Eribulin-based antibody-drug conjugates and methods of use

Abstract

To provide linker toxins and antibody-drug conjugates that bind to human oncology antigen targets such as folate receptor alpha and / or exhibit anti-tubulin drug activity.SOLUTION: The present invention provides an antibody-drug conjugate of Formula (I): Ab-(L-D)p (I), wherein Ab is an internalizing antibody or internalizing antigen-binding fragment thereof which targets a tumor cell; D is eribulin; L is a cleavable linker that covalently attaches Ab to D; and p is an integer from 1 to 20.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 302,562, filed March 2, 2016, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to antibody drug conjugates (ADCs) that bind to human tumor antigen targets, such as the folate receptor alpha, and / or provide anti-tubulin drug activity. The disclosure further relates to methods and compositions useful in the treatment and diagnosis of cancers that express the folate receptor alpha and / or are amenable to treatment by disruption of tubulin. [Background technology]

[0003] Cancer is one of 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 lung cancer (1.59 million deaths); liver cancer (745,000 deaths); stomach cancer (723,000 deaths); colorectal cancer (694,000 deaths); breast cancer (521,000 deaths); and esophageal cancer (400,000 deaths). The number of new cancer cases is predicted to increase by approximately 70% over the next 20 years, to approximately 22 million new cancer cases per year (World Cancer Report 2014).

[0004] Microtubules are dynamic, fibrous cytoskeletal proteins involved in various cellular functions, including intracellular internalization and transport, cell signaling, and maintaining cell shape. Microtubules also play an important role in mitotic cell division by forming the mitotic spindle, which is necessary for separating chromosomes into two daughter cells. The biological function of microtubules in all cells is largely regulated by their polymerization dynamics, which occurs through the reversible, noncovalent attachment of α- and β-tubulin dimers to both ends of the microtubule. This dynamic behavior and the resulting control over microtubule length are crucial for proper spindle function. Even slight changes in microtubule dynamics can trigger the spindle checkpoint, halting cell cycle progression in mitosis and subsequently leading to cell death (Mukhtar et al. (2014) Mol. Cancer Ther. 13:275-84). Due to their rapid cell division, cancer cells are generally more sensitive than normal cells to compounds that bind to tubulin and disrupt its normal function. For this reason, tubulin inhibitors and other microtubule-targeting drugs have become a promising class of drugs for treating cancer (Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9:790-803).

[0005] Folate receptor alpha (FRA) is a glycophosphatidylinositol (GPI)-linked membrane protein that binds folate. The role of FRA in the biology of normal and cancerous tissues is not fully understood, but it is highly overexpressed in a high percentage of epithelial-derived ovarian cancers (O'Shannessy et al. (2013) Int. J. Gynecol. Pathol. 32(3):258-68) and in a certain percentage of non-small cell lung cancers (Christoph et al. (2014) Clin. Lung Cancer 15(5):320-30). FRA also has limited expression in normal tissues. These properties make FRA an attractive target for cancer immunotherapy.

[0006] The proto-oncogene human epidermal growth factor receptor 2 (HER2) encodes a transmembrane tyrosine kinase receptor belonging to the human epidermal growth factor receptor (EGFR) family (King et al. (1985) Science 229:974-6). Overexpression of HER2 allows constitutive activation of growth factor signaling pathways, such as the PI3K-AKT-mTOR pathway, thereby serving as an oncogenic driver in several cancers, including approximately 20% of invasive breast cancers (Slamon et al. (1989) Science 244:707-12; Gajria and Chandarlapaty (2011) Expert Rev. Anticancer Ther. 11:263-75). HER2 amplification mediates a transformed phenotype, making it another promising target for cancer treatment.

[0007] This disclosure provides, in part, novel compounds with biological activity against tumor cells, which can inhibit tumor growth in mammals and may be useful for treating human cancer patients.

[0008] More specifically, the present disclosure relates to antibody-drug conjugate compounds that can bind to, internalize, and kill tumor cells (e.g., FRA-expressing tumor cells). Antibody-drug conjugate compounds are disclosed that include a linker that connects the drug moiety to the antibody moiety. Antibody-drug conjugate (ADC) compounds can be represented by Formula I: Ab-(LD) p (I) [wherein Ab is an internalizing antibody or an internalizing antigen-binding fragment thereof that targets tumor cells; D is eribulin; L is a cleavable linker covalently linking Ab to D; and p is an integer from 1 to 20.

[0009] In some embodiments, the linker is stable extracellularly such that it remains intact when the ADC is in extracellular conditions but can be cleaved upon internalization into a cell, e.g., a cancer cell. In some embodiments, the eribulin drug moiety is cleaved from the antibody moiety when the ADC enters a cell that expresses an antigen specific for the antibody moiety of the ADC, cleavage releasing the unmodified form of eribulin. In some embodiments, the linker comprises a cleavable moiety that is positioned such that, upon cleavage, no portion of the linker or antibody portion remains attached to the eribulin drug moiety.

