WO2025199464A1

WO2025199464A1 - Anti-trop2 antibody-drug conjugates and methods of use

Info

Publication number: WO2025199464A1
Application number: PCT/US2025/020961
Authority: WO
Inventors: Jared Spidel; Earl F. Albone; Xin Cheng; Keiji FURUUCHI; Kazunobu Kira
Original assignee: Eisai R&D Management Co Ltd
Current assignee: Eisai R&D Management Co Ltd
Priority date: 2024-03-22
Filing date: 2025-03-21
Publication date: 2025-09-25
Anticipated expiration: 2026-09-22

Abstract

Antibodies, antigen-binding fragments, and conjugates (e.g., antibody-drug conjugates) thereof that bind TROP2 are disclosed, as well as improved linkers for antibody-drug conjugates. The disclosure further relates to methods and compositions for use in the treatment of, e.g., cancer by administering the compositions provided herein.

Description

ANTT-TROP2 ANTIBODY-DRUG CONJUGATES AND METHODS OF USE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/569,028, filed on March 22, 2024, the contents of which are incorporated by reference in their entirety.

[0002] The present disclosure relates to anti-TROP2 antibodies and antigen-binding fragments thereof, as well as conjugates such as antibody-drug conjugates (ADCs), e.g., those comprising eribulin, and their use in the treatment and diagnosis of cancers that express TROP2 and/or are amenable to treatment by disrupting tubulin or by administering a composition disclosed herein. [0003] Cancer is among the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. The most common causes of cancer death are cancers of: lung (1.59 million deaths); liver (745,000 deaths); stomach (723,000 deaths); colorectal (694,000 deaths); breast (521,000 deaths); and esophagus (400,000 deaths). The number of new cancer cases is expected to rise by about 70% over the next two decades, to approximately 22 million new cancer cases per year. World Cancer Report 2014. The incidence rate and mortality of rare cancers, such as bile duct cancer (also known as cholangiocarcinoma) have also been increasing worldwide in the past few decades. Banales et al. (2020) Nat Rev Gastroenterol Hepatol. 17:557-88. Unlike with more common types of cancers, the relative lack of awareness, knowledge, and effective treatment options for rare cancers, e.g., cholangiocarcinoma, has meant that patient prognosis has not improved substantially in the past decade. For example, recent reports suggest that the 5-year survival rate for cholangiocarcinoma is only 7-20%. Thus, there is an unmet need for more effective therapies for patients who suffer from rare cancer types, such as cholangiocarcinoma.

[0004] TROP2 (also known as tumor-associated calcium signal transducer 2) is a transmembrane glycoprotein encoded by the TACSTD2 gene in humans. Researchers have demonstrated that TROP2 is overexpressed in a variety of epithelial carcinomas, including, but not limited to, cholangiocarcinoma, thus making it an attractive target for antibody-based cancer therapy. Furthermore, TROP2 expression has been shown to be associated with increased tumor aggressiveness, metastasis, and decreased patient survival. Cubas et al. (2010) Mol. Cancer. 9:253. [0005] Recent studies have implicated TROP2 in multiple intracellular signaling pathways that regulate proliferation, survival, self-renewal, and invasion in cells. Shvartsur & Bonavida (2015) Genes & Cancer. 6(3-4):84-105. For example, through its role as a calcium signal transducer, it has been shown to result in the activation of the pro-oncogenic MAPK signaling pathway. Additionally, TROP2 may also play a role in deregulating stem cell functions through the Notch, Hedgehog, and Wnt/|3-catenin pathways. Goldenburg et al. (2018) Oncotarget. 9(48):28989-29006.

[0006] In addition to its role in pro-growth signaling pathways, TROP2 expression has been implicated in the epithelial-to-mesenchymal transition (EMT) process. EMT is a mechanism by which cells achieve phenotypic plasticity, and the process is closely associated with cancer metastasis and recurrence. He et al. (2017). Mol Cancer. 16:63. EMT is thought to be a key process by which cancer cells gain motility and the ability to migrate and invade other sites in the body. Studies have demonstrated that TROP2 is associated with the epithelial phenotype in certain types of cancers, including lung cancer, breast cancer, prostate cancer, and squamous cell carcinoma. Moreover, within these cancer types, TROP2-expressing epithelial cells lose TROP2 expression upon transitioning to a mesenchymal (i.e., invasive) state. Remsik et al. (2018) Oncotarget. 39(11): 1411-18. It is believed that TROP2 may promote EMT by binding and promoting nuclear accumulation of P-catenin, thereby activating downstream pro-metastatic pathways. Zhao et al. (2019) Cancer Med. 8(3): 1135-1147. TROP2 has also been shown to promote EMT in certain cancers through additional pro-oncogenic signaling axes, such as PI3K-AKT. Li et al. (2017) Oncotarget. 8(29):47052-47063. Indeed, inhibition and/or ablation of TROP2 expression has been shown to prevent the development of metastatic properties in cell lines across a broad spectrum of cancers, including gastric cancer, gall bladder cancer, breast cancer, prostate cancer, hepatocellular carcinoma, pancreatic cancer, and cholangiocarcinoma. Without wishing to be bound by theory, it is believed that inhibition of TROP2, e.g., binding by an anti-TROP2 antibody, antigen-binding fragment, and/or ADC, may reduce or prevent EMT in cancer cells. The role of TROP2 expression in EMT (and its converse pathway, mesenchymal-to-epithelial transition, or MET) is an active field of study and more research will be needed to completely elucidate the role of TROP2 in EMT and/or MET.

[0007] Given the prevalence of TROP2 expression in various cancer types, its role in promoting metastasis, and its association with poor clinical outcome, TROP2 is an attractive target for tumor antigen-specific drug delivery approaches, e.g., an antibody-mediated approach. Antibodies conjugated with cytotoxic compounds such as chemotherapeutics have also been explored to enhance the cell-killing activity of antibody-based drug delivery to tumor cells. Nevertheless, the need remains to provide suitable antibodies and/or ADCs that offer a combination of efficient tumor targeting, on- target effects, and/or reduced off-target effects.

[0008] Eribulin is a synthetic compound of the macrocyclic compound halichondrin B, which has been previously shown to exert potent anti-cancer properties through its disruption of tubulin and microtubule dynamics. Towle et al. (2001) Cancer Res. 61(3): 1013-21. Tubulin makes up dynamic filamentous cytoskeletal proteins that are involved in a variety of cellular functions, including intracellular migration and transport, cell signaling, the maintenance of cell shape, and cell division. Due to their rapid cell division, cancer cells are generally more sensitive to compounds that bind to tubulin and disrupt its normal function, as compared to normal cells. For this reason, tubulin inhibitors and other microtubule-targeted agents have become a promising class of drugs for the treatment of cancer. Dumontet and Jordan (2010) Nat. Rev. DrugDiscov. 9:790-803. Halinchondrin B and eribulin in particular have demonstrated notable anti-cancer activities in vitro and in vivo. Tan et al. (2009) Clin Cancer Res. 15(12): 4213-4219; Vahdat et al. (2009) J. Clin. Oncol. 27(18): 2954-2961. The mesylate salt of eribulin (eribulin mesylate) is currently marketed under the trade name Halaven™ for the treatment of patients with refractory metastatic breast cancer.

[0009] While uses of eribulin have been reported in the art, including in the context of ADCs, there remains an unmet need to deliver eribulin in a targeted fashion to particular tissues, e.g., cancer tissues that express TROP2. Likewise, there remains a need in the art for improved antibodies that bind TROP2 with superior properties, e.g. , with respect to antigen-binding and/or the ability to effectively deliver payloads such as eribulin to a target cell or tissue expressing TROP2.

SUMMARY OF THE INVENTION

[0010] In various embodiments, the present disclosure provides, in part, novel antibodies and antigenbinding fragments that are capable of specifically binding TROP2 and may be used alone or linked to one or more additional agents (e.g., as ADCs) and administered as part of pharmaceutical compositions. In some embodiments, the antibodies, antigen-binding fragments, and/or ADCs of the present disclosure may be used to slow, inhibit, and/or reverse tumor growth in mammals, and may be useful for treating human cancer patients.

[0011] The present disclosure more specifically relates, in various embodiments, to antibodies and antibody-drug conjugate compounds that are capable of binding and/or killing TROP2-expressing cells. In various embodiments, the compounds are also capable of internalizing into a target TROP2- expressing cell after binding. ADC compounds comprising a linker that attaches eribulin drug moiety to an antibody moiety are disclosed. An antibody moiety may be a full-length antibody or an antigenbinding fragment.

[0012] In various embodiments, the present disclosure provides an internalizing anti-TROP2 antibody or internalizing antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to human TROP2, and wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1 , LCDR2, and LCDR3), wherein the HCDR1 comprises an amino acid sequence of SEQ ID NO: 18, the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 19 or 20, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 21, the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 22 or 23, the LCDR2 comprises an amino acid sequence selected from SEQ ID NO: 24 or 25, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26, as defined by the Kabat numbering system; or the HCDR1 comprises an amino acid sequence of SEQ ID NO: 27, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 28, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 29, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 30, the LCDR2 comprises an amino acid sequence selected from SEQ ID NO: 31 or 32, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 33, as defined by the IMGT numbering system; or the HCDR1 comprises an amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 2 or 3, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 4, the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 5, 6, 7, or 8, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10, as defined by the Kabat numbering system; or the HCDR1 comprises an amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 12, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 13, the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 14 or 15, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 16, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 17, as defined by the IMGT numbering system.

[0013] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 18 (HCDR1), SEQ ID NO: 20 (HCDR2), and SEQ ID NO: 21 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 22 (LCDR1), SEQ ID NO: 24 (LCDR2), and SEQ ID NO: 26 (LCDR3), as defined by the Kabat numbering system. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 18 (HCDR1), SEQ ID NO: 20 (HCDR2), and SEQ ID NO: 21 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 23 (LCDR1), SEQ ID NO: 24 (LCDR2), and SEQ ID NO: 26 (LCDR3), as defined by the Kabat numbering system. In some embodiments, the anti-TROP2 antibody or antigenbinding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 27 (HCDR1), SEQ ID NO: 28 (HCDR2), and SEQ ID NO: 29 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 30 (LCDR1), SEQ ID NO: 31 (LCDR2), and SEQ ID NO: 33 (LCDR3), as defined by the IMGT numbering system.

[0014] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 5 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 14 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the anti-TROP2 antibody or antigenbinding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system.

[0015] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 67, 68,

69, 70, or 177, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 71, 72, 167, 73, 74, 75, 76, 187, 77, or 78. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 53, 54, 55, 56, 174, or 175, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 57, 58, 166, 59, 60, 61, 62, 63, 176, 64, 65, or 66.

[0016] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:

70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77.

[0017] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[0018] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[0019] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant region. [0020] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises an IgGl Fc domain or an IgGl Fc domain mutated to reduce binding to a Fey receptor (FcyR) as compared to an IgGl Fc-containing antibody with a wild-type IgGl Fc domain. In some embodiments, the mutated IgGl Fc domain comprises the mutations L234A, L235A, P238S, H268Q, and K274Q.

[0021] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[0022] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 118, 119, 122, 123, 126, 127, or 381 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 129, 130, 133, or 134. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 96, 97, 100, 101, 104, 105, 179, 181, 183, or 380 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 107, 110, 112, or 115.

[0023] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[0024] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises part of a bispecific or multi-specific binding construct. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment is linked to a therapeutic agent or detectable agent.

[0025] In various embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is an anti-TROP2 antibody or antigen-binding fragment thereof disclosed herein; D is a cytotoxic agent; L is a cleavable linker that covalently attaches Ab to D; and p is an integer from 1 to 8.

[0026] In some embodiments, the cytotoxic agent comprises eribulin or a salt thereof.

[0027] In some embodiments, p is from 2 to 8. In some embodiments, p is 2.

[0028] In some embodiments, the cleavable linker comprises a cleavable moiety that is positioned such that no part of the linker or the antibody or antigen-binding fragment remains bound to the cytotoxic agent upon cleavage.

[0029] In some embodiments, the cleavable linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety is cleavable by an enzyme. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin B. In some embodiments, the cleavable peptide moiety is cleavable by legumain.

[0030] In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the amino acid unit comprises valine-citrulline (Val-Cit). In some embodiments, the amino acid unit comprises valine-dimethylated lysine (Val-Lys(Me)₂). In some embodiments, the amino acid unit comprises alanine-dimethylated lysine (Ala-Lys(Me)₂). In some embodiments, the amino acid unit comprises valine-alanine (Vai- Ala). In some embodiments, the amino acid unit comprises asparagine (Asn). In some embodiments, the amino acid unit comprises aspartic acid (Asp). In some embodiments, the amino acid unit comprises methylated aspartic acid (Asp(OMe)).

[0031] In some embodiments, the cleavable linker attaches to the antibody or antigen-binding fragment via a maleimide (Mai or MAL) moiety. In some embodiments, the Mai moiety comprises a maleimidocaproyl (MC) moiety. In some embodiments, the Mai moiety comprises a dithiomaleimide (DTM) moiety. In some embodiments, the Mai moiety is reactive with a cysteine residue on the antibody or antigen-binding fragment. In some embodiments, the Mai moiety is joined to the antibody or antigen-binding fragment via a cysteine residue on the antibody or antigen-binding fragment.

[0032] In some embodiments, the cleavable linker comprises the Mai moiety and a cleavable peptide moiety. In some embodiments, the Mai moiety attaches the antibody or antigen-binding fragment to the cleavable peptide moiety in the linker. In some embodiments, the cleavable linker comprises Mal- Val-Cit. In some embodiments, the cleavable linker comprises Mal-Val-Lys(Me)₂. In some embodiments, the amino acid unit comprises Mal-Ala-Lys(Me)₂. In some embodiments, the cleavable linker comprises Mal-Val-Ala. In some embodiments, the cleavable linker comprises Mal-Asn. In some embodiments, the cleavable linker comprises Mal-Asp. In some embodiments, the cleavable linker comprises Mal-Asp(OMe).

[0033] In some embodiments, the cleavable linker comprises at least one spacer unit. In some embodiments, the spacer unit comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety comprises -(PEG)_m- and m is an integer from 1 to 10. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4.

[0034] In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the alkyl moiety comprises -(CH₂)_n- and n is an integer from 1 to 10. In some embodiments, n is 5.

[0035] In some embodiments, the spacer unit comprises “C₂”

[0036] In some embodiments, the spacer unit comprises C₂ conjugated to a PEG moiety, wherein the PEG moiety comprises -(PEG)_m- and m is an integer from 1 to 4. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. [0037] In some embodiments, the spacer unit comprises “C₂(PEG)₂” as shown in the following chemical structure:

[0038] In some embodiments, the spacer unit attaches to the antibody or antigen-binding fragment via the maleimide (Mai) moiety ("Mal-spacer unit"). In some embodiments, the cleavable linker comprises the Mal-spacer unit and the cleavable peptide moiety. In some embodiments, the Mal- spacer unit attaches the antibody or antigen-binding fragment to the cleavable peptide moiety in the linker.

[0039] In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises Mal-(PEG)₂-Val-Cit. In some embodiments, the Mal-spacer unit comprises Mal-(PEG)₃-Val-Cit. In some embodiments, the Mal-spacer unit comprises Mal-(PEG)₄- Val-Cit. In some embodiments, the Mal-spacer unit comprises Mal-(PEG)₂-Val-Lys(Me)₂. In some embodiments, the Mal-spacer unit comprises Mal-(PEG)₂-Ala-Lys(Me)₂. In some embodiments, the Mal-spacer unit comprises Mal-(PEG)₂-Val-Ala. In some embodiments, the Mal-spacer unit comprises Mal-(PEG)₂-Asn. In some embodiments, the Mal-spacer unit comprises Mal-(PEG)₂-Asp. In some embodiments, the Mal-spacer unit comprises Mal-(PEG)₂-Asp(OMe).

[0040] In some embodiments, the Mal-spacer unit comprises a C₂ moiety.

[0041] In some embodiments, the Mal-spacer unit comprises C₂ conjugated to a PEG moiety, wherein the PEG moiety comprises -(PEG)_m- and m is an integer from 0 to 4. In some embodiments, m is 2. [0042] In some embodiments, the Mal-spacer unit comprises a C₂(PEG)_m moiety. In some embodiments, the Mal-spacer unit comprises Mal-C₂(PEG)_m-Val-Cit. In some embodiments, the Mal- spacer unit comprises Mal-C₂(PEG)_m-Val-Lys(Me)₂. In some embodiments, the Mal-spacer unit comprises Mal-C₂(PEG)_m-Ala-Lys(Me)₂. In some embodiments, the Mal-spacer unit comprises Mal- C₂(PEG)_m-Val-Ala. In some embodiments, the Mal-spacer unit comprises Mal-C₂(PEG)_m-Asn. In some embodiments, the Mal-spacer unit comprises Mal-C₂(PEG)_m-Asp. In some embodiments, the Mal-spacer unit comprises Mal-C₂(PEG)_m-Asp(OMe). In some embodiments, m is an integer from 0 to 4. In some embodiments, m is 2.

[0043] In some embodiments, the cleavable moiety in the linker is directly joined to the cytotoxic agent.

[0044] In some embodiments, a spacer unit attaches the cleavable moiety in the linker to the cytotoxic agent. In some embodiments, cleavage of the conjugate releases the cytotoxic agent from the antibody and linker. In some embodiments, the spacer unit attaching the cleavable moiety in the linker to the cytotoxic agent is self-immolative. In some embodiments, the spacer unit attaching the cleavable moiety in the linker to the cytotoxic agent comprises a p-aminobenzyl (pAB). In some embodiments, the spacer unit attaching the cleavable moiety in the linker to the cytotoxic agent comprises a p- aminobenzyloxycarbonyl (pABC). In some embodiments, the pAB attaches the cleavable moiety in the linker to the cytotoxic agent. In some embodiments, the cytotoxic agent is eribulin. In some embodiments, the pAB covalently attaches to eribulin via a C-35 amine. In some embodiments, the cleavable moiety comprises Val-Cit, Vai-Ala, Val-Lys(Me)₂, Ala-Lys(Me)₂, Asn, Asp, or Asp(OMe). [0045] In some embodiments, the cleavable linker comprises Val-Cit-pABC. In some embodiments, the cleavable linker comprises Val-Ala-pABC. In some embodiments, the cleavable linker comprises Val-Lys(Me)₂-pABC.

[0046] In some embodiments, the cleavable linker comprises DTM-Asn.

[0047] In some embodiments, the cleavable linker comprises Mal-(PEG)₂-Val-Cit-pABC. In some embodiments, the cleavable linker comprises Mal-(PEG)₃-Val-Cit-pABC. In some embodiments, the cleavable linker comprises Mal-(PEG)₄-Val-Cit-pABC. In some embodiments, the cleavable linker comprises Mal-(PEG)₂-Val-Lys(Me)₂-pABC. In some embodiments, the cleavable linker comprises Mal-(PEG)₂-Val-Ala-pABC. In some embodiments, the cleavable linker comprises Mal-C₂(PEG)₂- Val-Cit-pABC. In some embodiments, the cleavable linker comprises Mal-C₂(PEG)₂-Val-Lys(Me)₂- pABC. In some embodiments, the cleavable linker comprises Mal-C₂(PEG)₂-Val-Ala-pABC. In some embodiments, the cleavable linker comprises Mal-C₂(PEG)₂-Ala-Lys(Me)₂-pABC.

[0048] In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 5 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 14 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system.

[0049] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[0050] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 96 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 96 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. [0051] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is an internalizing anti-TROP2 antibody or internalizing antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system;

D is eribulin;

L is a cleavable linker comprising Mal-(PEG)₂-Val-Cit-pABC; and p is an integer from 1 to 8.

[0052] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is an internalizing anti-TROP2 antibody or internalizing antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system;

D is eribulin;

[0053] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

D is eribulin;

L is a cleavable linker comprising Mal-(PEG)₂-Val-Ala-pABC; and p is an integer from 1 to 8.

[0054] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is an internalizing anti-TR0P2 antibody or internalizing antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system;

D is eribulin;

[0055] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

D is eribulin;

L is a cleavable linker comprising Mal-(PEG)₂-Asn; and p is an integer from 1 to 8.

[0056] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

D is eribulin;

[0057] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

D is eribulin;

L is a cleavable linker comprising Mal-C₂(PEG)_m-Val-Cit-pABC, wherein m is an integer from 0 to 4; and p is an integer from 1 to 8.

[0058] In some embodiments, m is 2.

[0059] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

D is eribulin;

[0060] In some embodiments, m is 2.

[0061] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

D is eribulin;

L is a cleavable linker comprising Mal-C₂(PEG)_m-Val-Ala-pABC, wherein m is an integer from 0 to 4; and p is an integer from 1 to 8.

[0062] In some embodiments, m is 2.

[0063] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

D is eribulin;

[0064] In some embodiments, m is 2.

[0065] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

D is eribulin;

L is a cleavable linker comprising Mal-C₂(PEG)_m-Asn, wherein m is an integer from 0 to 4; and p is an integer from 1 to 8.

[0066] In some embodiments, m is 2.

[0067] In some embodiments, the present disclosure provides an antibody-drug conjugate of Formula (I):

D is eribulin;

[0068] In some embodiments, m is 2. [0069] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[0070] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[0071] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[0072] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 96 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 96 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[0073] In some embodiments, p is 2.

[0074] In some embodiments, p is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). In some embodiments, p is determined by reversephase liquid chromatography-mass spectrometry (LC-MS).

[0075] In some embodiments, the cleavable linker covalently attaches to the cytotoxic agent via a C- 35 amine. In some embodiments, the cleavable linker covalently attaches to the antibody or antigenbinding fragment via a cysteine or a lysine.

[0076] In various embodiments, the present disclosure provides a composition comprising multiple copies of an antibody-drug conjugate disclosed herein. In some embodiments, the average p of the antibody-drug conjugates in the composition is about 1 to about 3. In some embodiments, the average p is about 2.

[0077] In various embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate disclosed herein and a pharmaceutically acceptable carrier. [0078] In various embodiments, the present disclosure provides one or more nucleic acid(s) encoding an antibody or antigen-binding fragment disclosed herein. In some embodiments, the present disclosure provides a host cell comprising the one or more nucleic acids. In some embodiments, the present disclosure provides a method of producing an antibody or antigen-biding fragment disclosed herein, comprising culturing the host cell under conditions sufficient to produce the antibody or antigen-binding fragment.

[0079] In various embodiments, the present disclosure provides a method of producing an antibodydrug conjugate, comprising reacting an antibody or antigen-binding fragment disclosed herein with a cleavable linker joined to eribulin under conditions that allow conjugation.

[0080] In other embodiments, the present disclosure provides an antibody-drug conjugate of Formula (III): wherein: Ab is an antibody or antigen-binding fragment thereof;

D is a cytotoxic agent;

Y is a cleavable moiety;

Z is absent or a spacer unit; and p is an integer from 1 to 8.

[0081] In some embodiments, the cytotoxic agent comprises eribulin or a salt thereof.

[0082] In some embodiments, p is from 2 to 8. In some embodiments, p is 2.

[0083] In some embodiments, the bond to the Ab is on a cysteine residue of the antibody or antigenbinding fragment. In some embodiments, the bond to the Ab is on a cysteine-80 residue on the light chain of the antibody or antigen-binding fragment. In some embodiments, the bond to the Ab is on a cysteine- 149 residue on the light chain of the antibody or antigen-binding fragment. In some embodiments, the bond to the Ab is on a cysteine- 118 residue on the heavy chain of the antibody or antigen-binding fragment. In some embodiments, the bond to the Ab is on a cysteine- 140 residue on the heavy chain of the antibody or antigen-binding fragment. In some embodiments, the bond to the Ab is on a cysteine-239 residue on the heavy chain of the antibody or antigen-binding fragment.

[0084] In some embodiments, the cleavable moiety comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety is cleavable by an enzyme. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin or legumain. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin B.

[0085] In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the amino acid unit comprises valine-citrulline (Val-Cit), valine-dimethylated lysine (Val-Lys(Me)₂), alanine-dimethylated lysine (Ala-Lys(Me)₂), valine-alanine (Vai- Ala), asparagine (Asn), aspartic acid (Asp), or methylated aspartic acid (Asp(OMe)). In some embodiments, the amino acid unit comprises Val-Cit. In some embodiments, the amino acid unit comprises Val-Ala. In some embodiments, the amino acid unit comprises Asn. In some embodiments, the amino acid unit comprises Asp.

[0086] In some embodiments, Z is absent, and the cleavable moiety is directly joined to the cytotoxic agent. In some embodiments, -Y-Z- comprises Asn. In some embodiments, -Y-Z- comprises Asp. In some embodiments, D comprises eribulin or a salt thereof.

[0087] In some embodiments, Z is a spacer unit which attaches the cleavable moiety to the cytotoxic agent.

[0088] In some embodiments, the spacer unit attaching the cleavable moiety to the cytotoxic agent is self-immolative. In some embodiments, the spacer unit attaching the cleavable moiety to the cytotoxic agent comprises a p-aminobenzyloxycarbonyl (pABC). In some embodiments, the cytotoxic agent is eribulin, and the pABC covalently attaches to eribulin via a C-35 amine.

[0089] In some embodiments, -Y-Z- comprises Val-Cit-pABC, Val-Ala-pABC, Val-Lys(Me)₂-pABC, or Ala-Lys(Me)₂-pABC. In some embodiments, -Y-Z- comprises Val-Cit-pABC. In some embodiments, -Y-Z- comprises Val-Ala-pABC. In some embodiments, D comprises eribulin or a salt thereof.

[0090] In some embodiments, Ab is an anti-TROP2 antibody or antigen-binding fragment disclosed herein.

[0091] In various embodiments, the present disclosure provides a composition comprising multiple copies of the antibody-drug conjugate of Formula (II) or Formula (III). In some embodiments, the average p of the antibody-drug conjugates in the composition is about 1 to about 3. In some embodiments, the average p is about 2.

[0092] In various embodiments, the present disclosure provides a compound of Formula (Ila): , wherein:

D is a cytotoxic agent;

Y is a cleavable moiety; and

Z is absent or a spacer unit.

[0093] In some embodiments, the cytotoxic agent comprises eribulin or a salt thereof.

[0094] In some embodiments, the cleavable moiety comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety is cleavable by an enzyme. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin or legumain. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin B.

[0095] In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the amino acid unit comprises valine-citrulline (Val-Cit), valine-dimethylated lysine (Val-Lys(Me)₂), alanine-dimethylated lysine (Ala-Lys(Me)₂), valine-alanine (Vai- Ala), asparagine (Asn), aspartic acid (Asp), or methylated aspartic acid (Asp(OMe)). In some embodiments, the amino acid unit comprises Val-Cit. In some embodiments, the amino acid unit comprises Val-Ala. In some embodiments, the amino acid unit comprises Asn.

[0096] In some embodiments, Z is absent and the cleavable moiety is directly joined to the cytotoxic agent. In some embodiments, -Y-Z- comprises Asn. In some embodiments, -Y-Z- comprises Asp. In some embodiments, D comprises eribulin or a salt thereof.

[0097] In some embodiments, Z is a spacer unit which attaches the cleavable moiety to the cytotoxic agent. In some embodiments, the spacer unit attaching the cleavable moiety to the cytotoxic agent is self-immolative. In some embodiments, the spacer unit attaching the cleavable moiety to the cytotoxic agent comprises a p-aminobenzyloxycarbonyl (pABC). In some embodiments, the pABC covalently attaches to eribulin via a C-35 amine. [0098] In some embodiments, -Y-Z- comprises Val-Cit-pABC, Val-Ala-pABC, or Val-Lys(Me)₂- pABC, Ala-Lys(Me)₂-pABC. In some embodiments, -Y-Z- comprises Val-Cit-pABC. In some embodiments, -Y-Z- comprises Val-Ala-pABC. In some embodiments, D comprises eribulin or a salt thereof.

[0099] In various embodiments, the present disclosure provides a method of preparing the antibodydrug conjugate of Formula (II). In some embodiments, the method comprises:

- providing one or more compounds of Formula (Ila) , wherein: D is the cytotoxic agent; Y is the cleavable moiety; and Z is absent or the spacer unit; and

- conjugating the one or more compounds of Formula (Ila) to an antibody or antigen-binding fragment, to provide an antibody-drug conjugate of Formula (II): wherein Ab is the antibody or antigen-binding fragment.

[00100] In various embodiments, the present disclosure provides a method of preparing the antibodydrug conjugate of Formula (III). In some embodiments, the method comprises:

- providing one or more compounds of Formula (Ila) , wherein: D is the cytotoxic agent; Y is the cleavable moiety; and Z is absent or the spacer unit;

- conjugating the one or more compounds of Formula (Ila) to an antibody or antigen-binding fragment, to provide an antibody-drug conjugate of Formula (II): wherein Ab is the antibody or antigen-binding fragment; and

- hydrolyzing the succinimide ring in the compound of Formula (II), to provide the compound of

Formula (III)

[00101] In some embodiments, the step of hydrolyzing the succinimide ring in the compound of Formula (II) comprises a pH from about 7.4 to about 9.2. In some embodiments, the pH is from about 7.4 to about 8.5. In some embodiments, the pH is about 7.4.

[00102] In some embodiments, the step of hydrolyzing the succinimide ring in the compound of Formula (II) comprises a temperature of about 37 °C. In some embodiments, the step of hydrolyzing the succinimide ring in the compound of Formula (II) is completed in about 24 hours or less.

[00103] In various embodiments, the present disclosure provides a method of treating a patient having or at risk of having a cancer that expresses TROP2, comprising administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment disclosed herein. In various embodiments, the present disclosure provides a method of treating a patient having or at risk of having a cancer that expresses TROP2, comprising administering to the patient a therapeutically effective amount of an antibody-drug conjugate disclosed herein. In various embodiments, the present disclosure provides a method of treating a patient having or at risk of having a cancer that expresses TROP2, comprising administering to the patient a therapeutically effective amount of a composition disclosed herein. In various embodiments, the present disclosure provides a method of treating a patient having or at risk of having a cancer that expresses TROP2, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the TROP2-expressing cancer is a cholangiocarcinoma, bladder cancer, breast cancer, cervical carcinoma, colorectal cancer, esophageal cancer, gastric cancer, glioma, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer. In some embodiments, the TROP2-expressing cancer is a cholangiocarcinoma, breast cancer, or NSCLC. In some embodiments, the patient is intolerant, non-responsive, or poorly responsive to treatment with eribulin when administered alone. [00104] In various embodiments, the present disclosure provides a method of reducing or inhibiting growth of a TROP2 -expressing tumor, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment disclosed herein. In various embodiments, the present disclosure provides a method of reducing or inhibiting growth of a TROP2-expressing tumor, comprising administering a therapeutically effective amount of an antibody-drug conjugate disclosed herein. In various embodiments, the present disclosure provides a method of reducing or inhibiting growth of a TROP2 -expressing tumor, comprising administering a therapeutically effective amount of a composition disclosed herein. In various embodiments, the present disclosure provides a method of reducing or inhibiting growth of a TROP2-expressing tumor, comprising administering a therapeutically effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the tumor is a TROP2-expressing cholangiocarcinoma, bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer. In some embodiments, the tumor is a cholangiocarcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

[00105] FIG. 1 A shows FACS analysis of folate receptor alpha (FRA), HER2, and TROP2 expression on human breast cancer HCC1954 cell line. FIGs. IB and 1C show average tumor volume and body weight change after single-dose treatment with the indicated compound in a human breast cancer HCC-1954 xenograft model.

[00106] FIG. 2A shows average tumor volumes in NCI-H2110 NSCLC xenograft models upon single-dose treatment with anti-TROP2-162-46.2-SN-38 or anti-FRa-eribulin. FIGs. 2B and 2C show average tumor volumes and body weight for various doses of anti-TROP2-162-46.2-eribulin ADC compared to the unconjugated antibody or eribulin.

[00107] FIGs. 3A and 3B show average tumor volume and relative body weight normalized by day of treatment with a single dose of each compound at the indicated concentration in a WHIM5 TNBC PDx model.

[00108] FIGs. 4A and 4B show average tumor volume and weight in CTG-1258 or CTG-1388 upon single-dose treatment of the indicated compound.

[00109] FIG. 5 shows pre- and post-immunization blood samples from rabbits diluted and analyzed for human TROP2 reactivity by ELISA.

[00110] FIG. 6A shows analysis of overlapping epitopes on hTROP2-Fc for each indicated antibody pair. FIG. 6B shows groupings of antibodies with similar overlapping profiles. FIG. 6C shows analysis of overlapping epitopes on hTROP2-Fc for each indicated antibody pair. FIG. 6D shows grouping of antibodies with similar overlapping profiles.

[00111] FIG. 7 shows correlation between TROP2 affinity and IC₅₀ in BxPC3 cells.

[00112] FIG. 8 shows rabbit CDRs grafted onto the closest human germline frameworks. The frameworks (FR) and CDRs are shown above each clone as defined by Kabat (top) and IMGT (bottom). Residues that may impact antigen binding were retained in FRH2 and FRL1. Rabbit residues are in bold, humanized residues are underlined.

[00113] FIGs. 9A and 9B show in vitro cytotoxicity screening of humanized anti-TROP2 ADCs in a variety of cell types. FIG. 9C shows ADC IC₅₀ values and max killing (%) for the identified ADCs in AsPCl, BxPC3, and SNU-308 cells. Fig. 9D shows percent cytotoxicity at different ADC concentrations for AsPCl, BxPC3, and SNU-308 cells.

[00114] FIG. 10 shows internalization of anti-TROP2 antibodies using NCI-H2110 and MDA-MB- 231 cell lines. [00115] FIG. 11 shows rabbit CDRs grafted onto the closest human germline frameworks. The FRs and CDRs are shown above each clone as defined by Kabat (top) and IMGT (bottom). Residues that may impact antigen binding were retained in FRH2 and FRL1. Rabbit residues are in bold, humanized residues are underlined.

[00116] FIG. 12A shows the ECso of 16K21 Hl-3 and Ll-3, 17124 Hl-3 and Ll-3, and 20E16 Hl-3 and Ll-2 for binding human TROP2-ECD as determined by ELISA. FIG. 12B shows the EC₅₀ of each indicated clone for binding to human TROP2-ECD as determined by ELISA.

[00117] FIG. 13 shows polyspecificity assessment of control versus anti-TROP2 antibodies. Insulin, ssDNA, dsDNA, cardiolipin, LPS, or KLH were coated on wells of a 96-well plate.

[00118] FIGs. 14A, 14B, 14C, 14D, 14E, 14F, and 14G show the thermal stability of chimeric (xi) and humanized (zu) anti-TROP2 antibodies analyzed by DSC.

[00119] FIG. 15 shows MAAS mutations.

[00120] FIG. 16A shows neutrophil differentiation over 14 days upon indicated cytokine treatment as monitored by FACS. FIG. 16B shows the percentage of viable CD66b+ cells after incubation for four days with various concentrations of anti-TROP2-ADCs with wild-type or mutant Fes in the presence or absence of Fc blocker as determined by FACS.

[00121] FIG. 17 shows immunogenicity of antibody clones predicted by Abzena’s iTope in silico immunogenicity assessment tool.

[00122] FIG. 18A shows a disulfide re-bridging strategy and the DTM-N-eribulin linker payload.

FIG. 18B shows IC₅₀ values of the indicated ADC in various cell lines.

[00123] FIG. 19 shows linker-eribulin conjugates.

[00124] FIG. 20 shows percent growth inhibition of TROP2-expressing cells treated with anti- TROP2-ADCs in mono- and co-cultured in vitro bystander assay.

[00125] FIG. 21 shows the thermal stability of the indicated linkers over time. HIC = hydrophobic interaction chromatography.

[00126] FIGs. 22A, 22B, 22C, 22D, and 22E show mouse plasma stability of RESPECT-L linkers. FIG. 22 A shows the average DAR of immunocaptured samples as analyzed by LC-MS. FIG. 22B shows percent change in DAR of immunocaptured samples. FIGs. 22C and 22D show free payload (ng/mL or percentage in plasma) in the immunodepleted plasma samples as analyzed by LC-MS after organic extraction of the free payload. FIG. 22E shows the percentage of hydrolysis of the linker for each sample as determined by LC-MS.

[00127] FIG. 23 shows average tumor volume in NSCLC NCI-H2110 xenograft model treated with a single dose of the indicated compounds at 10 mg/kg.

[00128] FIG. 24 shows average tumor volume in NSCLC NCI-H2110 xenograft model treated with subcutaneous injection of a single dose of 16K21H1L1-Eribulin, 17I24H1L1-Eribulin, or IMMU-132 (7.5 mg/kg). [00129] FIG. 25 shows the anti-tumor activity of 16K21HlLl-ADCs comparing RESPECT-L conjugation ((PEG)₂-VCP-eribulin) and re-bridging conjugation (DTM-N-eribulin).

[00130] FIGs. 26A, 26B, 26C, and 26D show the in vivo anti-tumor activity of various RESPECT-L linkers. The anti-tumor activity of 16K21HlLl-ADCs with various PEG lengths (FIG. 26A), 16K21H1L1- and 17I24HlLl-ADCs with various cathepsin-cleavable linkers (FIGs. 26B and26C), and 16K21HlLl-ADCs with legumain-cleavable linkers (FIG. 26D) was evaluated inNSCLC NCI- H2110 xenograft model.

[00131] FIGs. 27A, 27B, 27C, 27D, 27E, 27F, and 27G show in vitro cytotoxicity of humanized monoclonal antibodies and linkers.

[00132] FIGs. 28A, 28B, 28C, and 28D show IC₅₀ values for various cell lines treated with the indicated ADCs.

[00133] FIG. 29 shows in vivo analysis of the indicated anti-TROP2-eribulin ADCs.

[00134] FIG. 30 shows an analysis of hydrolysis conditions for various maleimide-conjugated eribulin linkers.

[00135] FIG. 31A shows percent hydrolysis for antibody-drug conjugates comprising a (PEG)₂ spacer unit conjugated to various cleavable moieties and at various pH. FIG. 31B shows percent hydrolysis for antibody-drug conjugates comprising a C₂(PEG)₂ spacer unit conjugated to various cleavable moieties and at various pH. FIG. 31C shows percent hydrolysis for antibody-drug conjugates comprising a C₂ spacer unit conjugated to various cleavable moieties and at various pH.

[00136] FIG. 32A shows percent hydrolysis for antibody-drug conjugates comprising a Val-Cit- pABC (VCP) cleavable moiety conjugated to various spacer units and at various pH. FIG. 32B shows percent hydrolysis for antibody-drug conjugates comprising an asparagine (N) cleavable moiety conjugated to various spacer units and at various pH.

[00137] FIGs. 33A shows ADC stability of ADCs comprising VCP and C₂(PEG)₁, C₂(PEG)₂, (PEG)₂, or C₂ in buffer or plasma. FIG. 33B shows ADC stability of ADCs comprising N and C₂(PEG)₂, C₂(PEG)₁, (PEG)₂, or C₂ in buffer or plasma. “% DAR” represents the percentage of intact ADC relative to the initial DAR at the start of the experiment.

[00138] FIG. 34 shows in vitro cytotoxicity of humanized monoclonal antibodies conjugated to various linkers.

[00139] FIG. 35A shows in vivo tumor volume results after treatment with ADCs comprising C₂(PEG)₂. FIG. 35B shows in vivo tumor volume results after treatment with ADCs comprising (PEG)2. FIG. 35C shows in vivo tumor volume results after treatment with ADCs comprising C₂. DETAILED DESCRIPTION

[00140] The disclosed compositions and methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure.

[00141] Throughout this text, the descriptions refer to compositions and methods of using said compositions. Where the disclosure describes or claims a feature or embodiment associated with a composition, such a feature or embodiment is equally applicable to the methods of using said composition. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of using a composition, such a feature or embodiment is equally applicable to the composition.

[00142] When a range of values is expressed, it includes embodiments using any particular value within the range. Further, reference to values stated in ranges includes each and every value within that range. All ranges are inclusive of their endpoints and combinable. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The use of “or” will mean “and/or” unless the specific context of its use dictates otherwise. All references cited herein are incorporated by reference for any purpose. Where a reference and the specification conflict, the specification will control.

[00143] It is to be appreciated that certain features of the disclosed compositions and methods, which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

Definitions

[00144] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[00145] As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.

[00146] The terms “about” or “approximately” in the context of numerical values and ranges refer to values or ranges that approximate or are close to the recited values or ranges such that the embodiment may perform as intended, such as having a desired amount of nucleic acids or polypeptides in a reaction mixture, as is apparent to the skilled person from the teachings contained herein. In some embodiments, “about” means plus or minus 10% of a numerical amount. [00147] The term “agent” is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. The term “therapeutic agent,” “cytotoxic agent,” “drug,” or “drug moiety” refers to an agent that is capable of modulating a biological process and/or has biological activity.

[00148] The terms “antibody-drug conjugate,” “antibody conjugate,” “immunoconjugate,” and “ADC” are used interchangeably, and refer to a compound or derivative thereof that is linked to an antibody (e.g., an anti-TROP2 antibody) and may be defined by the generic formula: Ab-(L-D)_p (Formula (I)), wherein Ab = an antibody moiety (i.e., antibody or antigen-binding fragment), L = a linker moiety, D = a drug moiety, and p = the number of drug moieties per antibody moiety. In some embodiments, the linker L includes a cleavable moiety between the antibody or antigen-binding fragment and the therapeutic compound. In some embodiments, the linker L includes a cleavable moiety that can be attached to either or both the antibody or antigen-binding fragment and to the therapeutic compound, e.g., by spacer unit(s). Exemplary cleavable linkers are described and exemplified herein.

[00149] The term “antibody” is used in the broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The heavy chain (HC) of an antibody is composed of a heavy chain variable domain (VH) and a heavy chain constant region (CH). The light chain (LC) is composed of a light chain variable domain (VL) and a light chain constant domain (CL). As used herein, the terms “domain” and “region” maybe used interchangeably (e.g., the term “variable domain” may be used interchangeably with the term “variable region” and understood to refer to the same part of the antibody). For the purposes of this application, the mature heavy chain and light chain variable domains each comprise three complementarity determining regions (CDR1, CDR2, and CDR3; also referred to as “hypervariable regions”) within four framework regions (FR1, FR2, FR3, and FR4) arranged from N terminus to C terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs may be identified according to the Kabat and/or IMGT numbering systems. Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991); International ImMunoGeneTics Information System (IMGT®). An “antibody” can be naturally occurring or man-made, such as monoclonal antibodies produced by conventional hybridoma technology. The term “antibody” includes full-length monoclonal antibodies and full- length polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fv, and single chain antibodies. An antibody can be any one of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., isotypes IgGl, IgG2, IgG3, IgG4). An antibody of any of the aforementioned classes or subclasses can also comprise one of two functionally similar classes of light chains: IgK (also referred to herein as “Ig kappa” or “kappa”) and IgX (also referred to herein as “Ig lambda” or “lambda”). The term antibody encompasses human antibodies, chimeric antibodies, humanized antibodies and any modified immunoglobulin molecule containing an antigen recognition site, so long as it demonstrates the desired biological activity.

[00150] The term “bystander killing” or “bystander effect” refers to the killing of target-negative cells in the presence of target-positive cells, wherein killing of target-negative cells is not observed in the absence of target-positive cells. Cell-to-cell contact, or at least proximity between target-positive and target-negative cells, enables bystander killing. This type of killing is distinguishable from “off-target killing,” which refers to the indiscriminate killing of target-negative cells. “Off-target killing” may be observed in the absence of target-positive cells.

[00151] The term “chimeric antibody,” as used herein, refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. In some instances, the variable regions of both heavy and light chains correspond to the variable regions of antibodies derived from one species with the desired specificity, affinity, and activity while the constant regions are homologous to antibodies derived from another species (e.g., human) to minimize an immune response in the latter species.

[00152] The term “human antibody,” as used herein, refers to an antibody produced by a human or an antibody having an amino acid sequence of an antibody produced by a human.

[00153] As used herein, the term “humanized antibody” refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions 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. The humanized antibody can be further modified by the substitution of residues, either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or activity. The humanized antibody can also be further modified by the substitution of residues in the Fc domain to reduce its binding to various cell receptors, such as a Fey receptor (FcyR), and other immune molecules.

[00154] The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495 or may be made by recombinant DNA methods. See, e.g., U.S. Pat. No. 4,816,567. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, e.g., Clackson et al. (1991) Nature 352:624-8, and Marks et al. (1991) J. Mol. Biol. 222:581-97.

[00155] The monoclonal antibodies described herein specifically include “chimeric” antibodies, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in an antibody 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 an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity.

[00156] The term “antigen-binding fragment” or “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TROP2). Antigen-binding fragments preferably also retain the ability to internalize into an antigen-expressing cell. In some embodiments, antigen-binding fragments also retain immune effector activity. It has been shown that fragments of a full-length antibody can perform the antigenbinding function of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” or “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and Cm domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and Cm domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment, which comprises a single variable domain, e.g. , a VH domain, see, e.g. , Ward et al. (1989) Nature 341 :544-6; and Winter et al., WO 90/05144; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). See, e.g., Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” or “antigen-binding portion” of an antibody, and are known in the art as an exemplary type of binding fragment that can internalize into cells upon binding. See, e.g., Zhu et al. (2010) 9:2131-41; He et al. (2010) J. Nucl. Med. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402. In certain embodiments, scFv molecules may be incorporated into a fusion protein. Other forms of single chain antibodies such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites. See, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-8; and Poljak et al. (1994) Structure 2: 1121-3. Antigen-binding fragments are obtained using conventional techniques known to those of skill in the art, and the binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as are intact antibodies. Antigen-binding fragments may be prepared, e.g. , by cleavage of the intact protein, e.g. , by protease or chemical cleavage.

[00157] The term “anti-TROP2 antibody” or “antibody that specifically binds TROP2” refers to any form of antibody or fragment thereof that specifically binds TROP2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they specifically bind TROP2. Preferably the anti-TROP2 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. 17124, 16K21, and 20E16 are exemplary internalizing anti-human TROP2 antibodies. Sacituzumab and datopotamab are also exemplary internalizing anti-human TROP2 antibodies. As used herein, the terms “specific,” “specifically binds,” and “binds specifically” refer to the selective binding of the antibody to the target antigen or epitope over alternative antigens or epitopes. Antibodies can be tested for specificity of binding by comparing binding to an appropriate antigen to binding to an irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen with at least 5 times, or preferably 7 or 10 times, more affinity than to an irrelevant antigen or antigen mixture, then it is considered to be specific, e.g., as measured by surface plasmon resonance, e.g., BIAcore® analysis. In one embodiment, a specific antibody is one that binds the TROP2 antigen, but does not bind (or exhibits minimal binding) to other antigens.

