula of which is “Anti-FRα-(Succinimid-3-yl-N)- CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O-C(=O)-NH- CH2-C(═O)-(NH-Exa)”

[0813] To prepare this ADC, the same procedure was used as described above. After buffer exchange with 10 mM pH5.0 acetate buffer using a Spin-X UF500 MWCO 30,000 concentrator, the final antibody concentration was measured to be 4.41 mg / mL for a total of 0.25 mL. 6.0 µL TCEP (10 mM) was added at a molar ratio 8:1 (TCEP: Anti-FRα). Reduction was done in a 37 °C water bath for exactly 2 hours. After reduction, the tube was cooled on ice followed by addition of the linker-toxin (Mal-PEG4-VCP-Gly-Exa) DMF solution (12.2 µL, 6.1 mM) at 10:1 linker-toxin to antibody molar ratio. The tube was kept at a refrigerated temperature for 2 hours. The sample was then purified by buffer exchange using the same Spin-X UF500 concentrator.

[0814] Physicochemical characterization: Final obtained ADC protein concentration measured by NanoDrop at 280 nm was 3.38 mg / mL for a total of 0.3 mL. DAR was measured by RP-HPLC to be 7.71, and the yield was 92%.

[0815] Production of Reference ADC: hmGFP ADC

[0816] hmGFP-Maleimide Caproic-PEG4-Val-Cit-PABC-Gly-Exatecan (hmGFP-MC-PEG4- VCP-Gly-Exa) ADC Preparation, the formula of which is “hmGFP-(Succinimid-3-yl-N)- CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2-CH2-C(═O)-Val-Cit-NH-phenyl-CH2-O- C(=O)-NH-CH2-C(═O)-(NH-Exa)”

[0817] The nontargeting control antibody hmGFP is a humanized mouse anti-GFP antibody (IgG1 / kappa) developed internally. The same procedure was used as described above. After buffer exchange three times with 10 mM pH5.0 acetate buffer using a Pierce Protein concentrator (MWCO 30,0002-6 mL), the final hmGFP concentration was measured to be 12.63 mg / mL for a total of 0.5 mL.34.1 µL TCEP (10 mM) was added at molar ratio 8:1 (TCEP: hmGFP). Reduction was done in a 37 °C water bath for exactly 2 hours. After reduction, the tube was cooled to room temperature followed by addition of the MC-PEG4-VCP-Gly-Exa DMF solution (59.4 µL, 6.46 mM) at 9:1 linker-toxin to hmGFP antibody molar ratio. The tube was kept at room temperature for 1.5 hours. The sample was them purified by buffer exchange using the same Pierce Protein concentrator.

[0818] Physicochemical characterization: Final obtained ADC protein concentration measured by NanoDrop One Spectrophotometer at 280 nm was 10.83 mg / mL for a total of 0.5 mL. DAR was measured by RP-HPLC to be 7.93, and the yield was 86%. Example 3: Evaluation of the stability of the Linker-Toxin and ADCs in vitro

[0819] In Vitro Linker-Toxin Stability Test:

[0820] Stability in 1×PBS (pH7.4) was performed by mixing 20 µL 10 mM linker-toxin DMF solution in 0.4 mL 1×PBS. The sample was then placed in a 37 °C incubator. Samples (50 µL) were taken periodically and stored in a -20 °C freezer until analysis. The RP-HPLC method was modified by lowing the column temperature to 45 °C and a shallow gradient program with 40% B at 20 min was utilized to analyze all samples.

[0821] For linker-toxin stability test in human plasma or serum, the linker-toxin was first capped with N-acetyl cysteine and then mixed with human plasma or serum. Briefly, 20 µL 10 mM linker- toxin solution in DMF was transferred into a tube, aqueous solution of N-acetyl cysteine (10 µL, 100 mM) and 1×PBS (5 µL) were then added to the tube. The tube was kept at room temperature for 30 min before addition of 0.4 mL human plasma or serum. The sample was then placed in a 37 °C water bath followed by sampling periodically. Human plasma or serum stability samples were treated by mixing with equal volume of acetonitrile followed by agitation and centrifugation. The top clear solution was then injected onto HPLC column for analysis.