[0010] In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety. In some embodiments, ADCs comprising a cleavable peptide moiety demonstrate lower aggregation levels, improved antibody:drug ratios, increased on-target killing of cancer cells, decreased off-target killing of non-cancer cells, and / or higher drug loading (p) relative to ADCs comprising alternative cleavable moieties. In some embodiments, the addition of a cleavable moiety increases cytotoxicity and / or potency relative to non-cleavable linkers. In some embodiments, the increased potency and / or cytotoxicity is in cancers that express moderate levels of the antigen targeted by the antibody portion of the ADC (e.g., moderate FRA expression). In some embodiments, the cleavable peptide moiety is enzymatically cleavable and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is a cathepsin and the linker is a cathepsin-cleavable linker. In certain embodiments, enzyme-cleavable linkers (e.g., cathepsin-cleavable linkers) exhibit one or more of the improved properties described above when compared to alternative cleavage mechanisms.

[0011] In some embodiments, the cleavable peptide moiety in the linker comprises an amino acid unit. In some embodiments, the amino acid unit comprises valine-citrulline (Val-Cit). In some embodiments, ADCs comprising Val-Cit demonstrate increased stability, reduced off-target cell killing, increased on-target cell killing, lower aggregation levels, and / or higher drug loading relative to ADCs comprising alternative amino acid units or alternative cleavable moieties.

[0012] In some embodiments, the linker comprises at least one spacer unit that connects the antibody moiety to the cleavable moiety. In some embodiments, the spacer unit in the linker may comprise at least one polyethylene glycol (PEG) moiety. The PEG moiety may be, for example, -(PEG) m -, where m is an integer from 1 to 10. In some embodiments, the spacer unit in the linker comprises (PEG). In some embodiments, ADCs comprising short spacer units (e.g., (PEG)) demonstrate lower aggregation levels and / or higher drug loading relative to ADCs comprising long spacer units (e.g., (PEG)).

[0013] In some embodiments, the spacer unit in the linker is attached to the antibody portion of the ADC via a maleimide moiety (Mal). In some embodiments, ADCs comprising linkers attached to the antibody portion via a Mal demonstrate higher drug loading relative to ADCs comprising linkers attached to the antibody portion via an alternative moiety. In some embodiments, the Mal in the linker is reactive with a cysteine ​​residue on the antibody portion. In some embodiments, the Mal in the linker is connected to the antibody portion via a cysteine ​​residue. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the linker is Mal-(PEG) m, e.g., Mal-(PEG)2. In some embodiments, the linker comprises Mal-(PEG)2. In some embodiments, a Mal-spacer unit connects the antibody moiety to a cleavable moiety in the linker. In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety, e.g., an amino acid unit. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit.

[0014] In some embodiments, the cleavable moiety in the linker is directly connected to the eribulin drug moiety of the ADC, and the cleavable moiety is connected directly or via a spacer unit. In some embodiments, a spacer unit also connects the cleavable moiety in the linker to the eribulin drug moiety. In some embodiments, the spacer unit connecting the cleavable moiety in the linker to the eribulin drug moiety is self-immolative. In some embodiments, the self-immolative spacer is capable of releasing unmodified eribulin in target cells. In some embodiments, the self-immolative spacer unit comprises p-aminobenzyl alcohol. In some embodiments, the self-immolative spacer unit comprises p-aminobenzyloxycarbonyl (pAB). pAB in the linker, in some embodiments, connects the cleavable moiety to the eribulin drug moiety. In some embodiments, the cleavable moiety is a cleavable peptide moiety, e.g., an amino acid unit. In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and a spacer unit connecting the linker to the antibody moiety via Mal.

[0015] In some embodiments, p is an integer of 1 to 6, 2 to 5, or preferably 3 to 4. In some embodiments, p is 4. In some embodiments, a group of ADCs is provided, wherein the average p in the group is about 4 (e.g., 3.5 to 4.5, e.g., about 3.8). In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit-pAB. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit-pAB, and p is 4. In some embodiments, a group of ADCs is provided, wherein each ADC comprises a Mal-(PEG)2-Val-Cit-pAB linker, and the average p in the group is about 4 (e.g., 3.5 to 4.5, e.g., about 3.8).

[0016] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment (Ab or Ab portion) of the ADC is an anti-folate receptor alpha (FRA) antibody or internalizing antibody fragment and is capable of binding to FRA-expressing tumor cells (i.e., the ADC targets FRA-expressing cells). In some embodiments, ADCs comprising an anti-FRA Ab portion and a cleavable peptide portion demonstrate lower aggregation levels, improved antibody:drug ratios, increased on-target killing of cancer cells, decreased off-target killing of non-cancer cells, high drug loading (p), increased cytotoxicity, and / or increased potency relative to non-cleavable linkers or alternative cleavage mechanisms. In some embodiments, the increased potency and / or cytotoxicity is in cancers that express moderate levels of the antigen targeted by the antibody portion of the ADC (e.g., moderate FRA expression). In some embodiments, the cleavable peptide portion is enzymatically cleavable and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is cathepsin and the linker is a cathepsin-cleavable linker. In certain embodiments, the enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the improved properties described above when compared to alternative cleavage mechanisms. In some embodiments, the linker is Mal-(PEG) m -Val-Cit-pAB.