[00158] The term “cancer” refers to the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, bile duct cancer (e.g., cholangiocarcinoma), esophageal cancer, nasopharyngeal cancer, cancer of the peritoneum, hepatocellular cancer (e.g., hepatocellular carcinoma), gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, osteosarcoma, skin cancer (e.g., melanoma), colon cancer, colorectal cancer, endometrial or uterine cancer, ovarian cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancers.

[00159] The terms “cancer cell” and “tumor cell” refer to individual cells or the total population of cells derived from a tumor, including both non-tumorigenic cells and cancer stem cells. As used herein, the term “tumor cell” will be modified by the term “non-tumorigenic” when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.

[00160] The term “chemotherapeutic agent” or “anti-cancer agent” is used herein to refer to a chemical compound that is effective in treating cancer regardless of mechanism of action. Inhibition of metastasis or angiogenesis is frequently a property of a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include alkylating agents, for example, nitrogen mustards, ethyleneimine compounds, and alkyl sulphonates; antimetabolites, for example, folic acid, purine or pyrimidine antagonists; anti-mitotic agents, for example, anti-tubulin agents such as eribulin or eribulin mesylate (Halaven™) or derivatives thereof, vinca alkaloids, and auristatins; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, for example, DNA minor groove binders; and growth factor receptor antagonists. In addition, chemotherapeutic agents include antibodies, biological molecules, and small molecules. A chemotherapeutic agent may be a cytotoxic or cytostatic agent. The term “cytostatic agent” refers to an agent that inhibits or suppresses cell growth and/or multiplication of cells.

[00161] The term “co-administration” or administration “in combination with” one or more therapeutic agents includes concurrent and consecutive administration in any order.

[00162] The term “cytotoxic agent” refers to a substance that causes cell death primarily by interfering with a cell’s expression activity and/or functioning. Examples of cytotoxic agents include, but are not limited to, anti-mitotic agents, such as eribulin, auristatins (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), maytansinoids (e.g., maytansine), dolastatins, duostatins, cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine), taxanes, taxols, and colchicines; anthracyclines (e.g., daunorubicin, doxorubicin, dihydroxyanthracindione); cytotoxic antibiotics (e.g., mitomycins, actinomycins, duocarmycins (e.g., CC-1065), auromycins, duomycins, calicheamicins, endomycins, phenomycins); alkylating agents (e.g., cisplatin); intercalating agents (e.g., ethidium bromide); topoisomerase inhibitors (e.g., exactecan, irinotecan, etoposide, tenoposide); radioisotopes, such as At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212 or 213, P32, and radioactive isotopes of lutetium (e.g., Lu 177); and toxins of bacterial, fungal, plant or animal origin (e.g., ricin (e.g., ricin A-chain), diphtheria toxin, Pseudomonas exotoxin A (e.g., PE40), endotoxin, mitogellin, combrestatin, restrictocin, gelonin, alpha-sarcin, abrin (e.g. , abrin A-chain), modeccin (e.g., modeccin A-chain), curicin, crotin, Sapaonaria officinalis inhibitor, glucocorticoid).

[00163] An “effective amount” of an ADC as disclosed herein is an amount sufficient to perform a specifically stated purpose, for example to produce a therapeutic effect after administration, such as a reduction in tumor growth rate or tumor volume, a reduction in a symptom of cancer, or some other indicia of treatment efficacy. An effective amount can be determined in a routine manner in relation to the stated purpose. The term “therapeutically effective amount” refers to an amount of an ADC effective to treat a disease or disorder in a subject. In the case of cancer, a therapeutically effective amount of ADC can reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and/or relieve one or more symptoms. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[00164] The term “epitope” refers to the portion of an antigen capable of being recognized and specifically bound by an antibody. When the antigen is a polypeptide, epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of the polypeptide. The epitope bound by an antibody may be identified using any epitope mapping technique known in the art, including X-ray crystallography for epitope identification by direct visualization of the antigen-antibody complex, as well as monitoring the binding of the antibody to fragments or mutated variations of the antigen, or monitoring solvent accessibility of different parts of the antibody and the antigen. Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based oligo-peptide scanning, limited proteolysis, site-directed mutagenesis, high- throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry. See, e.g., Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev. Proteomics 2:745- 56.

[00165] Competitive binding and epitope binning can also be used to determine antibodies sharing identical or overlapping epitopes. Competitive binding can be evaluated using a cross-blocking assay, such as the assay described in “Antibodies, A Laboratory Manual,” Cold Spring Harbor Laboratory, Harlow and Lane (1st edition 1988, 2nd edition 2014). In some embodiments, competitive binding is identified when a test antibody or binding protein reduces binding of a reference antibody or binding protein to a target antigen such as TROP2 (e.g., a binding protein comprising CDRs and/or variable domains selected from those identified in Tables 2, 4, and 6), by at least about 50% in the crossblocking assay (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any percentage in between), and/or vice versa. In some embodiments, competitive binding can be due to shared or similar (e.g., partially overlapping) epitopes, or due to steric hindrance where antibodies or binding proteins bind at nearby epitopes. See, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66). In some embodiments, competitive binding can be used to sort groups of binding proteins that share similar epitopes, e.g., those that compete for binding can be “binned” as a group of binding proteins that have overlapping or nearby epitopes, while those that do not compete are placed in a separate group of binding proteins that do not have overlapping or nearby epitopes.

[00166] The term “eribulin,” as used herein, refers to a synthetic compound of halichondrin B. The term “eribulin drug moiety” refers to the component of an ADC that has the structure of eribulin, and is attached to the linker of the ADC, e.g., via its C-35 amine. The terms encompass salts thereof. Eribulin is a microtubule dynamics inhibitor, which is thought to bind tubulin and induce cell cycle arrest at the G2/M phase by inhibiting mitotic spindle assembly. The term “eribulin mesylate” refers to the mesylate salt of eribulin, which is marketed under the trade name Halaven™. Compositions and methods of inhibiting tumor growth in patients comprising administering eribulin are disclosed in U.S. Patent No. 10,322,192, which is incorporated herein by reference in its entirety for all eribulin structures and methods of synthesizing those structures. The structure of eribulin is shown below:

[00167] “Fey receptor,” “Fc-gamma receptor,” or “FcyR” refers to a cell surface protein generally found on immune cells of various types, e.g., neutrophils. The binding of an Fc region of an antibody to a Fey receptor may induce different effector functions, for example antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), which can have unwanted side effects, for example cell or tissue damage, effector cell activation, or cytokine release. The binding of an Fc region of an antibody to a Fey receptor on a neutrophil may induce cell damage, e.g., neutropenia, through non-antigen-mediated uptake of the antibody.

[00168] The term “homolog” in the context of an antibody or antigen-binding fragment refers to a protein sequence having amino acid residues that are the same or similar at corresponding positions. A homolog may refer to a similar sequence from a different species. Sequence homology, as used herein, refers to a sequence having amino acid residues that are the same or similar at corresponding positions in a reference sequence. Sequence homology may be described by percent identity with a reference sequence. Preferably, sequence homology may be determined by an algorithm such as PILEUP, BLAST, or gapped BLAST, described in further detail below for amino acid sequence identity.

[00169] The terms “IgGl Fc,” “IgGl Fc domain” or “IgGl Fc-containing antibody” as used herein refer to an antibody having at least an IgGl CH2 and CH3 domain, as identified by SEQ ID NO: 164 and SEQ ID NO: 165, respectively.

[00170] “Wild-type IgGl Fc domain” refers to a human IgGl Fc domain that comprises the amino acid sequence of SEQ ID NO: 163 or a fragment thereof.

[00171] The term “inhibit” or “inhibition of,” as used herein, means to reduce by a measurable amount, and can include but does not require complete prevention or inhibition.

[00172] “Internalizing” as used herein in reference to an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that is capable of being taken through the cell's lipid bilayer membrane to an internal compartment (i.e., “internalized”) upon binding to the cell, preferably into a degradative compartment in the cell. For example, an internalizing anti-TROP2 antibody is one that is capable of being taken into the cell after binding to TROP2 on the cell membrane.

[00173] The term “KD” refers to the equilibrium dissociation constant of a particular antibodyantigen interaction. KD is calculated by ka/kd. The rate can be determined using standard assays, such as a BIAcore® or ELISA assay.

[00174] The term “k_on” or “k_a” refers to the on-rate constant for association of an antibody to the antigen to form the antibody/antigen complex. The rate can be determined using standard assays, such as a BIAcore® or ELISA assay.

[00175] The term “k_off” or “k_d” refers to the off-rate constant for dissociation of an antibody from the antibody/antigen complex. The rate can be determined using standard assays, such as a BIAcore® or ELISA assay.

[00176] A “linker” or “linker moiety” is any chemical moiety that is capable of covalently joining a compound, usually a drug moiety such as a chemotherapeutic agent, to another moiety such as an antibody moiety. Linkers can be susceptible to or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and/or disulfide bond cleavage, at conditions under which the compound or the antibody remains active. A “cleavable linker” is any linker that comprises a cleavable moiety and can thus be susceptible to cleavage. A cleavable moiety can be a cleavable peptide moiety. The term “cleavable peptide moiety” refers to any chemical bond linking amino acids (natural or synthetic amino acid derivatives) that can be cleaved by an agent that is present in the intracellular environment.

[00177] The term ^up” or “antibody: drug ratio” or “drug-to-antibody ratio” or “DAR” refers to the number of drug moieties per antibody moiety, i.e., drug loading, or the number of -L-D moieties per antibody or antigen-binding fragment (Ab) in ADCs of Formula (I). In compositions comprising multiple copies of ADCs of Formula (I), “p” refers to the average number of -L-D moieties per antibody or antigen-binding fragment, also referred to as average drug loading.

[00178] A “pharmaceutical composition” refers to a preparation which is in such form as to permit administration and subsequently provide the intended biological activity of the active ingredients) and/or to achieve a therapeutic effect, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The pharmaceutical composition may be sterile.

[00179] A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH- adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like. [00180] “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans.

[00181] By “protein,” as used herein, is meant at least two covalently attached amino acids. The term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, the two or more covalently attached amino acids are attached by a peptide bond. The protein may be made up of naturally occurring amino acids and peptide bonds, for example when the protein is made recombinantly using expression systems and host cells. Alternatively, the protein may include synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine), or peptidomimetic structures, e.g., peptoids. Peptoids are an exemplary class of peptidomimetics whose side chains are appended to the nitrogen atom of the peptide backbone, rather than to the a-carbons (as they are in amino acids), and have different hydrogen bonding and conformational characteristics in comparison to peptides. See, e.g., Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367. As such, peptoids can be resistant to proteolysis or other physiological or storage conditions, and effective at permeating cell membranes. Such synthetic amino acids may be incorporated in particular when the antibody is synthesized in vitro by conventional methods well known in the art. In addition, any combination of peptidomimetic, synthetic, and naturally occurring residues/structures can be used. “Amino acid” also includes imino acid residues, such as proline and hydroxyproline. The amino acid “R group” or “side chain” may be in either the (L)- or the (S)-configuration. In a specific embodiment, the amino acids are in the (L)- or (S)-configuration.

[00182] For amino acid sequences, sequence identity and/or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al. (1984) Nucl. Acid Res. 12:387-95, preferably using the default settings, or by inspection.

[00183] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle (1987) J. Mol. Evol. 35:351-60; the method is similar to that described by Higgins and Sharp (1989) CABIOS 5:151-3. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. [00184] Another example of a useful algorithm is the BLAST algorithm, described in: Altschul et al. (1990) J. Mol. Biol. 215:403-10; Altschul et al. (1997) Nucleic Acids Res. 25:3389-402; and Karin et al. (1993) Proc. Natl. Acad. Sci. USA 90:5873-87. A particularly useful BLAST program is the WU- BLAST-2 program which was obtained from Altschul et al. (1996) Methods in Enzymology 266:460- 80. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

[00185] An additional useful algorithm is gapped BLAST as reported by Altschul et al. (1993) Nucl. Acids Res. 25:3389-402. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions, charges gap lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to about 22 bits.

[00186] Generally, proteins disclosed herein and variants thereof (e.g., variants that retain function of the original protein), including variants of TROP2, variants of tubulin sequences, and variants of antibody variable domains (including individual variant CDRs), have amino acid homology, similarity, or identity of at least 80%, and more typically homologies or identities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100% or 100%.

[00187] In a similar manner, “percent (%) nucleic acid sequence identity” with respect to the nucleic acid sequence of the antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the antigen-binding protein. A specific method uses the BLASTN module of WU- BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

[00188] As used herein, the term “solvent” refers to any liquid in which the product is at least partially soluble (solubility of product > 1 g/L).

[00189] As used herein, the term “isomer” refers to compounds with identical molecular formula but distinct spatial arrangement of atoms or bonds. Isomers include stereoisomers, cis-trans isomers, atropisomers, and tautomers.

[00190] As used herein, the term “stereoisomer” refers to both enantiomers and diastereomers. [00191] It will be appreciated that certain compounds of this invention may exist as separate stereoisomers or enantiomers and/or mixtures of those stereoisomers or enantiomers. As used in the chemical structures disclosed herein, a “wedge” “hash” bond to a stereogenic atom indicates a chiral center of known absolute stereochemistry (i.e., one stereoisomer). As used herein, a stereogenic atom that is notated with an (7?) or (5) indicates the stereochemical designation of the stereogenic atom under the Cahn-Ingold-Prelog convention. As used in the chemical structures disclosed herein, a (“straight”) bond to a stereogenic atom indicates where there is a mixture (e.g., a racemate or enrichment). As used herein, two (“straight”) bonds to a double-bonded carbon indicates that the double bond possesses the E/Z stereochemistry as drawn.

[00192] Certain compounds disclosed herein may exist as tautomers and both tautomeric forms are intended, even though only a single tautomeric structure is depicted. [00193] The terms “subject” and “patient” are used interchangeably herein to refer to any animal, such as any mammal, including but not limited to, humans, non-human primates, rodents, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[00194] The term “target-negative” or “target antigen-negative” refers to the absence of target antigen expression by a cell or tissue. The term “target-positive” or “target antigen-positive” refers to the presence of target antigen expression. For example, a cell or a cell line that does not express a target antigen may be described as target-negative, whereas a cell or cell line that expresses a target antigen may be described as target-positive.

[00195] The term “tumor associated calcium signal transducer 2,” “Trop2,” or “TROP2” as used herein, refers to any native form of human TROP2. The term encompasses full-length TROP2 (e.g., NCBI Reference Sequence: NP 002344.2; SEQ ID NO: 161), as well as any form of human TROP2 that results from cellular processing. The term also encompasses naturally occurring variants of TROP2, including but not limited to allelic variants and isoforms. TROP2 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

[00196] The terms “tumor” and “neoplasm” refer to any mass of tissue that results from excessive cell growth or proliferation, either benign or malignant, including precancerous lesions.

[00197] As used herein, “to treat” or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality. As is readily appreciated in the art, full eradication of disease is preferred but not a requirement for a treatment act. “Treatment” or “treat,” as used herein, refers to the administration of a described ADC to a subject, e.g., a patient. The treatment can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve, or affect the disorder, the symptoms of the disorder, or the predisposition toward the disorder, e.g., a cancer.

[00198] As used herein, “valine-citrulline-pABC,” “Val-Cit-pABC,” and “VCP,” are used interchangeably. As used herein, “valine-alanine-pABC,” “Val-Ala-pABC,” and “VAP” are used interchangeably.

[00199] Should the name of a compound conflict with the structure of the compound anywhere in the present application, the structure supersedes the name and is intended to be controlling.

Anti-TROP2 Antibodies and Antigen-Binding Fragments

[00200] The present disclosure provides antibodies that specifically bind to TROP2 and may be used alone, e.g., formulated as therapeutic or diagnostic antibody compositions, e.g., for use in treating or detecting TROP-2 expressing cancers. The antibodies may be provided packaged or prepared for therapeutic use as antibodies, antigen-binding fragments thereof, or as portions of ADCs. Antibodies include, but are not limited to, those listed in Table 1, as well as antibodies comprising CDRs and/or variable domains from the listed antibodies. [00201] The antibodies disclosed herein may bind to TROP2 with an affinity of ≤ 1 μM, ≤ 100 nM or ≤ 10 nM, or any amount in between, as measured by, e.g., BIAcore® analysis. In certain embodiments, the binding affinity is 1 pM to 500 pM. In some embodiments, the binding affinity is between 500 pM to 1 nM, or 1 nM to 10 nM.

[00202] In some embodiments, the antibodies are four-chain antibodies (also referred to as an immunoglobulin), comprising two heavy chains and two light chains. In some embodiments, the antibodies are two-chain half bodies (one light chain and one heavy chain), or antigen-binding fragments of an immunoglobulin.

[00203] Any antibody or antigen-binding fragment described herein may comprise part of a bispecific or multi-specific binding construct, e.g., a construct that can bind to at least one different antigen or additional epitope on TROP2.

[00204] In some embodiments, the antibodies are internalizing antibodies or internalizing antigenbinding fragments thereof. In some embodiments, the internalizing antibodies bind to TROP2 expressed on the surface of a cell and enter the cell upon or after binding. In some embodiments, the drug moiety of the ADC is released from the antibody moiety of the ADC after the ADC enters and is present in a cell expressing TROP2 (i.e., after the ADC has been internalized).

[00205] The antibodies disclosed herein that specifically bind a TROP2 protein may comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) having amino acid sequences selected from the HC CDRs listed in Table 2, infra, as defined by the Kabat numbering system, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) having amino acid sequences selected from the LC CDRs listed in Table 2, infra, as defined by the Kabat numbering system. In some embodiments, the antibodies comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) having amino acid sequences selected from the HC CDRs listed in Table 4, infra, as defined by the IMGT numbering system, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) having amino acid sequences selected from the LC CDRs listed in Table 4, infra, as defined by the IMGT numbering system.

[00206] In some embodiments, an antibody disclosed herein comprises a VH domain having an amino acid sequence selected from SEQ ID NOs: 53 to 56, 67 to 70, 79 to 83, 168, 174, 175, and 177 listed in Table 6, infra. In some embodiments, the antibody comprises a VL domain having an amino acid sequence selected from SEQ ID NOs: 57 to 66, 71 to 78, 84 to 93, 166, 167, 176, and 187 listed in Table 6, infra.

[00207] In some embodiments, an antigen-binding fragment disclosed herein retains TROP2 binding. In some embodiments, the antigen-binding fragment retains TROP2 binding by comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences selected from the HC CDRs listed in Table 2, infra, as defined by the Kabat numbering system, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences selected from the LC CDRs listed in Table 2, infra, as defined by the Kabat numbering system. In some embodiments, the antigen-binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences selected from the HC CDRs listed in Table 4, infra, as defined by the IMGT numbering system, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences selected from the LC CDRs listed in Table 4, infra, as defined by the IMGT numbering system. In some embodiments, the antigen-binding fragments disclosed herein may retain TROP2 binding by comprising a VH domain comprising an amino acid sequence selected from SEQ ID NOs: 53 to 56, 67 to 70, 79 to 83, 168, 174, 175, and 177 listed in Table 6, infra, and a VL domain comprising an amino acid sequence selected from SEQ ID NOs: 57 to 66, 71 to 78, 84 to 93, 166, 167, 176, and 187 listed in Table 6, infra.

[00208] In some embodiments, the antibodies disclosed herein may comprise an IgG constant domain, e.g., an IgGl domain or an IgGl domain that has been modified to reduce binding to an Fc receptor, e.g., an Fey receptor (FcyR) as compared to a wild-type constant domain-containing (e.g., a wild-type IgGl-containing) antibody. Reduced binding to an Fc receptor, e.g., to an FcyR, can be measured as a comparison to the binding of the wild-type antibody without the modification to the same receptor. Reduced binding may be by at least about 10-fold, and preferably at least about 100- fold as compared to the antibody containing the wild-type constant domain. Reduced binding may be measured using any assay known in the art. For example, reduced binding may be measured using a fluorescence resonance energy transfer (FRET) assay.

[00209] In some embodiments, an antibody disclosed herein may comprise an IgGl comprising the mutations L234A, L235A, P238S, H268Q, and/or K274Q (e.g., comprising all of those mutations) according to the EU numbering. See, e.g., Wang et al. (2017) Protein Cell 9(l):63-73; Vafa et al. (2014) Methods 1; 65(1): 114-26; Tam et al. (2017) Antibodies 1; 6(3):12. Without being bound by theory, these mutations may reduce binding of the antibody to a Fey receptor (FcyR), which may reduce non-antigen mediated uptake of antibodies or ADCs by immune cells, such as neutrophils, thus reducing neutropenia. Reduced neutropenia may be measured using any assay known in the art. For example, reduced neutropenia may be measured using a flow cytometry assay, as illustrated in the Examples provided herein.

[00210] In some embodiments, an antibody that specifically binds a TROP2 protein comprises a heavy chain having an amino acid sequence selected from SEQ ID NOs: 94 to 105, 116 to 127, 136 to 150, 171 to 173, 178 to 186, 380, and 381 listed in Table 8, infra. In some embodiments, an antibody that specifically binds a TROP2 protein comprises a light chain having an amino acid sequence selected from SEQ ID NOs: 106 to 115, 128 to 135, 151 to 160, 169, 170, 188, and 189 listed in Table 8, infra.

[00211] Amino acid and nucleic acid sequences of exemplary antibodies of the present disclosure are set forth in Tables 1-9. Table 1. Antibodies

[00212] In some embodiments, the antibodies listed in Table 1 are humanized by replacing the rabbitspecific framework regions with human-specific framework regions. In some embodiments, a variant of a humanized antibody comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) selected from the CDR sequences listed in Table 2, infra, according to the Kabat system. For example, a humanized variant of 16K21 may comprise three heavy chain CDRs, wherein the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2 or SEQ ID NO: 3, and the HCDR3 comprises SEQ ID NO: 4. In some embodiments, a variant of a humanized antibody comprises three light chain CDRs (LCDR1, LCDR2, and LCDR3) selected from the CDR sequences listed in Table 2, infra, according to the Kabat system. For example, a humanized variant of 16K21 may comprise three light chain CDRs, wherein the LCDR1 comprises SEQ ID NO: 5, 6, 7, or 8, the LCDR2 comprises SEQ ID NO: 9, and the LCDR3 comprises SEQ ID NO: 10.

[00213] In some embodiments, a variant of a humanized antibody comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) selected from the CDR sequences listed in Table 4, infra, according to the IMGT system. For example, a humanized variant of 16K21 may comprise three heavy chain CDRs, wherein the HCDR1 comprises SEQ ID NO: 11, the HCDR2 comprises SEQ ID NO: 12, and the HCDR3 comprises SEQ ID NO: 13. In some embodiments, a variant of a humanized antibody comprises three light chain CDRs (LCDR1, LCDR2, and LCDR3) selected from the CDR sequences listed in Table 4, infra, according to the IMGT system. For example, a humanized variant of 16K21 may comprise three light chain CDRs, wherein the LCDR1 comprises SEQ ID NO: 14 or 15, the LCDR2 comprises SEQ ID NO: 16, and the LCDR3 comprises SEQ ID NO: 17.

Table 2. Amino acid sequences of inAb Kabat CDRs

Table 3. Nucleic acid sequences encoding mAb Kabat CDRs Table 4. Amino acid sequences of mAb IMGT CDRs

Table 5. Nucleic acid sequences encoding mAb IMGT CDRs

Table 6. Amino acid sequences of mAb variable regions

Table 7. Nucleic acid sequences encoding mAb variable regions

Table 8. Amino acid sequences of full-length mAb Ig chains

Table 9. Nucleic acid sequences of full-length mAb Ig chains

[00214] In some embodiments, the sequences of the heavy chain variable domains, light chain variable domains, full-length heavy chains, and full-length light chains may be “mixed and matched” to create variants of the anti-TROP2 antibodies, e.g., 16K21, 17124, or 20E16. Such “mixed and matched” anti-TROP2 antibodies can be tested using binding assays known in the art (e.g., ELISAs and other assays described in the Examples). For example, an amino acid sequence corresponding to a heavy chain variable domain from a particular set of heavy chain variable domains listed above in Table 6 for a specific antibody may be replaced with another amino acid sequence corresponding to a heavy chain variable domain option from the table. For example, the amino acid sequence of SEQ ID NO: 53 may be replaced with the amino acid sequence of SEQ ID NO: 54 to create a variant of 16K21. Similarly, the amino acid sequence of SEQ ID NO: 57 may be replaced with the amino acid sequence of SEQ ID NO: 62 to create another variant of 16K21 that comprises a RESPECT-L site. The same applies to the mixing and matching of light chain variable domains, full-length heavy chains, and full-length light chains. In various embodiments, the antibodies disclosed herein may comprise any set of heavy and light chain variable domains listed in the tables above (e.g., 16K21 heavy and light chain variable domains, 17124 heavy and light chain variable domains, or 20E16 heavy and light chain variable domains), or the set of six CDR sequences from the heavy and light chain set (e.g., three 16K21 heavy chain CDRs and three 16K21 light chain CDRs, three 17124 heavy chain CDRs and three 17124 light chain CDRs, or three 20E16 heavy chain CDRs and three 20E16 light chain CDRs). In some embodiments, the antibodies further comprise human heavy and light chain constant domains or fragments thereof. In various embodiments, the antibodies may comprise any set of full-length heavy chain and full-length light chain sequences listed in the tables above (e.g., 16K21 full-length heavy and light chains, 17124 full-length heavy and light chains, or 20E16 full- length heavy and light chains). In some embodiments, the antibodies may comprise a human IgG heavy chain constant domain and a human kappa light chain constant domain. In some embodiments, the antibodies may comprise a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant domain. In various embodiments, an antibody of the present invention comprises a human immunoglobulin G subtype 1 (IgGl) heavy chain constant domain with a human Ig kappa light chain constant domain. In some embodiments, the constant domain is a modified version of a human constant domain, e.g., comprising one or more of L234A, L235A, P238S, H268Q, and/or K274Q modifications of a human IgGl heavy chain constant domain.

[00215] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 18, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 20, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 21; light chain CDR1 (LCDR1) comprising SEQ ID NO: 22, light chain CDR2 (LCDR2) comprising SEQ ID NO: 24, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 26, as defined by the Kabat numbering system.

[00216] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 20, HCDR3 comprising SEQ ID NO: 21; LCDR1 comprising SEQ ID NO: 23, LCDR2 comprising SEQ ID NO: 24, and LCDR3 comprising SEQ ID NO: 26, as defined by the Kabat numbering system.

[00217] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 27, HCDR2 comprising SEQ ID NO: 28, HCDR3 comprising SEQ ID NO: 29; LCDR1 comprising SEQ ID NO: 30, LCDR2 comprising SEQ ID NO: 31, and LCDR3 comprising SEQ ID NO: 33, as defined by the IMGT numbering system.

[00218] In other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 4; LCDR1 comprising SEQ ID NO: 5, LCDR2 comprising SEQ ID NO: 9, and LCDR3 comprising SEQ ID NO: 10, as defined by the Kabat numbering system.

[00219] In other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 4; LCDR1 comprising SEQ ID NO: 8, LCDR2 comprising SEQ ID NO: 9, and LCDR3 comprising SEQ ID NO: 10, as defined by the Kabat numbering system.

[00220] In other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 11, HCDR2 comprising SEQ ID NO: 12, HCDR3 comprising SEQ ID NO: 13; LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 16, and LCDR3 comprising SEQ ID NO: 17, as defined by the IMGT numbering system.

[00221] In other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 11 , HCDR2 comprising SEQ ID NO: 12, HCDR3 comprising SEQ ID NO: 13; LCDR1 comprising SEQ ID NO: 15, LCDR2 comprising SEQ ID NO: 16, and LCDR3 comprising SEQ ID NO: 17, as defined by the IMGT numbering system.

[00222] In yet other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 34, HCDR2 comprising SEQ ID NO: 37, HCDR3 comprising SEQ ID NO: 38; LCDR1 comprising SEQ ID NO: 39, LCDR2 comprising SEQ ID NO: 41, and LCDR3 comprising SEQ ID NO: 43, as defined by the Kabat numbering system.

[00223] In yet other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three fight chain CDRs as follows: HCDR1 comprising SEQ ID NO: 34, HCDR2 comprising SEQ ID NO: 37, HCDR3 comprising SEQ ID NO: 38; LCDR1 comprising SEQ ID NO: 40, LCDR2 comprising SEQ ID NO: 42, and LCDR3 comprising SEQ ID NO: 43, as defined by the Kabat numbering system.

[00224] In yet other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 44, HCDR2 comprising SEQ ID NO: 46, HCDR3 comprising SEQ ID NO: 47; LCDR1 comprising SEQ ID NO: 50, LCDR2 comprising SEQ ID NO: 51, and LCDR3 comprising SEQ ID NO: 52, as defined by the IMGT numbering system.

[00225] In yet other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 44, HCDR2 comprising SEQ ID NO: 46, HCDR3 comprising SEQ ID NO: 48; LCDR1 comprising SEQ ID NO: 50, LCDR2 comprising SEQ ID NO: 51, and LCDR3 comprising SEQ ID NO: 52, as defined by the IMGT numbering system. [00226] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

69, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 72; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:

70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 177, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 177, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 177, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 177, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77.

[00227] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 95% identical to the above-mentioned heavy chain variable region amino acid sequences and a light chain variable region amino acid sequence that is at least 95% identical to the above-mentioned light chain variable region amino acid sequences. In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 69 or 70 and a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 72, 73, 76, or 77.

[00228] In other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 95% identical to the above-mentioned heavy chain variable region amino acid sequences and a light chain variable region amino acid sequence that is at least 95% identical to the above-mentioned light chain variable region amino acid sequences. In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 55, 56, or 175 and a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 58, 61, 63, or 66.

[00229] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[00230] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63. [00231] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[00232] In yet other embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 80, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 85; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 80, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 90; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 80, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 88; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 80, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 93; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 82, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 85; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 82, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 90; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 82, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 88; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 82, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 93. In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 95% identical to the above-mentioned heavy chain variable region amino acid sequences and a light chain variable region amino acid sequence that is at least 95% identical to the above-mentioned light chain variable region amino acid sequences. In some embodiments, an anti- TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 80 or 82 and a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 85, 88, 90, or 93.

[00233] In various embodiments, any of the anti-TROP2 antibodies disclosed herein may comprise a human IgGl Fc domain. In some embodiments, an anti-TROP2 antibody comprises a human IgGl Fc domain that is modified to reduce binding to a FcyR as compared to an IgGl Fc-containing antibody with a wild-type IgGl Fc domain. In some embodiments, the anti-TROP2 antibodies comprise a mutated human IgGl Fc domain that comprises one or more (e.g., all of) L234A, L235A, P238S, H268Q, and K274Q modifications to a human IgGl heavy chain constant domain. [00234] In various embodiments, the anti-TROP2 antibodies comprise a human Ig kappa light chain constant region. In various embodiments, the anti-TROP2 antibodies comprise a human Ig lambda light chain constant region.

[00235] In some embodiments, an anti-TROP2 antibody provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 118, 119, 122, 123, 126, 127, 184, 185, 186, or 381, and a light chain comprising an amino acid sequence selected from SEQ ID NO: 129, 130, 133, or 134.

[00236] In some embodiments, an anti-TROP2 antibody provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 96, 97, 100, 101, 104, 105, 179, 181, 183, or 380, and a light chain comprising an amino acid sequence selected from SEQ ID NO: 107, 110, 112, or 115.

[00237] In some embodiments, an anti-TROP2 antibody provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 137, 139, 142, 144, 147, or 149, and a light chain comprising an amino acid sequence selected from SEQ ID NO: 152, 155, 157, or 160.

[00238] In some embodiments, an anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 126 and the light chain amino acid sequence of SEQ ID NO: 129; or the heavy chain amino acid sequence of SEQ ID NO: 122 and the light chain amino acid sequence of SEQ ID NO: 133; or the heavy chain amino acid sequence of SEQ ID NO: 126 and the light chain amino acid sequence of SEQ ID NO: 130; or the heavy chain amino acid sequence of SEQ ID NO:

122 and the light chain amino acid sequence of SEQ ID NO: 134; or the heavy chain amino acid sequence of SEQ ID NO: 127 and the light chain amino acid sequence of SEQ ID NO: 129; or the heavy chain amino acid sequence of SEQ ID NO: 123 and the light chain amino acid sequence of SEQ ID NO: 133; or the heavy chain amino acid sequence of SEQ ID NO: 127 and the light chain amino acid sequence of SEQ ID NO: 130; or the heavy chain amino acid sequence of SEQ ID NO:

123 and the light chain amino acid sequence of SEQ ID NO: 134; or the heavy chain amino acid sequence of SEQ ID NO: 186 and the light chain amino acid sequence of SEQ ID NO: 129; or the heavy chain amino acid sequence of SEQ ID NO: 185 and the light chain amino acid sequence of SEQ ID NO: 133; or the heavy chain amino acid sequence of SEQ ID NO: 186 and the light chain amino acid sequence of SEQ ID NO: 130; or the heavy chain amino acid sequence of SEQ ID NO: 185 and the light chain amino acid sequence of SEQ ID NO: 134; or the heavy chain amino acid sequence of SEQ ID NO: 381 and the light chain amino acid sequence of SEQ ID NO: 129; or the heavy chain amino acid sequence of SEQ ID NO: 381 and the light chain amino acid sequence of SEQ ID NO: 133; or the heavy chain amino acid sequence of SEQ ID NO: 381 and the light chain amino acid sequence of SEQ ID NO: 130; or the heavy chain amino acid sequence of SEQ ID NO: 381 and the light chain amino acid sequence of SEQ ID NO: 134. In some embodiments, an anti- TROP2 antibody comprises a heavy chain amino acid sequence that is at least 95% identical to one of the above-mentioned heavy chain amino acid sequences and a light chain amino acid sequence that is at least 95% identical to one of the above-mentioned light chain amino acid sequences. In some embodiments, an anti-TROP2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 126,127, 186, or 381 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 129 or 130. In some embodiments, an anti-TROP2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 122,123, 185, or 381 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 133 or 134.

[00239] In some embodiments, an anti-TROP2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 104 and the light chain amino acid sequence of SEQ ID NO: 107; or the heavy chain amino acid sequence of SEQ ID NO: 104 and the light chain amino acid sequence of SEQ ID NO: 110; or the heavy chain amino acid sequence of SEQ ID NO: 105 and the light chain amino acid sequence of SEQ ID NO: 107; or the heavy chain amino acid sequence of SEQ ID NO: 105 and the light chain amino acid sequence of SEQ ID NO: 110; or the heavy chain amino acid sequence of SEQ ID NO: 183 and the light chain amino acid sequence of SEQ ID NO: 107; or the heavy chain amino acid sequence of SEQ ID NO: 183 and the light chain amino acid sequence of SEQ ID NO: 110; or the heavy chain amino acid sequence of SEQ ID NO: 380 and the light chain amino acid sequence of SEQ ID NO: 107; or the heavy chain amino acid sequence of SEQ ID NO: 380 and the light chain amino acid sequence of SEQ ID NO: 110; or the heavy chain amino acid sequence of SEQ ID NO: 100 and the light chain amino acid sequence of SEQ ID NO: 112; or the heavy chain amino acid sequence of SEQ ID NO: 100 and the light chain amino acid sequence of SEQ ID NO: 115; or the heavy chain amino acid sequence of SEQ ID NO: 101 and the light chain amino acid sequence of SEQ ID NO: 112; or the heavy chain amino acid sequence of SEQ ID NO: 101 and the light chain amino acid sequence of SEQ ID NO: 115; or the heavy chain amino acid sequence of SEQ ID NO: 181 and the light chain amino acid sequence of SEQ ID NO: 112; or the heavy chain amino acid sequence of SEQ ID NO: 181 and the light chain amino acid sequence of SEQ ID NO: 115; or the heavy chain amino acid sequence of SEQ ID NO: 96 and the light chain amino acid sequence of SEQ ID NO: 112; or the heavy chain amino acid sequence of SEQ ID NO: 96 and the light chain amino acid sequence of SEQ ID NO: 115; or the heavy chain amino acid sequence of SEQ ID NO: 97 and the light chain amino acid sequence of SEQ ID NO: 112; or the heavy chain amino acid sequence of SEQ ID NO: 97 and the light chain amino acid sequence of SEQ ID NO: 115; or the heavy chain amino acid sequence of SEQ ID NO: 179 and the light chain amino acid sequence of SEQ ID NO: 112; or the heavy chain amino acid sequence of SEQ ID NO: 179 and the light chain amino acid sequence of SEQ ID NO: 115; or the heavy chain amino acid sequence of SEQ ID NO: 380 and the light chain amino acid sequence of SEQ ID NO: 112; or the heavy chain amino acid sequence of SEQ ID NO: 380 and the light chain amino acid sequence of SEQ ID NO: 115. In some embodiments, an anti-TROP2 antibody comprises a heavy chain amino acid sequence that is at least 95% identical to one of the above-mentioned heavy chain amino acid sequences and a light chain amino acid sequence that is at least 95% identical to one of the above-mentioned light chain amino acid sequences. In some embodiments, an anti-TROP2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 104, 105, 183, or 380 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 107 or 110. In some embodiments, an anti-TROP2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 100, 101, 181, or 380 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 112 or 115.

[00240] In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti- TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 181 and the light chain amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 183 and the light chain amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 380 and the light chain amino acid sequence of SEQ ID NO: 110.

[00241] In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 100 and a light chain amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 96 and a light chain amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti- TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 100 and the light chain amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-TROP2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 96 and the light chain amino acid sequence of SEQ ID NO: 110.

[00242] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 4; LCDR1 comprising SEQ ID NO: 8, LCDR2 comprising SEQ ID NO: 9, and LCDR3 comprising SEQ ID NO: 10, as defined by the Kabat numbering system. In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 11, HCDR2 comprising SEQ ID NO: 12, HCDR3 comprising SEQ ID NO: 13; LCDR1 comprising SEQ ID NO: 15, LCDR2 comprising SEQ ID NO: 16, and LCDR3 comprising SEQ ID NO: 17, as defined by the IMGT numbering system. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115 ; a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115; or a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115.

[00243] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 4; LCDR1 comprising SEQ ID NO: 8, LCDR2 comprising SEQ ID NO: 9, and LCDR3 comprising SEQ ID NO: 10, as defined by the Rabat numbering system. In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 11, HCDR2 comprising SEQ ID NO: 12, HCDR3 comprising SEQ ID NO: 13; LCDR1 comprising SEQ ID NO: 15, LCDR2 comprising SEQ ID NO: 16, and LCDR3 comprising SEQ ID NO: 17, as defined by the IMGT numbering system. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment comprises a heavy chain amino acid sequence of SEQ ID NO: 100 and a light chain amino acid sequence of SEQ ID NO: 115.

[00244] In some embodiments, the disclosed anti-TROP2 antibodies demonstrate an increased safety profile due to substitution of residues in the Fc domain. In some embodiments, the disclosed anti- TROP2 antibodies comprise an IgGl Fc domain that has been mutated to reduce binding to a Fey receptor (FcyR) as compared to an IgGl Fc-containing antibody with a wild-type IgGl Fc domain. These substitutions reduce the ability of the antibody to bind to various cell receptors, such as a Fey receptor (FcyR), and other immune molecules, as compared to an anti-TROP2 antibody without these substitutions. Without being bound by theory, the reduced binding of an antibody to a FcyR may reduce non-antigen mediated uptake by neutrophils, thereby reducing neutropenia in a treated subject. [00245] In some embodiments, the disclosed anti-TROP2 antibodies demonstrate superior properties (e.g., improved stability {e.g., shelf and/or serum stability), formulatability, antigen-binding specificity (e.g., epitope and/or affinity), safety profile, in vivo anti-tumor activity) compared to other TROP2 antibodies. In some embodiments, the disclosed anti-TROP2 antibodies demonstrate superior antigen-binding specificity compared to other TROP2 antibodies. In some embodiments, the disclosed anti-TROP2 antibodies demonstrate superior site-specific linker-payload conjugation compared to other TR0P2 antibodies. In some embodiments, the disclosed anti-TROP2 antibodies demonstrate a superior safety profile in vivo compared to other TR0P2 antibodies.

[00246] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof that comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system, demonstrates superior properties (e.g., improved stability (e.g., shelf and/or serum stability), formulatability, antigen-binding specificity (e.g., epitope and/or affinity), safety profile, in vivo anti-tumor activity, antigen-binding specificity, site-specific linker-payload conjugation, safety profile in vivo) compared to other TROP2 antibodies.

[00247] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof that comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system, demonstrates superior properties (e.g., improved stability (e.g., shelf and/or serum stability), formulatability, antigen-binding specificity (e.g., epitope and/or affinity), safety profile, in vivo anti-tumor activity, antigen-binding specificity, site-specific linkerpayload conjugation, safety profile in vivo) compared to other TROP2 antibodies.

[00248] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66 demonstrates superior properties (e.g., improved stability (e.g., shelf and/or serum stability), formulatability, antigen-binding specificity (e.g., epitope and/or affinity), safety profile, in vivo anti-tumor activity, antigen-binding specificity, site-specific linker-payload conjugation, safety profile in vivo) compared to other TROP2 antibodies.

[00249] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66 demonstrates superior properties (e.g., improved stability (e.g., shelf and/or serum stability), formulatability, antigen-binding specificity (e.g., epitope and/or affinity), safety profile, in vivo anti-tumor activity, antigen-binding specificity, site-specific linker-payload conjugation, safety profile in vivo) compared to other TROP2 antibodies.

[00250] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof that comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115 demonstrates superior properties (e.g., improved stability (e.g., shelf and/or serum stability), formulatability, antigen-binding specificity (e.g., epitope and/or affinity), safety profile, in vivo anti-tumor activity, antigen-binding specificity, site-specific linker-payload conjugation, safety profile in vivo) compared to other TR0P2 antibodies. [00251] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof that comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115 demonstrates superior properties (e.g., improved stability (e.g, shelf and/or serum stability), formulatability, antigen-binding specificity (e.g., epitope and/or affinity), safety profile, in vivo anti-tumor activity, antigen-binding specificity, site-specific linker-payload conjugation, safety profile in vivo) compared to other TROP2 antibodies. [00252] In some embodiments, an anti-TROP2 antibody or antigen-binding fragment thereof that comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 115 demonstrates superior properties (e.g., improved stability (e.g., shelf and/or serum stability), formulatability, antigen-binding specificity (e.g., epitope and/or affinity), safety profile, in vivo anti-tumor activity, antigen-binding specificity, site-specific linker-payload conjugation, safety profile in vivo) compared to other TROP2 antibodies. [00253] In any of the antibodies discussed above, the heavy chain amino acid sequence may lack the C-terminal lysine.

[00254] In various embodiments, amino acid substitutions may be made while retaining the binding affinity and/or specificity of an antibody disclosed herein and/or to provide one or more additional beneficial property, e.g., by making one or more changes in framework, constant domain, and/or CDR sequences. In some embodiments, the substitutions are of single residues. For instance, in some embodiments, the anti-TROP2 antibodies comprise a human IgGl Fc domain that comprises amino acid substitutions to reduce binding to a FcyR as compared to an IgGl Fc-containing antibody with a wild-type IgGl Fc domain. In some embodiments, the anti-TROP2 antibodies comprise a mutated human IgGl Fc domain that comprises one or more of the substitutions selected from N297Q, N297A, L234G/L235G, L234A/L235A, L234A/L235A/D265S, L234A/L235A/P329G, L234A/L235A/P238S/H268Q/K274Q, L235G/G236R, G236R/L328R, and/or L234S/L235T/G236R. In some embodiments, the anti-TROP2 antibodies comprise a mutated human IgGl Fc domain that comprises the substitutions L234A, L235A, P238S, H268Q, and K274Q. Insertions usually will be on the order of from about 1 to about 20 amino acid residues, although considerably larger insertions may be tolerated as long as biological function is retained (e.g., binding to TROP2). Deletions usually range from about 1 to about 20 amino acid residues, although in some cases deletions may be much larger. Substitutions, deletions, insertions, or any combination thereof may be used to arrive at a final derivative or variant. Generally, these changes are done on a few amino acids to minimize the alteration of the molecule, particularly the immunogenicity and specificity of the antigen-binding protein. However, larger changes may be tolerated in certain circumstances. Conservative substitutions are generally made in accordance with the following chart depicted as Table 10. Table 10. Exemplary Conservative Amino Acid Substitutions

[00255] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Table 10. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide’s properties are those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

[00256] In various embodiments where variant antibody sequences are used in an ADC, the variants typically exhibit the same qualitative biological activity and will elicit the same immune response, although variants may also be selected to modify the characteristics of the antigen-binding proteins as needed. For example, the anti-TROP2 antibodies provided herein may comprise a human IgGl Fc domain that is mutated to reduce binding to a FcyR as compared to an IgGl Fc-containing antibody with a wild-type IgGl Fc domain. Alternatively, the variant may be designed such that the biological activity of the antigen-binding protein is altered. For example, glycosylation sites may be altered or removed, as discussed herein.

[00257] Various anti-TROP2 antibodies may be used with the ADCs disclosed herein to target cancer cells. As shown below, the linker-toxins in the ADCs disclosed herein are surprisingly effective with the anti-TR0P2 antibodies also disclosed herein. These antibodies may be used with the linkers and toxin (e.g., eribulin) disclosed herein. In some embodiments, the TROP2-targeting antibody moiety is 17124 or a variant disclosed herein. In other embodiments, the TROP2-targeting antibody moiety is 16K21 or a variant disclosed herein. In yet other embodiments, the TROP2 -targeting antibody moiety is 20E16 or a variant disclosed herein. In some embodiments, while the disclosed linkers and toxin (eribulin) are surprisingly effective with several different TROP2-targeting antibodies, the TROP2 targeting antibody moieties such as 16K21 or a variant disclosed herein provided particularly improved antibody/ADC stability, manufacturability, tumor targeting, treatment efficacy, and reduced off-target killing. Improved treatment efficacy can be measured in vitro or in vivo, and may include reduced tumor growth rate and/or reduced tumor volume.

Linkers

[00258] In various embodiments, the anti-TROP2 antibodies and antigen-binding fragments disclosed herein may be joined to a drug moiety (e.g., a cytotoxic payload) by a linker to create an antibodydrug conjugate (ADC).

[00259] In some embodiments, the linker in an ADC is stable extracellularly in a sufficient manner to be therapeutically effective. In some embodiments, the linker is stable outside a cell, such that the ADC remains intact when present in extracellular conditions (e.g., prior to transport or delivery into a cell). The term “intact,” used in the context of an ADC, means that the antibody moiety remains attached to the drug moiety (e.g., eribulin). As used herein, “stable,” in the context of a linker or ADC comprising a linker, means that no more than 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linkers (or any percentage in between) in a sample of ADC are cleaved (or in the case of an overall ADC are otherwise not intact) when the ADC is present in extracellular conditions when evaluated over a set period of time. In some embodiments, the linkers in ADCs disclosed herein are chosen to remain stable for more than about 48 hours, more than 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours.