[0822] Stability of several linker-toxins was accessed in both pH7.4 phosphate buffer (1×PBS, Thermo Fisher) and in human plasma or serum at 37 °C according to the procedures described above. An extra step was taken to conduct linker-toxin stability in human plasma or serum. Due to presence of various proteins in human plasma or serum, the linker-toxins were first capped with N-acetyl cysteine to prevent them from conjugating to serum / plasma proteins.

[0823] It was noted that Maleimide caproic-Val-Cit-PABC-Exatecan (MC-VCP-Exa) had limited water solubility. When the 10 mM DMF solution was added to 1×PBS, precipitation formed immediately and the precipitate was present during the entire study period. For deruxtecan and MC-PEG4-VCP-Gly-Exa, slight cloudiness was also observed when their DMF solutions were added to the buffer. However, once placed in the 37 °C incubator, both solutions are clear. Stability results of deruxtecan and MC-PEG4-VCP-Gly-Exa in 1×PBS are shown in FIG.1.

[0824] FIG. 1 showed that deruxtecan and MC-PEG4-VCP-Gly-Exa had nearly identical stability profiles. It shows that both can be quickly degraded in pH7.4 PBS buffer at 37 °C, most likely due to hydrolysis of the exatecan lactone structure.

[0825] However, unlike in phosphate buffer, no cloudiness formed when the samples were mixed with human plasma. The stability profiles in human plasma at 37 °C of the three linker- toxins above described were shown in FIG. 2. Results showed that these three linker-toxins hadnearly the same stability profile, indicating again that the instability is most likely due to the exatecan lactone structure and not the linker. Thus, in terms of stability, those linker-toxins are indistinguishable.

[0826] In Vitro ADC Stability Test:

[0827] Each ADC solution described above was added to plasma or serum and mixed well so that the final ADC concentration was in the range of 1-2 mg / mL. The tube was then placed in a 37 °C water bath followed by 50 µL sampling taken periodically afterward. The samples were kept at -20 °C freezer until analysis. In order to measure accurate stability changes including DAR changes of the ADC molecule, a RP-HPLC method was developed. The method utilizes the fact that high amount salt can precipitate the ADC thus it can be used to purify the ADC plasma or serum sample. With this RP-HPLC method, only the light chain with 1 toxin and the heavy chain with 3 toxins are used for stability determination although other potential ADC species (heavy chain with either 1 toxin or 2 toxins) may be present. Briefly, once the stability samples were thawed, ammonium sulfate solution (22.5 µL, 4M) was added to each sample and the sample was vortexed thoroughly. The tube was centrifuged at 10,000 rpm for 2 minutes and the top aqueous layer was carefully removed. A diluted ammonium sulfate solution (50 µL, a mixture of 2.25 mL 4M ammonium sulfate and 5.0 mL 10 mM pH5.0 acetate buffer) was added to the tube. The tube was vortex thoroughly and centrifuged again. The top aqueous layer was discarded and the pellet was redissolved in freshly added 50 µL 10mM pH5.0 acetate buffer. The sample was then analyzed using RP-HPLC method.

[0828] The results of stability of several ADCs in human plasma at 37 °C are shown in FIG.3A and FIG.3B. FIG.3A showed that Trodelvy® is less stable than hRS7-derextecan and hRS7-MC- PEG4-VCP-Gly-Exa for both light chain with 1 toxin and heavy chain with 3 toxins. On the other hand, hRS7-derextecan and hRS7-MC-PEG4-VCP-Gly-Exa display comparable stability in human plasma. FIG. 3B showed that both Tras-MC-PEG4-VCP-Gly-Exa and Anti-FRα-MC- PEG4-VCP-Gly-Exa have comparable stability to Enhertu®. Therefore, since deruxtecan and MC- PEG4-VCP-Gly-Exa display comparable stability in both pH7.4 phosphate buffer and human plasma, their ADCs with different antibodies will exhibit comparable stability.