[0017] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment binds to folate receptor alpha (FRA) and targets FRA-expressing tumor cells. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs, wherein the heavy chain CDRs, as defined by the Kabat numbering system, comprise a heavy chain CDR1 consisting of SEQ ID NO:2, a heavy chain CDR2 consisting of SEQ ID NO:3, and a heavy chain CDR3 consisting of SEQ ID NO:4; the three light chain CDRs, as defined by the IMGT numbering system, comprise a heavy chain CDR1 consisting of SEQ ID NO:13, a heavy chain CDR2 consisting of SEQ ID NO:14, and a heavy chain CDR3 consisting of SEQ ID NO:15; and the light chain CDRs, as defined by the IMGT numbering system, comprise a light chain CDR1 consisting of SEQ ID NO:16, a light chain CDR2 consisting of SEQ ID NO:17, and a light chain CDR3 consisting of SEQ ID NO:18. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises human framework sequences. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a heavy chain variable domain of SEQ ID NO: 23 and a light chain variable domain of SEQ ID NO: 24. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment competes for binding to and / or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO: 23 and a light chain variable domain of SEQ ID NO: 24. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment binds to an epitope comprising alanine-histidine-lysine-aspartic acid (AHKD) (SEQ ID NO: 365) (O'Shannessy et al., (2011) Oncotarget 2:1227-43). In some embodiments, the internalizing antibody or internalizing antigen-binding fragment binds to an epitope comprising NTSQEAHKDVSYL (SEQ ID NO: 366).

[0018] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is an internalizing anti-FRA antibody or internalizing antigen-binding fragment. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs, wherein, as defined by the Kabat numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 2, a heavy chain CDR2 consisting of SEQ ID NO: 3, and a heavy chain CDR3 consisting of SEQ ID NO: 4; the three light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 7, a light chain CDR2 consisting of SEQ ID NO: 8, and a light chain CDR3 consisting of SEQ ID NO: 9; or, as defined by the IMGT numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 13, a heavy chain CDR2 consisting of SEQ ID NO: 14, and a heavy chain CDR3 consisting of SEQ ID NO: 15; the light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 16, a light chain CDR2 consisting of SEQ ID NO: 17, and a light chain CDR3 consisting of SEQ ID NO: 18; the linker comprises Mal-(PEG)2-Val-Cit-pAB; and p is 4. In some embodiments, a group of such ADCs is provided, wherein p is about 4 (e.g., about 3.8). In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a heavy chain variable domain of SEQ ID NO: 23 and a light chain variable domain of SEQ ID NO: 24. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment competes for binding to and / or binds to the same epitope as an antibody comprising the heavy chain variable domain of SEQ ID NO: 23 and the light chain variable domain of SEQ ID NO: 24. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment binds to an epitope comprising SEQ ID NO: 365. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment binds to an epitope comprising SEQ ID NO: 366.

[0019] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment binds to human epidermal growth factor receptor 2 (her2) and targets her2-expressing tumor cells. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs, wherein, as defined by the Kabat numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 71, a heavy chain CDR2 consisting of SEQ ID NO: 72, and a heavy chain CDR3 consisting of SEQ ID NO: 73; the three light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 74, a light chain CDR2 consisting of SEQ ID NO: 75, and a light chain CDR3 consisting of SEQ ID NO: 76; or, as defined by the IMGT numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 191, a heavy chain CDR2 consisting of SEQ ID NO: 192, and a heavy chain CDR3 consisting of SEQ ID NO: 193; and the light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 194, a light chain CDR2 consisting of SEQ ID NO: 195, and a light chain CDR3 consisting of SEQ ID NO: 196. In some embodiments, the antibody or internalizing antigen-binding fragment comprises human framework sequences. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a heavy chain variable domain of SEQ ID NO: 27 and a light chain variable domain of SEQ ID NO: 28. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment competes for binding to and / or binds to the same epitope as an antibody comprising the heavy chain variable domain of SEQ ID NO: 27 and the light chain variable domain of SEQ ID NO: 28.