[00260] Whether a linker is stable extracellularly can be determined, for example, by including an ADC in plasma for a predetermined time period (e.g., 2, 4, 6, 8, 16, or 24 hours) and then quantifying the amount of free drug moiety present in the plasma. Stability may allow the ADC time to localize to target tumor cells and prevent the premature release of the drug, which could lower the therapeutic index of the ADC by indiscriminately damaging both normal and tumor tissues. In some embodiments, the linker is stable outside of a target cell and releases the drug moiety from the ADC once inside of the cell, such that the drug moiety can bind to its target (e.g., to microtubules). Thus, an effective linker will: (i) maintain the specific binding properties of the antibody moiety; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety via stable attachment to the antibody moiety; (iii) remain stable and intact until the ADC has been transported or delivered to its target site; and (iv) allow for the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage.

[00261] Linkers may impact the physico-chemical properties of an ADC. As many cytotoxic agents are hydrophobic in nature, Unking them to the antibody with an additional hydrophobic moiety may lead to aggregation. ADC aggregates are insoluble and often limit achievable drug loading onto the antibody, which can negatively affect the potency of the ADC. Protein aggregates of biologies, in general, have also been linked to increased immunogenicity. As shown below, linkers disclosed herein result in ADCs with low aggregation levels and desirable levels of drug loading. In various embodiments, a linker is conjugated to the antibody or antigen-binding fragment through a cysteine. In various embodiments, a linker is conjugated to the antibody or antigen-binding fragment through a lysine. Suitable methods for conjugating linkers of the present disclosure to an antibody include the technologies for directed attachment to a lysine on a heavy chain of an antibody and to a cysteine on the light chain of an antibody, e.g., as disclosed in PCT applications WO 2017/213267, WO 2017/106643, and WO 2016/205618, all of which are herein incorporated by reference in their entireties.

[00262] In some embodiments, the linker is a cleavable linker. Cleavable linkers are designed to release the drug when subjected to certain environmental factors, e.g., when internalized into the target cell. A cleavable linker refers to any linker that comprises a cleavable moiety. As used herein, the term “cleavable moiety” refers to any chemical bond that can be cleaved. Suitable cleavable chemical bonds are well known in the art and include, but are not limited to, acid labile bonds, protease/peptidase labile bonds, photolabile bonds, disulfide bonds, and esterase labile bonds. Linkers comprising a cleavable moiety can allow for the release of the drug moiety from the ADC via cleavage at a particular site in the linker.

[00263] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker sufficiently releases the drug moiety from the antibody moiety in the intracellular environment to activate the drug and/or render the drug therapeutically effective. In some embodiments, the drug moiety is not cleaved from the antibody moiety until the ADC enters a cell that expresses an antigen specific for the antibody moiety of the ADC, and the drug moiety is cleaved from the antibody moiety upon entering the cell. In some embodiments, the linker comprises a cleavable moiety that is positioned such that no part of the linker or the antibody moiety remains bound to the drug moiety upon cleavage. Exemplary cleavable linkers include acid labile linkers, protease/peptidase-sensitive linkers, photolabile linkers, dimethyl-, disulfide-, or sulfonamide- containing linkers.

[00264] In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker can be, e.g., a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker refers to any linker that comprises a cleavable peptide moiety. The term “cleavable peptide moiety” refers to any chemical bond linking amino acids (natural or synthetic amino acid derivatives) that can be cleaved by an agent that is present in the intracellular environment. In some embodiments, a cleavable peptide linker is more stably conjugated to an antibody disclosed herein compared to an acid labile linker.

[00265] In some embodiments, the linker is an enzyme-cleavable linker and a cleavable peptide moiety in the linker is cleavable by the enzyme. In some embodiments, the cleavable peptide moiety is cleavable by a lysosomal enzyme, e.g., cathepsin or legumain (also known as asparaginyl endopeptidase or vacuolar processing enzyme). In some embodiments, the linker is a cathepsin- cleavable linker. In some embodiments, the linker is a legumain-cleavable linker. In some embodiments, the linker is a plasmin-cleavable tinker. In some embodiments, the cleavable peptide moiety in the tinker is cleavable by a lysosomal cysteine cathepsin, such as cathepsin B, C, F, H, K, L, O, S, V, X, or W. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin B. An exemplary dipeptide that may be cleaved by cathepsin B is valine-citrulline (Val-Cit). Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69. Another exemplary dipeptide that may be cleaved by cathepsin B is valine-alanine (Val-Ala). Fu and Ho (2002) Antib. Ther. 1 (2):33-43. In some embodiments, a dipeptide that may be cleaved by cathepsin B comprises at least one methyl group, e.g., two methyl groups. An exemplary dipeptide comprising two methyl groups that may be cleaved by cathepsin B is valine-dimethylated lysine (Val-Lys(Me)₂). Another exemplary dipeptide comprising two methyl groups that may be cleaved by cathepsin B is alanine-dimethylated lysine (Ala-Lys(Me)₂).

[00266] In some embodiments, the cleavable peptide moiety in the linker is cleavable by a lysosomal cysteine endopeptidase, such as legumain. An exemplary monopeptide that may be cleaved by legumain is asparagine (Asn). Another exemplary monopeptide that may be cleaved by legumain is aspartic acid (Asp). In some embodiments, a cleavable monopeptide that is cleaved by legumain comprises a methyl group, e.g., methylated aspartic acid (Asp(OMe)).

[00267] In some embodiments, a cleavable peptide moiety cleavable by legumain may be referred to as “DTM-N” (dithiomaleimide-Asn) and have the structure as shown below:

[00268] In some embodiments, the cleavable peptide moiety in the linker is cleavable by a serine protease, such as plasmin. In some embodiments, a peptide that may be cleaved by plasmin may comprise phenylalanine-lysine (Phe-Lys). [00269] In some embodiments, the linker or the cleavable peptide moiety in the linker comprises an amino acid unit. In some embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug moiety from the ADC upon exposure to one or more intracellular proteases, such as one or more lysosomal enzymes. Doronina et al. (2003) Nat. Biotechnol. 21:778-84; Dubowchik and Walker (1999) Pharm. Therapeutics 83:67-123. Exemplary amino acid units include, but are not limited to, monopeptides, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary monopeptides include, but are not limited to, asparagine (Asn) and aspartic acid (Asp). Exemplary dipeptides include, but are not limited to, valine-citrulline (Val-Cit), alanine-asparagine (Ala-Asn), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), alanine-lysine (Ala-Lys), alanine-valine (Ala-Vai), valine-alanine (Vai- Ala), valine-lysine (Val-Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucinecitrulline (Ile-Cit), tryptophan-citrulline (Trp-Cit), and phenylalanine-alanine (Phe-Ala). Exemplary tripeptides include, but are not limited to, alanine-alanine-asparagine (Ala-Ala-Asn), glycine-valine- citrulline (Gly- Val-Cit), glutamic acid-valine -citrulline, glycine-glycine-glycine (Gly-Gly-Gly), phenylalanine-phenylalanine-lysine (Phe-Phe-Lys), alanine-phenylalanine-lysine (Ala-Phe-Lys), and glycine-phenylalanine-lysine (Gly-Phe-Lys). Exemplary tetrapeptides include, but are not limited to, glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly). Other exemplary amino acid units include, but are not limited to, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-N9-tosyl-Arg, and Phe-N9-Nitro-Arg, as described in, e.g., U.S. Pat. No. 6,214,345. In some embodiments, an amino acid unit comprises amino acid residues comprising at least one methyl group, e.g., a monomethyl or dimethyl group. Exemplary amino acid units that comprise amino acid residues comprising at least one methyl group include, but are not limited to, methylated aspartic acid (Asp(OMe)), alanine-dimethylated lysine (Ala-Lys(Me)₂), and valine-dimethylated lysine (Val-Lys(Me)₂). In some embodiments, the amino acid unit in the linker comprises Val-Cit. In some embodiments, the amino acid unit in the linker comprises Val-Ala. An amino acid unit may comprise amino acid residues that occur naturally and/or minor amino acids and/or non-naturally occurring amino acids, such as citrulline. Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor- associated protease, a lysosomal protease such as cathepsin B, C, D, or S, or legumain.

[00270] In some embodiments, the linker in an ADC disclosed herein may comprise an antibody attachment moiety. An antibody attachment moiety may be used, for example, to link the antibody moiety to the linker, which in turn may link to the drug moiety, e.g., indirectly through a cleavable moiety (e.g., a cleavable peptide).

[00271] In some embodiments, the linker comprises an antibody attachment moiety comprising a maleimide moiety (Mai). The term “maleimide moiety,” as used herein, means a compound that contains a maleimide group and that is reactive with a sulfhydryl group, e.g., a sulfhydryl group of a cysteine residue on the antibody moiety. Other functional groups that are reactive with sulfhydryl groups (thiols) and may therefore be used in place of a Mai include, but are not limited to, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

[00272] In some embodiments, the linker attaches to the antibody or antigen-binding fragment via a Mai moiety. In some embodiments, the Mai moiety is reactive with a cysteine residue on the antibody or antigen-binding fragment. In some embodiments, the Mai moiety is joined to the antibody or antigen-binding fragment via the cysteine residue.

[00273] In some embodiments, the Mai moiety comprises at least one, e.g., two or more, sulfidereactive groups. In some embodiments, the Mai moiety is reactive with two adjacent reduced cysteine residues on the antibody or antigen-binding fragment. In some embodiments, the Mai moiety is joined to the antibody or antigen-binding fragment via two cysteine residues.

[00274] In some embodiments, the Mai moiety comprises a maleimidocaproyl (MC) moiety. In some embodiments, the linker attaches to the antibody or antigen-binding fragment via an MC moiety. In some embodiments, the MC moiety is reactive with a cysteine residue on the antibody or antigenbinding fragment. In some embodiments, the MC moiety is joined to the antibody or antigen-binding fragment via the cysteine residue.

[00275] In some embodiments, the Mai moiety comprises a dithiomaleimide (DTM). In some embodiments, the linker attaches to the antibody or antigen-binding fragment via a DTM moiety. In some embodiments, the DTM moiety is reactive with a cysteine residue on the antibody or antigenbinding fragment. In some embodiments, the DTM moiety is joined to the antibody or antigenbinding fragment via the cysteine residue.

[00276] In some embodiments, the linker comprises the Mai moiety and a cleavable peptide moiety. In some embodiments, the Mai moiety attaches the antibody moiety to the cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the amino acid unit comprises Val-Cit. In some embodiments, the amino acid unit comprises Val-Lys(Me)₂. In some embodiments, the amino acid unit comprises Val-Ala. In some embodiments, the amino acid unit comprises Asn. In some embodiments, the amino acid unit comprises Asp. In some embodiments, the amino acid unit comprises Asp(OMe). In some embodiments, the amino acid unit comprises Ala-Lys(Me)₂. In some embodiments, the linker comprises Mal-Val-Cit. In some embodiments, the linker comprises Mal-Val-Lys(Me)₂. In some embodiments, the linker comprises Mal-Val-Ala. In some embodiments, the linker comprises Mal- Asn. In some embodiments, the linker comprises Mal-Asp. In some embodiments, the linker comprises Mal-Asp(OMe). In some embodiments, the linker comprises Mal-Ala-Lys(Me)₂.

[00277] In some embodiments, any of the linkers in ADCs disclosed herein may comprise at least one spacer unit joining the antibody moiety to the drug moiety. In some embodiments, the spacer unit joins a cleavage site (e.g., a cleavable peptide moiety) in the linker to the antibody moiety. In some embodiments, the spacer unit joins a cleavage site (e.g., a cleavable peptide moiety) in the linker to the drug moiety. In some embodiments, the linker, and/or spacer unit in the linker, is substantially hydrophilic. A hydrophilic linker may be used to reduce the extent to which the drug may be pumped out of resistant cancer cells through multiple drug resistance (MDR) or functionally similar transporters. In some embodiments, the linker includes one or more polyethylene glycol (PEG) moieties, e.g., 1, 2, 3, or 4 PEG moieties.

[00278] In some embodiments, the spacer unit in the linker comprises one or more PEG moieties. In some embodiments, the spacer unit comprises -(PEG)_m-, and m is an integer from 1 to 4. In some embodiments, m ranges from 1 to 4; or from 2 to 4. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, the spacer unit comprises (PEG)₁, (PEG)₂, (PEG)₃, or (PEG)₄. In some embodiments, the spacer unit comprises (PEG)₂.

[00279] In some embodiments, the spacer unit in the linker comprises an alkyl moiety. In some embodiments, the spacer unit comprises -(CH₂)_n-, and n is an integer from 1 to 10 (i.e., n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n is an integer from 1 to 6. In some embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, the spacer unit comprises -CH₂-CH₂-.

[00280] In some embodiments, the spacer unit comprises (“C₂”).

[00281] In some embodiments, the spacer unit comprises (C₂) and -(PEG)_m-, and m is an integer from 1 to 4. In some embodiments, the spacer unit comprises conjugated to -(PEG)_m-, and m is an integer from 1 to 4. In some embodiments, m ranges from 1 to 4; from 2 to 4; or from 1 to 3. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4.

[00282] In some embodiments, the spacer unit comprises (C₂) conjugated to (PEG)₂.

[00283] In some embodiments, the spacer unit comprises

(“C₂(PEG)_m”), wherein m is 0-3. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.

[00284] In some embodiments, the spacer unit comprises

(“C₂(PEG)₂”).

[00285] In some embodiments, using a linker comprising a spacer unit comprising C₂ may provide benefits over other linkers when conjugated to any of a variety of different antibodies or antigen- binding fragments. In some embodiments, a linker comprising C₂ conjugated to -(PEG)_m-, wherein m is an integer from 0 to 4 (e.g., (PEG)₁, (PEG)₂, or (PEG)₃) demonstrates superior properties when conjugated to any of a variety of different antibodies or antigen-binding fragments. In some embodiments, the spacer unit comprising C₂ is or comprises C₂(PEG)_m, wherein m is 0-3. In some embodiments, using a linker comprising a spacer unit comprising C₂(PEG)_m, wherein m is 0-3, may provide benefits, as compared to alternative linkers not comprising a unit of C₂(PEG)_m, wherein m is 0-3, when conjugated to any of a variety of different antibodies or antigen-binding fragments. These benefits may include, e.g., improved conjugation stability, improved plasma stability, and/or in vivo anti-tumor activity compared to other linkers comprising alternative spacer units. In some embodiments, without being bound by theory, benefits of using a linker comprising C₂(PEG)_m, wherein m is 0-3, conjugated to eribulin may include improved conjugation stability, improved plasma stability, and/or in vivo anti-tumor activity. In some embodiments, a linker comprising C₂(PEG)_m, wherein m is 0-3, and VCP conjugated to eribulin demonstrates superior properties when conjugated to an anti-TROP2 antibody disclosed herein. In some embodiments, a linker comprising C₂(PEG)_m, wherein m is 0-3, and N conjugated to eribulin demonstrates superior properties when conjugated to an anti-TROP2 antibody disclosed herein. In some embodiments, m is 2. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Example 7.

[00286] A spacer unit may be used, for example, to link the antibody moiety to the drug moiety, either directly or indirectly. In some embodiments, the spacer unit links the antibody moiety to the drug moiety directly. In some embodiments, the antibody moiety and the drug moiety are attached via a spacer unit comprising one or more PEG moieties (e.g., (PEG)₂, (PEG)₃, or (PEG)₄). In some embodiments, the spacer unit links the antibody moiety to the drug moiety indirectly. In some embodiments, the spacer unit links the antibody moiety to the drug moiety indirectly through a cleavable moiety (e.g., a cleavable peptide) and/or an antibody attachment moiety to join the spacer unit to the antibody moiety, e.g., a maleimide moiety or a carbobenzoxy-L-glutaminyl-glycine moiety. [00287] In some embodiments, the spacer unit attaches to the antibody moiety (i.e., the antibody or antigen-binding fragment) via a maleimide moiety (Mai). A spacer unit that attaches to the antibody or antigen-binding fragment via a Mai is referred to herein as a “Mal-spacer unit.” In some embodiments, the Mal-spacer unit is reactive with a cysteine residue on the antibody or antigenbinding fragment. In some embodiments, the Mal-spacer unit is joined to the antibody or antigenbinding fragment via the cysteine residue. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises C₂. In some embodiments, the Mal-spacer unit comprises C₂(PEG)₁. In some embodiments, the Mal-spacer unit comprises C₂(PEG)₂. In some embodiments, the Mal-spacer unit comprises C₂(PEG)₃.

[00288] In some embodiments, the linker comprises the Mal-spacer unit and a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the amino acid unit comprises Val-Cit. In some embodiments, the amino acid unit comprises Val-Lys(Me)₂. In some embodiments, the amino acid unit comprises Ala-Lys(Me)₂. In some embodiments, the amino acid unit comprises Vai-Ala. In some embodiments, the amino acid unit comprises Asn. In some embodiments, the amino acid unit comprises Asp. In some embodiments, the amino acid unit comprises Asp(OMe). In some embodiments, the linker comprises the Mal-spacer unit and an amino acid unit. In some embodiments, the linker comprises Mal-(PEG)_m and an amino acid unit, wherein m is 2 to 4; or 2, 3, or 4. In some embodiments, the linker comprises Mal-(PEG)₂- Val-Cit. In some embodiments, the linker comprises Mal-(PEG)₃-Val-Cit. In some embodiments, the linker comprises Mal-(PEG)₄-Val-Cit. In some embodiments, the linker comprises Mal-(PEG)₂-Val- Lys(Me)₂. In some embodiments, the linker comprises Mal-(PEG)₂-Ala-Lys(Me)₂. In some embodiments, the linker comprises Mal-(PEG)₂-Val-Ala. In some embodiments, the linker comprises Mal-(PEG)₂-Asn. In some embodiments, the linker comprises Mal-(PEG)₂-Asp. In some embodiments, the linker comprises Mal-(PEG)₂-Asp(OMe). In some embodiments, the linker comprises Mal-C₂- Val-Cit. In some embodiments, the linker comprises Mal-C₂-VCP. In some embodiments, the linker comprises Mal-C₂(PEG)₁-Val-Cit. In some embodiments, the linker comprises Mal-C₂(PEG)₁-Val-Lys(Me)₂. In some embodiments, the linker comprises Mal-C₂(PEG)₁- Ala-Lys(Me)₂. In some embodiments, the linker comprises Mal-C₂(PEG)₁-Val-Ala. In some embodiments, the linker comprises Mal-C₂(PEG)₁-Asn. In some embodiments, the linker comprises Mal-C₂(PEG)₁-Asp. In some embodiments, the linker comprises Mal-C₂(PEG)₁-Asp(OMe). In some embodiments, the linker comprises Mal-C₂(PEG)₂-Val-Cit. In some embodiments, the linker comprises Mal-C₂(PEG)₂-Val-Lys(Me)₂. In some embodiments, the linker comprises Mal-C₂(PEG)₂- Ala-Lys(Me)₂. In some embodiments, the linker comprises Mal-C₂(PEG)₂-Val-Ala. In some embodiments, the linker comprises Mal-C₂(PEG)₂-Asn. In some embodiments, the linker comprises Mal-C₂(PEG)₂-Asp. In some embodiments, the linker comprises Mal-C₂(PEG)₂-Asp(OMe). In some embodiments, the linker comprises Mal-C₂(PEG)₃-Val-Cit. In some embodiments, the linker comprises Mal-C₂(PEG)₃-Val-Lys(Me)₂. In some embodiments, the linker comprises Mal-C₂(PEG)₃- Ala-Lys(Me)₂. In some embodiments, the linker comprises Mal-C₂(PEG)₃-Val-Ala. In some embodiments, the linker comprises Mal-C₂(PEG)₃-Asn. In some embodiments, the linker comprises Mal-C₂(PEG)₃-Asp. In some embodiments, the linker comprises Mal-C₂(PEG)₃-Asp(OMe).

[00289] In various embodiments, the cleavable moiety in the linker is joined directly to the drug moiety and/or to the antibody moiety. In other embodiments, a spacer unit is used to attach the cleavable moiety in the linker to the drug moiety and/or to the antibody moiety. In various embodiments, the drug moiety is eribulin. In various embodiments, the eribulin is attached to the cleavable moiety in the linker by a spacer unit. In some embodiments, the eribulin is attached to the cleavable moiety in the linker by a self-immolative spacer unit. In some embodiments, the eribulin is attached to the cleavable moiety in the linker by a self-immolative spacer unit, the cleavable moiety comprises an amino acid unit, and a further spacer unit comprising PEG joins the cleavable moiety to the antibody moiety. In some embodiments, the eribulin is attached to the cleavable moiety in the linker by a self-immolative spacer unit, the cleavable moiety comprises an amino acid unit, and a further spacer unit comprising C₂(PEG)_m, wherein m is 0 to 4 (e.g., C₂(PEG)₂), joins the cleavable moiety to the antibody moiety. In some embodiments, the eribulin is joined to an anti-TROP2 antibody via a Mal-spacer unit in the linker joined to a cleavable peptide moiety and a pAB or pABC self-immolative spacer unit.

[00290] A spacer unit may be “self-immolative” or “non-self-immolative.” A “non-self-immolative” spacer unit is one in which part or all of the spacer unit remains bound to the drug moiety upon cleavage of the linker. Examples of non-self-immolative spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. Non-self-immolative spacer units may eventually degrade over time but do not readily release a linked native drug entirely under cellular conditions. A “self-immolative” spacer unit allows for release of the native drug moiety under intracellular conditions. A “native drug” is one where no part of the spacer unit or other chemical modification remains after cleavage/degradation of the spacer unit.

[00291] Self-immolation chemistry is known in the art and could be readily selected for the disclosed ADCs. In various embodiments, the spacer unit attaching the cleavable moiety in the linker to the drug moiety (e.g., eribulin) is self-immolative, and undergoes self-immolation concurrently with or shortly before/after cleavage of the cleavable moiety under intracellular conditions.

[00292] In certain embodiments, the self-immolative spacer unit in the linker comprises a p- aminobenzyl unit. In some embodiments, a p-aminobenzyl alcohol (pAB-OH) is attached to an amino acid unit or other cleavable moiety in the linker via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the pAB-OH and the drug moiety. Hamann et al. (2005) Expert Opin. Ther. Patents 15:1087-103. In some embodiments, the self-immolative spacer unit is or comprises p-aminobenzyl (pAB). In some embodiments, the self-immolative spacer unit is or comprises p-aminobenzyloxycarbonyl (pABC). Without being bound by theory, it is thought that the self-immolation of pAB or pABC involves a spontaneous 1,6-elimination reaction. Jain et al. (2015) Pharm Res 32:3526-40.

[00293] In various embodiments, the structure of the p-aminobenzyl (pAB) used in the disclosed ADCs is shown below:

[00294] In various embodiments, the structure of the p-aminobenzyloxycarbonyl (pABC) used in the disclosed ADCs is shown below:

[00295] In various embodiments, a linker (e.g., one comprising a spacer, e.g., comprising a self- immolative spacer unit) attaches the cleavable moiety in the linker to the C-35 amine on eribulin. In some embodiments, the self-immolative spacer unit comprises pAB. In some embodiments, the pAB attaches the cleavable moiety in the linker to the C-35 amine on eribulin. In some embodiments, the pAB undergoes self-immolation upon cleavage of the cleavable moiety, and eribulin is released from the ADC in its native, active form.

[00296] In some embodiments, the self-immolative spacer unit comprises pABC. In some embodiments, the pABC attaches the cleavable moiety in the linker to the C-35 amine on eribulin. In some embodiments, the pABC undergoes self-immolation upon cleavage of the cleavable moiety, and eribulin is released from the ADC in its native, active form. In some embodiments, the pABC undergoes self-immolation upon cleavage of a cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises amino acid unit-pABC. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pABC (VCP). In some embodiments, the amino acid unit is Val-Lys(Me)₂. In some embodiments, the linker comprises Val-Lys(Me)₂-pABC. In some embodiments, the amino acid unit is Ala-Lys(Me)₂. In some embodiments, the linker comprises Ala- Lys(Me)₂-pABC. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pABC (VAP). In some embodiments, the amino acid unit is Asn. In some embodiments, the linker comprises Asn-pABC. In some embodiments, the amino acid unit is Asp. In some embodiments, the linker comprises Asp-pABC. In some embodiments, the amino acid unit is Asp(OMe). In some embodiments, the linker comprises Asp(OMe)-pABC.

[00297] In various aspects, the antibody moiety of the ADC is conjugated to the drug moiety via a linker, wherein the linker comprises a Mal-spacer unit, a cleavable amino acid unit, and a pAB or a pABC. In some embodiments, the spacer unit comprises a PEG moiety. In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the spacer unit comprises C₂. In some embodiments, the spacer unit comprises C₂(PEG)₁. In some embodiments, the spacer unit comprises C₂(PEG)₂. In some embodiments, the spacer unit comprises C₂(PEG)₃. In some embodiments, the linker comprises Mal-(PEG)₂-amino acid unit-pABC. In some embodiments, the linker comprises Mal-(PEG)₂-Val-Cit-pABC. In some embodiments, the linker comprises Mal-(PEG)₃-Val-Cit-pABC. In some embodiments, the linker comprises Mal-(PEG)₄-Val-Cit-pABC. In some embodiments, the linker comprises Mal-(PEG)₂-Val-Ala-pABC. In some embodiments, the linker comprises Mai- (PEG)₂-Val-Lys(Me)₂-pABC. In some embodiments, the linker comprises Mal-(PEG)₂-Ala-Lys(Me)₂- pABC. In some embodiments, the linker comprises Mal-C₂(PEG)_m-amino acid unit-pABC. In some embodiments, the linker comprises Mal-C₂(PEG)_m-Val-Cit-pABC. In some embodiments, the linker comprises Mal-C₂(PEG)_m -Val-Ala-pABC. In some embodiments, the linker comprises Mal- C₂(PEG)₂-Val-Lys(Me)_m -pABC. In some embodiments, m is an integer from 0 to 4. In some embodiments, m is 2.

[00298] In various embodiments, the linker is designed to facilitate bystander killing (the killing of neighboring cells) through cleavage after cellular internalization and diffusion of the linker-drug moiety and/or the drug moiety alone to neighboring cells. In some embodiments, the linker promotes cellular internalization. In some embodiments, the linker is designed to minimize cleavage in the extracellular environment and thereby reduce toxicity to off-target tissue (e.g., non-cancerous tissue), while preserving ADC binding to target tissue and bystander killing of cancerous tissue that does not express an antigen targeted by the antibody moiety of an ADC, but surrounds target cancer tissue expressing that antigen. In some embodiments, a linker comprising a maleimide moiety (Mai), a polyethylene glycol (PEG) moiety, valine-citrulline (Val-Cit or “VC”), and a pABC provides these functional features. In some embodiments, a linker comprising a maleimide moiety (Mai), a polyethylene glycol (PEG) moiety, valine-alanine (Val-Ala or “VA”), and a pABC provides these functional features. In some embodiments, a linker comprising a maleimide moiety (Mai), C₂(PEG)_m, valine-citrulline (Val-Cit or “VC”), and a pABC provides these functional features. In some embodiments, a linker comprising a maleimide moiety (Mai), C₂(PEG)_m, valine-alanine (Val-Ala or “VA”), and a pABC provides these functional features. In some embodiments, a linker comprising a maleimide moiety (Mai), C₂(PEG)_m, and Asn provides these functional features. In some embodiments, m is 0, 1, 2, or 3. In some embodiments, m is 2. In some embodiments, the drug moiety is eribulin.

[00299] In some embodiments, the antibody moiety is conjugated to the drug moiety via a linker comprising a maleimide moiety (Mai), a polyethylene glycol (PEG) moiety, and a cleavable amino acid moiety. In some embodiments, the antibody moiety is conjugated to the drug moiety via a linker comprising a maleimide moiety (Mai), a polyethylene glycol (PEG) moiety, a cleavable amino acid moiety, and a pABC.

[00300] In some embodiments, the antibody moiety is conjugated to the drug moiety via a linkerpayload conjugate of Formula (Ila): , wherein: D is the drug moiety (e.g., a cytotoxic agent, e.g., erihulin); Y is a cleavable moiety; and Z is absent or a spacer unit. In some embodiments, the drug moiety is eribulin. In some embodiments, Y comprises a cleavable amino acid moiety. In some embodiments, Y comprises Val-Cit. In some embodiments, Y comprises Vai-Ala. In some embodiments, Y comprises aspartic acid (Asp). In some embodiments, Y comprises asparagine (Asn). In some embodiments, Y comprises Val-Lys(Me)₂. In some embodiments, Y comprises Ala- Lys(Me)₂. In some embodiments, Y comprises Asp(OMe). In some embodiments, Z is absent. In some embodiments, Z comprises pAB. In some embodiments, Z comprises pABC.

[00301] In some embodiments, Y comprises Val-Cit and Z comprises pABC. In some embodiments, Y comprises Val-Ala and Z comprises pABC. In some embodiments, Y comprises Val-Lys(Me)₂ and Z comprises pABC. In some embodiments, Y comprises Ala-Lys(Me)₂ and Z comprises pABC. In some embodiments, Y comprises Asp and Z is absent. In some embodiments, Y comprises Asn and Z is absent. In some embodiments, Y comprises Asp(OMe) and Z is absent. In some embodiments, D comprises eribulin.

[00302] In some embodiments of linker-payload conjugates disclosed herein, the drug moiety is eribulin. The structures of exemplary linkers attached to eribulin that may be joined to TROP2 antibodies (e.g. , antibodies as disclosed herein) are provided in Table 11.

[00303] In some embodiments, the ADC comprises a cleavable linker and an internalizing anti- TROP2 antibody or internalizing antigen-binding fragment thereof as disclosed herein. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 5 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 14 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system.

[00304] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. [00305] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 107. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 105 and a light chain comprising an amino acid sequence of SEQ ID NO: 107. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 105 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 107. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 107. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti- TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 112. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 101 and a light chain comprising an amino acid sequence of SEQ ID NO: 112. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 101 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 112. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 112. In some embodiments, the anti-TR0P2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00306] In some embodiments, the internalizing antibody is 16K21. In some embodiments, the 16K21 antibody comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 4; LCDR1 comprising SEQ ID NO: 8, LCDR2 comprising SEQ ID NO: 9, and LCDR3 comprising SEQ ID NO: 10, as defined by the Kabat numbering system; or three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 11, HCDR2 comprising SEQ ID NO: 12, HCDR3 comprising SEQ ID NO: 13; LCDR1 comprising SEQ ID NO: 15, LCDR2 comprising SEQ ID NO: 16, and LCDR3 comprising SEQ ID NO: 17, as defined by the IMGT numbering system. In some embodiments, the 16K21 antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, the 16K21 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115. In some embodiments, p is from 1 to 8, or 2 to 8. In some embodiments, p is from 2 to 4. In some embodiments, p is 2.

[00307] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73.

[00308] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 126 and a light chain comprising an amino acid sequence of SEQ ID NO: 129. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 122 and a light chain comprising an amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 126 and a light chain comprising an amino acid sequence of SEQ ID NO: 130. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 122 and a light chain comprising an amino acid sequence of SEQ ID NO: 134. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 127 and a light chain comprising an amino acid sequence of SEQ ID NO: 129. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 123 and a light chain comprising an amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 127 and a light chain comprising an amino acid sequence of SEQ ID NO: 130. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 123 and a light chain comprising an amino acid sequence of SEQ ID NO: 134.

[00309] In some embodiments, the internalizing antibody is 16K21. In some embodiments, p is from 1 to 8, or 2 to 8. In some embodiments, p is from 2 to 4. In some embodiments, p is 2.

Antibody-Drug Conjugates

[00310] In various embodiments, an anti-TROP2 antibody moiety (including an antigen-binding fragment thereof) disclosed herein may be conjugated (i.e., covalently attached, e.g., by a linker) to a drug moiety, wherein the drug moiety when not conjugated to an antibody moiety has a cytotoxic or cytostatic effect. In some embodiments, the drug moiety exhibits reduced or no cytotoxicity when bound in an antibody-drug conjugate but resumes cytotoxicity after cleavage from the linker and antibody moiety. In various embodiments, the drug moiety exhibits reduced or no bystander killing when bound in an antibody-drug conjugate (e.g., using a non-cleavable linker) but exhibits increased bystander killing after cleavage from an antibody-drug conjugate (e.g., an antibody-drug conjugate having a cleavable Val-Cit, Val-Ala, Asp, or Asn cleavable moiety). [00311] The development and production of an ADC for use as a human therapeutic agent, e.g., as an oncologic agent, may require more than the identification of an antibody capable of binding to a desired target or targets and attaching to a drug used on its own to treat cancer. Linking the antibody to the drug may have significant and unpredictable effects on the activity of one or both of the antibody and the drug, effects which will vary depending on the type of linker and/or drug chosen. In some embodiments, therefore, the components of the ADC are selected to (i) retain one or more therapeutic properties exhibited by the antibody and drug moieties in isolation; (ii) maintain the specific binding properties of the antibody moiety; (iii) optimize drug loading and drug-to-antibody ratios; (iv) allow delivery, e.g., intracellular delivery, of the drug moiety via stable attachment to the antibody moiety; (v) retain ADC stability as an intact conjugate until transport or delivery to a target site; (vi) minimize aggregation of the ADC prior to or after administration; (vii) allow for the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage in the cellular environment; (viii) exhibit in vivo anti-cancer treatment efficacy comparable to or superior to that of the antibody and drug moieties in isolation; (ix) minimize off-target killing by the drug moiety; and/or (x) exhibit desirable pharmacokinetic and pharmacodynamics properties, formulatability, and toxicologic/immunologic profiles. Screening each of these properties may be needed to identify an improved ADC for therapeutic use. Ab et al. (2015) Mol. Cancer Ther. 14:1605-13.

[00312] The ADC compounds of the present disclosure may selectively deliver an effective dose of a cytotoxic or cytostatic agent to cancer cells or to tumor tissue. It has been discovered that the disclosed ADCs have potent cytotoxic and/or cytostatic activity against cells expressing TROP2. In some embodiments, the cytotoxic and/or cytostatic activity of the ADC is dependent on TROP2 expression level on a cell surface. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells expressing a high level of TROP2, as compared to cancer cells expressing the same antigen at a low level. Exemplary high TROP2-expressing cancers include but are not limited to bladder cancer, breast cancer, cervical carcinoma, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioma, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, and skin cancer. In some embodiments, the disclosed ADCs are particularly effective at killing high TROP2-expressing cancers such as cholangiocarcinoma, breast cancer, or NSCLC.

[00313] In some embodiments, the disclosed ADCs also demonstrate bystander killing activity, but low off-target cytotoxicity. Without being bound by theory, the bystander killing activity of an ADC may be particularly beneficial where its penetration into a solid tumor is limited and/or target antigen expression among tumor cells is heterogeneous. In some embodiments, an ADC comprising a cleavable linker is particularly effective at bystander killing and/or demonstrates improved bystander killing activity, as compared to comparable treatment with an ADC comprising a non-cleavable linker. [00314] In some embodiments, the disclosed ADCs also demonstrate an increased safety profile due to substitution of residues in the Fc domain of the anti-TROP2 antibody. In some embodiments, the disclosed ADCs comprise an anti-TROP2 antibody that comprises an IgGl Fc domain that has been mutated to reduce binding to a Fey receptor (FcyR) as compared to an IgGl Fc-containing antibody with a wild-type IgGl Fc domain. These substitutions reduce the ability of the ADC to bind to various cell receptors, such as a Fey receptor (FcyR), and other immune molecules, as compared to an ADC comprising an anti-TROP2 antibody without these substitutions. Without being bound by theory, the reduced binding of an ADC to a FcyR may reduce non-antigen mediated uptake by neutrophils, thereby reducing neutropenia in a treated subject.

[00315] In some embodiments, the disclosed ADCs demonstrate superior properties (e.g., antigenbinding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile) compared to other TROP2 ADCs (e.g., DS- 1062a or IMMU-132). In some embodiments, the disclosed ADCs have superior antigen-binding specificity compared to other TROP2 ADCs (e.g., DS-1062a or IMMU-132). In some embodiments, the disclosed ADCs have superior site-specific linker-payload conjugation compared to other TROP2 ADCs (e.g., DS- 1062a or IMMU-132). In some embodiments, the disclosed ADCs have superior tolerability compared to other TROP2 ADCs (e.g., DS-1062a or IMMU-132). In some embodiments, the disclosed ADCs have superior in vitro cytotoxicity compared to other TROP2 ADCs (e.g., DS- 1062a or IMMU-132). In some embodiments, the disclosed ADCs have a superior safety profile (e.g., reduced Fey receptor interactions) compared to other TROP2 ADCs (e.g., DS-1062a or IMMU-132). In some embodiments, the disclosed ADCs have a superior property in more than one of the listed categories (e.g., all of them).

[00316] Provided herein are ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab) which targets a tumor cell, a drug moiety (D), and a linker moiety (L) that covalently attaches Ab to D. In certain aspects, the antibody or antigen-binding fragment is able to bind to a tumor-associated antigen (e.g., TROP2) with high specificity and high affinity. In certain embodiments, the antibody or antigen-binding fragment is internalized into a target cell upon binding, e.g., into a degradative compartment in the cell. Preferred ADCs are thus those that internalize upon binding to a target cell, undergo degradation, and release the drug moiety to kill cancer cells. The drug moiety may be released from the antibody and/or the linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

[00317] In various embodiments, an anti-TROP2 ADC disclosed herein is conjugated to eribulin via a linker comprising C₂. In some embodiments, using a linker comprising a spacer unit comprising C₂ may provide benefits over other linkers when conjugated to an anti-TROP2 ADC disclosed herein. In some embodiments, the spacer unit comprising C₂is or comprises C₂(PEG)_m, wherein m is 1-3 (e.g., C₂(PEG)₁, C₂(PEG)₂, or C₂(PEG)₃). In some embodiments, using a linker comprising a spacer unit comprising C₂(PEG)_m, wherein m is 1-3, may provide benefits, as compared to alternate linkers not comprising a unit of C₂(PEG)_m, wherein m is 1-3, when conjugated to an anti-TROP2 ADC disclosed herein. These benefits may include, e.g., improved conjugation stability, improved plasma stability, and/or in vivo anti-tumor activity compared to other linkers comprising alternative spacer units. In some embodiments, without being bound by theory, benefits of using an anti-TROP2 ADC conjugated to eribulin via a linker comprising C₂(PEG)_m, wherein m is 1-3, may include improved conjugation stability, improved plasma stability, and/or in vivo anti-tumor activity. In some embodiments, an anti-TROP2 ADC conjugated to eribulin via a linker comprising C₂(PEG)_m, wherein m is 1-3, and VCP demonstrates superior properties compared to other anti-TROP2-eribulin ADCs. In some embodiments, an anti-TROP2 ADC conjugated to eribulin via a linker comprising C₂(PEG)_m, wherein m is 1-3, and N demonstrates superior properties compared to other anti-TROP2-eribulin ADCs. In some embodiments, m is 2. Exemplary evidence of the superior benefits of such antibodydrug conjugates is shown in Example 7.

[00318] An exemplary ADC has Formula (I):

Ab-(L-D)_p (I) wherein Ab is an antibody moiety (i.e., an antibody or antigen-binding fragment), L is a linker moiety, D is a drug moiety, and p is the number of drug moieties per antibody moiety.

[00319] In some embodiments, the antibody-drug conjugate comprises a conjugate of Formula (II): wherein: Ab is an antibody or antigen-binding fragment thereof; D is a drug moiety (e.g., a cytotoxic agent); Y is a cleavable moiety; Z is absent or a spacer unit; and p is an integer from 1 to 8.

[00320] In some embodiments, the drug moiety is eribulin. In some embodiments, Y comprises a cleavable amino acid moiety. In some embodiments, Y comprises Val-Cit. In some embodiments, Y comprises Vai-Ala. In some embodiments, Y comprises aspartic acid (Asp). In some embodiments, Y comprises asparagine (Asn). In some embodiments, Y comprises Val-Lys(Me)₂. In some embodiments, Y comprises Ala-Lys(Me)₂. In some embodiments, Y comprises Asp(OMe). In some embodiments, Z is absent. In some embodiments, Z comprises pAB. In some embodiments, Z comprises pABC.

[00321] In some embodiments, Y comprises Val-Cit and Z comprises pABC. In some embodiments, Y comprises Val-Ala and Z comprises pABC. In some embodiments, Y comprises Asp and Z is absent. In some embodiments, Y comprises Val-Lys(Me)₂ and Z comprises pABC. In some embodiments, Y comprises Ala-Lys(Me)₂ and Z comprises pABC. In some embodiments, Y comprises Asn and Z is absent. In some embodiments, Y comprises Asp(OMe) and Z is absent. In some embodiments, D comprises eribulin. [00322] In some embodiments, the antibody-drug conjugate comprises a conjugate of Formula (III): , wherein: Ab is an antibody or antigen-binding fragment thereof; D is a drug moiety (e.g., a cytotoxic agent); Y is a cleavable moiety; Z is absent or a spacer unit; and p is an integer from 1 to 8.

[00323] In some embodiments, the drug moiety is eribulin. In some embodiments, Y comprises a cleavable amino acid moiety. In some embodiments, Y comprises Val-Cit. In some embodiments, Y comprises Val-Ala. In some embodiments, Y comprises aspartic acid (Asp). In some embodiments, Y comprises asparagine (Asn). In some embodiments, Y comprises valine-dimethylated lysine (Val- Lys(Me)₂). In some embodiments, Y comprises alanine-dimethylated lysine (Ala-Lys(Me)₂). In some embodiments, Y comprises methylated aspartic acid (Asp(OMe)). In some embodiments, Z is absent. In some embodiments, Z comprises pAB. In some embodiments, Z comprises pABC.

[00324] In some embodiments, Y comprises Val-Cit and Z comprises pABC. In some embodiments, Y comprises Val-Ala and Z comprises pABC. In some embodiments, Y comprises Val-Lys(Me)₂ and Z comprises pABC. In some embodiments, Y comprises Ala-Lys(Me)₂ and Z comprises pABC. In some embodiments, Y comprises Asp and Z is absent. In some embodiments, Y comprises Asn and Z is absent. In some embodiments, Y comprises Asp(OMe) and Z is absent. In some embodiments, D comprises eribulin.

[00325] In some embodiments, the present disclosure describes methods of preparing an antibodydrug conjugate of Formula (II). In some embodiments, the method comprises:

- providing one or more compounds of Formula (Ila) wherein: D is a cytotoxic agent; Y is a cleavable moiety; and Z is absent or a spacer unit; and

- conjugating the one or more compounds of Formula (Ila) to an antibody or antigen-binding fragment, to provide an antibody-drug conjugate of Formula (II): wherein Ab is the antibody or antigen-binding fragment. [00326] In some embodiments, the present disclosure describes methods of preparing an antibodydrug conjugate of Formula (III). In some embodiments, the method comprises:

- providing one or more compounds of Formula (Ila) , wherein: D is a cytotoxic agent; Y is a cleavable moiety; and Z is absent or a spacer unit;

- hydrolyzing the succinimide ring in the compound of Formula (II), to provide the compound of Formula (III)

[00327] In some embodiments, the step of hydrolyzing the succinimide ring in the compound of Formula (II) comprises a pH from about 7.4 to about 9.2. In some embodiments, the pH is about 7.4 to about 8.5. In some embodiments, the pH is about 7.4. In some embodiments, the step of hydrolyzing the succinimide ring in the compound of Formula (II) comprises a temperature of about 37 °C. In some embodiments, the step of hydrolyzing the succinimide ring in the compound of Formula (II) is completed in about 24 hours or less.

Drug Moieties

[00328] The drug moiety (D) of the ADCs described herein can be any chemotherapeutic agent.

Useful classes of chemotherapeutic agents include, for example, anti-tubulin agents, topoisomerase I inhibitor agents, or immunostimulatory agents. In certain embodiments, the drug moiety is an anti- tubulin agent. In some embodiments, the drug moiety for use in the ADCs disclosed herein is eribulin. In some embodiments, the drug moiety is a topoisomerase inhibitor agent.

[00329] In various embodiments, the drug moiety is eribulin. In these embodiments, the linker of the ADC is attached via the C-35 amine on eribulin. [00330] In certain embodiments, an intermediate, such as a precursor of a linker disclosed above, is reacted with the drug moiety under appropriate conditions. In certain embodiments, reactive groups are used on the drug and/or the intermediate or linker. The product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with the antibody or antigenbinding fragment under appropriate conditions, e.g., according to the methods discussed below. Alternatively, the linker or intermediate may first be reacted with the antibody or a derivatized antibody, and then reacted with the drug or derivatized drug.

[00331] A number of different reactions are available for covalent attachment of drugs and/or linkers to the antibody moiety. This is often accomplished by reaction of one or more amino acid residues of the antibody molecule, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and the various moieties of the aromatic amino acids. For instance, non-specific covalent attachment may be undertaken using a carbodiimide reaction to link a carboxy (or amino) group on a compound to an amino (or carboxy) group on an antibody moiety. Additionally, bifunctional agents such as dialdehydes or imidoesters may also be used to link the amino group on a compound to an amino group on an antibody moiety. Also available for attachment of drugs to binding agents is the Schiff base reaction. This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the binding agent. Isothiocyanates may also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the skilled artisan and within the scope of the present disclosure.

[00332] To accomplish site-specific conjugation of linkers and/or drug moieties to an antibody or antigen-binding fragment thereof, in some embodiments, a microbial transglutaminase is used to conjugate the linker or an intermediate comprising an acyl donor substrate with the antibody or a derivatized antibody, e.g., at an engineered lysine residue as an acyl acceptor. In some embodiments, a linker comprising a thiol-reactive group is used to generate a conjugated antibody or antigenbinding fragment, e.g., by reacting with the antibody or antigen-binding fragment at a cysteine at amino acid position 80, 118, 140, 149, or 239.

[00333] Methods to accomplish site-specific conjugation of linkers and/or drug moieties to an antibody or antigen-binding fragment for the production of ADCs are known in the art and disclosed in PCT applications WO 2017/213267, WO 2017/106643, and WO 2016/205618, all herein incorporated by reference in their entirety.

Drug Loading

[00334] Drug loading is represented by p, and is also referred to herein as the drug-to-antibody ratio (DAR). In some embodiments, drug loading may range from 1-20 (i.e., 1-20 copies of the linkerpayload conjugate attached to each antibody moiety), e.g., 1 to 8 drug moieties per antibody moiety. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is an integer from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is an integer from 1 to 2. In some embodiments, p is an integer from 2 to 3. In some embodiments, p is 1 , 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p is 2. In some embodiments, drug loading may be expressed as an average loading in a population of antibodies, e.g., an average loading of about 1-8, e.g., about 2-4.