[0829] Additional in vitro ADC stability studies were conducted in human serum and in cynomolgus monkey serum, the results of which were shown in FIG. 4 and FIG. 5, respectively. Again, the results showed that Trodelvy® was less stable than both hRS7-derextecan and hRS7- MC-PEG4-VCP-Gly-Exa and that hRS7-derextecan and hRS7-MC-PEG4-VCP-Gly-Exa had comparable stability profiles.Example 4: Binding Affinity of ADC

[0830] TROP2 Antigen and hRS7 Antibody interaction kinetic testing was carried out on the BLItz system using an Anti-IgG Fc Capture (AHC) biosensor, in order to detect the binding affinity to TROP2 antigen of hRS7 in ADC. After baseline equilibration with kinetic buffer for 30 seconds, a solution of 100nM hRS7, hRS7-MC-PEG4-VCP-Gly-Exa, or Trodelvy® in kinetic buffer was loaded onto the biosensor for 120 seconds followed by another equilibration for 30 seconds with kinetic buffer. TROP2 ECD at 250nM, 125nM, 62.5nM and 31.3nM was added onto the biosensor for 120 seconds. The system then performed dissociation in kinetic buffer for 240 seconds to allow the antigen to dissociate into solution.

[0831] As shown in Table 5, the hRS7-MC-PEG4-VCP-Gly-Exa, hRS7 antibody and Trodelvy® exhibited very similar binding affinity to Trop2, indicating the binding affinity to antigen was not impacted by conjugation in ADC. Table 5 Human Trop2 CompoundExample 5: Internalization of ADC

[0832] JIMT-1 is a cell line with TROP2 antigen expressed on cell membrane surface. Humanized antibodies (hmGFP and hRS7, as primary antibodies) and corresponding ADCs are mixed with a pH sensor fluorescent dye (pHAb) labelled anti-human IgG antibody (secondary antibody) before being used to treat JIMT-1 cells. hmGFP does not bind to any mammalian cell surface targets, serving as a negative control.

[0833] JIMT-1 cells were then incubated at 37°C for 18h to allow for internalization, followed by detection of fluorescence using flow cytometer.

[0834] The anti-human IgG secondary antibody was labelled with pH-sensitive dye pHAb, which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. When the primary antibodies along with the secondary anti-human antibody bind to the cell membranesurface receptor without internalization, there is no fluorescence because of the neutral pH of the media. However, when antibodies are internalized, i.e., entering inside of the cells, the pHAb becomes fluorescent when trafficking into endosome and lysosome, due to low pH in these vehicles.

[0835] The results of internalization assay were shown in FIG. 6A-6B, Black lines indicate the population of JIMT-1 cells treated only with secondary antibody. Gray lines indicate the population of JIMT-1 cells treated with primary antibody or ADC and secondary antibody.

[0836] As shown in FIG 6A, Gray lines almost overlap with the black lines. Little / no shift of the histograms indicates that no fluorescent signal was observed from secondary anti-human IgG antibodies, and thus neither hmGFP antibody nor hmGFP-ADC was internalized into JIMT-1 cells.

[0837] As shown in FIG.6B, big shifts of gray lines from black line were observed in the JIMT- 1 cells for both hRS7 antibody and hRS7 ADC. Thus, the pHAb fluorescence was observed in the populations of JIMT-1 cells incubated with hRS7 antibody or hRS7-MC-PEG4-VCP-Gly-Exa and secondary antibody, respectively, indicating that both hRS7 antibody and hRS7 ADC was internalized into JIM-T1 cells.