[0020] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is an internalizing anti-her2 antibody or internalizing antigen-binding fragment. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs, wherein, as defined by the Kabat numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 71, a heavy chain CDR2 consisting of SEQ ID NO: 72, and a heavy chain CDR3 consisting of SEQ ID NO: 73; and the three light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 74, a light chain CDR2 consisting of SEQ ID NO: 75, and a light chain CDR3 consisting of SEQ ID NO: 76. R3; or as defined by the IMGT numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 191, a heavy chain CDR2 consisting of SEQ ID NO: 192, and a heavy chain CDR3 consisting of SEQ ID NO: 193; the light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 194, a light chain CDR2 consisting of SEQ ID NO: 195, and a light chain CDR3 consisting of SEQ ID NO: 196; the linker comprises Mal-(PEG)2-Val-Cit-pAB; and p is 4. In some embodiments, a group of such ADCs is provided, wherein p is about 4 (e.g., about 3.8). In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a heavy chain variable domain of SEQ ID NO: 27 and a light chain variable domain of SEQ ID NO: 28. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain. In some embodiments, the internalizing antibody or internalizing antigen binding competes for binding to and / or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:27 and a light chain variable domain of SEQ ID NO:28.

[0021] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment binds to mesothelin (MSLN) and targets MSLN-expressing tumor cells. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) and three light chain CDRs, wherein, as defined by the Kabat numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 65, a heavy chain CDR2 consisting of SEQ ID NO: 66, and a heavy chain CDR3 consisting of SEQ ID NO: 67; the three light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 68, a light chain CDR2 consisting of SEQ ID NO: 69, and a light chain CDR3 consisting of SEQ ID NO: 70; or, as defined by the IMGT numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 185, a heavy chain CDR2 consisting of SEQ ID NO: 186, and a heavy chain CDR3 consisting of SEQ ID NO: 187; and the light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 188, a light chain CDR2 consisting of SEQ ID NO: 189, and a light chain CDR3 consisting of SEQ ID NO: 190. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a heavy chain variable domain of SEQ ID NO: 25 and a light chain variable domain of SEQ ID NO: 26. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment competes for binding to and / or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO: 25 and a light chain variable domain of SEQ ID NO: 26.

[0022] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is an internalizing anti-MSLN antibody or internalizing antigen-binding fragment. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs, wherein, as defined by the Kabat numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 65, a heavy chain CDR2 consisting of SEQ ID NO: 66, and a heavy chain CDR3 consisting of SEQ ID NO: 67; and the three light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 68, a light chain CDR2 consisting of SEQ ID NO: 69, and a light chain CDR3 consisting of SEQ ID NO: 70. R3; or as defined by the IMGT numbering system, the heavy chain CDRs comprise a heavy chain CDR1 consisting of SEQ ID NO: 185, a heavy chain CDR2 consisting of SEQ ID NO: 186, and a heavy chain CDR3 consisting of SEQ ID NO: 187; the light chain CDRs comprise a light chain CDR1 consisting of SEQ ID NO: 188, a light chain CDR2 consisting of SEQ ID NO: 189, and a light chain CDR3 consisting of SEQ ID NO: 190; the linker comprises Mal-(PEG)2-Val-Cit-pAB; and p is 4. In some embodiments, a group of such ADCs is provided, wherein p is about 4 (e.g., about 3.8). In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a heavy chain variable domain of SEQ ID NO: 25 and a light chain variable domain of SEQ ID NO: 26. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain. In some embodiments, the internalizing antibody or internalizing antigen binding competes for binding to and / or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:25 and a light chain variable domain of SEQ ID NO:26.

[0023] Also provided herein are compositions comprising multiple copies of any of the described ADCs, wherein the average drug loading (average p) of the ADCs in the composition is about 3 to 4, or about 3.5 to about 4.5, or about 4. In some embodiments, the average p is about 3.2 to 3.8. In some embodiments, the average p is about 3.6 to 4.4.

[0024] Also provided herein is a composition comprising -LD, where D is eribulin; and L is a cleavable linker covalently bonded to D. In some embodiments, the cleavable linker is covalently bonded to the C-35 amine on eribulin. In some embodiments, the cleavable linker comprises Val-Cit. In some embodiments, the cleavable linker comprises a PEG spacer unit. In some embodiments, the cleavable linker comprises Mal-(PEG)2-Val-Cit-pAB.

[0025] Further provided herein are pharmaceutical compositions comprising an ADC and a pharmaceutically acceptable diluent, carrier, and / or excipient.

[0026] Another aspect of the disclosure includes therapeutic and diagnostic uses of the described ADC compounds and compositions, for example, in the treatment of cancer. Another aspect includes methods of treating cancers that express an antigen targeted by the antibody portion of the ADC, such as FRA. In various embodiments, methods are provided for killing or inhibiting the growth of tumor or cancer cells by administering a therapeutically effective amount and / or regimen of any one of the described ADCs. Another aspect includes methods of using the disclosed ADCs to detect tumor or cancer cells that express FRA and to screen cancer patients who will respond to treatment with the described ADCs. In some embodiments, the cancer is gastric cancer, serous ovarian cancer, clear cell ovarian cancer, non-small cell lung cancer, colorectal cancer, triple-negative breast cancer, endometrial cancer, serous endometrial cancer, lung carcinoid, or osteosarcoma. Methods of producing the described ADCs are also disclosed. [Brief explanation of the drawings]

[0027] [Figure 1]