[00335] Drug loading may be limited by the number of attachment sites on the antibody moiety. In some embodiments, the linker moiety (L) of the ADC attaches to the antibody moiety through a chemically active group on one or more amino acid residues on the antibody moiety. For example, the linker may be attached to the antibody moiety via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N or C terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl group of one or more cysteine residues). The site to which the linker is attached can be a natural residue in the amino acid sequence of the antibody moiety, or it can be introduced into the antibody moiety, e.g., by DNA recombinant technology (e.g., by introducing a cysteine or lysine residue into the amino acid sequence) or by protein biochemistry (e.g. , by reduction, pH adjustment, or hydrolysis). In some embodiments, the linker is attached to the antibody moiety via a cysteine residue. In some embodiments, the linker is attached to the antibody moiety via a lysine residue.

[00336] In some embodiments, the number of drug moieties that can be conjugated to an antibody moiety is limited by the number of free cysteine residues. For example, where the attachment is a cysteine thiol group, an antibody may have only one or a few cysteine thiol groups, or may have only one or a few sufficiently reactive thiol groups through which a linker may be attached. Generally, antibodies do not contain many free and reactive cysteine thiol groups that may be linked to a drug moiety. Indeed, most cysteine thiol residues in antibodies exist as disulfide bridges. Over-attachment of linker-toxin to an antibody may destabilize the antibody by reducing the cysteine residues available to form disulfide bridges. Therefore, an optimal drug:antibody ratio should increase potency of the ADC (by increasing the number of attached drug moieties per antibody) without destabilizing the antibody moiety. In some embodiments, an optimal ratio may be about 2.

[00337] In some embodiments, a linker attached to an antibody moiety through a Mai moiety may provide a ratio of about 2. In some embodiments, an ADC comprising Mal-(PEG)₂-Val-Cit-pABC- eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has aratio of about 2. . In some embodiments, an ADC comprising Mal-C₂-Val-Cit-pABC-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-C₂(PEG)₁-Val-Cit-pABC-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-C₂(PEG)₂- Val-Cit-pABC-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-C₂(PEG)₃-Val-Cit-pABC-eribulin joined to an anti-TR0P2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-(PEG)₂- Vai- Ala-pABC-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-C₂(PEG)₁-Val- Ala-pABC-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-C₂(PEG)₂- Val-Ala-pABC-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-C₂(PEG)₃-Val-Ala-pABC-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has aratio of about 2. In some embodiments, an ADC comprising Mal-(PEG)₂-N-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal- C₂-N-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-C₂(PEG)₁-N-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-C₂(PEG)₂-N-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has a ratio of about 2. In some embodiments, an ADC comprising Mal-C₂(PEG)₃-N-eribulin joined to an anti-TROP2 antibody as disclosed herein (e.g., 16K21) has aratio of about 2. In some embodiments, the anti-TROP2 antibody comprises three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 4; LCDR1 comprising SEQ ID NO: 8, LCDR2 comprising SEQ ID NO: 9, and LCDR3 comprising SEQ ID NO: 10, as defined by the Kabat numbering system; or three heavy chain CDRs and three light chain CDRs as follows: HCDR1 comprising SEQ ID NO: 11, HCDR2 comprising SEQ ID NO: 12, HCDR3 comprising SEQ ID NO: 13; LCDR1 comprising SEQ ID NO: 15, LCDR2 comprising SEQ ID NO: 16, and LCDR3 comprising SEQ ID NO: 17, as defined by the IMGT numbering system. In some embodiments, the anti-TROP2 antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[00338] In some embodiments, an antibody moiety is exposed to reducing conditions prior to conjugation in order to generate one or more free cysteine residues. An antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. Unpaired cysteines may be generated through partial reduction with limited molar equivalents of TCEP, which preferentially reduces the interchain disulfide bonds which link the light chain and heavy chain (one pair per H-L pairing) and the two heavy chains in the hinge region (two pairs per H-H pairing in the case of human IgGl) while leaving the intrachain disulfide bonds intact. Stefano et al. (2013) Methods Mol. Biol. 1045:145-71. In some embodiments, disulfide bonds within the antibodies are reduced electrochemically, e.g., by employing a working electrode that applies an alternating reducing and oxidizing voltage. This approach can allow for on-line coupling of disulfide bond reduction to an analytical device (e.g. , an electrochemical detection device, an NMR spectrometer, or a mass spectrometer) or a chemical separation device (e.g., a liquid chromatograph (e.g., an HPLC) or an electrophoresis device (see, e.g., U.S. Publ. No. 20140069822)). In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups on amino acid residues, such as lysine or cysteine.

[00339] The drug loading of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partial or limiting reductive conditions for cysteine thiol modification; and/or (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine or lysine residues is modified for control of the number and/or position of linker-payload conjugate attachments.

[00340] In some embodiments, free cysteine residues are introduced into the amino acid sequence of the antibody moiety. For example, cysteine engineered antibodies can be prepared wherein one or more amino acids of a parent antibody are replaced with a cysteine amino acid. Any form of antibody may be so engineered, i.e., mutated. For example, a parent Fab antibody fragment may be engineered to form a cysteine engineered Fab referred to as a “ThioFab.” Similarly, a parent monoclonal antibody may be engineered to form a “ThioMab.” A single site mutation yields a single engineered cysteine residue in a ThioFab, whereas a single site mutation yields two engineered cysteine residues in a ThioMab, due to the dimeric nature of the IgG antibody. DNA encoding an amino acid sequence variant of the parent polypeptide can be prepared by a variety of methods known in the art (see, e.g. , the methods described in W02006/034488). These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may also be constructed by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. ADCs of Formula (I) include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids. See, e.g., Lyon et al. (2012) Methods Enzymol. 502: 123-38. In some embodiments, one or more free cysteine residues are already present in an antibody moiety, without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody moiety to a drug moiety.

[00341] Where more than one nucleophilic group reacts with a drug-linker intermediate or a linker moiety reagent followed by drug moiety reagent, in a reaction mixture comprising multiple copies of the antibody moiety and linker moiety, then the resulting product can be a mixture of ADC compounds with a distribution of one or more drug moieties attached to each copy of the antibody moiety in the mixture. In some embodiments, the drug loading in a mixture of ADCs resulting from a conjugation reaction ranges from 1 to 8 drug moieties attached per antibody moiety. The average number of drug moieties per antibody moiety (i.e., the average drug loading, or average p) may be calculated by any conventional method known in the art, e.g., by mass spectrometry (e.g., reverse- phase LC-MS), and/or high-performance liquid chromatography (e.g., HIC-HPLC). In some embodiments, the average number of drug moieties per antibody moiety is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). In some embodiments, the average number of drug moieties per antibody moiety is determined by reverse-phase liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties per antibody moiety is from about 1 to about 8; from about 1 to about 6; from about 1 to about 3; or from about 1 to about 2. In some embodiments, the average number of drug moieties per antibody moiety is about 2.

[00342] Individual ADC compounds having particular DAR ratios, or “species,” may be identified in the mixture, e.g., by mass spectroscopy and separated, e.g., by ultra-performance liquid chromatography (UPLC) or HPLC, e.g., hydrophobic interaction chromatography (HIC-HPLC). In certain embodiments, a homogeneous or nearly homogenous ADC with a single loading value may be isolated from the conjugation mixture, e.g., by electrophoresis or chromatography.

[00343] The present disclosure includes methods of producing the described ADCs. Briefly, the ADCs comprise an antibody or antigen-binding fragment as the antibody moiety, a drug moiety, and a linker that joins the drug moiety and the antibody moiety. In some embodiments, the ADCs are prepared using a linker having reactive functionalities for covalently attaching to the drug moiety and to the antibody moiety. For example, in some embodiments, a cysteine thiol of an antibody moiety forms a bond with a reactive functional group of a linker or a drug-linker intermediate (e.g., a maleimide moiety) to make an ADC. In some embodiments, an engineered lysine residue of an antibody moiety forms a bond with an acyl donor substrate of a linker or a linker intermediate (e.g., a carbobenzoxy-L-glutaminyl-glycine moiety), the linker or linker intermediate comprising a reactive group that can be conjugated to a functional agent (e.g., a cleavable peptide comprising eribulin). [00344] In some embodiments, an ADC is produced by contacting an antibody moiety with a linker and a drug moiety in a sequential manner, such that the antibody moiety is covalently linked to the linker first, and then the pre-formed antibody-linker intermediate reacts with the drug moiety. The antibody-linker intermediate may or may not be subjected to a purification step prior to contacting the drug moiety. In other embodiments, an ADC is produced by contacting an antibody moiety with a linker-payload conjugate pre-formed by reacting a linker with a drug moiety. The pre-formed linkerpayload conjugate may or may not be subjected to a purification step prior to contacting the antibody moiety. In other embodiments, the antibody moiety contacts the linker and the drug moiety in one reaction mixture, allowing simultaneous formation of the covalent bonds between the antibody moiety and the linker, and between the linker and the drug moiety. In some embodiments, an ADC is produced by reacting an antibody moiety with a linker-payload conjugate, e.g., Mal-(PEG)₂-Val-Cit- pABC-eribulin, under conditions that allow conjugation. The conditions that allow conjugation may involve any biochemical methods known in the art for conjugating an ADC. These conditions include, but are not limited to, incubation at room temperature in a suitable buffer (e.g., 1 x DBPS, 0.1 M Tris- Glycine at pH 7.4, or 10% propylene glycol:90% 1 x DPBS). The conjugation conditions may or may not include the presence of an enzyme (e.g., transglutaminase).

[00345] The ADCs prepared according to the methods described above may be subjected to one or more purification steps. The purification step may involve any biochemical methods known in the art for purifying proteins, or any combination of methods thereof. These include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge or isoelectric point-based chromatography, mixed mode chromatography, e.g., CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, desalting chromatography, or any combination thereof.

Therapeutic Uses

[00346] Disclosed herein are methods of using the disclosed antibodies and ADCs in treating a subject for a disorder, e.g., an oncologic disorder. Antibodies and/or ADCs may be administered alone or in combination with a second therapeutic agent, and may be administered in any pharmaceutically acceptable formulation, dosage, and dosing regimen. Treatment efficacy may be evaluated for toxicity as well as indicators of efficacy and adjusted accordingly. Efficacy measures include, but are not limited to, a cytostatic and/or cytotoxic effect observed in vitro or in vivo, reduced tumor volume, tumor growth inhibition, and/or prolonged survival.

[00347] Methods of determining whether an antibody or ADC exerts a cytostatic and/or cytotoxic effect on a cell are known. For example, the cytotoxic activity of an antibody or ADC can be measured by: exposing mammalian cells expressing a target protein of the antibody or ADC (e.g., TROP2) in a cell culture medium; culturing the cells for a period of time, e.g., from about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays may also be used to measure viability (proliferation), cytotoxicity, growth inhibition, and induction of apoptosis (caspase activation) of the antibody or ADC.

[00348] In some embodiments, to determine cytotoxicity, necrosis or apoptosis (programmed cell death) may be measured. Necrosis is typically accompanied by increased permeability of the plasma membrane, swelling of the cell, and rupture of the plasma membrane. Apoptosis is typically characterized by membrane blebbing, condensation of cytoplasm, and the activation of endogenous endonucleases. Determination of any of these effects on cancer cells indicates that an antibody or ADC is useful in the treatment of cancers.

[00349] Cell viability may be measured, e.g., by determining in a cell the uptake of a dye such as neutral red, trypan blue, Crystal Violet, or ALAMAR™ blue. See, e.g., Page et al. (1993) Inti. J. Oncology 3:473-6. In such an assay, the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically. In certain embodiments, in vitro potency of prepared antibodies or ADCs is assessed using a Crystal Violet assay. Crystal Violet is a triaryhnethane dye that accumulates in the nucleus of viable cells. In this assay, cells are exposed to the antibodies or ADCs or control agents for a defined period of time, after which, cells are stained with crystal violet, washed copiously with water, then solubilized with 1% SDS and read spectrophotometrically. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytoxicity. Skehan et al. (1990) J. Natl. Cancer Inst. 82:1107-12.

[00350] Apoptosis can be quantitated, for example, by measuring DNA fragmentation. Commercial photometric methods for the quantitative in vitro determination of DNA fragmentation are available. Examples of such assays, including TUNEL (which detects incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals).

[00351] Apoptosis may also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring uptake of certain dyes (e.g., a fluorescent dye such as acridine orange or ethidium bromide). A method for measuring apoptotic cell number has been described by Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992) pp. 3.17.1-3.17.16). Cells also can be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide) and the cells observed for chromatin condensation and margination along the inner nuclear membrane. Other morphological changes that can be measured to determine apoptosis include, e.g., cytoplasmic condensation, increased membrane blebbing, and cellular shrinkage.

[00352] In some embodiments, the present disclosure provides a method of killing, inhibiting or modulating the growth of, or interfering with the metabolism of, a cancer cell or tissue by disrupting tubulin. The method may be used with any subject where disruption of tubulin provides a therapeutic benefit.

[00353] In various embodiments, the disclosed antibodies and/or ADCs may be administered to affect any cell or tissue that expresses TROP2, such as a TROP2-expressing cancer cell or tissue. An exemplary embodiment comprises a method of inhibiting TROP2 -mediated cell signaling or a method of killing a cell. The method may be used with any cell or tissue that expresses TROP2, such as a cancerous cell or a metastatic lesion. Non-limiting examples of TROP2-expressing cancers include cholangiocarcinoma (bile duct cancer), bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, and skin cancer. Non-limiting examples of TROP2- expressing cells include HuCCTl and SNU-308 human biliary tract cancer cells, BxPC3 human pancreatic adenocarcinoma cells, and cells comprising a recombinant nucleic acid encoding TROP2 or a portion thereof. Without being bound by theory, the anti-TROP2 antibodies and ADCs disclosed herein may be particularly effective at treating TROP2-expressing cancers by targeting TROP2 expressing cells for degradation or immune clearance, and/or by delivering a drug payload (e.g., eribulin) to cells. In some embodiments, an ADC may be used to deliver a drug payload (e.g., eribulin) to cells at the initial stages of the epithelial-to-mesenchymal transition (EMT). Without being bound by theory, cancer cells in an epithelial state may express high levels of TROP2. High TROP2 expression in cells may promote proliferation, migration, and metastasis by modulating various cellular signaling pathways, e.g., the PI3K/Akt and/or Wnt/p-catenin pathways. Due to changes in these cellular signaling pathways, cells may progress from an epithelial to mesenchymal state with a concurrent loss of TROP2 expression. These cells may then become immune to treatment with anti-TROP2 antibodies and/or ADCs. In some embodiments, the anti-TROP2 antibodies and ADCs disclosed herein provide for therapeutic effects of binding to TROP2, as well as the cytotoxic effects of delivering payloads such as eribulin, to cells that have not yet begun to metastasize, thus inhibiting cancer cells at the early stage of their growth and development.

[00354] Exemplary methods disclosed herein include the steps of contacting a cell with an antibody and/or ADC as described herein (eg., by administering the antibody and/or ADC to a subject by a suitable route of administration), in an effective amount, e.g., an amount sufficient to kill the cell. The method can be used on cells in culture, e.g., in vitro, ex vivo, or in situ. For example, cells that express TROP2 (e.g., cells collected by biopsy of a tumor or metastatic lesion; cells from an established cancer cell line; or recombinant cells), can be cultured in vitro in culture medium and the contacting step can be affected by adding the antibody or ADC to the culture medium. The method will result in killing of cells expressing TROP2, including in particular tumor cells expressing TROP2.

Alternatively, the antibody or ADC can be administered to a subject by any suitable administration route (e.g., intravenous, subcutaneous, or direct contact with a tumor tissue) to have an effect in vivo. [00355] The in vivo effect of a disclosed antibody and/or ADC can be evaluated in a suitable animal model. For example, xenogenic cancer models can be used, wherein cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice. Klein et al. (1997) Nature Med. 3:402-8. Efficacy may be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like. In some embodiments, the anti-TROP2 antibodies and ADCs disclosed herein are more efficacious at inhibiting tumor growth compared to xenograft-bearing mice treated with alternate treatments.

[00356] In vivo assays that evaluate the promotion of apoptosis may also be used. In one embodiment, xenografts from tumor-bearing mice treated with an antibody or ADC disclosed herein are examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice, or mice treated with alternate therapeutic compositions. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of a treatment.

[00357] In various embodiments, provided herein are methods of treating TROP2-expressing cancer. The antibodies and ADCs disclosed herein can be administered to a non-human mammal or human subject for therapeutic purposes. The therapeutic methods may entail administering to a mammal having a tumor, e.g., a tumor expressing TROP2, a biologically effective amount of an antibody disclosed herein or an ADC comprising a selected chemotherapeutic agent (e.g., erihulin) linked to that antibody.

[00358] In some embodiments, a method of treating a patient having or at risk of having a cancer that expresses TROP2 is provided, comprising administering to the patient a therapeutically effective amount of an antibody and/or ADC of the present disclosure. In some embodiments, the patient is non-responsive or poorly responsive to treatment with a drug moiety (e.g., eribulin) when administered alone, and the patient is administered an antibody or ADC disclosed herein. In other embodiments, the patient is intolerant to treatment with a drug moiety (e.g., eribulin) when administered alone. For instance, to treat a cancer, a patient may require doses of eribulin that lead to systemic toxicity, which are overcome by targeted delivery of the antibodies and/or ADCs disclosed herein to a TROP2-expressing cancer, thereby reducing off-target killing.

[00359] Another exemplary embodiment is a method of reducing or inhibiting growth of a TROP2- expressing tumor, comprising administering a therapeutically effective amount of an antibody and/or ADC disclosed herein. In some embodiments, the treatment is sufficient to reduce or inhibit the growth of the patient's tumor, reduce the number or size of metastatic lesions, reduce tumor load, reduce primary tumor load, reduce invasiveness, prolong survival time, and/or maintain or improve the quality of life. In some embodiments, the tumor is resistant or refractory to treatment with a drug moiety (e.g., eribulin) when administered alone.

[00360] In various embodiments, the methods disclosed herein treat a cholangiocarcinoma, bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer.

[00361] Moreover, antibodies and/or ADCs of the present disclosure may be administered to a nonhuman mammal expressing TROP2 for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be usefill for evaluating the therapeutic efficacy of the disclosed antibodies and ADCs (e.g., testing of dosages and time courses of administration).

[00362] In some embodiments, the efficacy of an antibody or ADC may be evaluated by contacting a tumor sample from a subject with the antibody or ADC and evaluating tumor growth rate or volume. In some embodiments, when an antibody or ADC has been determined to be effective, it may be administered to the subject. In some embodiments, the efficacy of an antibody or ADC may be evaluated by contacting a subject with the antibody or ADC and monitoring tumor growth rate or volume.

[00363] The antibodies and ADCs disclosed herein may be administered at a suitable dosage to a patient in need thereof. Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.

[00364] In various embodiments, treatment involves single bolus or repeated administration of the ADC preparation via an acceptable route of administration. [00365] The above therapeutic approaches can also be combined with any one of a wide variety of additional surgical, chemotherapy, or radiation therapy regimens.

[00366] Further provided herein are therapeutic uses of the disclosed antibodies and/or ADCs. An exemplary embodiment is the use of an antibody and/or ADC in the treatment of a TROP2 -expressing cancer, such as cholangiocarcinoma, bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer. Methods for identifying subjects having cancers that express TROP2 are known in the art and may be used to identify suitable patients for treatment with a disclosed ADC.

[00367] Another exemplary embodiment is the use of an ADC or an antibody or antigen-binding fragment as disclosed herein in the manufacture of a medicament for the treatment of a TROP2- expressing cancer, such as cholangiocarcinoma, bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer.

Pharmaceutical compositions and formulations

[00368] An antibody or ADC used in the practice of the foregoing methods may be formulated into a pharmaceutical composition suitable for administration to a subject, e.g., a human subject. In some embodiments, the pharmaceutical composition comprises the antibody and/or ADC and a pharmaceutically acceptable carrier suitable for the desired delivery method. Suitable carriers include any material that, when combined with an antibody or ADC disclosed herein, allows that antibody or ADC to retain its anti-tumor function and is generally non-reactive with the patient's immune system. Pharmaceutically acceptable carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, mesylate salt, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the pharmaceutical composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the ADC.

[00369] The pharmaceutical compositions described herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. [00370] Pharmaceutical compositions may be solubilized and administered via any route capable of delivering the pharmaceutical composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. In some embodiments, administration of an antibody-drug conjugate disclosed herein is intravenous. Pharmaceutical compositions can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol preservative) or in sterile water prior to injection. Administration can be either systemic or local. Pharmaceutical compositions may comprise an antibody and/or ADC or a pharmaceutically acceptable salt thereof, e.g., a mesylate salt.

[00371] In various embodiments, kits for use in the laboratory and the therapeutic applications described herein are within the scope of the present disclosure. Such kits may comprise an antibody or ADC disclosed herein and a carrier, package, or container. The carrier, package, or container may be compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the containers) comprising one of the separate elements to be used in a method disclosed herein, and/or a label or insert comprising instructions for use, such as a use described herein. Kits may further comprise one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.

[00372] A label may be present on or with the container to indicate that the pharmaceutical composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic, or laboratory application. A label may also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and/or other information may also be included on an insert(s) or label(s), which is included with or on the kit. The label may be on or associated with the container. A label may be on a container when letters, numbers, or other characters forming the label are molded or etched into the container itself. A label may be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label may indicate that the pharmaceutical composition is used for diagnosing or treating a condition, such as a cancer as described herein.

Exemplary Embodiments of the Disclosure

[00373] In various embodiments, the present disclosure provides novel antibody-drug conjugates capable of specifically binding TROP2.

[00374] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin. [00375] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00376] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-(PEG)₂-N, and a drug moiety comprising eribulin.

[00377] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00378] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂(PEG)₁-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00379] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂(PEG)₁-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00380] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂(PEG)₁-N, and a drug moiety comprising eribulin.

[00381] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00382] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00383] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂-N, and a drug moiety comprising eribulin.

[00384] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂(PEG)₂-N, and a drug moiety comprising eribulin.

[00385] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂(PEG)₃-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00386] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂(PEG)₃-Val-Ala-pABC, and a drug moiety comprising eribulin. [00387] In some embodiments, the antibody-drug conjugate comprises any anti-TROP2 antibody or antigen-binding fragment disclosed herein, a linker comprising Mal-C₂(PEG)₃-N, and a drug moiety comprising eribulin.

[00388] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00389] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00390] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00391] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00392] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00393] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00394] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 380, a linker comprising Mal-(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00395] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00396] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00397] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00398] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00399] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-(PEG)₂-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00400] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-(PEG)₂-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00401] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-(PEG)₂-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00402] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-(PEG)₂-N, and a drug moiety comprising eribulin.

[00403] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-(PEG)₂-N, and a drug moiety comprising eribulin.

[00404] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-(PEG)₂-N, and a drug moiety comprising eribulin.

[00405] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-(PEG)₂-N, and a drug moiety comprising eribulin.

[00406] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-(PEG)₂-N, and a drug moiety comprising eribulin.

[00407] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-(PEG)₂-N, and a drug moiety comprising eribulin.

[00408] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 380, a linker comprising Mal-(PEG)₂-N, and a drug moiety comprising eribulin. [00409] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00410] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00411] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂-Val-Cit-pABC, and a drug moiety comprising eribulin. [00412] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂-Val-Cit-pABC, and a drug moiety comprising eribulin. [00413] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00414] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00415] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 380, a linker comprising Mal-C₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00416] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₁-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00417] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂(PEG)₁-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00418] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂(PEG)₁-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00419] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂(PEG)₁-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00420] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₁-Val-Cit- pABC, and a drug moiety comprising eribulin.

[00421] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₁-Val-Cit- pABC, and a drug moiety comprising eribulin.

[00422] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 380, a linker comprising Mal-C₂(PEG)₁-Val-Cit- pABC, and a drug moiety comprising eribulin.

[00423] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00424] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00425] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00426] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00427] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₂-Val-Cit- pABC, and a drug moiety comprising eribulin.

[00428] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₂-Val-Cit- pABC, and a drug moiety comprising eribulin.

[00429] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 380, a linker comprising Mal-C₂(PEG)₃-Val-Cit- pABC, and a drug moiety comprising eribulin.

[00430] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₃-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00431] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂(PEG)₃-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00432] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂(PEG)₃-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00433] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂(PEG)₃-Val-Cit-pABC, and a drug moiety comprising eribulin.

[00434] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₃-Val-Cit- pABC, and a drug moiety comprising eribulin.

[00435] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₃-Val-Cit- pABC, and a drug moiety comprising eribulin.

[00436] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 380, a linker comprising Mal-C₂(PEG)₃-Val-Cit- pABC, and a drug moiety comprising eribulin.

[00437] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₁-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00438] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂(PEG)₁-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00439] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂(PEG)₁-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00440] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂(PEG)₁-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00441] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₁-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00442] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₁-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00443] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₁-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00444] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00445] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00446] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00447] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂(PEG)₂-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00448] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₂-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00449] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₂-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00450] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₂-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00451] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₃-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00452] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂(PEG)₃-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00453] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂(PEG)₃-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00454] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂(PEG)₃-Val-Ala-pABC, and a drug moiety comprising eribulin.

[00455] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₃-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00456] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₃-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00457] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₃-Val-Ala- pABC, and a drug moiety comprising eribulin.

[00458] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂-N, and a drug moiety comprising eribulin.

[00459] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂-N, and a drug moiety comprising eribulin.

[00460] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂-N, and a drug moiety comprising eribulin.

[00461] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂-N, and a drug moiety comprising eribulin.

[00462] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂-N, and a drug moiety comprising eribulin.

[00463] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂-N, and a drug moiety comprising eribulin.

[00464] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂-N, and a drug moiety comprising eribulin.

[00465] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₁-N, and a drug moiety comprising eribulin. [00466] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂(PEG)₁-N, and a drug moiety comprising eribulin.

[00467] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂(PEG)₁-N, and a drug moiety comprising eribulin.

[00468] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂(PEG)₁-N, and a drug moiety comprising eribulin.

[00469] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₁-N, and a drug moiety comprising eribulin.

[00470] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₁-N, and a drug moiety comprising eribulin.

[00471] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₁-N, and a drug moiety comprising eribulin.

[00472] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₂-N, and a drug moiety comprising eribulin.

[00473] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂(PEG)₂-N, and a drug moiety comprising eribulin.

[00474] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂(PEG)₂-N, and a drug moiety comprising eribulin.

[00475] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂(PEG)₂-N, and a drug moiety comprising eribulin.

[00476] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₂-N, and a drug moiety comprising eribulin.

[00477] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₂-N, and a drug moiety comprising eribulin.

[00478] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₂-N, and a drug moiety comprising eribulin.

[00479] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₃-N, and a drug moiety comprising eribulin.

[00480] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal-C₂(PEG)₃-N, and a drug moiety comprising eribulin.

[00481] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal-C₂(PEG)₃-N, and a drug moiety comprising eribulin.

[00482] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal-C₂(PEG)₃-N, and a drug moiety comprising eribulin.

[00483] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₃-N, and a drug moiety comprising eribulin.

[00484] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 179 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₃-N, and a drug moiety comprising eribulin.

[00485] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment comprising a heavy chain amino acid sequence of SEQ ID NO: 380 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₃-N, and a drug moiety comprising eribulin.

[00486] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-(PEG)₂-Val-Cit- pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit- pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal- C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala- pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile). In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti- TR0P2 antibody or antigen-binding fragment thereof comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system; a linker comprising Mal-(PEG)₂-Val-Cit-pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala- pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal- C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin, may include improved antigen-binding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, and safety profile. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Examples 3 and 4.

[00487] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mal- (PEG)₂-Val-Cit-pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal- C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit- pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal- C₂(PEG)₃-Val-Ala-pABC, or Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile). In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti-TROP2 antibody or antigen-binding fragment thereof comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system; a linker comprising Mal-(PEG)₂-Val-Cit- pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit- pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal- C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala- pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin, may include improved antigen-binding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, and safety profile. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Examples 3 and 4.

[00488] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal- (PEG)₂-Val-Cit-pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal- C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit- pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal- C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile). In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti-TROP2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; a linker comprising Mal- (PEG)₂-Val-Cit-pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal- C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit- pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal- C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin, may include improved antigen-binding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, and safety profile. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Examples 3 and 4.

[00489] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal- (PEG)₂-Val-Cit-pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal- C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit- pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal- C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile). In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti-TROP2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66; a linker comprising Mal- (PEG)₂-Val-Cit-pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal- C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit- pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal- C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin, may include improved antigen-binding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, and safety profile. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Examples 3 and 4.

[00490] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-(PEG)₂-Val-Cit-pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal- (PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile). In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti-TROP2 antibody or antigen-binding fragment thereof comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115; a linker comprising Mal-(PEG)₂-Val-Cit-pABC, Mal-(PEG)₂-Val-Ala- pABC, Mal-(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val- Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal- C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-Val-Cit-pABC, and a drug moiety comprising eribulin, may include improved antigenbinding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, and safety profile. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Examples 3 and 4. [00491] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system, a linker comprising Mal-C₂(PEG)₂-Val- Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁- Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal- C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala- pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, in vivo anti-tumor activity). In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti-TROP2 antibody or antigen-binding fragment thereof comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 3, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 4; light chain CDR1 (LCDR1) comprising SEQ ID NO: 8, light chain CDR2 (LCDR2) comprising SEQ ID NO: 9, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 10, as defined by the Kabat numbering system; a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂- Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal- C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit- pABC, Mal-C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin, may include improved antigen-binding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, and/or in vivo anti-tumor activity. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Example 7.

[00492] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, fight chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and fight chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system, a linker comprising Mai- C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val- Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal- C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, in vivo anti-tumor activity). In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti- TR0P2 antibody or antigen-binding fragment thereof comprising three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 11, heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 12, heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 13; light chain CDR1 (LCDR1) comprising SEQ ID NO: 15, light chain CDR2 (LCDR2) comprising SEQ ID NO: 16, and light chain CDR3 (LCDR3) comprising SEQ ID NO: 17, as defined by the IMGT numbering system; a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala- pABC, Mal-C₂(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val- Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal- C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin, may include improved antigen-binding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, and/or in vivo anti-tumor activity. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Example 7.

[00493] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61, a linker comprising Mal- C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val- Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal- C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, in vivo anti-tumor activity). In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti- TR0P2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, Mal- C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val- Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala-pABC, or Mal- C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin, may include improved antigenbinding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, and/or in vivo anti-tumor activity. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Example 7.

[00494] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66, a linker comprising Mal- C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val- Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal- C₂(PEG)₃-Val-Ala-pABC, or Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, in vivo anti-tumor activity In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti- TROP2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66; a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, Mal- C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val- Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala-pABC, Mal- C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin, may include improved antigenbinding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, and/or in vivo anti-tumor activity. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Example 7.

[00495] In some embodiments of antibody-drug conjugates disclosed herein, the antibody-drug conjugate comprises an anti-TROP2 antibody or antigen-binding fragment thereof which comprises a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115, a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal- C₂(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal-C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N, and a drug moiety comprising eribulin. Without being bound by theory, one or more of these antibody-drug conjugates may demonstrate superior properties over other ADCs, e.g., those using other antibodies and/or linkers, e.g., as compared to other ADCs disclosed herein (e.g., improved antigen-binding specificity (e.g., epitope and/or affinity), site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, in vivo anti-tumor activity). In some embodiments, without being bound by theory, benefits of using an antibody-drug conjugate comprising an anti-TROP2 antibody or antigen-binding fragment thereof comprising a heavy chain amino acid sequence of SEQ ID NO: 181 and a light chain amino acid sequence of SEQ ID NO: 115; a linker comprising Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal-C₂-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Cit-pABC, Mal-C₂(PEG)₁-Val-Ala-pABC, Mal- C₂(PEG)₁-N, Mal-C₂(PEG)₂-Val-Cit-pABC, Mal-C₂(PEG)₂-Val-Ala-pABC, Mal-C₂(PEG)₂-N, Mal- C₂(PEG)₃-Val-Cit-pABC, Mal-C₂(PEG)₃-Val-Ala-pABC, Mal-C₂(PEG)₃-N, or Mal-C₂-N-pABC, and a drug moiety comprising eribulin, may include improved antigen-binding specificity, site-specific linker-payload conjugation, tolerability, in vitro cytotoxicity, safety profile, conjugation stability, plasma stability, and/or in vivo anti-tumor activity. Exemplary evidence of the superior benefits of such antibody-drug conjugates is shown in Example 7.

[00496] 1. An internalizing anti-TROP2 antibody or internalizing antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to human TROP2, and wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein

(i) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 18, the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 19 or 20, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 21, the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 22 or 23, the LCDR2 comprises an amino acid sequence selected from SEQ ID NO: 24 or 25, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26, as defined by the Kabat numbering system; or

(ii) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 27, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 28, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 29, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 30, the LCDR2 comprises an amino acid sequence selected from SEQ ID NO: 31 or 32, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 33, as defined by the IMGT numbering system; or (iii) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 2 or 3, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 4, the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 5, 6, 7, or 8, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10, as defined by the Kabat numbering system; or

(iv) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 11 , the HCDR2 comprises an amino acid sequence of SEQ ID NO: 12, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 13, the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 14 or 15, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 16, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 17, as defined by the IMGT numbering system.

[00497] 2. The anti-TROP2 antibody or antigen-binding fragment of embodiment 1, wherein the antibody or antigen-binding fragment comprises

(i) three HCDRs comprising amino acid sequences of SEQ ID NO: 18 (HCDR1), SEQ ID NO: 20 (HCDR2), and SEQ ID NO: 21 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 22 (LCDR1), SEQ ID NO: 24 (LCDR2), and SEQ ID NO: 26 (LCDR3), as defined by the Kabat numbering system; or

(ii) three HCDRs comprising amino acid sequences of SEQ ID NO: 18 (HCDR1), SEQ ID NO: 20 (HCDR2), and SEQ ID NO: 21 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 23 (LCDR1), SEQ ID NO: 24 (LCDR2), and SEQ ID NO: 26 (LCDR3), as defined by the Kabat numbering system; or

(iii) three HCDRs comprising amino acid sequences of SEQ ID NO: 27 (HCDR1), SEQ ID NO: 28 (HCDR2), and SEQ ID NO: 29 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 30 (LCDR1), SEQ ID NO: 31 (LCDR2), and SEQ ID NO: 33 (LCDR3), as defined by the IMGT numbering system.

[00498] 3. The anti-TROP2 antibody or antigen-binding fragment of embodiment 1 , wherein the antibody or antigen-binding fragment comprises

(i) three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 5 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system; or

(ii) three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system; or

(iii) three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 14 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system; or

(iv) three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system.

[00499] 4. The anti-TROP2 antibody or antigen-binding fragment of embodiment 1, wherein the antibody or antigen-binding fragment comprises

(i) a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 67, 68, 69, 70, or 177, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 71, 72, 167, 73, 74, 75, 76, 187, 77, or 78; or

(ii) a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 53, 54, 55, 56, 174, or 175, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 57, 58, 166, 59, 60, 61, 62, 63, 176, 64, 65, or 66.

[00500] 5. The anti-TROP2 antibody or antigen-binding fragment of embodiment 4, wherein the antibody or antigen-binding fragment comprises

(i) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72; or

(ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76; or

(iii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73; or

(iv) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 69, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77; or

(v) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72; or

(vi) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76; or

(vii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73; or

(viii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 70, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77; or

(ix) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 177, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72; or

(x) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 177, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 76; or (xi) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 177, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73; or

(xii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 177, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 77.

[00501] 6. The anti-TROP2 antibody or antigen-binding fragment of embodiment 4, wherein the antibody or antigen-binding fragment comprises

(i) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; or

(ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63; or

(iii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; or

(iv) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66; or

(v) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; or

(vi) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63; or

(vii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; or

(viii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66; or

(ix) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 58; or

(x) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 63; or

(xi) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61; or

(xii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[00502] 7. The anti-TROP2 antibody or antigen-binding fragment of embodiment 6, wherein the antibody or antigen-binding fragment comprises

(i) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 ; or

(ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66. [00503] 8. The anti-TROP2 antibody or antigen-binding fragment of any one of embodiments 1 to 7, wherein the antibody or antigen-binding fragment comprises a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant region.

[00504] 9. The anti-TROP2 antibody or antigen-binding fragment of any one of embodiments 1 to 8, wherein the antibody or antigen-binding fragment comprises an IgGl Fc domain or an IgGl Fc domain mutated to reduce binding to a Fey receptor (FcyR) as compared to an IgGl Fc-containing antibody with a wild-type IgGl Fc domain.

[00505] 10. The anti-TROP2 antibody or antigen-binding fragment of embodiments 9, wherein the mutated IgGl Fc domain comprises the mutations L234A, L235A, P238S, H268Q, and K274Q.

[00506] 11. The anti-TROP2 antibody or antigen-binding fragment of any one of embodiments 1 to 10, wherein the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[00507] 12. The anti-TROP2 antibody or antigen-binding fragment of embodiment 1, wherein the antibody or antigen-binding fragment comprises

(i) a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 118, 119, 184, 122, 123, 185, 126, 127, 186, or 381 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 129, 130, 133, or 134; or

(ii) a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 96, 97, 100, 101, 104, 105, 179, 181, 183, or 380 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 107, 110, 112, or 115.

[00508] 13. The anti-TROP2 antibody or antigen-binding fragment of embodiment 12, wherein the antibody or antigen-binding fragment comprises

(i) a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115; or

(ii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 115; or

(iii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00509] 14. The anti-TROP2 antibody or antigen-binding fragment of any one of embodiments 1 to 13, wherein the antibody or antigen-binding fragment comprises part of a bispecific or multispecific binding construct.

[00510] 15. The anti-TROP2 antibody or antigen-binding fragment of any one of embodiments 1 to 14, wherein the antibody or antigen-binding fragment is linked to a therapeutic agent or detectable agent.

[00511] 16. An antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is an anti-TROP2 antibody or antigen-binding fragment thereof of any one of embodiments 1 to 13;

D is a cytotoxic agent;

L is a cleavable linker that covalently attaches Ab to D; and p is an integer from 1 to 8.

[00512] 17. The antibody-drug conjugate of embodiment 16, wherein the cytotoxic agent comprises an anti-tubulin agent, preferably wherein the anti-tubulin agent is eribulin or a salt thereof. [00513] 18. The antibody-drug conjugate of embodiment 16 or embodiment 17, wherein p is from 2 to 8.

[00514] 19. The antibody-drug conjugate of embodiment 18, wherein p is 2.

[00515] 20. The antibody-drug conjugate of any one of embodiments 16 to 19, wherein the cleavable linker comprises a cleavable moiety that is positioned such that no part of the tinker or the antibody or antigen-binding fragment remains bound to the cytotoxic agent upon cleavage.

[00516] 21. The antibody-drug conjugate of any one of embodiments 16 to 20, wherein the cleavable linker comprises a cleavable peptide moiety.

[00517] 22. The antibody-drug conjugate of embodiment 21 , wherein the cleavable peptide moiety is cleavable by an enzyme.

[00518] 23. The antibody-drug conjugate of embodiment 21 or embodiment 22, wherein the cleavable peptide moiety is cleavable by cathepsin.

[00519] 24. The antibody-drug conjugate of embodiment 23, wherein the cleavable peptide moiety is cleavable by cathepsin B.

[00520] 25. The antibody-drug conjugate of embodiment 21 or embodiment 22, wherein the cleavable peptide moiety is cleavable by legumain.

[00521] 26. The antibody-drug conjugate of any one of embodiments 21 to 25, wherein the cleavable peptide moiety comprises an amino acid unit.

[00522] 27. The antibody-drug conjugate of embodiment 26, wherein the amino acid unit comprises valine-citrulline (Val-Cit).

[00523] 28. The antibody-drug conjugate of embodiment 26, wherein the amino acid unit comprises valine-dimethylated lysine (Val-Lys(Me)₂).

[00524] 29. The antibody-drug conjugate of embodiment 26, wherein the amino acid unit comprises alanine-dimethylated lysine (Ala-Lys(Me)₂).

[00525] 30. The antibody-drug conjugate of embodiment 26, wherein the amino acid unit comprises valine-alanine (Vai- Ala).

[00526] 31. The antibody-drug conjugate of embodiment 26, wherein the amino acid unit comprises asparagine (Asn).

[00527] 32. The antibody-drug conjugate of embodiment 26, wherein the amino acid unit comprises aspartic acid (Asp). [00528] 33. The antibody-drug conjugate of embodiment 26, wherein the amino acid unit comprises methylated aspartic acid (Asp(OMe)).

[00529] 34. The antibody-drug conjugate of any one of embodiments 16 to 33, wherein the cleavable linker attaches to the antibody or antigen-binding fragment via a maleimide (Mai) moiety.

[00530] 35. The antibody-drug conjugate of embodiment 34, wherein the Mai moiety comprises a maleimidocaproyl (MC) moiety.

[00531] 36. The antibody-drug conjugate of embodiment 34, wherein the Mai moiety comprises a dithiomaleimide (DTM) moiety.

[00532] 37. The antibody-drug conjugate of any one of embodiments 34 to 36, wherein the Mai moiety is reactive with a cysteine residue on the antibody or antigen-binding fragment.

[00533] 38. The antibody-drug conjugate of any one of embodiments 34 to 37, wherein the Mai moiety is joined to the antibody or antigen-binding fragment via a cysteine residue on the antibody or antigen-binding fragment.

[00534] 39. The antibody-drug conjugate of any one of embodiments 34 to 38, wherein the cleavable linker comprises the Mai moiety and a cleavable peptide moiety.

[00535] 40. The antibody-drug conjugate of embodiment 39, wherein the Mai moiety attaches the antibody or antigen-binding fragment to the cleavable peptide moiety in the linker.

[00536] 41. The antibody-drug conjugate of any one of embodiments 34 to 40, wherein the cleavable linker comprises Mal-Val-Cit.

[00537] 42. The antibody-drug conjugate of any one of embodiments 34 to 40, wherein the cleavable linker comprises Mal-Val-Lys(Me)₂.

[00538] 43. The antibody-drug conjugate of any one of embodiments 34 to 40, wherein the cleavable linker comprises Mal-Ala-Lys(Me)₂.

[00539] 44. The antibody-drug conjugate of any one of embodiments 34 to 40, wherein the cleavable linker comprises Mal-Val-Ala.

[00540] 45. The antibody-drug conjugate of any one of embodiments 34 to 40, wherein the cleavable linker comprises Mal-Asn.

[00541] 46. The antibody-drug conjugate of any one of embodiments 34 to 40, wherein the cleavable linker comprises Mal-Asp.

[00542] 47. The antibody-drug conjugate of any one of embodiments 34 to 40, wherein the cleavable linker comprises Mal-Asp(OMe).

[00543] 48. The antibody-drug conjugate of any one of embodiments 16 to 40, wherein the cleavable linker comprises at least one spacer unit, and wherein the spacer unit attaches to the antibody or antigen-binding fragment via the maleimide (Mai) moiety ("Mal-spacer unit").

[00544] 49. The antibody-drug conjugate of embodiment 48, wherein the Mal-spacer unit comprises a polyethylene glycol (PEG) moiety. [00545] 50. The antibody-drug conjugate of embodiment 49, wherein the PEG moiety comprises - (PEG)_m- and m is an integer from 1 to 10.

[00546] 51. The antibody-drug conjugate of embodiment 50, wherein m is 2.

[00547] 52. The antibody-drug conjugate of embodiment 50, wherein m is 3.

[00548] 53. The antibody-drug conjugate of embodiment 50, wherein m is 4.

[00549] 54. The antibody-drug conjugate of embodiment 48, wherein the Mal-spacer unit comprises an alkyl moiety.

[00550] 55. The antibody-drug conjugate of embodiment 54, wherein the alkyl moiety comprises

-(CH₂)_n- and n is an integer from 1 to 10.

[00551] 56. The antibody-drug conjugate of embodiment 55, wherein n is 5.

[00552] 57. The antibody-drug conjugate of embodiment 48, wherein the Mal-spacer unit comprises C₂

[00553] 58. The antibody-drug conjugate of embodiment 48, wherein the Mal-spacer unit comprises C₂(PEG)_m, wherein m is an integer from 0 to 4.

[00554] 59. The antibody-drug conjugate of embodiment 58, wherein m is 1.

[00555] 60. The antibody-drug conjugate of embodiment 58, wherein m is 3.

[00556] 61. The antibody-drug conjugate of embodiment 58, wherein the spacer unit comprises C₂(PEG)₂.

[00557] 62. The antibody-drug conjugate of any one of embodiments 48 to 61, wherein the cleavable linker comprises the Mal-spacer unit and the cleavable peptide moiety.

[00558] 63. The antibody-drug conjugate of embodiment 62, wherein the Mal-spacer unit attaches the antibody or antigen-binding fragment to the cleavable peptide moiety in the linker.

[00559] 64. The antibody-drug conjugate of embodiment 62 or claim 63, wherein the Mal-spacer unit comprises a PEG moiety.

[00560] 65. The antibody-drug conjugate of embodiment 62 or claim 63, wherein the Mal-spacer unit comprises Mal-(PEG)₂-Val-Cit.

[00561] 66. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-(PEG)₃-Val-Cit.

[00562] 67. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-(PEG)₄-Val-Cit.

[00563] 68. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-(PEG)₂-Val-Lys(Me)₂. [00564] 69. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-(PEG)₂-Ala-Lys(Me)₂.

[00565] 70. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-(PEG)₂-Val-Ala.

[00566] 71. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-(PEG)₂-Asn.

[00567] 72. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-(PEG)₂-Asp.

[00568] 73. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-(PEG)₂-Asp(OMe).

[00569] 74. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises C₂.

[00570] 75. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises a C₂(PEG)_m moiety, wherein m is an integer from 0 to 4.

[00571] 76. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-C₂(PEG)_m-Val-Cit, wherein m is an integer from 0 to 4.

[00572] 77. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-C₂(PEG)_m-Val-Lys(Me)₂, wherein m is an integer from 0 to 4.

[00573] 78. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-C₂(PEG)_m-Ala-Lys(Me)₂, wherein m is an integer from 0 to 4.

[00574] 79. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-C₂(PEG)_m-Va1-Ala, wherein m is an integer from 0 to 4.

[00575] 80. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-C₂(PEG)_m-Asn, wherein m is an integer from 0 to 4.

[00576] 81. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-C₂(PEG)_m-Asp, wherein m is an integer from 0 to 4.

[00577] 82. The antibody-drug conjugate of embodiment 62 or embodiment 63, wherein the Mal- spacer unit comprises Mal-C₂(PEG)_m-Asp(OMe) , wherein m is an integer from 0 to 4.