[0838] These results suggested that the toxin conjugation processes of the antibody did not impair the internalization function of the antibody. Example 6: In vitro cytotoxicity

[0839] Analysis of the potency of the linker-toxin and ADCs was carried out in vitro using a cell-based assay. Briefly, cancer cells, expressing the target antigens, were adhered to an assay plate and then the ADCs were serially diluted and added to the plate and allowed to incubate for 3-5 days. Following incubation, a fluorescent, resazurin sodium (from Thermo Scientific, Cat. No. B2118706, Lot N21I030) solution was added to the wells and then the signal was measured. IC50and max cell killing were calculated for each of the ADCs.

[0840] All ADCs and liker-toxins were tested for their cytotoxicity in vitro using the general experimental procedure described above. SK-BR-3 (from ATCC, Cat No. HTB-30, Lot 70040519), NCI-87 (from ATCC, Cat No. CRL-5822, Lot 70033475) and KB cells (from ECACC via Millipore Sigma, Cat. No. 94050408, Lot 18B048) were used in the assay. In addition, clinically available Enhertu® and Trodelvy® were also tested as a control. Results were shown in Table 6. Table 6. Cytotoxicity of Exatecan, Exatecan Linker-Toxins, and ADCsIC50Maximum Growth Test Article DAR Cell Line (nM) Inhibition

[0841] Table 6 showed that exatecan mesylate had strong cytotoxicity against SK-BR-3, NCI- N87, and KB cancer cell lines.

[0842] Both linker-toxins of Mal-PEG4-VCP-Gly-Exa and MC-PEG4-VCP-Gly-Exa exhibited little cytotoxic against any of these tested cell lines, indicating that both linker-toxins were stable and there was no toxin released during the test period.

[0843] Once the linker-toxin was conjugated to the targeting antibody, the resulting ADCs were very cytotoxic to their respective cell lines: for example, IC50 is 0.134 nM for Tras-MC-PEG4- VCP-Gly-Exa on SK-BR-3, 0.369 nM for hRS7-MC-PEG4-VCP-Gly-Exa on NCI-N87, and 0.213 nM for Anti-FRα-MC-PEG4-VCP-Gly-Exa on KB. In comparison, the respective non-targeting ADC, hmGFP-MC-PEG4-VCP-Gly-Exa, has very weak cytotoxicity against target cell line.

[0844] In addition, hRS7-MC-PEG4-VCP-Gly-Exa or hRS7-Mal-PEG4-VCP-Gly-Exa is significantly more cytotoxic against NCI-N87 than Trodelvy®. Tras-Mal-PEG4-VCP-Gly-Exa, DAR 7.92 and Tras-MC-PEG4-VCP-Gly-Exa, DAR 5.75 are more cytotoxic against SK-BR-3 than Enhertu®.

[0845] Moreover, Tras-MC-PEG4-VCP-Gly-Exa appears more effective against SK-BR-3 cells than Tras-MC-VCP-Exa, indicating that linker-toxin of MC-PEG4-VCP-Gly-Exa may be superior to MC-VCP-Exa in terms of anticancer activity. Example 7: Evaluation of Antitumor Effect of ADCs in vivo

[0846] Tumor growth inhibition study in mice: female athymic nude-Foxn1nu mice, 6-7 weeks of age, were obtained from Envigo RMS LLC (Indianapolis, IN) or other places. They were acclimated for a week and then were inoculated subcutaneously with cancer cells in 50% Matrigel. When tumors reached 100–200 mm3in size, mice were randomized into groups of 5 animals per group and dosed intravenously with saline, dextrose solution or ADCs at various doses. Tumor measurement was taken twice a week after test article injection. Relative Tumor Volume (RTV) was calculated according to the following formula: RTV = TVn / TV0, where TVnis the tumor volume at day n and TV0 is the tumor volume at day 0. The tumor growth inhibition (TGI) is calculated using the formula TGI (%) = (1 – T / C) × 100, where T / C is determined by calculating T / C = (mean RTV of treated group) / (mean RTV of control group).