[0033] Figure 1 shows one method used to prepare MORAb-003 ADC as disclosed in certain embodiments. In this approach, unpaired cysteines are generated by partial reduction with a limited molar equivalent of the non-thiol reducing agent TCEP. This approach preferentially reduces the interchain disulfide bonds connecting the light and heavy chains (one pair per HL pairing) and the two heavy chains in the hinge region (two pairs per HH pairing in the case of human IgG1), while leaving the intrachain disulfide bonds intact. [Figure 2] 1 shows a method for synthesizing maleimide-(PEG)2-Val-Cit-pAB-eribulin (mal-(PEG)2-VCP-eribulin), as disclosed in certain embodiments. [Figure 3] SDS-PAGE analysis of MORAb-003 under reduced conditions is shown. Lanes are indicated on the right side of the figure. Lane M corresponds to the protein standard; lane 1 corresponds to untreated MORAb-003; lane 2 corresponds to 5.3 mg / mL reduced with 70.6 μM TCEP; lane 3 corresponds to 5.3 mg / mL MORAb-003 reduced with 141.2 μM TCEP; lane 4 corresponds to 1.5 mg / mL MORAb-003 reduced with 20 μM TCEP; and lane 5 corresponds to 1.5 mg / mL MORAb-003 reduced with 40 μM TCEP. The identity of each band is indicated on the gel at the bottom right. "H" indicates heavy chain. "L" indicates light chain. [Figure 4] 1 shows SDS-PAGE analysis of MORAb-003 under reducing conditions. Lane 1 corresponds to a protein standard; lane 2 corresponds to untreated MORAb-003; lane 3 corresponds to MORAb-003 treated at a ratio of 1:1 MORAb-003:TCEP; lane 4 corresponds to MORAb-003 treated at a ratio of 1:2 MORAb-003:TCEP; lane 5 corresponds to MORAb-003 treated at a ratio of 1:3 MORAb-003:TCEP; and lane 6 corresponds to MORAb-003 treated at a ratio of 1:4 MORAb-003:TCEP. [Figure 5]Non-reducing SDS-PAGE analysis of selected MORAb-003 ADCs, including M-MMAE (lane 2), M-DM1 (lane 3), M-0026 (lane 4), M-0260 (lane 5), M-0267 (lane 6), M-0272 (lane 7), M-0285 (lane 8), M-0292 (lane 9), M-027-0381 (lane 10), and M-0284 (lane 11), is shown. [Figure 6] Figure 6A shows the results of a bystander cytotoxicity assay for MORAb-003-maleimide-PEG2-Val-Cit-pAB-eribulin (M3-VCP-eribulin, or "MORAb-202"). Figure 6B shows the results of a bystander cytotoxicity assay for MORAb-003-maleimide-(CH2)5-Val-Cit-pAB-ER-001150828 (M3-ER-61318). Figure 6C shows the results of a bystander cytotoxicity assay for MORAb-003-PEG-pAB-duostatin3 (M3-027-0285). The information provided in each figure legend indicates the cell line: agent tested (cell line(s) cultured, seeding density of 1st / 2nd cell lines). [Figure 7] 7A and 7B show the drug-to-antibody ratio (DAR) distributions of the ADCs MORAb-003-VCP-eribulin (FIG. 7A) and MORAb-003-0285 (FIG. 7B) relative to unconjugated MORAb-003, as disclosed in certain embodiments. The numbers above each peak indicate the DAR of the individual species. [Figure 8] 1 shows the results of a cytotoxicity assay, i.e., competition between MORAb-003-VCP-eribulin and unconjugated MORAb-003 (2 μM) in IGROV1 or SJSA-1 cells. [Figure 9] Shown are body weight dynamics in each group of CD-1 mice (group mean and SEM) treated with vehicle (PBS) or a single intravenous dose of 10, 20, 40, or 80 mg / kg of MORAb-202. [Figure 10]Shown are body weight dynamics in each group of CD-1 mice (group mean and SEM) treated intravenously with PBS or 0.4, 0.8, 1.6, or 3.2 mg / kg eribulin according to a q4d x 3 dosing regimen (doses administered once every 4 days for a total of 3 doses). [Figure 11] Figure 1 shows tumor growth kinetics in groups of CB17-SCID mice implanted with hNSCLC NCI-H2110 cells (group mean and SEM) and treated with a single intravenous dose of PBS, 1, 2.5, or 5 mg / kg MORAb-003-VCP-eribulin (MORAb-202), or 5 mg / kg MORAb-003-0285. [Figure 12] Shown are tumor volumes of individual CB17-SCID mice implanted with hNSCLC NCI-H2110 cells, as well as group means and SEM, at day 17. Groups were treated with a single intravenous dose of PBS, 1, 2.5, or 5 mg / kg MORAb-003-VCP-eribulin (MORAb-202), or 5 mg / kg MORAb-003-0285. [Figure 13] Shown are body weight dynamics (group means and SEM) for each group of NCI-H2110-implanted