[00578] 83. The antibody-drug conjugate of any one of embodiments 75 to 82, wherein m is 2.

[00579] 84. The antibody-drug conjugate of any one of embodiments 20 to 83, wherein the cleavable moiety in the linker is directly joined to the cytotoxic agent, or wherein a spacer unit attaches the cleavable moiety in the linker to the cytotoxic agent.

[00580] 85. The antibody-drug conjugate of embodiment 84, wherein cleavage of the conjugate releases the cytotoxic agent from the antibody and linker.

[00581] 86. The antibody-drug conjugate of embodiment 84 or embodiment 85, wherein the spacer unit attaching the cleavable moiety in the linker to the cytotoxic agent is self-immolative. [00582] 87. The antibody-drug conjugate of any one of embodiments 84 to 86, wherein the spacer unit attaching the cleavable moiety in the linker to the cytotoxic agent comprises a p-aminobenzyl (pAB).

[00583] 88. The antibody-drug conjugate of any one of embodiments 84 to 86, wherein the spacer unit attaching the cleavable moiety in the linker to the cytotoxic agent comprises a p- aminobenzyloxycarbonyl (pABC).

[00584] 89. The antibody-drug conjugate of embodiment 87 or embodiment 88, wherein the pAB or pABC attaches the cleavable moiety in the linker to the cytotoxic agent.

[00585] 90. The antibody-drug conjugate of embodiment 87 or embodiment 88, wherein the cytotoxic agent is eribulin and the pAB or the pABC covalently attaches to eribulin via a C-35 amine.

[00586] 91. The antibody-drug conjugate of embodiment 89 or embodiment 90, wherein the cleavable moiety comprises Val-Cit, Vai-Ala, Val-Lys(Me)₂, Ala-Lys(Me)₂, Asn, Asp, or Asp(OMe).

[00587] 92. The antibody-drug conjugate of embodiment 91, wherein the cleavable linker comprises Val-Cit-pABC.

[00588] 93. The antibody-drug conjugate of embodiment 91, wherein the cleavable linker comprises Val-Ala-pABC.

[00589] 94. The antibody-drug conjugate of embodiment 91 , wherein the cleavable linker comprises Val-Lys(Me)₂-pABC.

[00590] 95. The antibody-drug conjugate of embodiment 91, wherein the cleavable linker comprises Ala-Lys(Me)₂-pABC.

[00591] 96. The antibody-drug conjugate of embodiment 36, wherein the cleavable linker comprises DTM-Asn.

[00592] 97. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-(PEG)₂-Val-Cit-pABC.

[00593] 98. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-(PEG)₃-Val-Cit-pABC.

[00594] 99. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-(PEG)₄-Val-Cit-pABC.

[00595] 100. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-(PEG)₂-Val-Lys(Me)₂-pABC.

[00596] 101. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-(PEG)₂-Ala-Lys(Me)₂-pABC.

[00597] 102. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-(PEG)₂-Val-Ala-pABC.

[00598] 103. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-C₂(PEG)_m-Val-Cit-pABC, wherein m is an integer from 0 to 4. [00599] 104. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-C₂(PEG)_m-Val-Lys(Me)₂-pABC, wherein m is an integer from 0 to 4.

[00600] 105. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-C₂(PEG)_m-Ala-Lys(Me)₂-pABC, wherein m is an integer from 0 to 4.

[00601] 106. The antibody-drug conjugate of embodiment 34, wherein the cleavable linker comprises Mal-C₂(PEG)_m-Val-Ala-pABC, wherein m is an integer from 0 to 4.

[00602] 107. The antibody-drug conjugate of any one of embodiments 103 to 106, wherein m is 2.

[00603] 108. The antibody-drug conjugate of any one of embodiments 16 to 107, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system.

[00604] 109. The antibody-drug conjugate of any one of embodiments 16 to 107, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system.

[00605] 110. The antibody-drug conjugate of embodiment 108 or embodiment 109, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61.

[00606] 111. The antibody-drug conjugate of embodiment 108 or embodiment 109, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61.

[00607] 112. The antibody-drug conjugate of embodiment 108 or embodiment 109, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[00608] 113. The antibody-drug conjugate of embodiment 108 or embodiment 109, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[00609] 114. The antibody-drug conjugate of embodiment 110, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 96 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. [00610] 115. The antibody-drug conjugate of embodiment 111, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00611] 116. The antibody-drug conjugate of embodiment 112, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 96 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00612] 117. The antibody-drug conjugate of embodiment 113, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00613] 118. The antibody-drug conjugate of embodiment 110, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00614] 119. The antibody-drug conjugate of embodiment 111, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00615] 120. The antibody-drug conjugate of embodiment 112, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00616] 121. The antibody-drug conjugate of embodiment 113, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00617] 122. The antibody-drug conjugate of embodiment 110, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00618] 123. The antibody-drug conjugate of embodiment 111, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00619] 124. The antibody-drug conjugate of embodiment 112, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00620] 125. The antibody-drug conjugate of embodiment 113, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00621] 126. The antibody-drug conjugate of embodiment 111, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. [00622] 127. The antibody-drug conjugate of embodiment 113, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00623] 128. An antibody-drug conjugate of Formula (I):

D is eribulin;

L is a cleavable linker comprising Mal-Val-Cit-pABC; and p is an integer from 1 to 8.

[00624] 129. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00625] 130. An antibody-drug conjugate of Formula (I):

D is eribulin;

L is a cleavable linker comprising Mal-Val-Ala-pABC; and p is an integer from 1 to 8.

[00626] 131. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00627] 132. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00628] 133. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00629] 134. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00630] 135. An antibody-drug conjugate of Formula (I): Ab-(L-D)_p (I) wherein Ab is an internalizing anti-TROP2 antibody or internalizing antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system;

D is eribulin;

[00631] 136. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00632] 137. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00633] 138. An antibody-drug conjugate of Formula (I):

D is eribulin; L is a cleavable linker comprising Mal-C₂(PEG)_m-Val-Cit-pABC, wherein m is an integer from 0 to 4; and p is an integer from 1 to 8.

[00634] 139. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00635] 140. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00636] 141. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00637] 142. An antibody-drug conjugate of Formula (I): Ab-(L-D)_p (I) wherein Ab is an internalizing anti-TR0P2 antibody or internalizing antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system;

D is eribulin;

[00638] 143. An antibody-drug conjugate of Formula (I):

D is eribulin;

[00639] 144. The antibody-drug conjugate of any one of embodiments 138 to 143, wherein m is 2.

[00640] 145. The antibody-drug conjugate of any one of embodiments 128 to 144, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61.

[00641] 146. The antibody-drug conjugate of any one of embodiments 128 to 144, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[00642] 147. The antibody-drug conjugate of any one of embodiments 128 to 144, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61.

[00643] 148. The antibody-drug conjugate of any one of embodiments 128 to 144, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[00644] 149. The antibody-drug conjugate of embodiment 145, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00645] 150. The antibody-drug conjugate of embodiment 145, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00646] 151. The antibody-drug conjugate of embodiment 145, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00647] 152. The antibody-drug conjugate of embodiment 145, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00648] 153. The antibody-drug conjugate of embodiment 146, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00649] 154. The antibody-drug conjugate of embodiment 146, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 183 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00650] 155. The antibody-drug conjugate of embodiment 146, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00651] 156. The antibody-drug conjugate of embodiment 146, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00652] 157. The antibody-drug conjugate of embodiment 147, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00653] 158. The antibody-drug conjugate of embodiment 147, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 110.

[00654] 159. The antibody-drug conjugate of embodiment 147, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 96 and a light chain comprising an amino acid sequence of SEQ ID NO: 110. [00655] 160. The antibody-drug conjugate of embodiment 148, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00656] 161. The antibody-drug conjugate of embodiment 148, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 104 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00657] 162. The antibody-drug conjugate of embodiment 148, wherein the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 96 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00658] 163. The antibody-drug conjugate of any one of embodiments 128 to 162, wherein p is 2. [00659] 164. The antibody-drug conjugate of any one of embodiments 16 to 163, wherein p is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC).

[00660] 165. The antibody-drug conjugate of any one of embodiments 16 to 163, wherein p is determined by reverse-phase liquid chromatography-mass spectrometry (LC-MS).

[00661] 166. The antibody-drug conjugate of any one of embodiments 16 to 165, wherein the cytotoxic agent is eribulin and the cleavable linker covalently attaches to the eribulin via a C-35 amine.

[00662] 167. The antibody-drug conjugate of any one of embodiments 16 to 36, wherein the cleavable linker covalently attaches to the antibody or antigen-binding fragment via a cysteine or a lysine.

[00663] 168. A composition comprising multiple copies of the antibody-drug conjugate of any one of embodiments 16 to 167, wherein the average p of the antibody-drug conjugates in the composition is about 1 to about 3.

[00664] 169. The composition of embodiment 168, wherein the average p is about 2.

[00665] 170. A composition comprising multiple copies of an antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is an internalizing anti-TROP2 antibody or internalizing antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system; or three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system;

D is eribulin; L is a cleavable linker comprising Mal-(PEG)i-Val-Cit-pABC; and p is the average number of L-D moieties per Ab, wherein the average p of the antibody-drug conjugates in the composition is from about 1 to about 8; optionally from about 1 to about 3; optionally about 2.

[00666] 171. A composition comprising multiple copies of an antibody-drug conjugate of Formula (I):

D is eribulin;

L is a cleavable linker comprising Mal-(PEG)₂-Val-Ala-pABC; and p is the average number of L-D moieties per Ab, wherein the average p of the antibody-drug conjugates in the composition is from about 1 to about 8; optionally from about 1 to about 3; optionally about 2.

[00667] 172. A composition comprising multiple copies of an antibody-drug conjugate of Formula (I):

D is eribulin;

L is a cleavable linker comprising Mal-(PEG)₂-Asn; and p is the average number of L-D moieties per Ab, wherein the average p of the antibody-drug conjugates in the composition is from about 1 to about 8; optionally from about 1 to about 3; optionally about 2. [00668] 173. A composition comprising multiple copies of an antibody-drug conjugate of Formula

(I):

D is eribulin;

L is a cleavable linker comprising Mal-C₂(PEG)_m-Val-Cit-pABC, wherein m is 0, 1, 2, or 3; and p is the average number of L-D moieties per Ab, wherein the average p of the antibody-drug conjugates in the composition is from about 1 to about 8; optionally from about 1 to about 3; optionally about 2.

[00669] 174. A composition comprising multiple copies of an antibody-drug conjugate of Formula

(I):

D is eribulin;

L is a cleavable linker comprising Mal-C₂(PEG)_m-Val-Ala-pABC, wherein m is 0, 1, 2, or 3; and p is the average number of L-D moieties per Ab, wherein the average p of the antibody-drug conjugates in the composition is from about 1 to about 8; optionally from about 1 to about 3; optionally about 2.

[00670] 175. A composition comprising multiple copies of an antibody-drug conjugate of Formula

(I):

Ab-(L-D)p (I) wherein Ab is an internalizing anti-TR0P2 antibody or internalizing antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system; or three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system;

D is eribulin;

L is a cleavable linker comprising Mal-C₂(PEG)_m-Asn, wherein m is 0, 1, 2, or 3; and p is the average number of L-D moieties per Ab, wherein the average p of the antibody-drug conjugates in the composition is from about 1 to about 8; optionally from about 1 to about 3; optionally about 2.

[00671] 176. The composition of any one of embodiments 173 to 175, wherein m is 2.

[00672] 177. The composition of any one of embodiments 170 to 176, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61.

[00673] 178. The composition of any one of embodiments 170 to 176, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

[00674] 179. The composition of embodiment 178, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00675] 180. The composition of embodiment 178, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 179 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

[00676] 181. The composition of embodiment 178, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a fight chain comprising an amino acid sequence of SEQ ID NO: 115.

[00677] 182. One or more nucleic acid(s) encoding the antibody or antigen-binding fragment of any one of embodiments 1 to 13.

[00678] 183. A host cell comprising the one or more nucleic acid(s) of embodiment 182.

[00679] 184. A method of producing an antibody or antigen-biding fragment of any one of embodiments 1 to 13, comprising culturing the host cell of embodiment 183 under conditions sufficient to produce the antibody or antigen-binding fragment. [00680] 185. A method of producing the antibody-drug conjugate of any one of embodiments 16 to

167 or the composition of any one of embodiments 168 to 181, comprising reacting an antibody or antigen-binding fragment of any one of embodiments 1 to 13 with a cleavable linker joined to eribulin under conditions that allow conjugation.

[00681] 186. An antibody-drug conjugate Formula (III):

Ab is an antibody or antigen-binding fragment thereof;

D is a cytotoxic agent;

Y is a cleavable moiety;

Z is absent or a spacer unit; and p is an integer from 1 to 8.

[00682] 187. The antibody-drug conjugate of embodiment 186, wherein the cytotoxic agent comprises eribulin or a salt thereof.

[00683] 188. The antibody-drug conjugate of embodiment 186 or embodiment 187, wherein p is from 2 to 8; optionally wherein p is 2.

[00684] 189. The antibody-drug conjugate of any one of embodiments 186 to 188, wherein the bond to the Ab is on a cysteine residue of the antibody or antigen-binding fragment.

[00685] 190. The antibody-drug conjugate of embodiment 189, wherein the bond to the Ab is on a cysteine-80 residue on the light chain of the antibody or antigen-binding fragment.

[00686] 191. The antibody-drug conjugate of embodiment 189, wherein the bond to the Ab is on a cysteine-149 residue on the light chain of the antibody or antigen-binding fragment.

[00687] 192. The antibody-drug conjugate of embodiment 189, wherein the bond to the Ab is on a cysteine- 118 residue on the heavy chain of the antibody or antigen-binding fragment.

[00688] 193. The antibody-drug conjugate of embodiment 189, wherein the bond to the Ab is on a cysteine- 140 residue on the heavy chain of the antibody or antigen-binding fragment.

[00689] 194. The antibody-drug conjugate of embodiment 189, wherein the bond to the Ab is on a cysteine-239 residue on the heavy chain of the antibody or antigen-binding fragment.

[00690] 195. The antibody-drug conjugate of any one of embodiments 186 to 194, wherein the cleavable moiety comprises a cleavable peptide moiety.

[00691] 196. The antibody-drug conjugate of embodiment 195, wherein the cleavable peptide moiety is cleavable by an enzyme; optionally wherein the cleavable peptide moiety is cleavable by cathepsin or legumain; optionally wherein the cleavable peptide moiety is cleavable by cathepsin B.

[00692] 197. The antibody-drug conjugate of embodiment 195 or embodiment 196, wherein the cleavable peptide moiety comprises an amino acid unit. [00693] 198. The antibody-drug conjugate of embodiment 197, wherein the amino acid unit comprises valine-citrulline (Val-Cit), valine-dimethylated lysine (Val-Lys(Me)₂), alaninedimethylated lysine (Ala-Lys(Me)₂), valine-alanine (Vai-Ala), asparagine (Asn), aspartic acid (Asp), or methylated aspartic acid (Asp(OMe)).

[00694] 199. The antibody-drug conjugate of embodiment 198, wherein the amino acid unit comprises Val-Cit.

[00695] 200. The antibody-drug conjugate of embodiment 198, wherein the amino acid unit comprises Val-Ala.

[00696] 201. The antibody-drug conjugate of embodiment 198, wherein the amino acid unit comprises Asn.

[00697] 202. The antibody-drug conjugate of embodiment 198, wherein the amino acid unit comprises Asp.

[00698] 203. The antibody-drug conjugate of any one of embodiments 186 to 198, 201, and 202, wherein Z is absent, and the cleavable moiety is directly joined to the cytotoxic agent.

[00699] 204. The antibody-drug conjugate of any one of embodiments 186 to 200, wherein Z is a spacer unit which attaches the cleavable moiety to the cytotoxic agent.

[00700] 205. The antibody-drug conjugate of embodiment 204, wherein the spacer unit attaching the cleavable moiety to the cytotoxic agent is self-immolative.

[00701] 206. The antibody-drug conjugate of embodiment 204 or claim 205, wherein the spacer unit attaching the cleavable moiety to the cytotoxic agent comprises a p-aminobenzyloxycarbonyl (pABC).

[00702] 207. The antibody-drug conjugate of embodiment 206, wherein the cytotoxic agent is eribulin, and the pABC covalently attaches to eribulin via a C-35 amine.

[00703] 208. The antibody-drug conjugate of embodiment 206 or embodiment 207, wherein -Y-Z- comprises Val-Cit-pABC, Val-Ala-pABC, Val-Lys(Me)₂-pABC, or Ala-Lys(Me)₂-pABC.

[00704] 209. The antibody-drug conjugate of any one of embodiments 186 to 208, wherein Ab is an anti-TROP2 antibody or antigen-binding fragment of any one of claims 1 to 13.

[00705] 210. The antibody-drug conjugate of any one of embodiments 186 to 199 and 204 to 209, wherein -Y-Z- comprises Val-Cit-pABC.

[00706] 211. The antibody-drug conjugate of any one of embodiments 186 to 198, 200, and 204 to 209, wherein -Y-Z- comprises Val-Ala-pABC.

[00707] 212. The antibody-drug conjugate of any one of embodiments 186 to 198, 201, 203, and 209, wherein -Y-Z- comprises Asn.

[00708] 210. The antibody-drug conjugate of any one of embodiments 210 to 212, wherein D comprises eribulin or a salt thereof. [00709] 211. A composition comprising multiple copies of the antibody-drug conjugate of any one of embodiments 186 to 210, wherein the average p of the antibody-drug conjugates in the composition is about 1 to about 3.

[00710] 212. The composition of embodiment 211, wherein the average p is about 2.

[00711] 213. A compound of Formula (Ila): , wherein:

D is a cytotoxic agent;

Y is a cleavable moiety; and

Z is absent or a spacer unit.

[00712] 214. The compound of embodiment 213, wherein the cytotoxic agent comprises eribulin or a salt thereof.

[00713] 215. The compound of embodiment 213 or claim 214, wherein the cleavable moiety comprises a cleavable peptide moiety.

[00714] 216. The compound of embodiment 215, wherein the cleavable peptide moiety is cleavable by an enzyme; optionally wherein the cleavable peptide moiety is cleavable by cathepsin or legumain; optionally wherein the cleavable peptide moiety is cleavable by cathepsin B.

[00715] 217. The compound of any one of embodiments 213 to 216, wherein the cleavable peptide moiety comprises an amino acid unit.

[00716] 218. The compound of embodiment 217, wherein the amino acid unit comprises valinecitrulline (Val-Cit), valine-dimethylated lysine (Val-Lys(Me)₂), alanine-dimethylated lysine (Ala- Lys(Me)₂), valine-alanine (Vai- Ala), asparagine (Asn), aspartic acid (Asp), or methylated aspartic acid (Asp(OMe)).

[00717] 219. The compound of embodiment 218, wherein the amino acid unit comprises Val-Cit.

[00718] 220. The compound of embodiment 218, wherein the amino acid unit comprises Vai-Ala.

[00719] 221. The compound of embodiment 218, wherein the amino acid unit comprises Asn.

[00720] 222. The compound of embodiment 218, wherein the amino acid unit comprises Asp.

[00721] 223. The compound of any one of embodiments 213 to 218 and 222, wherein Z is absent and the cleavable moiety is directly joined to the cytotoxic agent.

[00722] 224. The compound of any one of embodiments 213 to 220, wherein Z is a spacer unit which attaches the cleavable moiety to the cytotoxic agent.

[00723] 225. The compound of embodiment 224, wherein the spacer unit attaching the cleavable moiety to the cytotoxic agent is self-immolative. [00724] 226. The compound of embodiment 224 or embodiment 225, wherein the spacer unit attaching the cleavable moiety to the cytotoxic agent comprises a p-aminobenzyloxycarbonyl (pABC).

[00725] 227. The compound of embodiment 226, wherein the cytotoxic agent is eribulin, and the pABC covalently attaches to eribulin via a C-35 amine.

[00726] 228. The compound of embodiment 226 or embodiment 227, wherein -Y-Z- comprises Val-Cit-pABC, Val-Ala-pABC, Val-Lys(Me)₂-pABC, or Ala-Lys(Me)₂-pABC.

[00727] 229. The compound of any one of embodiments 213 to 219 and 224 to 228, wherein -Y-Z- comprises Val-Cit-pABC.

[00728] 230. The compound of any one of embodiments 213 to 218, 220, and 224 to 228, wherein -Y-Z- comprises Val-Ala-pABC.

[00729] 231. The compound of any one of embodiments 213 to 218, 221, and 223, wherein -Y-Z- comprises Asn.

[00730] 232. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of embodiments 1 to 15, the antibody-drug conjugate of any one of embodiments 16 to 167 and 186 to 210, the composition of any one of embodiments 168 to 181, 211, and 212, or the compound of any one of embodiments 213 to 231 ; and a pharmaceutically acceptable carrier.

[00731] 233. A method of treating a patient having or at risk of having a cancer that expresses

TROP2, comprising administering to the patient a therapeutically effective amount of the antibody or antigen-binding fragment of any one of embodiments 1 to 15, the antibody-drug conjugate of any one of embodiments 16 to 167 and 186 to 210, the composition of any one of embodiments 168 to 181, 211, and 212, the compound of any one of embodiments 213 to 231, or the pharmaceutical composition of claim 232.

[00732] 234. The method of embodiment 233, wherein the TROP2-expressing cancer is a cholangiocarcinoma, bladder cancer, breast cancer, cervical carcinoma, colorectal cancer, esophageal cancer, gastric cancer, glioma, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer; optionally wherein the TROP2-expressing cancer is a cholangiocarcinoma, breast cancer, or NSCLC.

[00733] 235. A method of reducing or inhibiting growth of a TROP2-expressing tumor, comprising administering to the patient a therapeutically effective amount of the antibody or antigen-binding fragment of any one of embodiments 1 to 15, the antibody-drug conjugate of any one of embodiments 16 to 167 and 186 to 210, the composition of any one of embodiments 168 to 181, 211, and 212, the compound of any one of embodiments 213 to 231, or the pharmaceutical composition of claim 232.

[00734] 236. The method of embodiment 235, wherein the tumor is a TROP2-expressing cholangiocarcinoma, bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer; optionally wherein the tumor is a cholangiocarcinoma. [00735] 237. An antibody or antigen-binding fragment of any one of embodiments 1 to 15, an antibody-drug conjugate of any one of embodiments 16 to 167 and 186 to 210, a composition of any one of embodiments 168 to 181, 211, and 212, a compound of any one of embodiments 213 to 231, or a pharmaceutical composition of embodiments 232 for use in the manufacture of a medicament for the treatment of a TROP2-expressing cancer.

[00736] 238. The antibody-drug conjugate, composition, compound, or pharmaceutical composition for use of embodiment 237, wherein the TROP2-expressing cancer is a cholangiocarcinoma, bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer; optionally wherein the TROP2-expressing cancer is a cholangiocarcinoma.

[00737] 239. Use of an antibody or antigen-binding fragment of any one of embodiments 1 to 15, an antibody-drug conjugate of any one of embodiments 16 to 167 and 186 to 210, a composition of any one of embodiments 168 to 181, 211, and 212, a compound of any one of embodiments 213 to 231 , or a pharmaceutical composition of embodiment 232 in the treatment of a TROP2-expressing cancer.

[00738] 240. The use of embodiment 239, wherein the TROP2-expressing cancer is a cholangiocarcinoma, bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer; optionally wherein the TROP2-expressing cancer is a cholangiocarcinoma.

[00739] 241. A method of preparing the antibody-drug conjugate of Formula (III) of any one of embodiments 186 to 210, wherein the method comprises:

- conjugating the one or more compounds of Formula (Ila) to an antibody or antigen-binding fragment, to provide an antibody-drug conjugate of Formula (II): wherein Ab is the antibody or antigen-binding fragment; and - hydrolyzing the succinimide ring in the compound of Formula (II), to provide the compound of Formula (III)

[00740] 242. The method of embodiment 241, wherein the step of hydrolyzing the succinimide ring in the compound of Formula (II) comprises a pH from about 7.4 to about 9.2; optionally a pH from about 7.4 to about 8.5; optionally a pH about 7.4.

[00741] 243. The method of embodiment 241 or embodiment 242, wherein the step of hydrolyzing the succinimide ring in the compound of Formula (II) comprises a temperature of about 37 °C.

[00742] 244. The method of any one of embodiments 241 to 243, wherein the step of hydrolyzing the succinimide ring in the compound of Formula (II) is completed in about 24 hours or less.

[00743] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. Having now described the invention in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

EXAMPLES

Example 1: Anti-TROP2 ADC using antibody 162-46.2

Anti-Tumor Activity of an Anti-TROP2-Eribulin ADC in Human Breast Cancer HCC1954 Xenograft Models

[00744] The anti-tumor activity of an anti-TROP2-eribulin ADC was first analyzed using the commercially available mouse anti-TROP2 antibody 162-46.2. The Vh and VK of the antibody was chimerized with human IgGl (hlgGl) and kappa constant regions and conjugated with eribulin using a partial reduction method. The TROP2-positive human breast cancer cell line HCC1954 was used in an in vivo xenograft study. Similar expression levels of TROP2 and HER2 on the HCC1954 human breast cancer cell line were observed (FIG. 1 A), and the anti-TROP2-eribulin ADC was compared with trastuzumab-eribulin.

[00745] A single dose of anti-TROP2-162-46.2-eribulin (5 mg/kg), trastuzumab-eribulin (5 mg/kg), eribulin at the maximum tolerated dose (MTD, 3.2 mg/kg), or eribulin at the equivalent molar dose as 5 mg/kg anti-TROP-2 -eribulin (0.1 mg/kg) was administrated in mice bearing an HCC1954 xenograft. Durable anti-tumor activity of anti-TROP2-162-46.2-eribulin was observed (FIG. IB). Limited antitumor activity of trastuzumab-eribulin was seen (FIG. 1 A). Further, the equimolar equivalent dose of eribulin (0.1 mg/kg) showed no anti-tumor activity. The MTD dosage of eribulin at 3.2 mg/kg showed acute body weight loss (FIG. 1C), and no informative anti-tumor activity was obtained. There was no body weight loss observed in any other group. These data show the utility of TROP2 -targeted delivery of eribulin to tumors.

SN-38 as an Alternative Payload Assessment in NCI-H2110 NSCLC Xenograft Model [00746] SN-38 was evaluated as a potential alternative payload to eribulin, as this is the payload for the clinically approved IMMU-132. Anti-TROP2-162-46.2 antibody was conjugated to SN-38 in a manner similar to IMMU-132. No anti-tumor activity was seen for anti-TROP2-162-46.2-SN-38. Despite TROP2 expression being higher than FRa on NCI-H2110 cells (not shown), tumor reduction was observed for an anti-FRa-eribulin ADC (FIG. 2A). In a separate study in the NCI-H2110 xenograft model, anti-TROP2-l 62-46.2-eribulin was administered at 2.5, 5, or 10 mg/kg and compared with naked anti-TROP2 antibody (10 mg/kg) or eribulin (0.2 mg/kg is the molar equivalent of eribulin in TROP2-eribulin ADC at 10 mg/kg). No anti-tumor activity was seen for the naked antibody, eribulin, or the lowest dose of anti-TROP2-eribulin (FIG. 2B). However, increasing the dose to 5 and 10 mg/kg showed durable anti-tumor activity. There were no adverse events observed for any molecule tested.

[00747] To further compare the SN-38 and eribulin payloads, the anti-tumor activity in TNBC (Triple Negative Breast Cancer) PDx WHIM5 model was analyzed. A single dose of anti-TROP2- 162-46.2- SN-38 (5 mg/kg), anti-FRa-eribulin (5 mg/kg), or anti-TROP-162-46.2-eribulin ADCs was administrated. Durable anti-tumor activity of anti-FRa-eribulin and anti-TROP2-162-46.2-eribulin was observed, while little to no anti-tumor activity was seen for anti-TROP2-162-46.2-SN-38 (FIG. 3A). No bodyweight loss was observed (FIG. 3B). These data demonstrate the superiority of eribulin as a payload for anti-TROP2 ADCs. Differences between the potency of these ADCs may be related to differences in antigen expression levels.

Anti-Tumor Activity of anti-TROP2-162-46.2-Eribulin ADC in Human Bladder Cancer PDx Models [00748] The anti-tumor activity of anti -TROP2-162-46.2-eribulin ADC was compared with eribulin in various human bladder cancer PDx models since overexpression of TROP2 in the human bladder cancer correlated to poor prognosis. IHC staining showed strong intensity of TROP2 in CTG-1258 and positive staining of TROP2 in CTG-1388. In the CTG-1258 model, a single dose of anti-TROP2- 162-46.2-eribulin ADC at 5 mg/kg showed durable anti-tumor activity, while the MTD eribulin dosage (3.2 mg/kg) showed limited anti-tumor activity (FIG. 4A). Eribulin treatment (0.1 mg/kg, the molar equivalent of 5 mg/kg anti-TROP2- 162-46.2-eribulin) showed no anti-tumor activity (FIG. 4A). In the CTG-1388 model, both anti-TROP2- 162-46.2-eribulin ADC and the higher dose of eribulin (3.2 mg/kg) showed similar but limited anti-tumor activity, while no anti-tumor activity was achieved with lower eribulin treatment (0.1 mg/kg). It is likely that the CTG-1388 model is eribulin insensitive and/or TROP2 expression is lower than expression in CTG-1258. There was no body weight loss observed in anti-TROP2-l 62-46.2-eribulin ADC and eribulin (0.1 mg/kg), while MID eribulin (3.2 mg/kg) showed temporary body weight loss (FIGs. 4A and 4B).

Example 2: Generating Novel Anti-TROP2 Antibodies

TROP2 Immunization and B Cell Screening

[00749] Novel anti-TROP2 antibodies were produced by immunizing and boosting rabbits with human TROP2 extracellular domain fused to a mouse IgG2b Fc domain (hTROP2-Fc). The antibody titer for two rabbits was analyzed by ELISA for hTROP2-Fc reactivity at day 52 post-immunization and compared to the anti-TROP2 pre-immunization antibody titer (FIG. 5). After an additional boost, splenocytes and lymphocytes were harvested from rabbits at day 70 post immunization. The lymphocytes were seeded in 384-well plates at a density that ensured monoclonality of B cells. After 2 weeks, the supernatants were screened by ELISA for reactivity to hTROP2-Fc, the N-terminal deletion of human TROP2 extracellular domain (ECD) fused to a mouse IgG2b Fc domain (A147hTROP2-Fc), and an irrelevant Fc fusion protein. Clones reactive to the irrelevant Fc fusion protein were eliminated. Clones reactive to hTROP2 and/or A147hTROP2 were selected for further development. The RNA from B cells in TROP2-reactive wells was isolated, and the variable domains were PCR amplified and cloned into human IgGl (hlgGl) and human kappa constant region expression vectors to create rabbit-human chimeric hlgGl antibodies.

Recombinant Antibody Screening

[00750] The binding of the recombinant, chimeric antibodies was confirmed by screening the supernatants for reactivity to TROP2 in an ELISA assay. Recombinant antibodies were expressed in HEK293 cells and the supernatants were normalized to an antibody concentration of 1 μg/mL. The normalized supernatants were screened by ELISA for binding to hTROP2-Fc, A147hTROP2-Fc, cynomolgus TROP2 (cTROP2-Fc), mouse TROP2 (mTROP2-Fc), and rat TROP2 (rTROP2-Fc). TROP2 proteins were coated on 384-well plates and incubated with 1 μg/ml of antibody. Following binding and washing, an anti-human Fc-HRP detection antibody was added to wells. TMB substrate was used to visualize positive wells and the absorption at 450 nm is listed in Table 12. The absorbance value of the hTROP2 ELISA assay was used to rank the clones. The clones with a signal > 0.8 were selected for further development. In addition, 5 clones (38G3, 15H2, 20E16, 32010, and 83N21) were selected due to their cross-species reactivity. One clone, 90D7, was chosen because, despite a low signal in the hTROP2-Fc assay, the antibody concentration was below 1 μg/mL, and the low antibody concentration may have resulted in the low signal in the hTROP2-Fc ELISA. Table 12. Recombinant anti-TROP2 antibodies screen

[00751] The selected clones were screened for binding to cell surface-expressed hTROP2, cTROP2, mTR0P2, and rTROP2. HEK293 cells expressing each species of TROP2 were incubated with 1 μg/mL of each antibody (Table 13). Binding to the cells was detected with a FITC-anti-human Fc antibody, and the mean fluorescence intensity (MFI) was determined using a Guava easyCyte 8HT Flow Cytometer (ND = not determined). Clones that did not bind to cell surface-expressed hTROP2 were eliminated from the selection process. Table 13. Screening antibody hits for binding to cell surface TROP2

[00752] To narrow down the number of lead antibodies, clones were screened for overlapping epitopes to determine which antibodies bind the same or proximal epitopes. The Forte Octet instrument uses a shift in the wavelength of light to monitor a protein binding event. When a protein is coated onto a tip, the wavelength of light through the tip changes. Adding a second protein that binds to the coated protein causes a further shift in the wavelength of light, where a greater shift corresponds to more protein binding.

[00753] Analysis of overlapping epitopes was performed with two different formats. In the first format, each clone was incubated with hTROP2-Fc coated onto a tip and the binding was monitored. The complex was then incubated with a second antibody. A further shift in the wavelength indicated an additional binding event by the second antibody, demonstrating two antibodies binding different epitopes on the TROP2 protein. However, if no additional binding was seen, the two antibodies likely had overlapping epitopes. Clones 23H9, 66M7, 45D6, 58C17, 81E22, 11P2, 19B21, 5B13, and 32010 showed weak binding to hTROP2-Fc in this assay format and could not be binned. The remaining antibodies were analyzed in a matrix format. The antibody in each row was incubated with hTROP2- Fc and then the antibody in the column was added to this complex (FIG. 6A). Antibody 1 (rows) was incubated with hTROP2-Fc on sensor tips and the association was measured using a ForteBio Octet Red384 instrument. After binding equilibrated, each tip was incubated with all other anti-TROP2 hits (columns). The change in signal was measured and this value was divided by the signal on tips containing TROP2-Fc alone (no antibody 1) to determine the percent of maximum signal for each competing antibody. Antibody pairings with less than 80% of the maximum signal are highlighted as competing antibodies.

[00754] Since a clone competes with itself, the value obtained by pairing the same clone represents the signal of a single binding event on hTROP2-Fc. Antibody combinations with values similar to this control pairing were considered to have overlapping epitopes and are shaded in FIG. 6A. The white cells show an additional binding event upon the addition of the second antibody.

[00755] Each antibody clone was grouped in relation to how it overlapped each other clone, and seven bins were identified (FIG. 6B). The majority of antibodies demonstrated similar patterns regarding the antibodies they blocked, and these are represented as bin 1. Most antibodies overlapped with each other bin in some way, as seen with bins 1-5. Bin 6 only overlapped with two bins, and bin 7 only overlapped with bin 6. While the exact epitope of these bins has not been determined, Table 12 shows that these antibodies bind to the A147hTROP2 protein and thereby bind somewhere in the Cys- poor domain.

[00756] A second method was used to analyze lead antibodies, as well as IMMU-132 (sacituzumab) and DS-1062a (datopotamab). In this method, antibody 1 was first captured onto anti-Fc sensor tip. Unoccupied anti-Fc sites were then blocked with a blocking antibody and the tips were then incubated with TROP2-Fc. The antibody 1-TROP2 complexed tips were then incubated with antibody 2 and change in signal was monitored. A signal change during the last step indicated that both antibodies bound to TROP2 and were non-competing. Changes less than 0.2 (indicating no binding by the second antibody) are highlighted as competing antibodies in FIG. 6C. This format was less dependent on the association rate of antibody 1, as was the limitation of the first method. However, the association rate of antibody 2 can be limiting in this second method. If the association rate is too slow, little to no change in signal is seen. This was the case for clones 20E16, 45D6, and 55H19. When they were complexed with TROP2 as the first antibody, clones 16K21, 17124, and 78H3 could bind the little amount of TROP2 captured. However, when these clones were added to TROP2 already complexed with 16K21, 17124, or 78H3, the association of 20E16, 45D6, and 55H19 was too weak to be measured in this assay (FIG. 6C).

[00757] The overlapping epitopes identified with the second method were similar to those identified with the first method. The only difference is that both 20E16 and 78H3 bound TROP2 in the second method (FIG. 6D). The low association rate of 20E16 competing with the higher affinity 78H3 could have produced a false signal in the first method. The sacituzumab and datopotamab epitopes overlapped with all antibodies tested except for 17124 and 20E16. The epitopes for sacituzumab and datopotamab are clearly different from 16K21, 78H3, 45D6, and 55H19, as the bins for the latter antibodies differ from the former.

[00758] The binding kinetics of the clones were analyzed by BIAcore and the kinetics were compared to known anti-TROP2 antibodies and ADCs (Table 14). The KD of each antibody for hTROP was similar and typically sub-nanomolar. Only four clones (20E16, 45D6, 81E22, and 66M7) were approximately 1 nanomolar. 79G3 and 17124 displayed the lowest KD. The KDs for these clones are similar to two other established anti-TROP2 antibodies (Table 15). These data are consistent with Table 12 in regard to each clone’s species specificity.

Table 14. BIAcore analysis of anti-TROP2 mAbs

Table 15. BIAcore analysis of anti-TROP2 mAbs datopotamab and Sacituzumab

[00759] Purified antibodies were conjugated to eribulin and the ADC’s cytotoxicity on TROP2- positive cell lines (BxPC3, MCF7, and MDA-MB-231) was compared to a TROP2 -negative cell line (AsPCl). The concentration of ADC needed to reduce the number of cells to 50% of the untreated control was determined (IC₅₀; Table 16). Cells seeded at 5000 cells/well the day prior were incubated with various concentrations of ADC for 5 days. Cells were then stained with crystal violet and solubilized in lysis buffer. The absorbance at 570 nm was used to quantify the amount of crystal violet retained by the cells in each well. The percent of growth inhibition was calculated as (l-A570nm Treated /A570nm Untreated)xl00. The concentration of ADC required to reduce the number of cells to 50% of the control sample (IC₅₀) and max percentage of cell killing was determined and compared across all cell lines and ADC clones.

[00760] The IC₅₀ was plotted versus the KD affinity measured by BIAcore (FIG. 7). Clones for further development were selected based on low IC₅₀ and KD. Only the best clone within each epitope bin was selected. Most of the antibodies showed a correlation of high affinity and high cytotoxicity, and clones 16K21, 17124, 57H16, and 78H3 were selected for further analysis. However, several clones showed low affinity and high cytotoxicity (e.g., 55H19 and 20E16), and these clones were chosen for further development. 45D5 is a sister clone of 55H19, based on the antibody sequence. This clone was chosen for further analysis to further understand the impact minor differences in the CDRs have on the efficacy of the ADC.

Table 16. Cytotoxicity IC₅₀ of anti-TROP2 antibodies Humanization and Affinity Characterization

[00761] The top clones were humanized by CDR grafting whereby the CDRs from each VH and VL were grafted onto the closest human germline sequence in place of the human CDR sequences. Since there are several definitions of CDR lengths, the CDR sequences that were grafted encompassed both the IMGT and Kabat definitions (FIG. 8). This increased the likelihood that the entire paratope was grafted onto the human framework. Residues in the framework that may be critical for retaining affinity and/or stability were retained. These included residues 48-49 in FRH2 and residues 1-3 in FRL1. The cysteine at residue 80 was retained during humanization as a RESPECT-L conjugation site.

[00762] The affinity of purified chimeric and humanized antibodies was analyzed by BIAcore and compared to the affinity of the anti-TROP2 antibodies sacituzumab and datopotamab (Table 17). The binding kinetics of the purified chimeric antibodies are similar to that seen with the unpurified antibodies in Table 14. The dissociation constant (kd) of the humanized variant for each clone was similar to the parental chimeric antibody. However, for most of the antibodies, there was a decrease in the association constant (ka) for each clone. The overall KD for the humanized variants were within 5-6-fold of the chimeric, except for humanized 78H3, which showed a 9.5-fold loss in affinity. The KD for all clones except for 20E16 and 55H19 was below 1 nM.

Table 17. BIAcore of humanized clones

Table 18. BIAcore of humanized clone ADCs

[00763] Conjugation of a payload could affect antigen binding, depending on the payload linkage, the DAR, and the tertiary structure of the antibody-antigen complex. As with the chimeric anti-TROP2 antibodies, eribulin was conjugated to Cys80 of the humanized LCs at a DAR of ~2. The KD of the eribulin-conjugated humanized ADCs for each clone was compared to the unconjugated antibody, and no difference was seen (Table 18).

In vitro Screening of Lead ADCs

[00764] Eribulin conjugates of the humanized anti-TROP2 antibodies were screened for cytotoxicity across a broad panel of cell lines (FIG. 9). Wells seeded with 5000 cells/well one day earlier were incubated with various concentrations of ADC for 5 days. Cells stained with crystal violet and solubilized in lysis buffer. The absorbance at 570 nm was used to quantify the amount of crystal violet retained by the cells in each well. 16K21, 17124, 57H16, and 78H3 demonstrated the highest amount of cytotoxicity on TROP2-positive cells. 55H19 and 45D6 showed only mild cytotoxicity and were removed from the top leads. 20E16 displayed intermediate cytotoxicity. This antibody was chosen for further development because, unlike the other clones chosen, the binding kinetics of 20E16 showed a slow on-rate and slow off-rate. The effect an anti-TROP2 ADC with a slow on-rate would have on tumor growth in an in vivo model was not predictable.

[00765] Antibody internalization can affect the cytotoxicity of an ADC. If the internalization rate is too slow or not enough antibody can be internalized, the cytotoxic effect of the payload can be too low to kill the cell. The internalization rate of each antibody was tested on two cell types: a high- expressing cell line (MDA-MB-468) and a low-expressing cell line (NCI-H2110). The maximum amount of antibody internalized into the NCI-H2110 cells was lower than the MDA-MB-268 cells (FIG. 10). Nonetheless, similar trends were seen in both cell lines with the fastest internalizing antibody being 57H16 and the slowest antibodies being 45D6 and 55H19. Further Humanization of Lead Antibodies

[00766] 16K21, 17124, and 20E16 were further humanized and analyzed in silico for developability risks. The “human-ness” of the remaining clones was increased through a series of humanized variants targeting rabbit residues within CDRH1, FWRH2, CDRH2, FWRH3/CDRH3, FWRL1, and/or CDRL2 (FIG. 11).

[00767] The humanized HCs zul (Hl), zu2 (H2), and zu3 (H3) were paired with each of the humanized LCs Hl, H2, and H3 (except for 20E16). The antibodies were screened for antigen binding by ELISA and the new humanized variants were compared to HILI. All new humanized variants showed poorer binding to hTROP2-Fc than HILI (FIG. 12A). The HILI antibodies were purified by protein A chromatography, and unpurified supernatants were used for all other humanized clones. The only outlier for the highest EC₅₀ for the 16K21 clones was H2L1. There was little variation among the 17I24 clones. The H2 variants for 20E16 both showed high EC₅₀. Therefore, the human valine and serine residues at positions 48 and 49 in the VH of 16K21 and 20E16 may effect antigen binding.

[00768] Using these data, several more humanized clones were generated (FIG. 11). The CDRH2 of 16K21 was super-humanized in the context of both Hl and H2. 16K21L2 was used as the backbone for super humanized variants L4 and L5 targeting CDRL1 . As with 16K21, 17124 CDRH2 was humanized using both Hl and H2 variants. CDRL1 and CDRL2 of 17I24L2 were super humanized. 20E16H1 was used as the template to super humanize CDRH1, CDRH2, and FWRH3/CDRH3. 20E16L2 was used for super humanizing CDRL1 and CDRL2.

[00769] 16K21L2 was paired with each of the humanized heavy chains and L4 was only paired with H4 and H5. 17124 HC and LCs were paired as with 16K21. 20E16L2 was paired with Hl, 3, 4, 5, and 6, and L3 and 4 were paired with just H3. The supernatants of all pairings (including HILI) were analyzed for binding to hTROP2-Fc by ELISA (FIG. 12B). No differences in hTROP2 binding were seen with any 16K21 variants, and the super humanized CDRH2 and CDRL1 variants did not negatively impact binding. One more LC variant was generated for 16K21 for future studies. The two CDRL1 mutants were combined to generate L6 (FIG. 11). All 17124 HC and LC variants bound hTROP2 similarly except for L5. While CDRH2 and CDRL1 could be super humanized, CDRL2 could not.

[00770] The two most humanized HCs with the lowest IC₅₀s (16K21H3 & H4, 17I24H3 & H4, and 20E16H3 & H5), the humanized LC L2, and the most super-humanized LC (16K21L6, 17I24L4, and 20E16L5) were chosen for further characterization. In addition, residue Pl 05 in FWRH4 was humanized to Q105 in 16K21H5, 16K21H6, and 17I24H5. The HCs and LCs for each clone were paired together. The affinity of the purified antibodies for hTROP2 was measured by BIAcore (Table 19). There was little difference in affinity for any of the 16K21 humanized variants or the 20E 16 humanized clones. The 17I24L4 resulted in ~4-8-fold loss in affinity for hTROP2 compared to L2. 16K21-H5L2, -H5L6, -H6L2, and -H6L6 were analyzed for binding to cynomolgus and rat TROP2. There was little difference between the humanized variants binding to cynomolgus TROP2, and there was litle difference between each antibodies affinity for human and cynomolgus TROP2. Between the L2 and L6 variants, there was litle difference (within 2-fold) binding rat TROP2. However, H6 variants bound rat TROP2 3-6-fold beter than the H2 variants. Further, the H5 variants bound rat TROP ~15-fold less than human TROP2, while the H6 variants bound rat TROP2 3-5-fold less than human TROP2.

Table 19. BIAcore analysis of lead humanized anti-TROP2 clones

Polyspecificity Analysis

[00771] The lead humanized variants for 16K21, 17124, and20E16 were analyzed for polyspecificity to unrelated antigens. This assay includes proteins (e.g., insulin, keyhole limpet hemocyanin (KLH)), nucleic acids (e.g., ssDNA, dsDNA), glycans (e.g., lipopolysaccharide (LPS)), and lipids (e.g., cardiolipin) to determine whether the antibodies contain non-specific binding properties. Four clinical-stage antibodies with and two without polyspecificity were used as controls. See Jain, T., et al., Biophysical properties of the clinical-stage antibody landscape. Proc Natl Acad Sci USA, 2017. 114(5): p. 944-949. Following washing and blocking of the wells with BSA, test antibodies (100 nM) were added to the wells. Antigens were coated onto 96-well plates and sample antibodies (100 nM) were incubated with the antigens. Binding was assessed by ELISA. Briefly, following incubation, the wells were washed and the presence of antibody was detected using an anti-human antibody conjugated with HRP. After adding the TMB substrate, the absorbance at 450 nM was analyzed. The positive control antibodies bound to most or all of the antigens, while the negative control antibodies did not (FIG. 13). No binding was seen for any anti-TROP2 antibody, indicating a low risk for nonspecific-off target binding.