[0847] In vivo antitumor efficacy test in mice tumor model with NCI-N87 cancer cells

[0848] hRS7 ADCs:

[0849] Human gastric cancer line NCI-N87 cells purchased from ATCC were suspended in physiological saline, and the cells were subcutaneously transplanted to the right side of the body of each female nude mouse, and the mice were randomly grouped when the tumor reached 100- 200 mm3in sizes. Both Trodelvy® and hRS7-MC / Mal-PEG4-VCP-Gly-Exa were diluted in 5% dextrose to 2 mg / mL and were intravenously administered at 10 mg / kg to the tail vein of eachmouse on that day (Day 0). The same dose was given again a week later for a total of two injections per mouse during the experimental period. A 0.9% saline solution administration group was established as a control group.

[0850] The results were shown in FIG.7A and 7B. FIG.7A showed tumor volume changes with time for each group and FIG. 7B displayed mice body weight changes. hRS7-MC-PEG4-VCP- Gly-Exa exhibited a much better antitumor effect than Trodelvy®. By Day 39, mice from both control and Trodelvy® groups had to be euthanized due to their tumor sizes and their overall body conditions while hRS7-MC-PEG4-VCP-Gly-Exa treated group were in good condition until the end of the study at 63 days. There are no significant body weight changes for all the study groups, which indicated that the ADC is well-tolerated. The results for hRS7-Mal-PEG4-VCP-Gly-Exa, which were similar to that of hRS7-MC-PEG4-VCP-Gly-Exa, were also observed (data not shown).

[0851] Trastuzumab ADCs:

[0852] Similar to the earlier in vivo TGI study with NCI-N87 for hRS7 ADCs, NCI-N87 cells were subcutaneously transplanted to the right side of the body of each female nude mouse, and the mice were randomly grouped when the tumor reached 90-200 mm3in sizes. Both hmGFP-MC- PEG4-VCP-Gly-Exa and Tras-MC / Mal-PEG4-VCP-Gly-Exa were diluted in 5% dextrose to 1 mg / mL and were intravenously administered at 5 mg / kg to the tail vein of each mouse on that day. The same dose was given again a week later for a total of two injections per mouse during the experimental period.0.9% saline solution administration group was established as a control group.

[0853] The results were shown in FIG.7C and 7D. FIG.7C showed tumor volume changes for each group and FIG. 7D displayed their body weight changes. Tras-MC-PEG4-VCP-Gly-Exa exhibited strong antitumor effect while hmGFP-hmGFP-MC-PEG4-VCP-Gly-Exa has only modest antitumor efficacy. By Day 12, the Tras-MC-PEG4-VCP-Gly-Exa groups yielded a TGI about 90% and the TGI was kept above 90% until the end of the study at Day 35. No significant body weight changes for the any of the groups were observed during the entire study period. Similar results were also observed for Tras-Mal-PEG4-VCP-Gly-Exa (data not shown).

[0854] In vivo antitumor efficacy test in mice tumor model with MDA-MB-468 Cancer Cells

[0855] In another antitumor efficacy study, human breast cancer cell line MDA-MB-468 was utilized. The cells were cultured and subcutaneously transplanted to the right side of the body of each female nude mouse as in the earlier study. When the tumor reached 100-200 mm3in sizes, they were randomly grouped and were given test articles by intravenous injection through the tail vein as described above. A 5% dextrose group was established as a control. All test articles were prepared by dilution in 5% dextrose to either 1 mg / mL or 2 mg / mL followed by aseptic filtrationinto pre-sterilized and depyrogenated vials. Each test article was given twice on Day 0 and again on Day 7.