CB17-SCID mice treated with a single intravenous dose of PBS, 1, 2.5, or 5 mg / kg of MORAb-003-VCP-eribulin (MORAb-202), or 5 mg / kg of MORAb-003-0285. [Figure 14] Tumor growth kinetics in each group of NCI-H2110-implanted CB17-SCID mice (group mean and SEM) treated intravenously with vehicle (PBS) or 0.5, 0.2, 0.8, or 1.6 mg / kg eribulin according to a q4d x 3 dosing regimen are shown. [Figure 15] Shown are tumor volumes of individual NCI-H2110-implanted CB17-SCID mice, as well as group means and SEM, at day 24. Groups were treated intravenously with vehicle (PBS) or 0.5, 0.2, 0.8, or 1.6 mg / kg eribulin according to a q4d x 3 dosing regimen. [Figure 16]Shown are the weight change kinetics in each group of NCI-H2110-implanted CB17-SCID mice (group mean and SEM) treated intravenously with vehicle (PBS) or 0.5, 0.2, 0.8, or 1.6 mg / kg eribulin according to a q4d x 3 dosing regimen. [Figure 17] 1 shows the efficacy of MORAb-003-VCP-eribulin (MORAb-202) against IGROV1, OVCAR3, NCI-H2110, A431-A3, and SJSA-1 cells as measured by Crystal Violet cytotoxicity assay. [Figure 18] Tumor growth kinetics in each group of NCI-H2110-implanted CB17-SCID mice (group mean and SEM) treated with a single intravenous dose of PBS or 1, 2.5, or 5 mg / kg of MORAb-003-VCP-eribulin (MORAb-202) are shown. [Figure 19] Figures 19A and 19B show tumor growth kinetics (Figure 19A) and body weight change kinetics (Figure 19B) in each group of NSCLC PDx (LXFA-737) tumor-bearing mice (group mean and SEM) treated with a single intravenous dose of vehicle (PBS), 5 mg / kg MORAb-003, or 5 mg / kg MORAb-003-VCP-eribulin (MORAb-202). [Figure 20] Figures 20A and 20B show the individual tumor volume ratios (Figure 20A) and body weight change kinetics (Figure 20B) for each group of endometrial cancer PDx (Endo-12961) tumor-bearing mice (group means and SEM) treated with a single intravenous dose of PBS, 0.1 or 3.2 mg / kg eribulin, or 5 mg / kg MORAb-003-VCP-eribulin (MORAb-202). Figures 20C and 20D show the tumor growth kinetics (Figure 20C) and body weight change kinetics (Figure 20D) for each group of endometrial cancer PDx (Endo-10590) tumor-bearing mice (group means and SEM) treated with a single intravenous dose of PBS, 0.1 or 3.2 mg / kg eribulin, or 5 mg / kg MORAb-003-VCP-eribulin (MORAb-202). [Figure 21A]Immunohistochemical (IHC) staining of tumor tissue from TNBC PDx (OD-BRE-0631) tumor-bearing mice with an anti-human IgG antibody is shown. Tumor tissue from mice treated with a single intravenous dose of vehicle (right) or 5 mg / kg MORAb-003-VCP-eribulin (MORAb-202) (left) was collected and stained 5 days after treatment. [Figure 21B] IHC staining of tumor tissue from TNBC PDx (OD-BRE-0631) tumor-bearing mice with α-smooth muscle actin (SMA)-FITC antibody is shown. Tumor tissue from an untreated mouse was collected 2 days before treatment (left), and tumor tissue from a mouse treated with a single intravenous dose of 5 mg / kg MORAb-003-VCP-eribulin (MORAb-202) was collected 5 days after treatment (right). [Figure 21C] Figure 1 shows tumor growth kinetics in each group of TNBC PDx (OD-BRE-0631) tumor-bearing mice (group mean and SEM) treated with vehicle (PBS) or a single intravenous dose of 5 mg / kg MORAb-003-VCP-eribulin (MORAb-202). [Figure 22] Figure 1 shows differentiation of human bone marrow mesenchymal stem cells (BM-MSCs) in culture with MKN-74 cells after treatment with vehicle (PBS or ethanol), eribulin, MORAb-003, or MORAb-003-VCP-eribulin (MORAb-202), as measured by flow cytometry analysis. Stro-1 / CD105, CD34 / CD31, and NG2 are markers for MSCs, adipocytes, and pericytes, respectively. [Figure 23] Figure 1 shows a time course analysis of tumor tissue stained with α-smooth muscle actin (SMA)-FITC antibody from NCI-H2110-implanted CB17-SCID mice treated with vehicle (PBS) or a single intravenous dose of 5 mg / kg MORAb-003-VCP-eribulin (MORAb-202). Tumor tissue was collected and stained on day 0 and days 3, 5, 7, and 9 after treatment. Y-axis: % = [stained cells counted / total cells counted] x 100. X-axis: days (total cells counted). DETAILED DESCRIPTION OF THE INVENTION