Differential Scanning Calorimetry

[00772] The thermal stability of the lead antibodies was analyzed by differential scanning calorimetry (DSC). DSC is a thermoanalytical technique which was used to measure the heat change (ΔH) associated with the thermal unfolding and denaturation of various anti-TROP2 purified mAbs and F(ab’)₂ fragments. Differences in AH resulting from hydrophobic interactions, hydrogen bonding, conformational enthalpy and thus the folding and stability of the native antibodies can be used to predict sample similarity, comparability, and stability. Whole IgG molecules were analyzed to determine the Tm of the Fab, CH2, and CH3 domains. The CH2 domain has the lowest thermal stability and can be seen as a peak in the low to mid 70s °C. The CH3 domain is the most stable and is seen as a peak in the low to mid 80s °C. The Tm of Fab domain can vary. An unstable Fab domain may overlap with the CH2 domain, while a very stable Fab may overlap with the CH3 domain.

[00773] 16K21 humanized variants were compared to the chimeric antibody. The Tm of the humanized 16K21 variant Fabs is significantly increased compared to the chimeric Fab. The peak of the chimeric Fab overlapped with CH2 with a Tm of 70.17 °C. Humanizing the variable domains increased the Tm 2-12 °C (FIGs. 14A, B and C) The Tms of the humanized Fabs group according to the Vh sequence. The H4 and H5 Fabs were less stable (~72-74 °C) than the H3 and H6 Fabs (~75.5- 77 °C), and the H3 Fabs were less stable then the Hl Fab (82 °C). The L2 and L6 LC variants did not impact the Tm.

[00774] The thermal stability of the 16K21 protein A purified mAb (capped), decysteinylated (decapped), and DAR 2 RESPECT-L eribulin antibody-drug conjugate (ADC) for HlLl-IgRH12, H3L6-IgRH2 (wild-type IgGl Fc with T135K and K222A mutations), and H3Ll-IgRH12 were analyzed to determine if the process of producing the ADC affected the stability of the ADC. In previous studies, the MAAS mutations were shown to reduce FcyR interactions by destabilizing a loop in CH2, resulting in a lower Tm of the CH2 domain. Consistent this with these observations, the Tm of the CH2 domain for the IgRH12 variants are ~65-67 °C, while the IgRH2 CH2 Tm was ~72 °C (FIGs. 14C and D). The Fab and CH3 Tms of the capped, decapped, and ADC did not vary more than 1 °C, demonstrating the thermal stability of the ADC compared to the unconjugated mAb.

[00775] As with 16K21, the 17124 humanized variants were much more stable than the chimeric mAb (FIG. 14E). The H4 variant was less stable than H3. Unlike 16K21, L2 was less stable than L4. The most stable Fab was H3L4 (81.45 °C) and the least stable was H4L2 (77.68 °C). The most human variant was H4L4 with a Fab Tm of 78.95°C. The Tm of the Fab was within the range of other therapeutic mAbs and was chosen for further development based on the more human protein sequence. The H4 variant was further humanized to H5 to remove a potential immunogenic residue (P105Q in FWR4 (FIG. 11). The Tm of the H5L4 Fab was within 1 °C of the H4L4 Fab. The Tms of H5L4 ADC and decysteinylated intermediate were analyzed. As with 16K21, there was little variation between the capped mAb, decapped mAb, and the ADC (FIG. 14F).

[00776] The Tm of the chimeric 20E16 Fab (75.88 °C, Tml) was similar to each of the humanized Fabs (~75-78 °C, Tm2) (FIG. 14G). (Note that the chimeric mAb is wild-type CH2 and the peak overlaps with the Fab while the humanized variants are MAAS with a reduced Tm for CH2.)

Aggregation Analysis

[00777] The aggregation potential of the humanized clones was analyzed by size exclusion chromatography (SEC).

[00778] SEC analysis of the humanized variants showed that 17124 clones are the most monomeric (Table 20). 16K21 and 20E16 samples contained more fragmented antibodies than 17124. The 17124 samples contained low amounts of aggregate. Chimeric 16K21 was more aggregated than the humanized samples, and the aggregation seen in the humanized samples was at acceptable levels. All of the chimeric and humanized 20E16 samples showed high amounts of aggregate. Overall, the aggregation levels of 16K21 and 17124 humanized samples was similar to datopotamab and sacituzumab. Table 20. SEC Analysis of lead humanized anti-TROP2 clones

Elimination of FcyR Interactions

[00779] To reduce possible effects such as neutropenia from FcyR-ADC interactions, the top clones were formatted with the following mutations: L234A, L235A, P238S, H268Q, and K274Q (FIG. 15). This format is referred to as IgG14AAS. In addition to these Fc mutations, T135K and K222A mutations, allowing for transglutaminase-mediated site-specific conjugation, were also introduced. This format is referred to as IgRH12. This format also retains the RESPECT-L conjugation site, thereby allowing the antibodies to be conjugated at either K135 or Cys80.

[00780] The reformatted antibodies were analyzed by BIAcore of affinity to hTROP2. Although it is unlikely that Fc mutations could affect antigen binding, the humanized IgRH12 antibodies and eribulin ADCs were compared to the IgGl variants. No differences were seen between any of the antibody formats with or without eribulin conjugated (Table 21).

Table 21. BIAcore of IgRH12

[00781] To evaluate the effect of these ADCs on neutrophil development, a neutropenia assay was adapted from Zhao et al. (A Potential Mechanism for ADC-Induced Neutropenia: Role of Neutrophils in Their Own Demise. Mol Cancer Ther, 2017. 16(9): p. 1866-1876). Because neutrophils express high amounts of FcyRIIA and FcyRIIIA, which could potentially mediate nonspecific internalization and cytotoxicity, alternate versions of these ADCs prepared with FcyR mutations (Gigarad) were also tested.

[00782] Hematopoietic stem cells (HSC) were differentiated to neutrophils in vitro. Progression was monitored over the course of 2 weeks using the neutrophil marker CD66b and the stem cell markers CD34 and CD133 (FIG. 16A). Initially, most cells expressed the stem cell markers with no expression of CD66b. Naive neutrophils began to express CD66b on day 3. On average, 50% of cells expressed CD66b after 7 days of differentiation and expression was maintained through the end of the 2-week period. Conversely, CD34 and CD 133 expression was detected on a much smaller percentage of cells on day 3 and was largely absent on day 7.

[00783] IgGl and IgRH12 antibodies for 16K21H1L1, 17I24H1L1, 20E16H1L1, and 78H3H1L1 were conjugated to the RESPECT-L Cys80 site with (PEG)₂-VCP-eribulin at a DAR of 2. To investigate their effect on neutrophil development, ADCs were added at different concentrations on day 3 of differentiation, and flow cytometry was used to analyze CD66b expression after 4 days of treatment (day 7 of differentiation). The percent of viable cells positive for CD66b staining in relation to untreated cultures (represented as % control CD66b+) was used to measure cytotoxicity. The data showed that differentiating neutrophils were sensitive to all four IgGl ADCs, with IC₅₀ values in close range of one another at ~0.2 nM (FIG. 16B). 17124 and 20E16 IgRH12 ADCs showed a 150- and 44- fold reduction in neutrophil cytotoxicity, respectively, similar to the samples treated with an Fc blocker. 16K21 and 78H3 IgRH12 ADCs showed a more limited effect, with just ~2-fold increase in ICso- However, the Fc-blocked samples and the IgRH12 ADCs demonstrated a similar reduction in cytotoxicity compared to the IgGl ADC. The affinity of 17124 is about 3-fold lower than 16K21 and 78H3, and 20E16 is about 10-fold lower. Immunogenicity Assessment

[00784] The potential immunogenicity of 16K21 and 17124 was evaluated using EpiVax’s ISPRI in silico prediction. In silico-generated overlapping 9-mer frames were evaluated against a set of nine HLA-DR supertype alleles to identify potential HLA class Il-binding peptides. Each peptide is compared to a pre-defined matrix of HLA binding preferences for the HLA-DR alleles, and an EpiMatrix score is assigned indicating the likelihood of HLA-DR binding compared to previously published peptides. The EpiMatrix scores is adjusted for the presence of tolerogenic HLA Class II T cell epitopes, or Tregitopes, to generate a tReg Adjusted Epx Score (De Groot et al 2023). The EpiMatrix Score of an average protein is set to zero with scores above zero indicating the presence of excess HLA ligands and scores below zero indicating the presence of fewer potential HLA ligands than expected.

[00785] HC and LC variants for 16K21 and 17124 were analyzed. Hl and H3 variants were compared to elucidate the potential immunogenicity of 61(T/S)WA63 in rabbit and 61DSV63 in human. H3 and H4 variants were compared to elucidate the potential immunogenicity of 48IG49 in rabbit and 48VS49 in human. 16K21 H4 versus H5, 16K21 H3 versus H6, and 17124 H4 versus H5 were compared to elucidate the significance of Pl 05 in rabbit and QI 05 in human.

[00786] All 16K21 and 17124 HC and LC sequences showed very low immunogenicity potential (FIG. 17). In the case of both 16K21 and 17124, 61DSV63 reduced the immunogenicity potential. Interestingly, 48VS49 and Pl 05 lowered the immunogenicity risk in 16K21, but not 17124. Both light chains showed reduced immunogenicity potential when the CDRL1 was super-humanized.

Example 3: Evaluating ADC Linker Options

Cysteine Rebridging Linker

[00787] Traditional, stochastic conjugation uses reduced interchain cysteines in the hinge and HC-LC to attach linker payloads to the antibody. This results in a single payload conjugated to each cysteine, resulting in an ADC having no covalent bonds between the chains where there are payloads attached. An alternative linker payload (FIG. 18A) contains two sulfide-reactive groups that covalently attach to adjacent reduced cysteines, resulting in re-bridging of these cysteine residues.

[00788] The cytotoxicity of DTM-N-eribulin conjugated to 16K21H1L1 (DAR 1.52) was analyzed across multiple cell lines and compared to 16K21H1L1 conjugated with (PEG)₂-VCP-eribulin at Cys80 (FIG. 18B). Wells seeded with 5000 cells/well one day earlier were incubated with various concentrations of ADC for 5 days. Cells were stained with crystal violet and solubilized in lysis buffer. The absorbance at 570 nm was used to quantify the amount of crystal violet retained by the cells in each well. There was little difference in the in vitro cytotoxicity between the two linkerpayload conjugates. Both showed little cytotoxic effect on the TROP2 -negative cell line ASPC-1 and the eribulin-resistant lines SNU-245, SNU-478, and TFK1. The TROP2-expressing/eribulin-sensitive cell lines showed similar IC₅₀ values for both ADCs. RESPECT-L Linkers

[00789] Linker variants were generated to optimize the cleavable trigger mechanism, linker type, and linker length. The enzymatic cleavage of the linker for ADCs screened in the above sections was the substrate for the lysosomal enzyme cathepsin, valine-citrulline (VC). Other cathepsin substrates were incorporated into linkers to compare to VC-based linkers. Valine-dimethyllysine (VK(Me)₂), alaninedimethyllysine (AK(Me)₂), alanine-phenylalanine-lysine (aFK), and glycine-glycine-phenylalanine- glycine (GGFG)-based linkers were synthesized (FIG. 19). There are many enzymes within the endocytic pathway and triggering release of the payload earlier in the endocytic pathway or by enzymes more restricted to the endosomes or lysosome within cancer cells may provide less nontumor release of free payload. Legumain enzymes are endosomal enzymes that hydrolyze asparaginyl bonds. They are overexpressed in tumor cells, thus making them an attractive trigger release mechanism with the potential to reduce off-target toxicity. Asparagine (N), aspartic acid (D), O- methylated aspartic acid (D(OMe)), and alanine-alanine-asparagine (AAN and AANMeN)-based linkers were synthesized. In addition to the trigger mechanism, the linker length was optimized by varying the PEG length ((PEG)₁, (PEG)₂, (PEG)₃, or (PEG)₄) or the alkyl spacer between the maleimide and amide (C₂ versus C₆).

[00790] Each linker-payload conjugate was conjugated to the RESPECT-L site (Cys80 on the LC) on hlgGl antibodies with Fc mutation to minimize Fc-FcyR interactions. The DAR of each linker payload was determined by HIC-HPLC (Table 22). The DAR of all ADCs was 1.5 or higher.

[00791] Conjugation of a hydrophobic payload can affect the biophysical properties of the ADC by increasing the hydrophobicity and the propensity of the ADC to aggregate. The ADCs were analyzed by HIC-HPLC for changes in hydrophobicity compared to the unconjugated 16K21HlLl-IgG (Table 22). There was little difference in hydrophobicity between the (PEG)_n, C₂ , and C₆ linkers. The cathepsin substrate-based ADCs were more hydrophobic than the legumain linkers with the VCP ADCs being the most hydrophobic.

Table 22. Drug-to-Antibody Ratio and Change in Hydrophobicity for RESPECT-L linkers

[00792] The cleavage mechanism that triggers the immolative release of the eribulin payload was further investigated. The cathepsin-cleavable linkers VCP, VK(Me)₂, AK(Me)₂, and GGFG were compared with the legumain-cleavable linkers Asp (D), D(OMe), Asn (N), AAN, and AANMeN. The spacer used to compare each of these trigger mechanisms was (PEG)₂. The cytotoxicity of each ADC was analyzed using BxPC3 and SNU-308 cell lines. Eribulin-insensitive cells hSEAC and the TROP2-low AsPCl cell line were used as negative controls. The VCP linker showed the highest potency of all linkers tested on the TROP2 -positive, eribulin sensitive cell lines with an IC₅₀ of 2.44 pM on BxPC3 cells and 42.8 pM on SNU-308 cells (Table 23). Of the cathepsin-cleavable linkers, VK(Me)₂P also showed high potency with IC₅₀s of 8.83 pM and 148 pM on the same respective cell lines. The pABC group is essential for immolative release of the eribulin payload when cleaving the VK(Me)₂ trigger. When the pABC is absent in the VK(Me)₂ linker, no cytotoxic activity was seen. Substituting the valine with alanine in the K(Me)₂-based linker reduced potency ~6-fold on BxPC3 cells and ~2-fold on SNU-308 cells. The aFKP linker showed further reduced potency with IC₅₀s of 81.1 pM and 445 pM on the BxPC3 cells and SNU-308 cells, respectively. Finally, the GGFG linker used in the DS-1062a ADC showed an IC₅₀ of 137 pM on BxPC3 cells, but no potency on SNU-308 cells.

Table 23. In vitro cytotoxicity of RESPECT-L linkers

[00793] A comparison of the various legumain-cleavable linkers showed very little variation in the IC₅₀ on BxPC3 cells, ranging from 46.3 to 106 pM, with D(OMe) being an outlier at 611 pM (Table 23). A similar trend was seen with the SNU-308 cells with IC₅₀S ranging from 175 to 327 pM. However, the aspartic acid-based linkers showed no potency on this cell line.

[00794] To determine the effect of linker length and type on cytotoxicity of the ADC, linkers comprising VCP conjugated to C₂, (PEG)₂, (PEG)₃, and (PEG)₄; linkers comprising D/N conjugated to C₂, C₆, (PEG)₁, (PEG)₂, and (PEG)₃; and linkers comprising AANMeN conjugated to C₂ and (PEG)₂ were analyzed. Comparing the IC₅₀ values of the different linker lengths and types within each cleavage trigger, there was little difference in potency between the PEG-based and C-based spacers (Table 23). There was also little effect on cytotoxicity due to linker length. The (PEG)₂-VCP linker showed higher potency than the (PEG)₃-VCP and (PEG)₄-VCP linkers.

In Vitro Bystander Cytotoxicity Assay

[00795] The bystander effect of eribulin-based linkers was analyzed by co-culturing TROP2-positive (SK-BR-3) and TROP2-negative (HL-60) cell lines with (PEG)₂-VCP-eribulin, (PEG)₂-N-eribulin, and (PEG)₂-aFK-eribulin ADCs. If an ADC demonstrates a bystander effect, HL-60 cells show an increased growth inhibition sensitivity to the ADC when co-cultured with the TROP2-positive cells. HL-60 and SK-BR-3 cells expressing green or red fluorescent proteins, respectively, were analyzed for cell growth using an IncuCyte S3 live cell imager. As seen in FIG. 20, mono-cultured SK-BR-3 cells showed growth inhibition sensitivity to each ADC, while HL-60 cells showed ~ 100-fold less growth inhibition. Co-culturing HL-60 and SK-BR-3 cells showed no difference in the growth inhibition of SK-BR-3 cells. However, the HL-60 cells in the co-culture increased growth inhibition sensitivity 10-fold. These data demonstrate that the bystander effect of eribulin was independent of the payload release mechanism of the linker and is dependent on TROP2-expressing cells.

Thermal Stability Analysis of Various Linkers

[00796] The thermal stability of the antibody-linker-payload linkage was assessed for each ADC. The ADCs were formulated at 1 mg/mL in phosphate buffered saline (PBS) and stored at either 37 °C or 4 °C for three weeks. The DAR was measured by HIC at various timepoints (T = 0, 1, 7, 14, and 21(22) days) (FIG. 21). Overall, for the RESPECT-L linkers the change in DAR was less than 10% for most linkers. The change in DAR (PEG)₂-D, C₆-D, and (PEG)₂-D(OMe) was greater with a 12-15% less in DAR.

[00797] Aggregation and fragmentation of the ADC was measured over time using SEC-HPLC. There was little (< 5%) fragmentation for all the ADCs with the exception of the C₂-N and C₂- AANMeN ADCs (FIG. 21). Likewise, aggregation was low (<5%) for all ADCs except for C₆-D and (PEG)₃-D. The C₆-D and (PEG)₃-D ADCs were aggregated prior to incubation at 37 °C.

Mouse Plasma Stability Analysis of Various Linkers

[00798] The stability of 16K21HlLl-ADCs containing various linkers was analyzed in mouse plasma. Sample ADCs were spiked into mouse plasma and incubated at 37 °C. Samples were taken at 0, 2, 4, 6, 24, 48, 72, 168 and 240 hours. The antibodies (and ADCs) were removed from the samples by immunocapture and eluted into new tubes. The immunocaptured samples were analyzed for intact ADC and linker hydrolysis. The immunodepleted samples were analyzed for free payload.

[00799] Mass spectrometry was used to determine the average DAR of the ADCs at each timepoint (FIG. 22A). For all samples, the DAR decreased over time. The DAR at TO differed for each ADC and comparing the change in DAR required normalization of the samples to the TO timepoint. The percent change in DAR relative to TO showed similar changes in DAR over time for all samples (FIG. 22B). The D(OMe) linker showed the biggest reduction in DAR with a 9% change in DAR versus 6- 7% loss for the other linkers. The largest drop for D(OMe) was between 6 and 24 h. Between 24 h and 240 h, the rate of DAR reduction was similar to the other linkers.

[00800] The immunodepleted plasma samples were analyzed for free eribulin. Organic extraction was used to remove eribulin from the plasma samples. After removal of organic solvent, the amount of eribulin in each sample was determined by mass spectrometry (FIG. 22C). From these values, the percent change in free eribulin was calculated (FIG. 22D). Little free eribulin was seen in the (PEG)i- Asp and the (PEG)₂-Asp(OMe) samples. Although the (PEG)₂-VCP linker showed the largest accumulation of free eribulin over time, there was a less than 4% increase in eribulin over ten days.

[00801] Hydrolysis of conjugated maleimide stabilizes the conjugated linker-payload from release via retro-Michael addition, and thus is an important property to monitor during protracted incubation experiments. Linker hydrolysis was analyzed for each time point. Hydrolysis of the (PEG)₂-D(OMe) linker was rapid (FIG. 22E). Analysis of the mass spectrum showed more than one hydrolysis event for this sample. The remaining samples hydrolyzed similarly, though the (PEG)₂-Asn linker hydrolyzed slightly faster than (PEG)₂-VCP and (PEG)₂-D.

Example 4: Target Validation in vivo

Anti-TROP2-Eribulin in vivo efficacy

[00802] An in vivo NCI-H2110 NSCLC xenograft model was established to test the efficacy of lead anti-TROP2-ADC molecules. The humanized HILI variant of clones 16K21, 17124, 20E16, and 78H3 were formatted as wild-type hlgGl and the Fc mutant hIgG14AAS. The antibodies were conjugated to eribulin and purified at a DAR of 2. Sacituzumab (hlgGl) conjugated at DAR 2 with eribulin was also compared. Mice bearing tumors around 150 mm³ were injected intravenously with a single dose of 10 mg/kg of test antibodies. The size of the tumors was monitored for 54 days (FIG. 23). The tumor of the PBS control group grew to a volume of 2,000 mm³ for 32 days before the mice were sacrificed. Anti-TROP2-hIgGl-ADCs (wild-type Fc) showed an initial reduction in tumor volume, and then a similar, delayed, tumor growth. The exception was 78H3, which showed no tumor shrinkage and very little tumor growth inhibition. All hIgG14AAS variants displayed better tumor inhibition than their hlgGl counterparts. In this case, all clones reduced the tumor size. Again, 78H3 showed the weakest effect as the tumors quickly grew after the initial reduction in tumor size. 20E16- hIgG14AAS showed better efficacy than the hlgGl, but the tumor growth was only delayed. Both 16K21-hIgG14AAS and 17I24-hIgG14AAS inhibited tumor growth throughout most of the study. There was no body weight loss observed in all tested TROP2-eribulin ADCs at this concentration.

[00803] Clones 16K21H1L1 and 17I24H1L1 in the IgRH12-C80 background were compared to each other and IMMU-132, dosed at 7.5 mg/kg. As above, there was no difference in tumor shrinkage between the 16K21H1L1 and 17I24H1L1 clones. IMMU-132 demonstrated limited to no anti-tumor activity (FIG. 24).

Comparison o/in vivo anti-Tumor Activity of Rebridging and RESPECT-L Linkers

[00804] Clone 16K21H1L1 in the IgRH12-C80 background conjugated to DTM-N-eribulin or (PEG)₂-VCP-eribulin were analyzed for in vivo efficacy in the NCI-H2110 xenograft model dosed at 7.5 mg/kg. FIG. 25 shows that the DTM-N-eribulin conjugation demonstrated a moderate amount of anti-tumor activity. The RESPECT-L conjugation, however, almost completely reduced tumors. Data is shown as average tumor volume in NSCLC NCI-H2110 xenograft model treated with subcutaneous injection of a single dose of each ADC at 7.5 mg/kg.

Comparison ofm' vivo anti-Tumor Activity for Various RESPECT-L Linkers

[00805] The in vivo activity of 16K21HlLl-IgRH12-C80-eribulin ADCs with three different PEG linker lengths was analyzed in the NCI-H2110 xenograft model. Mice were injected subcutaneously with a single dose of each ADC at 7.5 mg/kg. Two lots of 16K21HlLl-(PEG)₂-VCP-eribulin were compared. The tumor volume was monitored for 15 weeks. After an initial reduction in tumor volume for both lots of the (PEG)₂ ADC, tumors began growing back by day 50 (FIG. 26A). The anti-tumor activity of the (PEG)₂ ADCs was much greater than the (PEG)a that demonstrated moderate antitumor activity. The (PEG)₄ ADC showed no anti-tumor activity. There was no significant body weight loss from any of ADCs observed.

[00806] The cathepsin-cleavable linker (PEG)₂-VCP was compared to two other cathepsin-cleavable linkers, VK(Me)₂P and GGFG. Unlike (PEG)₂-VCP, no anti-tumor activity was seen for VK(Me)₂P or GGFG (FIG. 26B). The DAR for VK(Me)₂P was very low at 0.62. Therefore, 17I24HlLl-IgRH12- C80 was also conjugated to VK(Me)₂P. The DAR of both the 17I21H1L1-(PEG)₂-VCP and - VK(Me)₂P ADCs were similar. In this experiment, no difference was seen between the two linkers, as both showed potent anti-tumor activity (FIG. 26C). While the VK(Me)₂? linker may demonstrate similar efficacy to the VCP linker, the GGFG linker used in DS- 1062a was not effective when combined with an eribulin payload in the NCI-H2110 model.

[00807] Finally, the anti-tumor activity of legumain cleavable linkers was examined in the NSCLC NCI-H2110 xenograft model. Three legumain substrates (Asn, Asp, and Asp(OMe)) were compared with the VCP linker. The anti-tumor activity of the Asn linker reduced the tumor volume for several weeks, but by day 50 the tumors started growing out more rapidly than the VCP-based ADC (FIG. 26D). Both Asp-based legumain linkers showed attenuated anti-tumor activity. There was no significant body weight loss observed from any of ADCs. Example 5: Optimization of lead humanized antibodies and lead linkers

[00808] With the identification of lead humanized antibodies (16K21-H3L6 and 17I24-H5L4) and lead linkers ((PEG)₂-VCP and (PEG)₂-N), combinations of these clones (compared to HILI variants) with these linkers were compared for their in vitro cytotoxic activity on six cell lines. In addition, DAR 4 of 17I24-H5L4 were generated (with an engineered cysteine at position 118: Al 18C) to compare (PEG)₂-VCP and (PEG)₂-N.

[00809] As shown in FIG. 27 A, little difference is seen in the cytotoxicity activity of 16K21-H1L1 and 16K21-H3L6 conjugated to either VCP or N linkers. 17I24H1L1 demonstrated 2-3-fold greater cytotoxicity than 17I24-H5L4. As with 16K21, there was little difference in the cytotoxic effect between the VCP and N linkers. The DAR 4 17I24-H5L4 variants increased cytotoxicity ~ 2-fold. [00810] In addition to the six cell lines evaluated in FIG. 27 A, twenty-five additional cell lines were investigated for sensitivity to anti-TROP2-ADCs. The cell lines tested spanned PDAC, pancreatic, head and neck carcinoma, cholangiocarcinoma, breast cancer, non-small cell lung cancer, cervical cancer, gastric cancer, bladder cancer, and normal cells. 16K21-H3L6 and 17I24-H5L4 were selected for further analysis. The (PEG)₂-VCP and (PEG)₂-N linkers were compared using 16K21-H3L6 and 17I24-H5L4. Finally, DAR2 and DAR4 were compared using 17I24-H5L4 with the (PEG)₂-VCP linker. 16K21-H3L6-IgRH2 (wild-type IgGl Fc) was compared to 16K21-H3L6-IgRH12 (IgG14AAS Fc) using the (PEG)₂-VCP linker. In addition, the anti-TROP2 ADCs were compared to IMMU-132 and DS- 1062a. To quantify the differences in cytotoxicity for each ADC component, the ratio of IC₅₀ values was calculated for each cell line. The higher the ratio above 1, the less cytotoxic the first variable, and the lower the number below 1, the more cytotoxic the first variable. For instance, if the ratio of IC₅₀ for an N linker to VCP linker for a particular cell line were 2, then the N linker would be 2-fold less cytotoxic than the VCP linker. If the ratio were 0.5, then the N linker would be 2-fold more cytotoxic than the VCP linker.

[00811] VCP- and N-based linkers were compared across all 31 cell lines (FIG. 27B-G). There was little difference (typically within 2-fold) in the cytotoxicity between the linkers within each cell fine and each clone as measured by the IC₅₀ as shown in FIG. 28A. Likewise, there was little difference between the site-specific conjugation technology. When comparing the DAR2 17I24-H5L4-C80 and the DAR4 17I25-H5L4-A118C-C80, an increase in cytotoxicity ranging from 1.5-3-fold was observed for the DAR4 ADC, except for NCI-H2444, where the DAR4 demonstrated an almost 8-fold increase in cytotoxicity (FIG. 28B). Little difference in the cytotoxicity of 16K21-H3L6 was observed for wild-type Fc versus MAAS (FIG. 28C). Finally, 16K21-H3L6 was more cytotoxic than 17I24-H5L4. The increase in cytotoxicity ranged from 2-7-fold for most cell lines (FIG. 28D).

[00812] Analysis of the cytotoxicity of the anti-TROP2 ADCs on cell lines grouped by cancer type showed no correlation to cytotoxicity and cancer type (FIG. 27B-G). There was little correlation between medium and high TROP2 expression and cytotoxicity (e.g., OE33 and CNE1), while eribulin-based ADCs showed little to no effect on TROP2-low cell lines (e.g.. ASPC1 andNCI- H1975).

In vivo efficacy of lead humanized variants

[00813] The humanized variants 16K21-H3L6 and 17I24-H5L4 were analyzed for in vivo efficacy when conjugated to (PEGji-VCP-eribulin. Within the 16K21 ADC cohort, the superhumanized H3L6 was compared to the CDR-grafted HILI . Similarly, 17124 superhumanized variant H5L4 was compared to the CDR-grafted HILI . Additionally, the DAR2 and DAR4 variants for 17I24-H5L4 were compared. Finally, the effect of a mutated Fc was analyzed.

[00814] Comparing the superhumanized and CDR-grafted variant, there was little difference in the anti-tumor activity between 16K21-H1L1 and 16K21-H3L6 (FIG. 29). The 17124 superhumanized variant demonstrated reduced anti-tumor efficacy compared to the HILI CDR grafted variant. The IgG14AAS backbone showed potent anti-tumor activity.

Example 6: Methods

[00815] The examples herein employed methods that are detailed below.

Methods for Identifying anti-TROP2 Antibodies

Generation of anti-TROP2 Antibodies

[00816] DNA encoding the human TROP2-ECD was gene synthesized and cloned into an expression vector. Additionally, DNA encoding an N-terminal deletion of human TROP2-ECD (A147hTROP2) fused to mouse IgG2b was gene synthesized and cloned into an expression vector. Proteins were harvested from transfected HEK cells and purified by protein A chromatography. Four rabbits were then immunized: 2 with the full length TROP2-ECD and 2 with the A147hTROP2. After initial immunization and two boosts, immune sera were taken at day 52 of the immunization protocol to determine the anti-TROP2 antibody titer. Immune sera were diluted and tested by ELISA using full length hTROP2-ECD-Fc coated at 1 μg/mL. Rabbits were given one additional boost and at the end of the study (day 70) the immunized rabbits were euthanized. Lymph nodes and spleen were harvested, and then cells were isolated and cryopreserved.

High Throughput Screening

[00817] Cryo-conserved rabbit lymph node cells (2.0 x 10⁷ cells) were thawed, then activated with 2.5 μg/mL of lectin from Phytolacca americana and recovered with DNAse I for one hour at 37 °C with 5% CO₂. The cells were seeded at 5 cells per well on a 384-well plate with feeder cells (CHOs expressing rabbit CD 154) and cultured in complete IMDM (IMDM supplemented with 10% FBS, 2 mM L-glutamine, IX MEM NEAA, 1 mM sodium pyruvate, 50 U/mL penicillin, 50 μg/mL streptomycin, 55 μM 2-Me) that contained 10.5 ng/rnL human IL-2 and 10.5 ng/mL human IL-21 cytokines (PeproTech).

[00818] On week two, the wells producing rabbit IgG antibody were identified by IgG FRET using 33 ng/mL europium cryptate-conjugated polyclonal anti -Rabbit IgG, and 133 ng/mL AlexaFlour 647- conjugated polyclonal goat anti-rabbit IgG Fey. The screen for antibodies to full length TROP2-ECD was performed with a primary ELISA screen using plates coated with 1 μg/mL full length TROP2- ECD. The hits were consolidated and anti-TROP2 binding was confirmed with the same ELISA. Additionally, the hits were screened by ELISA for binding to A147hTROP2 -Fc and counter- screened in an ELISA for Fc-specific binding using CD3-Fc.

[00819] ELISA plates (384-well high-binding Greiner #781061) were coated with 1 μg/mL of antigen in 50 mM carbonate-bicarbonate, pH 9.4 (Pierce #28382), and incubated overnight at 4 °C. Plates were washed three times with wash buffer using BioTek ELX405 plate washers. Wash buffer was made from PBS (Coming #46-013-CM) and 0.05% Tween20 (SeraCare #5460-0020). Plates were blocked with Assay Buffer made from PBS with 0.05% Tween20 and 1% BSA (Sigma A7906), and incubated overnight at 4 °C. Samples were added after aspirating the plates, then incubated overnight at 4 °C. Plates were washed three times, then HRP-conjugated goat anti-rabbit IgG H+L antibody (Jackson ImmunoResearch #111-035- 144) at a 1:10,000 dilution in Assay Buffer was added, and incubated for 1 hour at room temperature. Plates were washed three times and TMB substrate was added (SeraCare #5120-0078) and incubated for 20 minutes at room temperature. Plates were read at 370 nm on a BMG CLARIOstar plate reader. mRNA Gene Rescue of Rabbit anti-TROP2 Antibodies

[00820] Total RNA was isolated from wells producing rabbit IgG anti-TROP2 antibodies using RNAqueous™-96 Total RNA Isolation Kit (Ambion) according to the manufacturer’s instructions. cDNA was synthesized, and light and heavy chain variable regions were amplified by PCR using Platinum Taq one step RT-PCR kit (Invitrogen) according to the manufacturer’s instructions, using in-house primers. The light and heavy chain variable regions were amplified with nested primers using Platinum Taq Amplification Kit using a thermocycler at 40 cycles (1 min 94 °C, 1 min 54 °C, 1.5 min 68 °C). Amplified DNA template was visualized by gel electrophoresis, purified by QIAquick 96 PCR Purification Kit (Qiagen) and DNA sequence was determined by Genscript (Piscataway, NJ) using in-house primers. DNA Sequences were analyzed against V-gene and J-gene rabbit families (IMGT/V-QUEST) and against in-house In-Fusion primer database (Blastn). In-Fusion primers were either identified or designed containing a human Fc linker added to the 5’ end for the V and J gene primer. PCR was performed with Platinum Taq Amplification Kit using a thermocycler at 40 cycles (1 min 94 °C, 1 min 54 °C, 1.5 min 68 °C). Amplified In-Fusion DNA template was visualized on gel electrophoresis, purified by Qiagen’s QIAquick 96 PCR Purification Kit. Primers were synthesized by IDT (Coralville, IA). Gene Synthesis and Cloning

[00821] PCR-amplified variable domains included 15 base-pairs at the 5’ and 3’ ends homologous to the cloning site within the subcloning vector. PCR fragments were subcloned into an expression plasmid containing a human gamma or kappa constant region using an In-Fusion HD cloning kit (Clontech) according to the manufacture’s protocol. 1 μL of the In-Fusion reaction was transformed into Stellar Competent Cells (Clontech) according to the manufacturer’s protocol. Transformants were grown in 1 mL LB medium (Teknova) overnight at 37 °C on a microtiter plate shaker. The next day, cultures were miniprepped with a QIAprep 96 Turbo miniprep kit (Qiagen) according to the manufacture’s instruction.

[00822] The top anti-TROP2 antibody hits were subcloned by streaking several microliters of the HC and LC E. coli glycerol stocks onto LB + Amp agar plates (Teknova) and grown overnight at 37 °C. Several colonies were grown in LB + Amp medium overnight at 37 °C with shaking. Cultures were miniprepped with a QIAQuick miniprep kit according to the manufacture’s protocol. The HC and LC genes of subclones were then sequenced.

[00823] Humanized heavy and light variable domains were codon-optimized for expression in HEK293 cells and were synthesized by Thermo Fisher Scientific. The variable domains were synthesized with a Kozak translation initiation sequence and an Ig secretion leader sequence and included 15 base-pairs at the 5’ and 3’ ends homologous to the cloning site within the subcloning vector. The LCs were engineered to retain Cys80. PCR fragments synthesized by Thermo Fisher Scientific were subcloned into an expression plasmid containing a human gamma or kappa constant region using an InFusion HD cloning kit (Clontech). All clones were sequenced to confirm the presence and fidelity of the inserts.

Humanization

[00824] The amino acid sequences of anti-TROP2 mAbs were analyzed using a BLAST search against a human variable domain database at http://www.ncbi.nhn.nih.gov/igblast/ to identify the human sequence with highest homology to the rabbit sequences. The sequences corresponding to the antigen-binding domains as identified by Kabat and Chothia CDRH1, Chothia CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 were grafted into the framework (FWR) regions of the most homologous human framework.

[00825] The variable domains of the rabbit and CDR-grafted sequences were used to generate in silico structural models. Models were generated using Schrodinger’s BioLuminate software, following the homology domain protocol.

[00826] Humanized variants were generated as described above. Point mutations were made using Stratagene's QuikChange XL according to the manufacturer’s protocol. All clones were sequenced to confirm the presence of the mutation. Methods of antibody production

HEK Transient mAb Expression

[00827] expi293F cells (ThermoFisher) were cultured according to the manufacturer’s protocol. For each milliliter of 3 x 10⁶ expi293F cells to be transfected with ExpiFectamine (ThermoFisher), 333.3 ng HC plasmid and 333.3 ng LC plasmid was incubated for 5-10 min in 50 μL Opti-MEM (ThermoFisher). Likewise, 2.67 μL ExpiFectamine was incubated in 50 μL Opti-MEM. The ExpiFectamine solution was added to the DNA mixture and incubated for 20-30 min at room temperature. The DNA:ExpiFectamine mixture was added to the cells while swirling and incubated at 37 °C, 8% CO₂, shaking at 125 rpm. The following day, 5 μL of enhancer 1 and 50 μL of enhancer 2 per mL of cells were added to the transfection with continued incubation for another 7-10 days. After 48-72 h, cells were fed at a final concentration of 10 g/L yeastolate (BD Biosciences), 5 mM valeric acid (Sigma-Aldrich), and 1:100 CD Lipid Concentrate (ThermoFisher).

HEK Stable Cell Line mAb Expression

[00828] Stably transfected cell line pools were seeded at 0.5 x 10⁶ cells/mL in FreeStyle 293 expression medium. Cells were incubated at 37 °C, 8% CO₂, shaking at 125 rpm. Once the culture reached a cell density of ~4 x 10⁶ cells/ml (3-5 days), cultures were fed with final concentrations of 10 g/L Select Soytone (BD Biosciences), 5 mM valeric acid (Sigma Aldrich), and 1:100 CD Lipid Concentrate (ThermoFisher). When the cell viability was less than 50% (7-10 days), the cultures were centrifuged for 1 h at 8000 rpm in a Beckman JLA8.1000 rotor. The supernatant was then filtered through a 0.2 μm PES filter and stored at 4 °C or -20 °C until purification.

CHO Transient mAb Expression

[00829] expiCHO cells (ThermoFisher) were cultured according to the manufacturer’s protocol. For each milliliter of 6 x 10⁶ expiCHO cells to be transfected with ExpiFectamine CHO (ThermoFisher), 500 ng HC plasmid and 500 ng LC plasmid was mixed in Opti-PRO (ThermoFisher) in 40 μL total volume. Likewise, 3.2 μL ExpiFectamine CHO was mixed in 36.8 μL Opti-PRO. The ExpiFectamine CHO solution was added to the DNA mixture and incubated for 1-5 min at room temperature. The DNA:ExpiFectamine CHO mixture was added to the cells while swirling and incubated at 37 °C, 8% CO₂, shaking at 125 rpm. The following day, 6 μL of enhancer and 160 μL of feed per mL of cells were added to the transfection, and cells were transferred to 32 °C, 5% CO₂. At day 5, an additional 160 μL of feed per mL of cells was added. At day 12 to 14, the supernatants were harvested.

CHO Stable Cell Line mAb Expression

[00830] Stably transfected cell line pools were seeded at 4 x 10⁶ cells/mL in ExpiCHO Stable

Production media (ThermoFisher) + 100x Amphotericin B (ThermoFisher). Cells were incubated at 32 °C, 8% CO₂, shaking at 125 rpm. On day 3 and 10 post seeding, cells were fed with 2% Efficient Feed C+ (ThermoFisher) and 4 g/L glucose. When the cell viability was between 70% and 50% (2-3 weeks), the cultures were centrifuged for 30 min at 3500 rpm in an Allegra 6R (Beckman) centrifuge. The supernatant was then filtered through a 0.2 μm PES filter and stored at 4 °C or -20 °C until purification.

Protein A Antibody Purification

[00831] Purification was performed using an AKTA Express purification platform (GE Healthcare). A 5 mL Mabselect sure column (Cytivia) was equilibrated with 10 column volumes (CV) of 20 mM Sodium phosphate, 300 mM NaCl, pH 7.2 (10X PBS stock diluted) at a flow rate of 5 mL/min. The conditioned medium, filtered through a 0.2 μm membrane, was then loaded at 4 mL/min, followed by washing unbound material with 10 CV of Equilibration buffer at 5 mL/min. The sample was eluted using 5 CV of 0.1 M Glycine pH 2.9 at 3 mL/min. Eluted material was immediately injected onto a 26/10 HiPrep desalting column (GE Healthcare) equilibrated in IX phosphate-buffered saline (PBS) and eluted in the same buffer. Peak fractions were pooled and filtered. For higher volumes of supernatants, Mabselect columns were made with the appropriate size, and purification was done on Akta explorer or Avant, followed by a desalting purification or dialysis into IX PBS. Final recovery was determined using BCA assay (Thermo-Fisher Scientific). Purity was determined using SDS- PAGE analysis on 4-12% gradient SDS-PAGE gels with Coomassie staining following electrophoresis.

Decapping Purification

[00832] Purification was performed using an AKTA Explorer purification platform (GE Healthcare). Appropriate size Mabselect sure column (Cytivia) was equilibrated with 6 column volumes (CV) of 20 mM sodium phosphate, 10 mM EDTA, pH 7.2. The conditioned medium, filtered through a 0.2 μm membrane, was then loaded, followed by washing unbound material with 10 CV of equilibration buffer. The column was then washed with 20 mM sodium phosphate, 10 mM EDTA, 10 mM cysteine, pH 7.2 for 16 hours at a low flow rate (0.2-1 mL/min depending on column size). This step was followed by an additional wash with 20 mM Tris, pH 7.5 for 60 hours at a low flow rate that removed cysteine and reoxidized reduced interchain disulfides. The sample was eluted using 5 CV of 0.1 M glycine pH 2.9. Eluted material was then loaded onto a 26/10 HiPrep desalting column (GE Healthcare) equilibrated in IX PBS and eluted in the same buffer. Peak fractions were pooled and filtered. As an alternative, dialysis in IX PBS was also used (4x buffer changes). Final recovery was determined using BCA assay (Thermo-Fisher Scientific) according to the manufacturer’s protocol. Purity was determined using SDS-PAGE analysis on 4-12% gradient SDS-PAGE gels with Coomassie staining following electrophoresis. Antibody and Linker Payload Conjugation Methods

Maleimide Conjugation

[00833] Decapped RESPECT-L antibodies were prepared at 1 or 7 mg/mL and then incubated with maleimide-linker-payloads at molar ratio of 1:4 or 1:5 in lx DPBS buffer at room temperature for 30 min. The reaction was then purified by desalting using a HiTrap desalting column or Zeba spin desalting column, eluting with lx DPBS buffer.

[00834] Datopotamab and sacituzumab were partially reduced by TCEP at room temperature for 60- 80 minutes. Datopotamab at 5.66 mg/ml was first partially reduced by TCEP by mixing mAb with TCEP solution at a mAb:TCEP molar ratio of 1:3.6 at room temperature on an orbital shaker at 22 rpm for 60 minutes. The mAb was then conjugated to maleimide-GGFG-Dxd at a molar ratio of 1 :6 in lx DPBS at room temperature for 30 min, resulting in an average DAR of 4.0. Sacituzumab (4.5 mg/ml) was first partially reduced by TCEP at a mAb:TCEP molar ratio of 1:9.25 at room temperature on an orbital shaker at 22 rpm for 90 minutes. The mAb was conjugated with CL2A-SN- 38 at a molar ratio of 1 :8 in lx DPBS at room temperature for 30 min, resulting in an average DAR of 7.6.

ADC Analytical Methods

[00835] The HIC-HPLC was performed on an Agilent 1260 HPLC system with the TOSOH TSKgel butyl-NPR column (4.6 x 35 mm). The ADC was loaded to the column for 5 min of 100% buffer A equilibrating, then eluted with 20 minutes of linear gradient from 0-100% buffer B using mobile phases of 1.5 M ammonium sulfate, 25 mM sodium phosphate buffer at pH 7.0 (buffer A), and 25 mM sodium phosphate, 25% IPA (buffer B), followed with 5 min of 100% buffer B washing, at flow rate of 0.6 mL/min. The overall change in hydrophobicity (RRT) of the ADC was calculated based on the retention time of DAR2 peak divided by the retention time of unconjugated antibody on the HIC column. The higher RRT indicated the conjugate was more hydrophobic than the unconjugated antibody.

[00836] The SEC-HPLC was performed on an Agilent AdvanceBio SEC 300A column (2.7 μm, 7.8 x 300 mm) coupled with matched AdvanceBio guard column. Samples were eluted with mobile phase containing 0.1 M sodium phosphate, 0.15 M sodium chloride, 5% IPA at pH 7.4, for total 36 minutes at a flow rate of 0.5 mL/min.

[00837] The LC-MS was operated on a Waters Alliance e2695 HPLC coupled with SQD and 2998 PDA detectors or on a Waters Acquity Premier coupled with SQD 2. Samples were reduced with 20 mM DTT at 60 °C for 2 min, then separated by the Waters BioResolve RP mAb Polyphenyl column (4.5 x 100 mm) using 30 minutes of 25-75% B linear gradient of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B), flow rate at 1 mL/min. The linker-payload was only conjugated to the unpaired Cysteine 80 at light chain of the antibody, and the DAR was calculated based on the integrated peak intensity of identified light chain peaks. Payload Release Assays

ADC Buffer Stability

[00838] Buffer stability samples were prepared by diluting ADCs to 1 mg/mL in 25 mM Na Citrate buffer containing 100 mM sucrose, at pH 6.0. The samples were then stored at 37 °C for up to 504 hours. At each time point (T = 0, 24, 72, 168, 336, and 504 hours), an aliquot was removed and stored at -80 °C until analysis. Samples were analyzed by HIC-HPLC and SEC-HPLC. DAR was calculated by HIC-HPLC method.

ADC Plasma Stability

[00839] Plasma stability samples were prepared by diluting ADCs to 0.2 mg/mL in mouse plasma (BioIVT, BALB/C mouse plasma prepared using 3.8% sodium citrate as anticoagulant) and aliquoted into 0.1 mL time point samples. Samples were placed into 37 °C incubator and aliquots were removed at 0, 1, 4, 24, 72, 168, and 240 hours and frozen at -80 °C immediately after removal.