[0856] The results were shown in FIG.8A and FIG 8B. It was noted that even the nontargeting hmGFP ADC has some antitumor effect but its antitumor effect is markedly weaker than either Trodelvy® or hRS7-MC-PEG4-VCP-Gly-Exa probably due to non-specific internalization. For hRS7-MC-PEG4-VCP-Gly-Exa treated groups, tumor growth inhibition (TGI) was surprisingly good for both the 5 mg / kg and 10 mg / kg, reached 99% on Day 30 and beyond. Besides, the antitumor effect in hRS7-MC-PEG4-VCP-Gly-Exa treated groups was much better than that in Trodelvy® treated group, either at the dose of 5 mg / kg or 10 mg / kg.

[0857] In addition, these mice maintained their regular body weight gain with no noticeable toxicity as shown in FIG.8B.

[0858] In vivo antitumor efficacy test in mice tumor model with HCC1806 Cancer Cells

[0859] In yet another antitumor efficacy study, human breast cancer cell line HCC1806 was utilized. This cell line has moderate Trop2 expression on its cell surface. The cells were cultured and subcutaneously transplanted to the right side of the body of each female nude mouse. When the tumor reached 100-200 mm3in sizes, they were randomly grouped and were given test articles by intravenous injection through the tail vein as described above. A saline group was established as a control. All test articles were prepared by dilution in 5% dextrose to 1 mg / mL, 2 mg / mL, or 4 mg / mL followed by aseptic filtration into pre-sterilized and depyrogenated vials. Each test article was given twice on Day 0 and again on Day 7.

[0860] The results were shown in FIG.9A and FIG.9B. Just like in MDA-MB-468 cell line, the non-targeting hmGFP ADC had some antitumor effect but its antitumor effect was transient lasting about 3 weeks. Trodelvy® had only a moderate antitumor effect against HCC1806 cancer cells. However, hRS7-MC-PEG4-VCP-Gly-Exa at all three dosing levels of 5, 10, and 20 mg / kg shrank the tumor to a barely measurable level after 2 weeks and the antitumor effect lasted during the entire study period.

[0861] In fact, in hRS7-MC-PEG4-VCP-Gly-Exa treated groups, after Day 30, 2 in 5 mice in the 5 mg / kg group, 3 in 5 mice in the 10 mg / kg group, and 4 in 5 mice in the 20 mg / kg group had complete tumor suppression without any sign of tumor at all.

[0862] In addition, these mice maintained their regular body weight gain with no noticeable toxicity as shown in FIG.9B.

[0863] In vivo antitumor efficacy test in mice tumor model with KB Cancer Cells

[0864] In another antitumor efficacy study, human KB cancer cell line is utilized. Four to five- week-old female SCID mice are quarantined for 7 days prior to study. Mice are inoculated subcutaneously with cells in PBS. When average tumor volume reaches about 80-180 mm3insizes, mice are randomized (typically 5-10 mice per group) and treated by a single intravenous injection of investigated antibody-drug conjugates (anti-FRα-Mal-PEG4-VCP-Gly-Exa, anti-FRα- MC-PEG4-VCP-Gly-Exa) at the doses of 5mg / kg and 10mg / kg. A saline group is established as a control. Tumor volumes are measured every 3-5 days.

[0865] It is expected that anti-FRα-Mal-PEG4-VCP-Gly-Exa and anti-FRα-MC-PEG4-VCP- Gly-Exa are efficacious in the mice tumor model and exhibit effective anti-tumor activity.

Claims

1. An antibody-drug conjugate, comprising an antibody and an antitumor compound connected by a linker, wherein the linker and the antitumor compound are represented by the formula of -Q-L1-L2-L3-L4-(NH-Exa), wherein， Q is an attachment entity that conjugates to the antibody, L1 represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-, or a single bond, and n1 is an integer of 2 to 24, optionally, n1 is an integer of 4 to 12, L2 represents a peptide residue that is cleavable by cathepsin B, L3 represents a self-immolative spacer unit that may release the toxin without a separate hydrolysis step, L4 represents a natural or unnatural amino acid residue, wherein the natural or unnatural amino acid residue is connected to -(NH-Exa) by an amide bond formed between the amine residue of -(NH-Exa) and the carboxylic residue of the natural or unnatural amino acid residue, and -(NH-Exa) is a group represented by the following formula:amino group at position 1 is the connecting site.