[0028] The disclosed compositions and methods may be more readily understood by reference to the following detailed description in conjunction with the accompanying drawings, which form a part of this disclosure: It is understood that the disclosed compositions and methods are not limited to the specific compositions and methods described and / or illustrated herein, and that the terminology used herein is for the purpose of merely illustrating particular embodiments and is not intended to limit the compositions and methods described in the claims.

[0029] Throughout this text, descriptions refer to compositions and methods of using said compositions. Where this disclosure describes or claims features or embodiments related to compositions, such features or embodiments are equally applicable to methods of using said compositions. Similarly, where this disclosure describes or claims features or embodiments related to methods of using compositions, such features or embodiments are equally applicable to compositions.

[0030] When a range of values ​​is given, it includes embodiments using any specific value within that range. Moreover, reference to values ​​stated in ranges includes any and all values ​​within that range. All ranges are inclusive of their endpoints and are 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" means "and / or" unless the specific context in which it is used dictates otherwise. All references cited herein are incorporated by reference for all purposes. In the event of a conflict between a reference and the present specification, the present specification will control.

[0031] It should be appreciated that certain features of the compositions and methods of the present disclosure, which are, for clarity, described herein in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the compositions and methods of the present disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any subcombination.

[0032] definition Various terms relating to the described aspects 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 should be construed consistent with the definitions provided herein.

[0033] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context dictates otherwise.

[0034] The terms "about" or "approximately" in the context of numerical values ​​and ranges refer to a value or range that is close to or near the recited value or range such that an embodiment can be performed as intended, such as having a desired amount of nucleic acid or polypeptide in a reaction mixture, as one of ordinary skill in the art would know from the teachings contained herein. This is due, at least in part, to various characteristics of nucleic acid composition, age, race, sex, anatomical and physiological variations, and ecological imprecision. Thus, these terms include values ​​beyond those resulting from systematic error.

[0035] The terms "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably and refer to a compound or derivative thereof linked to an antibody (e.g., an anti-FRA antibody) and have the general formula: Ab-(LD) pwhere Ab = antibody moiety (i.e., antibody or antigen-binding fragment), L = linker moiety, D = drug moiety, and p = number of drug moieties per antibody moiety.

[0036] The term "antibody" is used in the broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, e.g., a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a combination of the above, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The heavy chain of an antibody comprises a heavy chain variable domain (V H ) and heavy chain constant region (C H The light chain consists of a light chain variable domain (V L ) and the light chain constant domain (C L ). For purposes of this application, mature heavy and light chain variable domains each comprise three complementarity determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4), arranged from N-terminus to C-terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An "antibody" may be naturally occurring or artificial, such as a monoclonal antibody produced by conventional hybridoma technology. The term "antibody" includes full-length monoclonal and polyclonal antibodies, as well as antibody fragments, e.g., Fab, Fab', F(ab')2, Fv, and single-chain antibodies. An antibody may belong to any one of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., isotypes IgG1, IgG2, IgG3, IgG4). The term further includes human antibodies, chimeric antibodies, humanized antibodies and any modified immunoglobulin molecule containing an antigen recognition site, so long as the desired biological activity is demonstrated.

[0037] 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 large number 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, monoclonal antibodies 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 can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al. (1991) Nature 352:624-8 and Marks et al. (1991) J. Mol. Biol. 222:581-97.

[0038] The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind to the target antigen and / or exhibit the desired biological activity.

[0039] The term "human antibody," as used herein, refers to an antibody produced by a human or an antibody having the amino acid sequence of an antibody produced by a human.

[0040] The term "chimeric antibody," as used herein, refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more species. In some instances, the variable regions of both the 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.

[0041] As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) and even human antibodies. Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin. A humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized antibodies may be further modified by substitution of residues either in the Fv framework regions and / or within the substituted non-human residues to refine and optimize antibody specificity, affinity, and / or activity.

[0042] 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., FRA). Antigen-binding fragments preferably also retain the ability to be internalized into antigen-expressing cells. In some embodiments, antigen-binding fragments also retain immune effector activity. It has been found that fragments of full-length antibodies can perform the antigen-binding function of the full-length antibody. Examples of binding fragments included within the term "antigen-binding fragment" or "antigen-binding portion" of an antibody include (i) Fab fragments, V L , V H , C L , and C H1(ii) a F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H1 (iv) a V fragment of one arm of an antibody; L and V H (v) an Fv fragment consisting of one variable domain, e.g., V H and (v) isolated complementarity-determining regions (CDRs). In addition, two domains of an Fv fragment, V ... L and V H are encoded by separate genes, but using recombinant methods, they can be synthesized to L and V H They can be connected by a synthetic linker that allows the domains to be produced as a single protein chain (known as a single-chain Fv (scFv)) that pairs to form a monovalent molecule. 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 included within the scope of 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 be internalized 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 fusion proteins. Other forms of single chain antibodies, such as diabodies, are also included. Diabodies are antibodies that are composed of a V H and V LBivalent diabodies are those in which the domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain to create 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 skilled in the art, and binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments can also be prepared by cleavage of the intact protein, for example, by protease or chemical cleavage.