Analysis by LC-MS

[00840] Magnetic beads (Dynabeads, Invitrogen, cat 65602) were prepared for immunocapture by washing 3 times with PBS, with collection of the magnetic beads by a DynaMag-2 magnet (Invitrogen, 12321D) after each wash. 40 μg of biotin anti-human Fc (Southern Biotech, 9040-08) per 200 μL stock beads was added to the beads and mixed at room temperature for 1 hour. Beads were washed 3 times with PBS, then brought back to original volume in PBS. 100 μL of plasma sample was added to 200 μL anti-human Fc-immobilized magnetic beads, mixed at room temperature for 2 hours, washed 1 time with PBST followed by 2 times with PBS, and resuspended in 100 μL 2% acetic acid. Beads were mixed at room temperature for 30 minutes. Supernatant was collected and neutralized with 50 μL IM ammonium bicarbonate. Samples were transferred to a 96-well 350 μL plate. 10 μL of lOx GlycoBuffer2, 1 μL of PNGase F enzyme (NEB, P0704S) and 4 μL of 100 mM DTT was added to each DAR4 and 4 μL of 100 mM DTT was added to each DAR2 eluted/neutralized sample and digested with microwave-assisted digestion (Rapid Enzyme Digestion System, Hudson Surface Technologies), 400W, 37 °C for 15 minutes. Samples were analyzed by LC-MS on a Waters Synapt G2 fitted with a Waters Acquity UPLC.

DAR was calculated as:

DAR = [(DPA cLC/(DPA cLC + DPA ucLC)) + (DPA cHC/(DPA cHC + DPA ucHC))] x2 DPA = deconvoluted peak area cLC = conjugated light chain ucLC = unconjugated light chain cHC = conjugated heavy chain ucHC = unconjugated heavy chain Hydrolysis

[00841] Hydrolysis samples were prepared by diluting ADCs in PBS to 0.3 mg/mL and buffer exchanging into pH 7.4, pH 8.5 and pH 9.2 using Zeba Desalting plates (Thermo, 89808). Samples were placed into 37 °C incubator and aliquots were removed at 1, 4, and 24 hours. Samples were immediately buffer exchanged into PBS and frozen at -80 °C until analysis. Samples were analyzed by LC-MS on a Waters Synapt G2 fitted with a Waters Acquity UPLC.

Cell Lines and Cell Culture

[00842] All cell lines were cultured according to standard mammalian tissue culture protocols and sterile technique. All tissue culture media and supplements were obtained from Invitrogen. The expression level of TROP2, HER2, or FRA were determined by flow cytometry using an anti-TROP2, anti-HER2, or anti-FRA antibody and an isotype control. Relative fluorescence intensity of each antigen was normalized to that of control IgG.

FACS Analysis

[00843] Purified anti-TROP2 antibodies were evaluated for surface rat, mouse, cynomolgus monkey, and human TROP2 binding by FACS. Briefly, Expi293 cells expressing rat, mouse, cynomolgus monkey, or human TROP2 on the cell surface were seeded at le5 cells/well in a 96-well round bottom plate. Cells were washed two times with 200 μL/well cold FACS buffer. Cells were then stained with purified antibodies diluted to 1 μg/mL, 0.5 jxg/mL, and 0.1 μg/mL in FACS buffer for 1 hour on ice with 100 μL/well. After the 1-hour incubation, cells were washed three times with 200 μL/well cold FACS buffer. The secondary antibody anti-human IgG AlexaFluor-488 was diluted 1:100 in FACS buffer and added 100 μL/well to stained cells as well as secondary only controls and cells were incubated on ice for 30 minutes. Cells were then washed three times in cold FACS buffer and resuspended in 100 μL cold FACS buffer. Stained cells were run on a Guava easycyte 8HT Flow Cytometer (Luminex) and the MFI was graphed.

Surface Plasmon Resonance (SPR) Binding Analysis

[00844] Anti-TROP2 antibody ligand samples were diluted in assay buffer (IX HBS-P+) at 1 (ig/ml. TROP2-ECD-Fc analytes were diluted in assay buffer at 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and 0 nM concentrations. The diluted ligands and analytes were centrifuged at 18,000 x g for 5 min, then transferred to a new tube prior to analysis.

[00845] All experiments were performed using single-cycle kinetics on the BIAcore T-200 instrument (Cytiva) and a CM5, anti-Hu Fc immobilized chip. Antibodies were captured on flow cells 2, 3, and 4 at a flow rate of 10 μL/min for a contact time of 40 sec. Dilutions of TROP2-ECD-Fc proteins were injected over all four flow cells at a flow rate of 30 μL/min for a contact time of 300 sec. Dissociation was followed for 1800 sec. Following each cycle, the surface was regenerated twice by a 30-sec injection of 3 M MgCl₂ at 30 μL/min, followed by a buffer wash and 180-sec stabilization period. Following running, data were fitted using a 1:1 Langmuir model using all concentrations using BIAE valuations .

In vitro Cytotoxic Assays

[00846] Cells were seeded at 5,000 cells/well in 96-well tissue culture plates, incubated at 37 °C, 5% CO₂ overnight, and then treated with 0.005-100 nM ADCs, treated for 5 days. At the end of the assay, the viable cells were stained with 0.2% crystal violet solution, washed with tap water, and dissolved in 100 μL of 1% SDS solution. The absorbance at 570 nm was analyzed using an M5 plate reader. Alternatively, the viable cells were lysed with Cell Titer Gio at a ratio of 1:5 (v/v) and incubated at RT for 20mins. Luminescence was then measured with a 0.1 s integration time using the BMG CLARIOStar. The percent of growth inhibition was calculated as (l-A_570nm Treated /A_570nm Untreated) x 100. The dose response curve was plotted as concentration (X-axis) vs. % growth inhibition (Y- axis) using GraphPad Prism, and curve fitted with nonlinear regression four-parameter fit. The IC₅₀ (the concentration of ADC required to reduce the number of cells to 50% of the control sample) was summarized for potency comparison.

Internalization Assay

[00847] 100 μL of cells were seeded at 10,000 cells/well (MDA-MB-468) or 15,000 cells/well (NCI- 112110) and incubated at 37 °C, 5% CO₂ overnight. Antibodies were labeled with IncuCyte Human FabFluor-pH Red antibody labeling reagent (Sartorius cat #4722) at a molar ratio of 1 :3 (test antibody:labeling Fab) in cell culture medium for 15 min, then added to the cells by replacing the medium. The cell plates were then immediately transferred to an IncuCyte S3 Live-Cell Analysis System placed inside a tissue culture incubator at 37 °C, 5% CO₂. Automated phase and red fluorescence micrographs were captured with a 20x objective every 30 minutes for 48 hours. When the Fab-mAb complex is internalized and processed via acidic lysosomes and endosomes, the red fluorescence area and signal intensity inside the cell increase. The total red object area (μm²/ well) was quantified for each time point using the IncuCyte software.

Polyspeciflcity Assay

[00848] Six different antigens (cardiolipin (50 μg/mL, C0563; Sigma), KLH (5 μg/mL, H8283; Sigma), LPS (10 μg/mL, InvivoGen), ssDNA 1 μg/mL, D8899; Sigma), dsDNA 1 μg/ml, D4522; Sigma), and insulin (5 μg/ml, 19278; Sigma)) were coated on ELISA plates (3369; Coming) individually at 100 μL per well overnight at 4 °C. Plates were blocked with IX assay buffer containing 5% BSA at room temperature for 1 h or overnight at 4 °C, followed by three washes with PBST (PBS + 0.05% Tween 20). 100 μL of antibody at 100, 10, 1, and 0.1 nM was added to each well and incubated at RT for 1 h, followed by three washes with PBST. 100 μL of 0.5 μg/mL HRP- conjugated goat anti-human IgG antibody solution was added to each well and incubated at RT for 1 h, followed by three washes with PBST. 100 μL of TMB ELISA peroxidase substrate was added to each well and incubated for 10-15 minutes. Plates were read at 320 nM using ELISA plate reader (Clarion Star).

Differential Scanning Calorimetry

[00849] DSC was performed using Malvern Instruments, model PEAQ-DSC, and the data were analyzed with MicroCai PEAQ software v.1.53. 335 μg (1 μg/μL) of anti-TROP2 mAb or F(ab’)₂ fragment was added, along with corresponding reference formulation buffer, to each half of a thermal cell. Both the sample and reference cells were maintained at equal temperature, pressure (70 psi), and volume (325 μL) as the temperature increased, linearly, from 20-100 °C at a scan rate of 60 °C/hour. The amount of measured energy required to keep sample and reference materials at equal temperature was then converted to Molar Heat Capacity (Cp). The Cp is the amount of heat required to increase the temperature of one mol of anti-TROP2 Mab or F(ab’)₂ by one degree at constant pressure (70 psi). The Cp measurements for each unfolding event of the CH2 domain, Fab, and CH3 domains were graphed and used to predict sample stability.

Thermal Stability Assay

[00850] The thermal stability of anti-TROP2-eribulin ADCs was evaluated by dilution to 1 mg/mL in lx DPBS buffer and aliquoting to 6 vials at 0.15 mL/vial. The samples were incubated at 37 °C for four time points at T = 1, 7, 14, and 21 days. Samples T = 0 and T = 21 at 4 °C were prepared as control samples. At each time point the samples were immediately stored at -80 °C. After all time points were collected, samples were thawed at the same time and the DAR was analyzed using HIC- HPLC and LC-MS analysis.

Neutropenia Assay

Expansion of CD34+ Stem Cells and Differentiation of Immature Neutrophils

[00851] Human CD34+ progenitor stem cells isolated from bone marrow were thawed into SFEM II medium containing CC100 cytokine cocktail (IX). Cells were seeded into 24-well plates (TPP; 5,000 c/well) and incubated at 37 °C, 5% CO₂ for 3 days. Expanded stem cells were then collected and a small number of cells were reserved for flow cytometry analysis using Guava easycyte 12HT. The remaining cells were diluted in SFEM II supplemented with rhSCF (50 ng/mL), rhFlt-3 (100 ng/mL), rhIL-3 (5 ng/mL), rhGM-CSF (5 ng/mL), and rhG-CSF (5 ng/mL). Cultures were seeded into 6-well plates (TPP; 75,000 c/well) and incubated for 3 days. Neutrophil Differentiation, Fc Blocking, and ADC-Induced Neutropenia Assay

[00852] Immature neutrophils were collected, centrifuged, and resuspended in SFEM II supplemented with rhIL-3 (5 ng/mL) and rhG-CSF (30 ng/mL). Half of the cells were treated with human IgG (100 μg/mL) for 15 min at room temperature. Cells were then seeded into 96-well round-bottom plates (TPP; 5,000 c/well). Five-fold serial dilutions of ADCs (500 nM - 1 pM) were added to the cells and the plates were incubated for 4 days. Flow cytometry analysis was performed using the indicated markers. The effect of ADCs on neutrophil development is represented by the percentage of CD66b+ cells within the viable cell population.

In vivo Study Protocol

[00853] Female NOD.CB17-SCID mice (NOD.CB17-Prkdcscid/J) at 5 weeks old (Jackson Lab) were implanted subcutaneously in their right hind flanks with indicated cell line (HCC-1954 or NCI- H2110) mixed 1 : 1 with Matrigel. Tumor volumes were measured 2-3 times per week and calculated using the following formula:

[00854] Timor Volume (mm³) = L (mm) x W (mm) x H (mm) x π/6

[00855] When tumors reached an average of ~250 mm³, the mice were randomized into defined groups and treated with one bolus intravenous injection of indicated compound or PBS. Relative tumor volume normalized by the size of tumor on Day 0 was calculated to evaluate anti-tumor activities. Bodyweight change was also evaluated.

In Vitro Bystander Cytotoxicity Assay

[00856] SK-BR-3-Nuclight Red cells (NR) (TROP2+) were seeded in 96-well round-bottom plates (12,000 c/well) and incubated at 37°C overnight. ADCs (1:4 serial dilutions) were added to the target cells and incubated at 37°C for 30 min. HL-60-Nuclight Green (NG) cells (TROP2-) were added to the added to the wells with or without SK-BR-3-NR cells (5,000 c/well). The plates were incubated for up to 5 days, capturing images every 2 hours using an IncuCyte S3 live cell imager and counting the number of nuclei/well. IC₅₀ values were calculated for percent growth inhibition of target and bystander cells.

Synthesis Methods

[00857] Examples 8-23 employed methods that are detailed below.

[00858] All reagents purchased from commercial sources were used without further purification unless otherwise noted. All reactions involving air or moisture sensitive reagents were performed under an inert atmosphere.

[00859] In the manufacturing examples and implementation examples, unless otherwise specified, the silica gel used in silica gel column chromatography includes Silica gel 60 (Merck KGaA, catalog code : 1.07734), Hi-Flash™ Column Silicagel (YAMAZEN CORPORATION), or Presep Silica Gel (WAK O). The purification silica gel used in ODS silica gel column chromatography is Universal Column O DS (YAMAZEN CORPORATION).

[00860] Proton magnetic resonance spectra were recorded primarily on JEOL 400 (JMTC-400/54/SS, ECZ400S), or JEOL 500 (JMTC-500/54/JJ, ECZ500RS). ¹H NMR data are in the form of delta values for major diagnostic protons, given in parts per million (ppm) relative to tetramethylsilane (TMS, 0 ppm) or chloroform (CHCl₃, 7.27 ppm), methanol (MeOH, 3.31 ppm), Dimethyl Sulfoxide (DMSO, 2.50 ppm) as an internal standard. The coupling constants are reported in the unit of Hertz (Hz).

Abbreviations for splitting patterns are as follows: s: singlet; d: doublet; t: triplet; q: quartet; quin: quintet, sext: sextet; dd: doublet of doublets; dt: doublet of triplets; dq: doublet of quartets; td: triplet of doublets; tq: triplet of quartets; tt: triplet of triplets; ddd: doublet of doublet of doublets; m: multiplet; and br s: broad single.

[00861] Mass spectral analysis was performed using a Waters UPLC™. The ionization method used was electrospray ionization (ESI).

[00862] Chemical names were determined using MarvinSketch 21.4, version 21.4.0 (ChemAxon Ltd.) or ChemDraw Prime, version 22.2.0.3300 (PerkinEhner Informatics, Inc.). Chemical symbols have their usual meanings.

Abbreviations

The following abbreviations may be used throughout the examples.

AcOH: Acetic acid aq: aqueous

Boe: tert-butyloxycarbonyl brine: Saturate aqueous sodium chloride

CD₃CN: Deuterated acetonitrile

CDCl₃: Deuterated chloroform

CD₃OD: Deuterated methanol

D₂O: Deuterium oxide

DCC: N,N'-Dicyclohexylcarbodiimide

DCM: Dichloromethane

DIEA: N,N-Diisopropylethylamine

DMAP: 4-Dimethylaminopyridine

DMF: N,N-Dimethylformamide

DMSO: Dimethyl sulfoxide

DMSO-d6: Deuterated dimethyl sulfoxide

EDCI: l-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide EEDQ: 1 -Ethoxycarbonyl-2-ethoxy- 1 ,2-dihydroquinoline ESI: Electrospray ionization EtOAc: Ethyl acetate

Et₂O: Diethyl ether g: gram(s) h: hour(s)

HATU: l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridiniuni 3-oxid hexafluorophosphate Hep: n-Heptane

HPLC: High Performance Liquid Chromatography

IPA: 2-Propanol

LC-MS: Liquid Chromatography-Mass spectrometry

MeCN: Acetonitrile min: minute(s)

NaHCO₃: sodium bicarbonate

ODS: octadecylsilyl prep HPLC: Preparative high-performance liquid chromatographyrt: room temperature

TEA: Triethylamine

TFA: Trifluoroacetic acid

THF: Tetrahydrofuran

TLC: Thin layer chromatography

Example 7: Spacer Studies for Linker-payload Conjugates

Analysis of Maleimide-Conjugated Eribulin Linkers for Hydrolysis Conditions

[00863] The effect of linker composition on hydrolysis rate and pH dependence of maleimide ring opening (hydrolysis) post-conjugation of eribulin RESPECT-L ADCs was analyzed. A summary of the linker-payload conjugate structures included in the study is presented in Table 24.

Table 24. Linker-payload conjugates for study

[00864] Linker-payload conjugates were subjected to hydrolysis conditions at pH 7.4, pH 8.5, pH 9.2, and pH 10.0, with samples extracted at 0, 1 , 4, and 24 hours for calculation of percent hydrolysis (light chain intact mass by LC-MS). The results of the study in FIG. 30 show that linker-payload conjugates that included a C₂ spacer unit in the linker had a much higher rate of hydrolysis and pH sensitivity when compared to the other spacer units. The C2 spacer demonstrated: (i) complete hydrolysis after 24 hours at pH 9.2 (as well as two other (PEG)₂-containing linker compounds), (ii) over 90% hydrolysis in all samples after 24 hours at pH 8.5 (with no other sample reaching above 40%), and hydrolysis ranging from 40-90% after 24 hours at pH 7.4 (with no other sample reaching above 20%).

C2, C₂(PEG)„, and (PEG)i Spacer Comparison

Analysis of Hydrolysis in buffer

[00865] With the surprising observation that the C₂ moiety increased the hydrolysis of the maleimide ring, it was hypothesized that the addition of a C₂ moiety between the maleimide ring and the (PEG)_n may promote rapid hydrolysis of the maleimide ring. The effect of C₂, C₂(PEG)_n (where n = 1, 2, or 3), and (PEG)₂ spacer units on hydrolysis rate and pH dependence of maleimide ring opening (hydrolysis) post-conjugation of eribulin DAR 2 RESPECT-L Cys 80 or DAR 4 Cys80/Al 18C ADCs was evaluated. Each spacer unit was tested with: (i) anti-TROP2 antibody 16K21-H1L1, (ii) a maleimide conjugation moiety, (iii) each of aspartic acid (D), asparagine (N), and Val-Cit-pABC cleavable moieties, and (iv) eribulin payload (FIG. 19). ADC compounds were subjected to hydrolysis conditions at pH 7.4, pH 8.5, and pH 9.2, with samples extracted at 0, 1, 4, and 24 hours for calculation of percent hydrolysis (light chain intact mass by LC-MS). The results in FIG. 31 show that C₂(PEG)_n spacers facilitated: (i) 25-40% hydrolysis after 4 hours and 75-100% hydrolysis after 24 hours at pH 7.4; (ii) 35-60% hydrolysis after 1 hour and 90-100% hydrolysis after 4 hours at pH 8.5; and (iii) 80-100% hydrolysis after 1 hour at pH 9.2. The C₂ spacer facilitated: (i) 15-60% hydrolysis after 4 hours and 75-100% hydrolysis after 24 hours at pH 7.4; (ii) 25-75% hydrolysis after 1 hour and 75-100% hydrolysis after 4 hours at pH 8.5; and (iii) 75-100% hydrolysis after 1 hour at pH 9.2. (PEG)₂ spacers facilitated: (i) less than 25% hydrolysis after 4 hours and less than 40% hydrolysis after 24 hours at pH 7.4; (ii) less than 25% hydrolysis after 1 hour and less than 50% hydrolysis after 4 hours at pH 8.5; and (iii) less than 25% hydrolysis after 1 hour and less than 75% hydrolysis when conjugated to Cys 80 after 4 hours at pH 9.2.

[00866] A second set of experiments were performed to test each spacer unit with: (i) anti-TROP2 antibody 16K21 -H6L6, (ii) a maleimide conjugation moiety, (iii) each of asparagine (N) and Val-Cit- pABC (VCP) cleavable moieties, and (iv) eribulin payload (FIG. 19). The results in FIG. 32 show that C₂(PEG)_n spacers facilitated: (i) >40% hydrolysis after 4 hours and 100% hydrolysis after 24 hours at pH 7.4; (ii) >75% hydrolysis after 1 hour and 100% hydrolysis after 4 hours at pH 8.5; and (iii) 100% hydrolysis after 1 hour at pH 9.2. The C₂ spacer facilitated: (i) 19-46% hydrolysis after 4 hours and 95-100% hydrolysis after 24 hours at pH 7.4; (ii) 80-94% hydrolysis after 1 hour and 100% hydrolysis after 4 hours at pH 8.5; and (iii) 100% hydrolysis after 1 hour at pH 9.2. (PEG)₂ spacers facilitated: (i) less than 10% hydrolysis after 4 hours and less than 30% hydrolysis after 24 hours at pH 7.4; (ii) less than 15% hydrolysis after 1 hour and less than 45% hydrolysis after 4 hours at pH 8.5; and (iii) less than 25% hydrolysis after 1 hour and less than 85% hydrolysis when conjugated to Cys 80 after 4 hours at pH 9.2. The hydrolysis rate was more rapid when conjugated to Al 18C, and hydrolysis reached 100% by 4 hours at pH 9.2.

Analysis of Hydrolysis in Plasma

[00867] The rapid hydrolysis of the C₂ and C₂(PEG)_n linkers in pH 7.4 buffer suggested that the maleimide ring could rapidly hydrolyze in plasma. The ADCs tested in the previous section were analyzed for maleimide ring hydrolysis in mouse plasma at 0, 1, 4, 24, 72, 168, and 240 hours. FIG. 31 shows that by 4 h, 39-52% hydrolysis has occurred in the C₂(PEG)_n linkers and > 95% hydrolysis has occurred by 24 h in the DAR 2 ADCs. In contrast, the (PEG)₂ linkers demonstrated 10-12% hydrolysis by 4 h and 34-42% hydrolysis by 24 h. As with the pH 7.2 buffered DAR 4 ADCs, the DAR 4-conjugated linkers showed more rapid hydrolysis with the C₂(PEG)₂-VCP linker, being 100% hydrolyzed at the Al 18C site and 81% hydrolyzed at the C80 site by 4 h. Both sites were 100% hydrolyzed by 24 h. The rate of hydrolysis at these two sites was slower for the C₂(PEG)₂-N linker with 12% hydrolyzed at Al 18C, but 74% hydrolyzed at C80. Both sites were 100% hydrolyzed by 24 h.

Antibody-linker stability analysis

[00868] The rapid and complete hydrolysis of conjugated C₂ and C₂(PEG)_n maleimide linkers incubated in buffer and plasma suggests that these linkers may be more resistant to the instability via retro-Michael addition demonstrated in non-C₂ based payloads, as seen in FIG. 22 and throughout the literature. As shown in FIG. 33, when incubated in buffer at pH 7.2, there was little change in the DAR over time (92-100%) and no correlation between reduction in DAR and the C₂, C₂(PEG)_n, and (PEG)₂ groups. However, when incubated in mouse plasma, differences in the stability of the ADC DAR were seen. By the end of the study at 240 h, the DAR of the (PEG)₂ ADCs was reduced to 79- 88% of the original DAR. The C₂ linkers retained the DAR at 97% of the original DAR. The C₂(PEG)_n variants also retained a high percentage of the original DAR, ranging from 93-98%.

[00869] Together, these data demonstrate that the C₂ group increases the hydrolysis of the maleimide ring when conjugated to an antibody, resulting in a more stable ADC. in vitro Potency Analysis

[00870] The effect of C₂, C₂(PEG)_n, and (PEG)₂ spacer units on the potency of anti-TROP2-eribulin ADCs was evaluated using BxPC3, NCI-H2110, and NCI-N87 cell lines. Each spacer unit was tested with: (i) the anti-TROP2 16K21-H6L6-14AAS-A118C-C80 antibody, (ii) a maleimide conjugation moiety, (iii) each of asparagine (N) and Val-Cit-pAB cleavable moieties, and (iv) an eribulin payload. FIG. 33 shows that in all three TROP2-expressing cell lines, the IC₅₀ and max cytotoxicity of the C₂(PEG)₂-based ADCs differ little from the PEG₂-based ADCs when coupled with either the VCP or N cleavage mechanism. in vivo Potency Analysis

[00871] The effect of C₂, C₂(PEG)₂, and (PEG)₂ spacer units on in vivo potency for eribulin RESPECT-L ADCs in an NCI-H2110 NOD.CB17-SCID mouse model was evaluated. Each spacer unit was tested with: (i) anti-TROP2 antibody, (ii) a maleimide conjugation moiety, (iii) each of asparagine (N), and Val-Cit-pABC cleavable moieties, and (iv) eribulin payload. Samples were collected every 3-5 days for 70-80 days, and analyzed for tumor volume (mm³) (with PBS as a control). Results are shown in FIG. 35A (C₂(PEG)₂), FIG. 35B ((PEG)₂), and FIG. 35C (C₂).

[00872] The results show that treatment with ADCs comprising C₂(PEG)₂ spacer units results in tumor volumes of less than 500 mm³ up to 50 days post treatment, less than 1000 mm³ up to 60 days post treatment, and less than 1250 mm³ up to 75 days post treatment. Treatment with ADCs comprising C₂(PEG)₂ with VCP or N cleavable moieties resulted in tumor volumes of less than 500 mm³ up to 75 days post treatment. Treatment with ADCs comprising (PEG)₂ spacer units resulted in tumor volumes of less than 500 mm³ up to 30 days post treatment, less than 1250 mm³ up to 40 days post treatment, and less than 2000 mm³ up to 60 days post treatment. Treatment with ADCs comprising C₂ resulted in tumor volumes of less than 500 mm³ up to 40 days post treatment, less than 1250 mm³ up to 60 days post treatment, and less than 1500 mm³ up to 70 days post treatment.

Example 8: Synthesis of Mal-(PEG)₂-Val-Lys(Me)₂-pABC-Eribulin (Scheme 1)

8.1 Preparation ofFmoc-K(MMT)-pAB-OH [00873] To a stirred solution of Fmoc-K(MMT)-OH (100 mg, 0.156 mmol) in DMF (1 mL) was added (4-aminophenyl)methanol (28.8 mg, 0.234 mmol), Hünig’s base (82 μL, 0.468 mmol), and HATU (107 mg, 0.281 mmol). The mixture was stirred at rt for 18 h. The crude mixture was directly purified with Yamazen (SiO₂, EtOAc 100% to EtOAc-MeOH 90%) to give Fmoc-K(MMT)-pAB-OH (87 mg).

[00874] ESI-MS (m/z): 746.35 [M+H]⁺.

8.2 Preparation ofFmoc-VK-pABC-Eribulin

[00875] To a stirred solution of Fmoc-K(MMT)-pAB-OH (87 mg, 0.117 mmol) in DMF (2 mL) was added Hünig’s base (41 μL, 0.233 mmol), DMAP (1.4 mg, 0.012 mmol), and 4-Nitrophenyl chloroformate (28.2 mg, 0.140 mmol). The mixture was stirred at rt for 1 hours. Then the mixture was quenched with a saturated aqueous solution of NaHCO₃ (500 μL). The mixture was extracted with ethylacetate (1 mL), 3 times. The combined organic layer was dried with Na₂SO₄. The mixture was filtered through a celite pad. The resulting filtrate was concentrated in vacuo to give intermediate A as a crude material (124 mg).

[00876] To a stirred solution of intermediate A (124 mg, 0.076 mmol) in DMF (5 mL) was added eribulin mesylate (112 mg, 0.136 mmol) and Hünig’s base (71 μL, 0.408 mmol). The reaction mixture was stirred at rt for 1 hour. Then the mixture was purified by ODS (30 to 50% MeCNH₂ O, containing 0.1% HCO₂H) to give coupling compound. To a stirred solution of coupling compound in DMF (5 mL) was added diethylamine (100 uL). The reaction mixture was stirred at rt for 2 hours. Then the mixture was concentrated in vacuo to give intermediate B as a crude material (97 mg).

[00877] To a stirred solution of intermediate B (97 mg, 0.076 mmol) in DMF (1 mL) was added Fmoc-V-OH (38.6 mg, 0.114 mmol), Hünig’s base (66 μL, 0.379 mmol) and HATU (57.6 mg, 0.152 mmol). The reaction mixture was stirred at rt for 18 hours. Then the mixture was quenched with water (500 μL). The mixture was extracted with ethylacetate (500 μL), 3 times. Combined organic layer was washed with brine, and concentrated in vacuo to give the residue. To a stirred solution of resulting residue in DCM (1 mL) was added dichloroacetic acid (50 μL). The reaction mixture was diluted by DMF (500 μL). Then DCM was concentrated in vacuo. The mixture in DMF was directly purified by ODS (10 to 70% MeCN/H₂O, containing 0.1% HCO₂H) to give Fmoc-VK-pABC-Eribulin (45 mg).

[00878] ESI-MS (m/z): 1328.51 [M+H]⁺. 8.3 Preparation of Fmoc-VK(Me)₂-pABC-Eribulin

[00879] To a stirred solution of Fmoc-VK-pABC-Eribulin (45 mg, 0.034 mmol) in DCM (1 mL) was added an aqueous solution of paraformaldehyde (200 uL) and NaHB(OAc)₃ (71.8 mg, 0.339 mmol). The reaction mixture was stirred at rt for 18 hours. Then the mixture was diluted with ethyl acetate. The mixture was filtered through a celite pad. The filtrate was washed with an aqueous solution of NaHCO₃ and brine. The organic layer was concentrated in vacuo. The residue was purified by ODS (30 to 50% MeCN/H₂O, containing 0.1% HCO₂H) to give Fmoc-VK(Me)₂-pABC-Eribulin (31 mg). [00880] ESI-MS (m/z): 1356.58 [M+H]⁺.

8.4 Preparation ofMalfPEGf-VK(Me)₂-pABC-Eribulin

[00881] To a stirred solution of Fmoc-VK(Me)₂-pABC-Eribulin in DMF (1 mL) was added diethylamine (100 μL). The mixture was stirred at rt for 2 hours. Then the mixture was concentrated in vacuo to give amine compound. To a stirred solution of amine compound in DMF (1 mL) was added 2,5-dioxopyrrolidine-l-yl 3-(2-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)ethoxy)ethoxy)propanoate (8.1 mg, 0.023 mmol) and Hünig’s base (10 μL, 0.057 mmol). The reaction mixture was stirred at rt for 2 hours. The reaction mixture was directly purified by ODS (10 to 50% MeCN/H₂O, containing 0.1% HCO₂H) to give Mal-(PEG)₂-VK(Me)₂-pABC-Eribulin with low purity. Title compound with low purity was re-purified by ODS (10 to 50% MeCN/H₂O, containing 0.1% HCO₂H) to give Mal-(PEG)₂-VK(Me)₂-pABC-Eribulin as a colorless solid (20.5 mg).

[00882] ESI-MS (m/z): 1373.56 [M+H]⁺.

Example 9: Synthesis of Mal-(PEG)₂-Ala-Lys(Me)₂-pABC-Eribulin (Scheme 2)

9.1 Preparation of Fmoc-K(Me)₂ -pAB-OH

[00883] To a solution of Fmoc-Lys(Me)₂-OH HC1 (565 mg, 1.305 mmol) and 4-aminobenzyl alcohol (321 mg, 2.61 mmol) in MeOH (1.8 mL) at 0 °C (ice bath) was added EEDQ (645 mg, 2.61 mmol) at rt. The mixture was warmed to rt and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was directly purified with SiO₂ (0 to 15% MeCN/AcOEt) to give Fmoc-K(Me)₂-pAB-OH (418 mg).

9.2 Preparation of Fmoc-AK(Me)₂-p AB-OH [00884] To a solution of Fmoc-K(Me)₂-pAB-OH (418 mg, 0.83 mmol) in DMF (6 ml) at rt was added piperidine (355 mg, 4.2 mmol). The reaction mixture was stirred at rt for 2 hours. The reaction mixture was concentrated under reduced pressure to give a cure residue.

[00885] To the residue were added DMF (6 ml), DIEA (0.363 ml, 2.1 mmol) and Fmoc-Ala-OH (311 mg, 1.00 mmol). HATU (634 mg, 1.7 mmol) was added to the mixture at rt. The reaction mixture was immediately cooled to 0 °C (ice bath). The reaction mixture was stirred at 0 °C for 30 minutes. The reaction mixture was concentrated under reduced pressure. The residue was purified with SiO₂ column (2 to 15% MeCN/AcOEt) to give Fmoc-AK(Me)₂-pAB-OH (320 mg).

9.3 Preparation ofMal-(PEG)2-AK(Me)₂-pAB-OH

[00886] To a solution of Fmoc-AK(Me)₂-pAB-OH (80 mg, 0.14 mmol) in DMF (1.5 ml, 0.14 mmol) at rt was added piperidine (0.069 ml, 0.70 mmol). The reaction mixture was stirred at rt for 30 minutes, after which the mixture was concentrated in vacuo to give a crude residue.

[00887] ESI-MS (m/z): 1136.85 [M+H]⁺.

[00888] To a solution of the crude residue in DMF (2.0 mL) at rt was added DIEA (0.036 ml, 0.21 mmol) at rt. To the solution above at rt was added Mal-(PEG)₂-NHS ester (74.2 mg, 0.21 mmol). The mixture was stirred for 30 min. The crude mixture was directly purified with ODS column (5 to 10% MeCN/EfeO, containing 0.1% HCO₂H) to give Mal-(PEG)₂-AK(Me)₂-pAB-OH (55 mg).

[00889] ESI-MS (m/z): 1518.02 [M+H]⁺. 9.4 Preparation ofMal-(PEG)2-AK(Me)₂-pABC-Eribulin

[00890] To the Mal-(PEG)₂-AK(Me)₂-pAB-OH (18.0 mg, 0.028 mmol) in DMF (0.5 mL) was added DIEA (12 mg, 0.091 mmol) and bis(4-nitrophenyl) carbonate (8.8 mg, 0.029 mmol) at 0 °C (ice bath). The reaction mixture was allowed to warm to rt and stirred for 3.5 hours. Bis(4-nitrophenyl) carbonate (8.8 mg, 0.029 mmol) was added to the reaction mixture at rt and stirred at rt for 2 hours. To the reaction mixture was added eribulin mesylate (15 mg, 0.018 mmol) in DMF (500 μl). The reaction mixture was stirred at rt. The reaction mixture was directly purified with ODS column (5 to 50% MeCN/H₂O, containing 0.1% HCO₂H) to give Mal-(PEG)₂-AK(Me)₂-pABC-Eribulin as a low purity fraction. This compound was re-purified via ODS column (5 to 30% MeCN/H₂O, containing 0.1% HCO₂H) to give Mal-(PEG)₂-AK(Me)₂-pABC-Eribulin as colorless oil (3.4 mg).

[00891] ESI-MS (m/z): 1346.03 [M+H]⁺.

Example 10: Synthesis of Mal-(PEG)₂-Val-Ala-pABC-Eribulin (Scheme 2)

[00892] Mal-(PEG)₂-Val-Ala-pABC-Eribulin was synthesized using steps similar to steps 93-9.4 of

Example 9, with Fmoc-Val-Ala-pABC-PNP replacing Fmoc-AK(Me)₂-pAB-OH in step 93.

Example 11: Synthesis of Mal-(PEG)₂-Val-Cit-pABC-Eribulin (Scheme 2)

[00893] Mal-(PEG)₂-Val-Cit-pABC-Eribulin was synthesized using steps similar to steps 93-9.4 of

Example 9, with Fmoc-Val-Cit-pABC-PNP replacing Fmoc-AK(Me)₂-pAB-OH in step 9.3. Example 12: Synthesis of Mal-C₂-Val-Cit-pABC-Eribulin

12.1 Preparation ofFmoc-VCP-Eribulin

[00894] To a mixture of eribulin mesylate (100 mg, 0.121 mmol) and DIEA (0.063 mL, 0.363 mmol) in DMF (1 mL) was added (9H-fluoren-9-yl)methyl ((S)-3-methyl-l-(((S)-l-((4-((((4- nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-l-oxobutan-2- yl)carbamate (93 mg, 0.121 mmol). The mixture was stirred at rt for 1 h. The reaction mixture was directly purified by ODS column (5 to 70% MeCN/H₂O, containing 0.1% HCO₂H) to afford the title compound (153 mg).

[00895] ESI-MS (m/z): 1358.0 [M+H]⁺

12.2 Preparation of Mal-C₂-VCP-Eribulin

[00896] Piperidine (14.6 μL, 0.147 mmol) was added to a solution of Fmoc-VCP-Eribulin (10 mg, 7.366 μmol) in DMF (300 μL) at rt. After being stirred for 1 h at rt, the reaction mixture was concentrated and azeotroped with toluene (1 mLx2). The residue was dissolved in DMF (300 μL). DIEA (3.86 μL, 0.022 mmol) and N-Succinimidyl Maleimidoacetate (1.9 mg, 7.366 μmol) were added to the mixture. After being stirred for 1 h at rt, the reaction mixture was directly purified with ODS column (5 to 25% MeCN/H₂O, containing 0.1% HCO₂H) to afford the title compound (4.59 mg).

[00897] ESI-MS (m/z): 1272.86 [M+H]⁺

Example 13: Synthesis of Mal-C₂-Val- Ala-p ABC-Eribulin

13.1 Preparation ofFmoc-Val-Ala-pABC-Eribulin

[00898] To a mixture of eribulin mesylate (55 mg, 0.067 mmol) and Fmoc-Val-Ala-pABC-PNP (54 mg, 0.08 mmol) in DMF (1000 μL) was added DIEA (0.035 mL, 0.20 mmol) at rt. The mixture was stirred at rt for 2 h. The reaction mixture was directly purified by ODS column (5 to 80% MeCN/H₂O, containing 0.1% HCO₂H) to afford Fmoc-Val-Ala-pABC-Eribulin (85 mg).

[00899] ESI-MS (m/z): 1290.8 [M+H₂O]⁺

13.2 Preparation ofNH₂-Val-Ala-pABC-Eribulin

[00900] Piperidine (66 μL, 0.67 mmol) was added to a solution of Fmoc-Val-Ala-pABC-Eribulin (85 mg, 0.067 mmol) in DMF (1.0 mL) at rt. After being stirred at rt for 1 h, the reaction mixture was concentrated under reduced pressure to give a title compound as a crude (70 mg).

[00901] ESI-MS (m/z): 1049.8 [M+H]⁺ 13.3 Preparation of Mal-C₂-Val-Ala-pABC-Eribulin

[00902] To a solution of crude NH₂-Val-Ala-pABC-Eribulin (35 mg, 0.33 mmol) and N-Succinimidyl Maleimidoacetate (10.1 mg, 0.04 mmol) in DMF (700 μL) at rt was added DIEA (18 μL, 0.10 mmol). The reaction mixture was stirred at rt for 90 min.

[00903] Flash chromatography of the reaction mixture on ODS silica gel (Cl 8, YAMAZEN UNIVERSAL Column ODS-SM, L size) using 5% to 80% MeCN/Water (containing 0.1% HCO₂H) gave the title compound (18 mg).

[00904] ESI-MS (m/z): 1187.77 [M+H]⁺

[00905] ¹H NMR (500 MHz, CDCl₃) δ ppm: 0.95 - 1.02 (m, 6 H) 1.06 - 1.15 (m, 4 H) 1.24 - 1.53 (m, 8 H) 1.67 - 1.80 (m, 4 H) 1.89 - 2.01 (m, 4 H) 2.07 - 2.14 (m, 1 H) 2.14 - 2.38 (m, 8 H) 2.41 - 2.56 (m, 3 H) 2.72 (dd, 7=16.20, 10.09 Hz, 1 H) 2.84 - 2.93 (m, 2 H) 3.18 (dt, 7=13.14, 6.27 Hz, 1 H) 3.28 (d, 7=3.06 Hz, 1 H) 3.34 - 3.41 (m, 1 H) 3.43 (s, 3 H) 3.57 (s, 1 H) 3.63 (br d, 7=11.00 Hz, 2 H) 3.82 (br dd, 7=9.48, 6.42 Hz, 1 H) 3.82 (br d, 7=9.17 Hz, 1 H) 3.89 - 4.00 (m, 3 H) 4.04 (dd, 7=6.11, 4.28 Hz, 1 H) 4.08 - 4.16 (m, 2 H) 4.20 (dd, 7=6.42, 4.58 Hz, 1 H) 4.27 - 4.41 (m, 5 H) 4.62 (t, 7=4.28 Hz, 1 H) 4.66 - 4.73 (m, 2 H) 4.82 (s, 1 H) 4.89 (s, 1 H) 4.94 (br s, 1 H) 5.00 - 5.11 (m, 3 H) 5.25 - 5.33 (m, 1 H) 6.36 - 6.45 (m, 1 H) 6.64 (s, 2 H) 6.67 - 6.73 (m, 1 H) 7.25 (br d, 7=8.56 Hz, 2 H) 7.53 (br d, 7=8.56 Hz, 2 H) 8.20 - 8.29 (m, 1 H).

Example 14: Synthesis of Mal-C₂-PEG₂-AK(Me)₂-pABC-Eribulin

14.1 Preparation ofFmoc-PEG₁-AK(Me)₂ -pAB-OH

[00906] To a stirred solution of Fmoc-AK(Me)₂-pAB-OH (155 mg, synthesized using steps similar to step 9.2 of Example 9), DMF (3 mL) was added piperidine (134 μL). The mixture was stirred at rt for 1 h, then the mixture was concentrated in vacuo to give amine compound. To a stirred solution of amine compound in DMF (3 mL) was added l-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan- 13-oic acid (162 mg, 0.406 mmol), HATU (206 mg, 0.541 mmol) and DIEA (236 μL, 1.353 mmol). The reaction mixture was stirred at rt for 3 h. The reaction mixture was directly purified by ODS- column (0 to 90% MeCN/H₂O, containing 0.1% HCO₂H) to give the title compound as colorless oil (183 mg).

[00907] ESI-MS (m/z): 732.12 [M+H]⁺.

Example 14.2 Preparation of Mal-C2-PEG₂-AK(Me)₂-pAB-OH

[00908] To a stirred solution of Fmoc-AK(Me)₂-pAB-OH (183 mg) in DMF (3 mL) was added diethylamine (492 μL). The mixture was stirred at rt for 1 h. The mixture was stirred at rt for 1 h, then the mixture was concentrated in vacuo to give an amine compound. To a stirred solution of amine compound in DMF (3 mL) was added N-Succinimidyl Maleimidoacetate (71.2 mg, 0.282 mmol) and DIEA (164 μL, 0.941 mmol). The reaction mixture was stirred at rt for Ih, the reaction mixture was directly purified by ODS-column (5 to 90% MeCN/H₂O, containing 0.1% HCO₂H) to give the title compound as colorless oil (70 mg).

[00909] ESI-MS (m/z): 647.62 [M+H]⁺. Example 14.3 Preparation of Mal-C2-PEG₂-AK(Me)₂-pABC-Eribulin

[00910] To a solution of Mal-C₂-PEG₂-AK(Me)₂-pAB-OH (35.2 mg) and DIEA (0.044 mL, 0.254 mmol) in DMF (1 mL) at 0 deg was added bis(4-nitrophenyl) carbonate (24.75 mg, 0.081 mmol). The reaction mixture was allowed to warm to rt for 3 h. Then, a solution of eribulin mesylate (42 mg, 0.051 mmol) in DMF (1 mL) was added to the mixture at rt. The reaction mixture was stirred at rt for 3 h. The reaction mixture was directly purified by ODS-column (5 to 50% MeCN/H₂O, containing 0.1% HCO₂H) to give title compound with low purity as colorless oil.

[00911] Title compound with low purity obtained above was purified by ODS-column (0 to 60% MeCN/H₂O, containing 0.1% HCO₂H) to give title compound as white solid (19.63 mg).

[00912] ES1-MS (m/z): 1402.54 [M+H]⁺.

Example 15: Synthesis of Mal-C₂-PEG₂-VCP-Eribulin

[00913] To a solution of Fmoc-VCP-Eribulin (50 mg, 0.037 mmol, synthesized using steps similar to step 12.1 of Example 12) in DMF (1.0 mL) was added diethylamine (0.038 mL, 0.37 mmol) at rt. After being stirred for 1 h at rt, the reaction mixture was concentrated.

[00914] ESI-MS (m/z): 1135.91 [M+H]⁺.

[00915] To a solution of the NH₂-VCP-Eribulin (crude residue) in DMF (1.5 mL) at rt was added 3- {2-[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanoic acid (22 mg, 0.055 mmol), HATU (24 mg, 0.063 mmol) and DIEA (24 mg, 0.18 mmol) atrt. The mixture was stirred atrt for 38 h. The reaction mixture was directly purified with ODS column (5 to 80% MeCN/H₂O, containing 0.1% HCO₂H) to give Fmoc-PEG₂-VCP-Eribulin (47 mg).

[00916] ESI-MS (m/z): 1518.02 [M+H]⁺.

[00917] To a solution of Fmoc-PEG₂-VCP-Eribulin (47 mg, 0.031 mmol) in DMF (1.5 mL) at rt was added diethylamine (32 μL, 0.31 mmol). After being stirred at rt for 1 h, the mixture was concentrated in vacuo to give NH₂-PEG₂-VCP-Eribulin as a crude residue.

[00918] ESI-MS (m/z): 1295.00 [M+H]⁺.

[00919] To the NH₂-PEG₂-VCP-Eribulin (crude residue) was added DMF (1.5 mL), DIEA (27 μL, 0.15 mmol) and N-Succinimidyl Maleimidoacetate (7.0 mg, 0.028 mmol) and stirred at rt for 1 h. The reaction mixture was directly purified with ODS column (3 to 80% MeCN/H₂O, containing 0.1% HCO₂H) to afford the title compound (30 mg).

[00920] ESI-MS (m/z): 1432.00 [M+H]⁺.

[00921] ¹H NMR (500 MHz, CDCl₃) δ(ppm): 0.95 (br t, 7=7.03 Hz, 6 H) 1.04 - 1.15 (m, 4 H) 1.26 -

1.63 (m, 9 H) 1.66 - 1.79 (m, 5 H) 1.89 - 2.00 (m, 5 H) 2.03 - 2.11 (m, 2 H) 2.05 - 2.36 (m, 7 H) 2.38 -

2.64 (m, 5 H) 2.71 (br dd, 7=15.89, 9.78 Hz, 1 H) 2.87 (br d, 7=8.56 Hz, 2 H) 3.07 - 3.24 (m, 3 H) 3.24 - 3.37 (m, 3 H) 3.42 (s, 3H) 3.44 - 3.54 (m, 3 H) 3.55 - 3.64 (m, 6 H) 3.70 (dt, 7=9.78, 4.89 Hz, 1 H) 3.75 - 3.87 (m, 2 H) 3.87 - 3.99 (m, 3 H) 4.04 (dd, 7=6.11,4.89 Hz, 1 H) 4.10 - 4.15 (m, 1 H) 4.18 - 4.22 (m, 1 H) 4.24 - 4.39 (m, 5 H) 4.44 (br t, 7=7.34 Hz, 1 H) 4.61 (t, 7=4.58 Hz, 1 H) 4.63 - 4.68 (m, 1 H) 4.70 (t, 7=4.28 Hz, 1 H) 4.81 (s, 1 H) 4.88 (s, 1 H) 4.94 (br s, 1 H) 4.98-5.15 (m, 4 H) 5.47 (br s, 1 H) 5.57 (br s, 1 H) 6.75 (s, 2 H) 7.25 (br d, 7=8.56 Hz, 2 H) 7.31 - 7.40 (m, 1 H) 7.53 (br d, 7=8.56 Hz, 2 H) 7.58 - 7.62 (m, 1 H) 7.94 - 7.94 (m, 1 H) 8.07 (br s, 1 H) 9.06 (br s, 1 H). Example 16: Synthesis of Mal-C₂-PEG₁-VCP-Eribulin

16.1 Preparation ofFmoc-PEG₁-VCP-Eribulin

[00922] To a solution of Fmoc-VCP-Eribulin (15.8 mg, 0.012 mmol, synthesized using steps similar to step 12.1 of Example 12) in DMF (400 μL) at rt was added piperidine (5.8 μL, 0.058 mmol). The reaction mixture was stirred at rt. After 105 min, all volatiles were removed azeotropically with toluene (three times). To the resulting residue were added DMF (400 μL), 3-[2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)ethoxy]propanoic acid (6.2 mg, 0.017 mmol), DIEA (6.1 μL, 0.035 mmol) and HATU (9.2 mg, 0.024 mmol) at rt. The mixture was stirred at rt. After 55 min, the reaction mixture was directly purified via ODS-column (10 to 80% MeCN/H₂O, containing 0.1% HCO₂H) to give the title compound (13.6 mg) as a white solid.