2. The antibody-drug conjugate according to claim 1, wherein Q conjugates to cysteine residues of the antibody and is derived from maleimidoacetic, maleimidopropionic, maleimidobutanoic, maleimidopentanoic, maleimidocaproic, or maleimido-methyl- cyclohexanecarboxylic.

3. The antibody-drug conjugate according to claim 1, wherein Q represents -(Succinimid- 3-yl-N)-(CH2)n2-C(═O)-, wherein n2 represents an integer of 2 to 8, and -(Succinimid-3-yl-N)-has a structure represented by the following formula:1which is connected to the antibody at position 3 thereof and is connected to a methylene group on the nitrogen atom at position 1.

4. The antibody-drug conjugate according to claim 3, wherein Q represents -(Succinimid- 3-yl-N)-(CH2)2-C(═O)- or -(Succinimid-3-yl-N)-(CH2)5-C(═O)-.

5. The antibody-drug conjugate according to claim 1, wherein L2 is a dipeptide or tripeptide residue.

6. The antibody-drug conjugate according to claim 5, wherein L2 is -Val-Cit-, -Val-Ala-, or -Phe-Lys-.

7. The antibody-drug conjugate according to claim 1, wherein L3 is -NH-phenyl-CH2-O- C(═O)- that has the following formula: .

8. The antibody-drug conjugate according to claim 1, wherein L4 is a natural α- amino acid residue, unnatural β-amino acid residue, or unnatural D-amino acid residue.

9. The antibody-drug conjugate according to claim 8, wherein L4 is a glycine residue, alanine residue, β-alanine residue, or D-alanine residue.

10. The antibody-drug conjugate according to claims 1, wherein the linker and the antitumor compound are represented by one of the following formulas: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2- C(═O)-Val-Cit-NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa),2-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2- C(═O)-Val-Ala-NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa), -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)-Val-Cit- NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa), or -(Succinimid-3-yl-N)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)-Val-Ala- NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa), Wherein, -(Succinimid-3-yl-N)- represents the following formula:to the antibody at position 3 thereof and is connected to a methylene group on the nitrogen atom at position 1, -(NH-Exa) represents the following formula:amino group at position 1 is the connecting position, and -NH-phenyl-CH2-O-C(═O)- represents the following formula:

3.

11. e compound represented by the following formula: Q’-L1- L2-L3-L4-(NH-Exa), wherein Q’ represents an attachment entity that can conjugate with amino acid residues on the antibody, L1 represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-, or a single bond, and n1 is an integer of 2 to 24, optionally, n1 is an integer of 4 to 12, L2 represents a peptide residue that is cleavable by cathepsin B, L3 represents a self-immolative spacer unit that may release the toxin without a separate hydrolysis step, L4 represents a natural or unnatural amino acid residue, wherein the natural or unnatural amino acid residue is connected to -(NH-Exa) by an amide bond formed between the amine residue of -(NH-Exa) and the carboxylic residue of the natural or unnatural amino acid residue, and -(NH-Exa) is a group represented by the following formula:amino group at position 1 is the connecting site.

12. The linker-toxin intermediate compound according to claim 11, wherein Q’ can conjugate to cysteine residues of the antibody.

13. The linker-toxin intermediate compound according to claim 12, wherein Q’ comprises (maleimido-N-yl)- group having the following formula:4w ogen atom is a connecting position.

14. The linker-toxin intermediate compound according to claim 12, wherein the Q’ is derived from the one selected from the group consisting of maleimidoacetic, maleimidopropionic, maleimidobutanoic, maleimidopentanoic, maleimidocaproic, and maleimido-methyl-cyclohexanecarboxylic.

15. The linker-toxin intermediate compound according to claim 12, wherein Q’ represents (maleimido-N-yl)-(CH2)n2-C(═O)-, and n2 is a integer of 2 to 5.

16. The linker-toxin intermediate compound according to claim 15 wherein Q’ represents (maleimido-N-yl)-CH2CH2-C(═O)- or (maleimido-N-yl)-CH2CH2CH2CH2CH2- C(═O)-.

17. The linker-toxin intermediate compound according to claim 11, wherein L1 represents -NH-(CH2-CH2-O)n1-CH2-CH2-C(═O)-, and n1 is an integer of 2 to 24, optionally, n1 is an integer of 4 to 12.

18. The linker-toxin intermediate compound according to claim 11, wherein L2is a dipeptide or tripeptide residue.

19. The linker-toxin intermediate compound according to claim 18, wherein L2is -Val- Cit-, -Val-Ala-，or -Phe-Lys-.

20. The linker-toxin intermediate compound according to claim 11, wherein L3is -NH- phenyl-CH2-O-C(═O)- that has the following formula:

521. The linker-toxin intermediate compound according to claim 11, wherein L4 is a natural α- amino acid residue, unnatural β-amino acid residue, or unnatural D-amino acid residue.

22. The linker-toxin intermediate compound according to claim 21, wherein L4 is a glycine residue, alanine residue, β-alanine residue, or D-alanine residue.

23. The linker-toxin intermediate compound according to claim 11, wherein, the linker- toxin intermediate compound has one of the following structures: (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)- Val-Cit-NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa), (maleimido-N-yl)-CH2CH2CH2CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)- Val-Ala-NH-phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa), (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)-Val-Cit-NH- phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa), or (maleimido-N-yl)-CH2CH2-C(═O)-NH-(CH2-CH2-O)4-CH2CH2-C(═O)-Val-Ala-NH- phenyl-CH2-O-C(═O)-NH-CH2-C(═O)-(NH-Exa), wherein (maleimido-N-yl)- represents the following formula:atom is the connecting position, -(NH-Exa) represents the following formula:6e amino group at position 1 is the connecting position, and -NH-phenyl-CH2-O-C(═O)- represents the following formula: .

24. The antibody-drug conjugate according to any one of claims 1-10, wherein the antibody targets a tumor cell, optionally the antibody is selected from an anti-HER2 antibody, an anti-TROP2 antibody, or an anti-FRα antibody.

25. The antibody-drug conjugate according to claim 24, wherein the anti-HER2 antibody is selected from Trastuzumab, Disitamab, Pertuzumab, Zanidatamab or Hertuzumab, the anti-TROP2 antibody is selected from Sacituzumab or Datopotamab, or the anti- FRα antibody is selected from Luveltamab, Farletuzumab or Mirvetuximab.

26. The antibody-drug conjugate according to claim 25, wherein the antibody comprises: (i) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC- CDR3 comprising the amino acid sequence of SEQ ID NO:6;7(ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an LC- CDR3 comprising the amino acid sequence of SEQ ID NO:16; or, (iii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and an LC- CDR3 comprising the amino acid sequence of SEQ ID NO:

26.

27. The antibody-drug conjugate according to claim 26, wherein the antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 8; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 17; and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 18; or (iii) a VH comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 27; and8a VL comprising the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

28.

28. The antibody-drug conjugate according to claim 25, wherein the antibody comprises: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and a light chain comprising the amino acid sequence of SEQ ID NO: 10; (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 19; and a light chain comprising the amino acid sequence of SEQ ID NO: 20; or (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and a light chain comprising the amino acid sequence of SEQ ID NO:

30.

29. A pharmaceutical composition containing the antibody-drug conjugate according to any one of claims 1-10, 24-28, or a salt thereof as an active component, and a pharmaceutically acceptable formulation component.

30. A method of treating cancer in an individual comprising administering to an individual the antibody-drug conjugate of any one of claims 1-10, 24-28, or the pharmaceutical composition of claim 29.

31. The method according to claim 30, wherein the cancer is lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, head and neck cancer, or esophageal cancer.9

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