[00923] ESI-MS (m/z): 1473.0 [M+H]⁺.

16.2 Preparation ofMal-C₂-PEG₁-VCP-Eribulin

[00924] To a solution of Fmoc-PEG₁-VCP-Eribulin (13.6 mg, 9.235 μmol) in DMF (1000 μL) at rt was added piperidine (4.56 μL, 0.046 mmol). The reaction mixture was stirred at rt. After 92 min, all volatiles were removed azeotropically with toluene (three times). To the resulting residue were added DMF (500 μL), DIEA (4.83 μL, 0.028 mmol) and N-succinimidyl maleimidoacetate (2.8 mg, 0.011 mmol) at rt. The reaction mixture was stirred at rt. After 45 min, the reaction mixture was quenched by addition of formic acid (1.05 μL, 0.028 mmol). The mixture was directly purified with ODS- column (10 to 70% MeCN/H₂O) to give the title compound (12.09 mg) as a white solid.

[00925] ESI-MS (m/z): 1388.0 [M+H]⁺.

[00926] ¹H NMR (500 MHz, CDCl₃) δ(ppm): 0.90 - 1.17 (m, 9 H), 1.23 - 2.37 (m, 49 H), 2.40 - 2.66 (m, 6 H), 2.68 - 2.76 (m, 1 H), 2.84 - 2.93 (m, 2 H), 3.12 - 3.39 (m, 6 H), 3.42 (s, 3 H), 3.46 - 3.85 (m, 8 H), 3.87 - 4.00 (m, 3 H), 4.04 (dd, J=6.30, 4.58 Hz, 1 H), 4.10 - 4.16 (m, 1 H), 4.16 - 4.26 (m, 1 H), 4.27 - 4.41 (m, 4 H), 4.61 (t, .7=4.58 Hz, 1 H), 4.65 - 4.75 (m, 2 H), 4.81 (br s, 1 H), 4.89 (br s, 1 H), 4.94 (br s, 1 H), 4.96 - 5.06 (m, 2 H), 5.08 (br s, 1 H), 5.34 - 5.68 (m, 2 H), 6.68 (br s, 1 H), 7.16 -

7.24 (m, 2 H), 7.52 (br d, .7=7.45 Hz, 2 H), 7.73 (br s, 1 H), 8.91 - 9.20 (m, 1 H).

Example 17: Synthesis of Mal-PEG₂-N-Eribulin

[00927] To a solution of Fmoc-N-Eribulin (33 mg, 0.031 mmol, synthesized using steps similar to step 19.1 of Example 19) in DMF (479 μL) was added diethylamine (128 μL, 1.24 mmol). After being stirred for 20 min, the mixture was concentrated. The residue was azeotropically dried with toluene (three times). The residue was dissolved in DMF (479 μL) and was added DIEA (27.0 μL, 0.155 mmol) and 2,5-dioxopyrrolidin-l-yl 3-{2-[2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)ethoxy] ethoxy }propanoate (16.45 mg, 0.046 mmol). The mixture was stirred for 2 h, then purified with ODS-column (MeCN/H₂ O, containing 0.1% HCO₂H) to give the title compound (19.48 mg).

[00928] ESI-MS (m/z): 1083.79 [M+H]⁺.

[00929] ¹H-NMR (500 MHz, METHANOL-d₄)) δ(ppm): 1.02 (q, J=12.23 Hz, 1 H), 1.11 (d, J=6.72 Hz, 3 H), 1.27 - 1.61 (m, 6 H), 1.68 - 1.91 (m, 6 H), 1.95 - 2.03 (m, 3 H), 2.05 - 2.25 (m, 4 H), 2.30 - 2.52 (m, 7 H), 2.65 - 2.77 (m, 4 H), 2.84 - 2.95 (m, 2 H), 3.24 - 3.29 (m, 2 H), 3.32 - 3.34 (m, 1 H), 3.35 (s, 1 H), 3.41 (s, 3 H), 3.55 - 3.65 (m, 6 H), 3.67 - 3.80 (m, 6 H), 3.81 - 3.89 (m, 3 H), 3.98 (br t, J=10.39 Hz, 1 H), 4.07 - 4.14 (m, 2 H), 4.18 (dd, J=6.42, 4.58 Hz, 1 H), 4.27 - 4.34 (m, 2 H), 4.48 (br d, J=11.00 Hz, 1 H), 4.61 (t, J=4.28 Hz, 1 H), 4.69 - 4.73 (m, 2 H), 4.82 - 4.84 (m, 1 H), 4.88 - 4.90 (m, 1 H), 5.04 (br s, 1 H), 5.14 (br s, 1 H), 6.83 (s, 2 H), 7.79 (t, J=5.81 Hz, 1 H), 8.06 - 8.26 (m, 2 H).

Example 18: Synthesis of Mal-C₂-N-Eribulin

[00930] To a solution of Fmoc-N-Eribulin (15 mg, 0.014 mmol, synthesized using steps similar to step 19.1 of Example 19) in DMF (0.20 mL) was added piperidine (12 mg, 0.14 mmol) at rt. After being stirred at rt for 10 min, the mixture was concentrated in vacuo. To the residue was added DMF (0.20 mL), DIEA (5.5 mg, 0.042 mmol), and N-Succinimidyl Maleimidoacetate (4.3 mg, 0.017 mmol) and stirred at rt for 1 h. The crude mixture was directly purified with ODS-column (10 to 50% MeCN/H₂O, containing 0.1% HCO₂H) to afford the title compound (10 mg).

[00931] ESI-MS (m/z): 981.63 [M+H]⁺.

[00932] 1H-NMR Spectrum (500 MHz, CDCl₃) δ(ppm): 1.06-1.14 (4H, m), 1.22-1.81 (*81H, m), 1.86-2.06 (5H, m), 2.07-2.38 (9H, m), 2.41-2.59 (4H, m), 2.70-2.77 (1H, m), 2.85-2.92 (2H, m), 2.97- 3.02 (1H, m), 3.19-3.26 (1H, m), 3.27-3.30 (1H, m), 3.43 (3H, s), 3.47-3.52 (1H, m), 3.62-3.67 (2H, m), 3.80-3.99 (4H, m), 4.03-4.06 (1H, m), 4.10-4.16 (1H, m), 4.18-4.39 (6H, m), 4.59-4.65 (1H, m), 4.68-4.73 (1H, m), 4.75-4.78 (1H, m), 4.80-4.85 (1H, m), 4.88-4.91 (1H, m), 4.94-5.00 (1H, m), 5.06- 5.14 (1H, m), 5.45-5.55 (1H, m), 5.79-5.88 (1H, m), 6.75-6.86 (2H, m), 7.18-7.22 (1H, m), 7.67-7.75 (1H, m). *overlapped withH₂O.

Example 19: Synthesis of Mal- C₂-PEG₁-N-Eribulin

19.1 Preparation ofFmoc-N-Eribulin

[00933] To a solution of eribulin mesylate (300 mg, 0.363 mmol) in DMF (5.0 mL) were added DIEA (190 μL, 1.09 mmol), Fmoc-Asn-OH (0.581 mmol) and HATU (207 mg, 0.545 mmol) at rt. The mixture was stirred at rt for 1 h. The reaction mixture was concentrated under reduced pressure and the residue was purified with ODS column (3 to 70% MeCN/H₂O, containing 0.1% HCO₂H) to give the title compound (353 mg).

[00934] ESI-MS (m/z): 1066.73 [M+H]⁺.

19.2 Preparation ofN-Eribulin

[00935] To a solution ofFmoc-N-Eribulin (353 mg, 0.331 mmol) in DMF (3.0 mL) was added piperidine (328 μL, 3.31 mmol) at rt. After being stirred at rt for 2 h, the mixture was concentrated in vacuo. The residue was directly purified with ODS column (2 to 50% MeCN/H₂O, containing 0.1% HCO₂H) to give the title compound (220 mg). [00936] ESI-MS (m/z): 844.63 [M+H]⁺.

19.3 Preparation ofFmoc-PEG₁-N-Eribulin

[00937] To a solution of 3-[2-({[(9H-fluoren-9yl)methoxy]carbonyl}amino)ethoxy]propanoic acid (12.5 mg, 0.035 mmol) and DIEA (14.7 μL, 0.084 mmol) in DMF (0.3 mL) was added HAITI (12.8 mg, 0.034 mmol) at 4 °C (ice bath). After being stirred for 10 min at rt, N-Eribulin (25 mg, 0.028 mmol) in DMF (0.3 mL) was added. The mixture was stirred at rt for 1 h. The crude mixture was directly purified with ODS column (5 to 80% MeCMH₂ O, containing 0.1% HCO₂H) to give the title compound (20.1 mg).

[00938] ESI-MS (m/z): 1181.70 [M+H]⁺.

19.4 Preparation ofNH2-PEG₁-N-Eribulin

[00939] To a solution of Fmoc-PEG₁-N-Eribulin (20 mg, 0.017 mmol) in DMF (0.30 mL) was added piperidine (16.8 μL, 0.17 mmol) at rt. After being stirred at rt for 30 min, the reaction mixture was concentrated in vacuo to give the title compound as a crude (16.3 mg).

[00940] ESI-MS (m/z): 959.72 [M+H]⁺

19.5 Preparation ofMal-C₂-PEG₁-N-Eribulin [00941] To the NH₂-PEG₁-N-Eribulin (16.3 mg) were added DMF (0.50 mL), DIEA (8.9 μL, 0.051 mmol), and N-succinimidyl maleimidoacetate (5.6 mg, 0.022 mmol) at rt and stirred at rt for 12 h. The reaction mixture was directly purified with ODS column (3 to 80% MeCN/H₂O, containing 0.1% HCO₂H) to afford the title compound (14.0 mg).

[00942] ESI-MS (m/z): 1096.71 [M+H]⁺.

[00943] ¹H-NMR Spectrum (500 MHz, CDCl₃) δ(ppm):1.04 - 1.15 (m, 4 H) 1.25 - 1.52 (m, 5 H) 1.53 - 1.64 (m, 2 H) 1.63 - 1.83 (m, 3 H) 1.85 - 2.03 (m, 5 H) 2.07 - 2.13 (m, 1 H) 2.13 - 2.38 (m, 7 H) 2.40

- 2.64 (m, 6 H) 2.73 (dd, J=16.32, 10.02 Hz, 1 H) 2.81 - 2.93 (m, 2 H) 3.00 (dd, J=16.04, 4.01 Hz, 1 H) 3.10 - 3.20 (m, 1 H) 3.28 (d, J=3.44 Hz, 1 H) 3.42 (s, 3 H) 3.43 - 3.60 (m, 5 H) 3.61 - 3.72 (m, 3 H) 3.73 - 3.80 (m, 1 H) 3.80 - 3.85 (m, 1 H) 3.89 - 4.00 (m, 3 H) 4.04 (dd, J=6.59, 4.30 Hz, 1 H) 4.09

- 4.17 (m, 1 H) 4.20 (dd, J=6.87, 4.58 Hz, 1 H) 4.22 - 4.40 (m, 5 H) 4.61 (t, J=4.30 Hz, 1 H) 4.70 (t, J=4.58 Hz, 1 H) 4.81 - 4.85 (m, 2 H) 4.89 (s, 1 H) 4.97 (d, J=1.72 Hz, 1 H) 5.08 (d, J=1.72 Hz, 1 H) 5.74 - 5.91 (m, 1 H) 6.00 - 6.18 (m, 1 H) 6.76 (s, 2 H) 7.24 (br s, 1 H) 7.66 - 7.78 (m, 1 H) 7.99 (d, J=8.02 Hz, 1 H).

Example 20: Synthesis of Mal-C₂-PEG₂-N-Eribulin

20.1 Preparation ofFmoc-PEG₂-N-Eribulin

[00944] To a solution of 3-{2-[2-({[(9HFluoren-9- yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanoic acid (18 mg, 0.044 mmol) and DIEA (19 μL, 0.11 mmol) in DMF (0.9 mL) was added HATU (16 mg, 0.043 mmol) at 4 °C (ice bath). After being stirred at rt for 10 min, N-Eribulin (30 mg, 0.034 mmol, synthesized using steps similar to step 19.2 of Example 19) was added at rt. The mixture was stirred at rt for 60 min. The crude mixture was directly purified with ODS column (5 to 80% MeCN/H₂O, containing 0.1% HCO₂H) to give the title compound (21 mg).

[00945] ESI-MS (m/z): 1225.63 [M+H]⁺. 20.2 Synthesis ofNH₂-PEG₂-N-Eribulin

[00946] To a solution of Fmoc-PEG₂-N-Eribulin (21 mg, 0.017 mmol) in DMF (0.60 mL) was added piperidine (15 mg, 0.17 mmol) at rt. After being stirred at rt for 30 min, the mixture was concentrated in vacuo and azeotroped with toluene (three times) to give the title compound as a crude product.

[00947] ESI-MS (m/z): 1004.14 [M+H]⁺.

20.3 Preparation ofMal-C₂-PEG₁-N-Eribulin

[00948] To NH₂-PEG₂-N-Eribulin (crude) was added DMF (0.6 mL), DIEA (15 μL, 0.087 mmol) and N-Succinimidyl Maleimidoacetate (5.7 mg, 0.023 mmol) at rt and stirred at rt for 8 h. The reaction mixture was directly purified with ODS-column (3 to 75% MeCN/H₂O, containing 0.1% HCO₂H) to afford the title compound (15 mg).

[00949] ESI-MS (m/z): 1140.57 [M+H]⁺.

[00950] ¹H-NMR Spectrum (500 MHz, CDCl₃) δ(ppm):1.04 - 1.16 (m, 4 H) 1.28 - 1.50 (m, 4 H) 1.54 - 1.64 (m, 2 H) 1.65 - 1.80 (m, 3 H) 1.86 - 2.01 (m, 4 H) 2.09 (dt, J=7.88, 3.79 Hz, 1 H) 2.14 - 2.35 (m, 8 H) 2.38 - 2.58 (m, 6 H) 2.58 - 2.65 (m, 1 H) 2.72 (dd, 7=16.04, 10.31 Hz, 1 H) 2.81 - 2.97 (m, 3 H) 3.21 (ddd, 7=13.60, 7.59, 5.73 Hz, 1 H) 3.27 (d, 7=2.86 Hz, 1 H) 3.42 (s, 3 H) 3.39 - 3.51 (m, 2 H) 3.54 - 3.68 (m, 8 H) 3.75 (ddd, 7=9.88, 6.16, 4.01 Hz, 1 H) 3.79 - 3.85 (m, 2 H) 3.86 - 3.93 (m, 2 H) 3.96 (br t, 7=10.60 Hz, 1 H) 4.04 (dd, 7=6.59, 4.30 Hz, 1 H) 4.08 - 4.16 (m, 1 H) 4.19 (dd, 7=6.30, 4.58 Hz, 1 H) 4.26 (s, 2 H) 4.27 - 4.39 (m, 3 H) 4.61 (t, 7=4.58 Hz, 1 H) 4.70 (t, 7=4.58 Hz, 1 H) 4.81 (d, 7=1.15 Hz, 1 H) 4.84 (dt, 7=8.16, 5.37 Hz, 1 H) 4.88 (s, 1 H) 4.93 - 4.99 (m, 1 H) 5.08 (d, 7=1.72 Hz, 1 H) 5.87 - 5.98 (m, 1 H) 6.18 - 6.30 (m, 1 H) 6.77 (s, 2 H) 7.21 - 7.26 (m, 1 H) 7.52 (br s, 1 H) 7.67 (br d, J=5.73 Hz, 1 H) 8.03 (br s, 1 H). Example 21: Synthesis of Mal-C₂-PEG₃-N-Eribulin

21.1 Preparation ofFmoc-PEG₃-N-Eribulin

[00951] To a solution of l-(9H-fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azahexadecan-16-oic acid (19 mg, 0.042 mmol) and DIEA (13 mg, 0.10 mmol) in DMF (400 μL) was added HATU (15 mg, 0.04 mmol) at 4 °C (ice bath). The reaction mixture was stirred at rt for 10 min. Then N-Eribulin (30 mg, 0.034 mmol, synthesized using steps similar to step 19.2 of Example 19) in DMF (400 μL) was added to the reaction mixture, then the reaction mixture was stirred at rt for 1 h. The reaction mixture was directly purified by ODS-column (5 to 90% MeCN/H₂O, containing 0.1% HCO₂H) to afford a title compound (30 mg).

[00952] ESI-MS (m/z): 1270.00 [M+H]⁺.

21.2 Preparation ofMal-C₂-PEG₃-N-Eribulin

[00953] To a solution of Fmoc-PEG₃-N-Eribulin (30 mg, 0.024 mmol) in DMF (0.55 mL) was added piperidine (20 mg, 0.24 mmol) at rt. After being stirred at rt for 10 min, the mixture was concentrated in vacuo. To the residue was added DMF (0.55 mL), DIEA (9.2 mg, 0.071 mmol), and N- Succinimidyl Maleimidoacetate (7.8 mg, 0.031 mmol) and stirred at rt for 1 h. The crude mixture was directly purified with ODS-column (5 to 70% MeCN/H₂O, containing 0.1% HCO₂H) to afford a title compound (19 mg).

[00954] ESI-MS (m/z): 1184.94 [M+H]⁺.

[00955] ¹H-NMR Spectrum (500 MHz, CDCl₃) δ(ppm): 1.05-1.15 (4H, m), 1.21-2.06 (*94H, m), 2.07-2.35 (10H, m), 2.39-2.63 (5H, m), 2.68-2.93 (5H, m), 3.17-3.26 (1H, m), 3.27-3.31 (1H, m), 3.38-3.52 (6H, m), 3.53-3.73 (12H, m), 3.74-3.78 (1H, m), 3.78-3.92 (4H, m), 3.94-4.00 (1H, m), 4.02-4.07 (1H, m), 4.10-4.16 (1H, m), 4.18-4.23 (1H, m), 4.22-4.27 (2H, m), 4.28-4.42 (3H. m), 4.59- 4.63 (1H, m), 4.68-4.72 (1H, m), 4.73-4.79 (1H, m), 4.79-4.84 (1H, m), 4.87-4.91 (1H, m), 4.94-5.00 (1H, m), 5.06-5.11 (1H, m), 6.09-6.17 (1H, m), 6.31-6.40 (1H, m), 6.76 (2H, s), 7.00-7.08 (1H, m), 7.43-7.47 (1H, m), 7.78-7.86 (1H, m). *overlapped with H₂O. Example 22: Synthesis of Mal- C₂-PEG₂-D-Eribulin

22.1 Preparation ofFmoc-D(OMe)-Eribulin

[00956] To a solution of eribulin mesylate (200 mg, 0.242 mmol) in DMF (1.5 mL) was added DIEA (127 μL, 0.726 mmol), Fmoc-Asp(OMe)-OH (179 mg, 0.484 mmol), and HATU (166 mg, 0.436 mmol). After being stirred for 2 h, the reaction mixture was purified with ODS column (0 to 80% MeCN/H₂O, containing 0.1% HCO₂H) to give the title compound (151 mg).

[00957] ESI-MS (m/z): 1082.73 [M+H]⁺.

22.2 Preparation ofD(OMe)-Eribulin

[00958] To a solution of Fmoc-D(OMe)-Eribulin (100 mg, 0.092 mmol) in DMF (0.9 mL) was added piperidine (92 μL, 0.93 mmol) at rt. After being stirred at rt for 1 h, the mixture was concentrated in vacuo to give a title compound as a crude mixture (79 mg).

[00959] ESI-MS (m/z): 860.02 [M+H]⁺.

[00960] To a solution of methyl D(OMe)-Eribulin (79 mg, 0.092 mmol) in MeOH (1.0 mL) and THF (0.5 mL) at rt was added 1.0 M Lithium hydroxide solution (110 μL, 0.11 mmol). The reaction mixture was stirred at rt. After 30 min, 110 μL of IN HCL aq and 1.0 mL of DMF was added to the reaction mixture, then concentrated under reduced pressure and the residue was purified with ODS- column (3 to 80% MeCN/EbO, containing 0.1% HCO₂H) to give the title compound (77 mg).

[00961] ESI-MS (m/z): 846.00 [M+H]⁺.

22.4 Synthesis of Mal-C₂-PEG₂-D-Eribulin

[00962] To a solution of (3-{2-[2-({[(9HFluoren-9- yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanoic acid (CAS No. 872679-70-4, 28 mg, 0.071 mmol) in DMF (0.72 mL) were added DIEA (23 mg, 0.18 mmol) and HATU (19 mg, 0.05 mmol) at 0 °C. After being stirred at 0 °C for 10 min, D-Eribulin (30 mg, 0.036 mmol) was added. The mixture was warmed to rt and stirred for 30 min. The crude mixture was directly purified with ODS-column (10 to 75% MeCN/H₂O, containing 0.1% HCO₂H) to give Fmoc-PEG₂-D-Eribulin.

[00963] To a solution of the Fmoc-PEG₂-D-Eribulin in DMF (0.72 mL) was added piperidine (30 mg, 0.36 mmol) at rt. After being stirred at rt for 10 min, the mixture was concentrated in vacuo.

[00964] To the residue was added DMF (0.72 mL), DIEA (11 mg, 0.09 mmol), and N-succinimidyl maleimidoacetate (11 mg, 0.043 mmol) and stirred at rt for 1 h. The crude mixture was directly purified with ODS-column (10 to 50% MeCN/EbO, containing 0.1% HCO₂H) to afford the title compound (17 mg).

[00965] ESI-MS (m/z): 1141.77 [M+H]⁺. [00966] ¹H-NMR Spectrum (500 MHz, CDCl₃) δ(ppm): 1.07-1.14 (4H, m), 1.18-2.06 (*55H, m), 2.07-2.35 (10H, m), 2.42-2.57 (5H, m), 2.69-2.78 (2H, m), 2.84-3.00 (3H, m), 3.14-3.24 (1H, m), 3.26-3.34 (1H, m), 3.37-3.71 (15H, m), 3.73-3.80 (2H, m), 3.81-3.87 (1H, m), 3.88-4.00 (3H, m), 4.03-4.08 (1H, m), 4.10-4.17 (1H, m), 4.18-4.22 (1H, m), 4.24-4.43 (5H, m), 4.60-4.64 (1H, m), 4.66- 4.74 (2H, m), 4.80-4.84 (1H, m), 4.87-4.92 (1H, m), 4.93-5.01 (1H, m), 5.02-5.13 (1H, m), 6.77 (2H, s), 6.79-6.88 (1H, m), 7.33-7.40 (1H, m), 7.58-7.65 (1H, m). *overlapped with H₂O

Example 23: Synthesis of Mal-C₂-PEG₂-Val-Ala-pABC-Eribulin

23.1 Preparation of Mal- C₂-PEG₁-CO₂tBu

[00967] To a solution of Amino-PEG₂-acid tert-Butyl Ester (440 mg, 1.89 mmol) in DMF (4.4 mL) at 0 °C were added N-succinimidyl maleimidoacetate (499 mg, 1.98 mmol) and DIEA (0.43 mL, 2.45 mmol). The reaction mixture was stirred at rt.

[00968] After 4 h, the reaction mixture was diluted with EtOAc (50 mL) and quenched with sat. NH₄Claq (50 mL), then the layers separated. The aqueous layer was extracted with EtOAc (50 mL). The combined organic extracts were successively washed with water and brine, dried over Na₂SO₄, filtered and concentrated in vacuo. Flash chromatography of the residue on silica gel (Y AMAZEN, Hi-FLASH™ column, Silicagel 40 μm) using 5% to 100% EtOAc/heptane gave the title compound (430 mg) as a colorless oil.

[00969] ESI-MS (m/z): 371.23 [M+H]⁺.

[00970] ¹H NMR (500 MHz, CDCl₃) δ ppm: 1.47 (s, 9 H) 2.52 (t, J=6.11 Hz, 2 H) 3.45-3.50 (m, 2 H) 3.55 - 3.57 (m, 2 H) 3.59-3.66 (m, 4 H) 3.77 (t, J=6.11 Hz, 2 H) 4.24 (s, 2 H) 6.61 - 6.68 (m, 1 H) 6.78 (s, 2 H).

23.2 Preparation ofMal-C₂-PEG₁-CO₂H

[00971] To a solution of Mal-C₂-PEG₂-CO₂tBu (220 mg, 0.59 mmol) in DCM (3 mL) at 0 °C (ice bath) was added TFA (3 mL). The reaction mixture was stirred at rt.

[00972] After 30 min, toluene (6 mL) was added to the reaction mixture then concentrated under reduced pressure (three times) to give the title compound as a solid.

[00973] ESI-MS (m/z): 315.19 [M+H]⁺.

[00974] To a solution of NH₂-Val-Ala-pABC-Eribulin (35 mg, 0.033 mmol, synthesized using steps similar to step 13.2 of 13) in DMF (700 μL) at rt were added Mal-C₂-PEG₂-CO₂H (12.6 mg, 0.04 mmol), DIEA (11.7 μL, 0.067 mmol) and HATU (16.5 mg, 0.043 mmol). The reaction mixture was stirred at rt for 30 min.

[00975] Flash chromatography of the reaction mixture on ODS-column using 5% to 80% MeCN/water (containing 0.1% HCO₂H) gave the title compound.

[00976] ESI-MS (m/z): 1345.9 [M+H]⁺.

[00977] ¹H NMR (500 MHz, CDCl₃) δ ppm: 0.86 (d, J=6.72 Hz, 1 H) 0.96 (dd, J=9.17, 6.72 Hz, 6 H) 1.08 - 1.13 (m, 4 H) 1.30 - 1.53 (m, 9 H) 1.68 - 1.80 (m, 6 H) 1.90 - 2.00 (m, 4 H) 2.07 - 2.36 (m, 6 H) 2.41 - 2.50 (m, 3 H) 2.51 - 2.57 (m, 1 H) 2.57 - 2.64 (m, 1 H) 2.72 (dd, J=16.20, 10.09 Hz, 1 H) 2.84 - 2.92 (m, 2 H) 3.18 (dt, .7=13.14, 6.27 Hz, 1 H) 3.22 - 3.26 (m, 1 H) 3.28 (d, .7=3.06 Hz, 1 H) 3.34 - 3.40 (m, 1 H) 3.43 (s, 3 H) 3.47 - 3.67 (m, 10 H) 3.71 - 3.76 (m, 1 H) 3.78 - 3.84 (m, 2 H) 3.89 - 4.00 (m, 3 H) 4.02 - 4.07 (m, 1 H) 4.09 - 4.16 (m, 1 H) 4.20 (dd, J=6.42, 4.58 Hz, 1 H) 4.28 - 4.43 (m, 6 H) 4.59 - 4.66 (m, 2 H) 4.70 (t, .7=4.58 Hz, 1 H) 4.80 - 4.84 (m, 1 H) 4.91 (br.s, 1 H) 4.94 (br.s, 1 H) 5.03 - 5.11 (m, 3 H) 5.24 - 5.30 (m, 1 H) 6.77 (s, 2 H) 6.83 - 6.90 (m, 1 H) 7.05 - 7.12 (m, 1 H) 7.30 (d, .7=8.56 Hz, 2 H) 7.38 - 7.42 (m, 1 H) 7.57 (d, .7=8.56 Hz, 2 H) 8.59 - 8.63 (m, 1 H).

Selected sequences

Claims

1. An anti-TR0P2 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to human TR0P2, and wherein the antibody or antigenbinding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein

(i) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 3, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 4, the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 8, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 9, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 10, as defined by the Kabat numbering system; or

(ii) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 12, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 13, the LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 15, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 16, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 17, as defined by the IMGT numbering system.

2. The anti-TROP2 antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 55 or 175, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 61 or 66.

3. The anti-TROP2 antibody or antigen-binding fragment of claim 1 or claim 2, wherein the antibody or antigen-binding fragment comprises

(i) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66; or

(ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

4. The anti-TROP2 antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a human IgGl heavy chain constant region.

5. The anti-TROP2 antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment comprises an IgGl Fc domain or an IgGl Fc domain mutated to reduce binding to a Fey receptor (FcyR) as compared to an IgGl Fc-containing antibody with a wild type IgGl Fc domain, optionally wherein the mutated IgGl Fc domain comprises one or more of the mutations L234A, L235A, P238S, H268Q, and K274Q.

6. The anti-TROP2 antibody or antigen-binding fragment of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

7. The anti-TROP2 antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises

(i) a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115;

(ii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115; or

(iii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

8. The anti-TROP2 antibody or antigen-binding fragment of any one of claims 1 to 7, wherein

(a) the antibody or antigen-binding fragment comprises part of a bispecific or multi-specific binding construct; and/or

(b) the antibody or antigen-binding fragment is linked to a therapeutic agent or detectable agent.

9. An antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is the anti-TROP2 antibody or antigen-binding fragment thereof of any one of claims 1 to 7;

D is a cytotoxic agent;

10. The antibody-drug conjugate of claim 9, wherein the cytotoxic agent comprises an antitubulin agent, preferably wherein the anti-tubulin agent is eribulin or a salt thereof, preferably eribulin mesylate.

11. The antibody-drug conjugate of claim 9 or claim 10, wherein p is from 2 to 8, optionally wherein p is 2.

12. The antibody-drug conjugate of any one of claims 9 to 11 , wherein cleavage of the conjugate releases the cytotoxic agent from the antibody and linker.

13. The antibody-drug conjugate of any one of claims 9 to 12, wherein the cleavable linker comprises a cleavable peptide moiety.

14. The antibody-drug conjugate of claim 13, wherein the cleavable peptide moiety is cleavable by an enzyme; optionally wherein the cleavable peptide moiety is cleavable by cathepsin or legumain; optionally wherein the cleavable peptide moiety is cleavable by cathepsin B.

15. The antibody-drug conjugate of claim 13 or claim 14, wherein the cleavable peptide moiety comprises an amino acid unit.

16. The antibody-drug conjugate of claim 15, wherein the amino acid unit comprises valinecitrulline (Val-Cit), valine-dimethylated lysine (Val-Lys(Me)₂), alanine-dimethylated lysine (Ala- Lys(Me)₂), valine-alanine (Val-Ala), asparagine (Asn), aspartic acid (Asp), or methylated aspartic acid (Asp(OMe)).

17. The antibody-drug conjugate of claim 16, wherein the amino acid unit comprises valinecitrulline (Val-Cit), valine-alanine (Val-Ala), or asparagine (Asn).

18. The antibody-drug conjugate of any one of claims 9 to 17, wherein the cleavable linker attaches to the antibody or antigen-binding fragment via a maleimide (Mai) moiety, optionally wherein the Mai moiety comprises a maleimidocaproyl (MC) moiety or a dithiomaleimide (DTM) moiety.

19. The antibody-drug conjugate of claim 18, wherein the Mai moiety is reactive with a cysteine residue on the antibody or antigen-binding fragment.

20. The antibody-drug conjugate of claim 18 or claim 19, wherein the Mai moiety is joined to the antibody or antigen-binding fragment via a cysteine residue on the antibody or antigen-binding fragment.

21. The antibody-drug conjugate of any one of claims 18 to 20, wherein the cleavable linker comprises the Mai moiety and a cleavable peptide moiety.

22. The antibody-drug conjugate of claim 21, wherein the Mai moiety attaches the antibody or antigen-binding fragment to the cleavable peptide moiety in the linker.

23. The antibody-drug conjugate of any one of claims 18 to 22, wherein the cleavable linker comprises Mal-Val-Cit, Mal-Val-Lys(Me)₂, Mal-Ala-Lys(Me)₂, Mal-Val-Ala, Mal-Asn, Mal-Asp, or Mal-Asp(OMe).

24. The antibody-drug conjugate of any one of claims 9 to 21 , wherein the cleavable linker comprises at least one spacer unit, wherein the spacer unit attaches to the antibody or antigen-binding fragment via the maleimide (Mai) moiety ("Mal-spacer unit").

25. The antibody-drug conjugate of claim 24, wherein the Mal-spacer unit comprises a polyethylene glycol (PEG) moiety, wherein the PEG moiety comprises -(PEG)_m- and m is an integer from 1 to 10.

26. The antibody-drug conjugate of claim 25, wherein m is 2, 3, or 4.

27. The antibody-drug conjugate of claim 24, wherein the Mal-spacer unit comprises an alkyl moiety, wherein the alkyl moiety comprises -(CH₂)_n- and n is an integer from 1 to 10, optionally wherein n is 5.

28. The antibody-drug conjugate of claim 24, wherein the Mal-spacer unit comprises C₂

29. The antibody-drug conjugate of claim 24, wherein the Mal-spacer unit comprises C₂(PEG)_m wherein m is an integer from 0 to 4.

30. The antibody-drug conjugate of claim 29, wherein m is 1, 2, or 3.

31. The antibody-drug conjugate of claim 29 or claim 30, wherein the Mal-spacer unit comprises

C₂(PEG)₂

32. The antibody-drug conjugate of any one of claims 24 to 31, wherein the cleavable linker comprises the Mal-spacer unit and the cleavable peptide moiety.

33. The antibody-drug conjugate of claim 32, wherein the Mal-spacer unit attaches the antibody or antigen-binding fragment to the cleavable peptide moiety in the linker.

34. The antibody-drug conjugate of claim 32 or claim 33, wherein the Mal-spacer unit comprises Mal-(PEG)₂-Val-Cit, Mal-(PEG)₃-Val-Cit, Mal-(PEG)₄-Val-Cit, Mal-(PEG)₂-Val-Lys(Me)₂,Mal- (PEG)₂-Ala-Lys(Me)₂, Mal-(PEG)₂-Val-Ala, Mal-(PEG)₂-Asn, Mal-(PEG)₂-Asp, Mal-(PEG)₂- Asp(OMe), Mal-C₂(PEG)_m-Val-Cit, Mal-C₂(PEG)_m-Val-Lys(Me)₂, Mal-C₂(PEG)_m-Val-Ala(Me)₂, Mal-C₂(PEG)_m-Val-Ala, Mal-C₂(PEG)_m-Asn, Mal-C₂(PEG)_m-Asp, or Mal-C₂(PEG)_m-Asp(OMe); wherein m is an integer from 0 to 4.

35. The antibody-drug conjugate of claim 34, wherein m is 2.

36. The antibody-drug conjugate of any one of claims 12 to 35, wherein the cleavable moiety in the linker is directly joined to the cytotoxic agent, or a spacer unit attaches the cleavable moiety in the linker to the cytotoxic agent.

37. The antibody-drug conjugate of claim 36, wherein the spacer unit attaching the cleavable moiety in the linker to the cytotoxic agent is self-immolative.

38. The antibody-drug conjugate of claim 36 or claim 37, wherein the spacer unit attaching the cleavable moiety in the linker to the cytotoxic agent comprises a p-aminobenzyloxycarbonyl (pABC).

39. The antibody-drug conjugate of claim 38, wherein the pABC attaches the cleavable moiety in the linker to the cytotoxic agent.

40. The antibody-drug conjugate of claim 39, wherein the cytotoxic agent is eribulin and the pABC covalently attaches to eribulin via a C-35 amine.

41. The antibody-drug conjugate of any one of claims 36 to 40, wherein the cleavable moiety comprises Val-Cit, Val-Ala, Val-Lys(Me)₂, Ala-Lys(Me)₂, Asn, Asp, or Asp(OMe).

42. The antibody-drug conjugate of claim 41 , wherein the cleavable linker comprises Val-Cit- pABC, Val-Ala-pABC, Val-Lys(Me)₂-pABC, or Ala-Lys(Me)₂-pABC.

43. The antibody-drug conjugate of claim 24, wherein the cleavable linker comprises DTM-Asn, Mal-(PEG)₂-Val-Cit-pABC, Mal-(PEG)₃-Val-Cit-pABC, Mal-(PEG)₄-Val-Cit-pABC, Mal-(PEG)₂- Val-Lys(Me)₂-pABC, Mal-(PEG)₂-Ala-Lys(Me)₂-pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂- Asn, Mal-(PEG)₂-Asp, Mal-(PEG)₂-Asp(OMe), Mal-C₂(PEG)_m-Val-Cit-pABC, Ma1-C₂(PEG)_m-Val- Lys(Me)₂-pABC, Mal-C₂(PEG)_m-Val-Ala(Me)₂-pABC, Mal-C₂(PEG)_m-Val-Ala-pABC, Mal- C₂(PEG)_m-Asn, Mal-C₂(PEG)_m-Asp, or Mal-C₂(PEG)_m-Asp(OMe); wherein m is an integer from O to 4.

44. The antibody-drug conjugate of claim 43, wherein the cleavable linker comprises Mal- (PEG)₂-Val-Cit-pABC, Mal-(PEG)₂-Val-Ala-pABC, Mal-(PEG)₂-Asn, MaJ-C₂(PEG)_m-Val-Cit- pABC, Mal-C₂(PEG)_m-Val-Ala-pABC, or Mal-C₂(PEG)_m-Asn; wherein m is an integer from 1 to 3.

45. The antibody-drug conjugate of claim 43 or claim 44, wherein m is 2.

46. The antibody-drug conjugate of any one of claims 9 to 45, wherein the antibody or antigenbinding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system.

47. The antibody-drug conjugate of any one of claims 9 to 45, wherein the antibody or antigenbinding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system.

48. The antibody-drug conjugate of claim 46 or claim 47, wherein the antibody or antigenbinding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

49. The antibody-drug conjugate of claim 46 or claim 47, wherein the antibody or antigenbinding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

50. The antibody-drug conjugate of claim 48, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

51. The antibody-drug conjugate of claim 49, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

52. The antibody-drug conjugate of claim 49, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

53. An antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is an anti-TROP2 antibody or antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system;

D is eribulin;

54. An antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is an anti-TROP2 antibody or antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system;

D is eribulin;

55. An antibody-drug conjugate of Formula (I):

Ab-(L-D)_p (I) wherein Ab is an anti-TROP2 antibody or antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 3 (HCDR2), and SEQ ID NO: 4 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3), as defined by the Kabat numbering system; D is eribulin;

L is a cleavable linker comprising Mal-(PEG)i-Val-Ala-pABC; and p is an integer from 1 to 8.

56. An antibody-drug conjugate of Formula (I):

D is eribulin;

57. An antibody-drug conjugate of Formula (I):

D is eribulin;

58. An antibody-drug conjugate of Formula (I):

D is eribulin;

59. An antibody-drug conjugate of Formula (I):

D is eribulin;

60. An antibody-drug conjugate of Formula (I):

D is eribulin;

61. An antibody-drug conjugate of Formula (I):

D is eribulin;

62. An antibody-drug conjugate of Formula (I): Ab-(L-D)_p (I) wherein Ab is an anti-TROP2 antibody or antigen-binding fragment thereof comprising three HCDRs comprising amino acid sequences of SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 15 (LCDR1), SEQ ID NO: 16 (LCDR2), and SEQ ID NO: 17 (LCDR3), as defined by the IMGT numbering system;

D is eribulin;

63. An antibody-drug conjugate of Formula (I):

D is eribulin;

64. An antibody-drug conjugate of Formula (I):

D is eribulin;

65. The antibody-drug conjugate of any one of claims 53 to 64, wherein m is 2.

66. The antibody-drug conjugate of any one of claims 53 to 65, wherein the antibody or antigenbinding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 175, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

67. The antibody-drug conjugate of any one of claims 53 to 65, wherein the antibody or antigenbinding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 66.

68. The antibody-drug conjugate of claim 66, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 181 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

69. The antibody-drug conjugate of claim 66, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 380 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

70. The antibody-drug conjugate of claim 67, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 100 and a light chain comprising an amino acid sequence of SEQ ID NO: 115.

71. The antibody-drug conjugate of any one of claims 53 to 70, wherein p is 2.

72. The antibody-drug conjugate of any one of claims 9 to 71 , wherein p is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) or reverse-phase liquid chromatography-mass spectrometry (LC-MS).

73. The antibody-drug conjugate of any one of claims 9 to 72, wherein the cytotoxic agent is eribulin and the cleavable linker covalently attaches to the eribulin via a C-35 amine.

74. The antibody-drug conjugate of any one of claims 9 and 53 to 73, wherein the cleavable linker covalently attaches to the antibody or antigen-binding fragment via a cysteine or a lysine.

75. A composition comprising multiple copies of the antibody-drug conjugate of any one of claims 9 to 74, wherein the average p of the antibody-drug conjugates in the composition is about 1 to about 3.

76. The composition of claim 75, wherein the average p is about 2.

77. One or more nucleic acid(s) encoding the antibody or antigen-binding fragment of any one of claims 1 to 7.

78. A host cell comprising the one or more nucleic acid(s) of claim 77.

79. A method of producing an antibody or antigen-biding fragment of any one of claims 1 to 7, comprising culturing the host cell of claim 78 under conditions sufficient to produce the antibody or antigen-binding fragment.

80. A method of producing the antibody-drug conjugate of any one of claims 9 to 74 or the composition of claim 75 or claim 76, comprising reacting an antibody or antigen-binding fragment of any one of claims 1 to 7 with a cleavable linker joined to eribulin under conditions that allow conjugation.

81. A compound of Formula (Ila): , wherein:

D is a cytotoxic agent;

Y is a cleavable moiety; and

Z is absent or a spacer unit.

82. The compound of claim 81 , wherein the cytotoxic agent comprises eribulin or a salt thereof.

83. The compound of claim 81 or claim 82, wherein the cleavable moiety comprises a cleavable peptide moiety, optionally wherein the cleavable peptide moiety is cleavable by an enzyme; further optionally wherein the cleavable peptide moiety is cleavable by cathepsin or legumain, optionally wherein the cleavable peptide moiety is cleavable by cathepsin B.

84. The compound of claim 83, wherein the cleavable peptide moiety comprises an amino acid unit.

85. The compound of claim 84, wherein the amino acid unit comprises valine-citrulline (Val-Cit), valine-dimethylated lysine (Val-Lys(Me)₂), alanine-dimethylated lysine (Ala-Lys(Me)₂), valinealanine (Vai-Ala), asparagine (Asn), aspartic acid (Asp), or methylated aspartic acid (Asp(OMe)).

86. The compound of claim 85, wherein the amino acid unit comprises Val-Cit or Val-Ala.

87. The compound of claim 85, wherein the amino acid unit comprises Asn or Asp.

88. The compound of any one of claims 81 to 85 and 87, wherein Z is absent and the cleavable moiety is directly joined to the cytotoxic agent.

89. The compound of any one of claims 81 to 86, wherein Z is a spacer unit which attaches the cleavable moiety to the cytotoxic agent.

90. The compound of claim 89, wherein the spacer unit attaching the cleavable moiety to the cytotoxic agent is self-immolative.

91. The compound of claim 89 or claim 90, wherein the spacer unit attaching the cleavable moiety to the cytotoxic agent comprises a p-aminobenzyloxycarbonyl (pABC).

92. The compound of claim 91 , wherein the cytotoxic agent is eribulin, and the pABC covalently attaches to eribulin via a C-35 amine.

93. The compound of claim 91 or claim 92, wherein -Y-Z- comprises Val-Cit-pABC, Val-Ala- pABC, Val-Lys(Me)₂-pABC, or Ala-Lys(Me)₂-pABC.

94. The compound of any one of claims 81 to 85, wherein -Y-Z- comprises Val-Cit-pABC, Val- Ala-pABC, or Asn.

95. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 8, the antibody-drug conjugate of any one of claims 9 to 74, the composition of claim 75 or claim 76, or the compound of any one of claims 81 to 94; and a pharmaceutically acceptable carrier.

96. A method of treating a patient having or at risk of having a cancer that expresses TROP2, comprising administering to the patient a therapeutically effective amount of the antibody or antigenbinding fragment of any one of claims 1 to 8, the antibody-drug conjugate of any one of claims 9 to 74, the composition of claim 75 or claim 76, the compound of any one of claims 81 to 94, or the pharmaceutical composition of claim 95.

97. The method of claim 96, wherein the TROP2-expressing cancer is a cholangiocarcinoma, bladder cancer, breast cancer, cervical carcinoma, colorectal cancer, esophageal cancer, gastric cancer, glioma, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer; optionally wherein the TROP2-expressing cancer is a cholangiocarcinoma, breast cancer, or NSCLC.

98. A method of reducing or inhibiting growth of a TROP2-expressing tumor, comprising administering to the patient a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 8, the antibody-drug conjugate of any one of claims 9 to 74, the composition of claim 75 or claim 76, the compound of any one of claims 81 to 94, or the pharmaceutical composition of claim 95.

99. The method of claim 98, wherein the tumor is a TROP2-expressing cholangiocarcinoma, bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer; optionally wherein the tumor is a cholangiocarcinoma.

100. Use of the antibody or antigen-binding fragment of any one of claims 1 to 8, the antibodydrug conjugate of any one of claims 9 to 75, the composition of claim 75 or claim 76, the compound of any one of claims 81 to 94, or the pharmaceutical composition of claim 95 in the manufacture of a medicament for the treatment of a TROP2-expressing cancer.

101. Use of an antibody or antigen-binding fragment of any one of claims 1 to 8, an antibody-drug conjugate of any one of claims 9 to 75, a composition of claim 75 or claim 76, a compound of any one of claims 81 to 95, or a pharmaceutical composition of claim 95 in the treatment of a TROP2- expressing cancer.

102. The use of claim 100 or claim 101, wherein the TROP2-expressing cancer is a cholangiocarcinoma, bladder cancer, breast cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, endometrial cancer, ovarian cancer, or skin cancer; optionally wherein the TROP2-expressing cancer is a cholangiocarcinoma.