[0043] "Internalization," as used herein in connection with an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that, upon binding to a cell, can be taken up (i.e., "internalized") through the lipid bilayer membrane of the cell into an internal compartment, preferably into a degradation compartment within the cell. For example, an internalizing anti-FRA antibody is one that can be taken up into the cell after binding to FRA on the cell membrane.

[0044] The term "folate receptor alpha" or "FRA," as used herein, refers to any naturally occurring form of human FRA. The term includes full-length FRA (e.g., NCBI Reference Sequence: NP_000793; SEQ ID NO: 19), as well as any form of human FRA that results from cellular processing. The term also includes naturally occurring variants of FRA, including, but not limited to, splice variants, allelic variants, and isoforms. FRA can be isolated from humans or produced recombinantly or by synthetic methods.

[0045] The terms "anti-FRA antibody" or "antibody that specifically binds to FRA" refer to any form of antibody or fragment thereof that specifically binds to FRA, including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they specifically bind to FRA. Preferably, the anti-FRA antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. MORAb-003 is an exemplary internalizing anti-human FRA antibody. As used herein, the terms "specific," "specifically binds," and "binds specifically" refer to the selective binding of an antibody to a target antigen epitope. An antibody can be tested for binding specificity by comparing binding to the appropriate antigen with binding to an irrelevant antigen or mixture of antigens under a given set of conditions. An antibody is considered specific if it binds to the appropriate antigen with at least 2-, 5-, 7-, and preferably 10-fold greater affinity than to the irrelevant antigen or mixture of antigens. In one embodiment, a specific antibody binds only to the FRA antigen and does not bind (or shows only weak binding to) other antigens.

[0046] The terms "human epidermal growth factor receptor 2," "her2," or "her2 / neu," as used herein, refer to any naturally occurring form of human her2. The term includes full-length her2 (e.g., NCBI Reference Sequence: NP_004439.2; SEQ ID NO: 21), as well as any form of human her2 that results from cellular processing. The term also includes naturally occurring variants of her2, including, but not limited to, splice variants, allelic variants, and isoforms. Her2 can be isolated from humans or produced recombinantly or by synthetic methods.

[0047] The term "anti-her2 antibody" or "antibody that specifically binds to her2" refers to any form of antibody or fragment thereof that specifically binds to her2, including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they specifically bind to her2. U.S. Patent No. 5,821,337 (incorporated herein by reference) provides exemplary her2-binding sequences, including exemplary anti-her2 antibody sequences. Preferably, the anti-her2 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. Trastuzumab is an exemplary internalizing anti-human her2 antibody.

[0048] The term "epitope" refers to the portion of an antigen that can be recognized and specifically bound by an antibody. When the antigen is a polypeptide, the epitope can be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of the polypeptide. Any epitope mapping technique known in the art can be used to identify the epitope to which an antibody binds, 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 mutant variants of the antigen, or monitoring the solvent accessibility of various portions of the antibody and 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).

[0049] Competitive binding and epitope binning can also be used to determine antibodies that share the same or overlapping epitopes. Cross-blocking assays, e.g., "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Harlow and Lane (1999), pp. 111-114, 2001. st Edition 1988, 2 nd Competitive binding can be assessed using assays such as those described in the 2014 edition. In some embodiments, competitive binding is identified if a test antibody or binding protein reduces binding of a reference antibody or binding protein (e.g., a binding protein comprising a CDR and / or variable domain selected from those identified in Tables 2, 4, and 6) to a target antigen, e.g., FRA or her2, by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any percentage therebetween) in a cross-blocking assay, 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 when antibodies or binding proteins bind at adjacent 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; for example, those that compete for binding can be "binned" into a group of binding proteins that have overlapping or adjacent epitopes, and those that do not compete into another group of binding proteins that do not have overlapping or adjacent epitopes.

[0050] "k on " or "k a The term "rate constant" refers to the rate constant for the association of an antibody and an antigen to form an antibody / antigen complex. This rate can be determined using standard assays, such as Biacore or ELISA assays.

[0051] "k off " or "k d The term "off rate constant" refers to the off rate constant for dissociation of an antibody from the antibody / antigen complex. This rate can be determined using standard assays, such as Biacore or ELISA assays.

[0052] "K D The term K refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. D is k a / k d This rate can be determined using standard assays, such as Biacore or ELISA assays.

[0053] The term "p" 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, for ADCs of Formula I, the number of -LD moieties per antibody or antigen-binding fragment (Ab). In compositions comprising multiple copies of an ADC of Formula I, "p" refers to the average number of -LD moieties per antibody or antigen-binding fragment, also referred to as the average drug loading.

[0054] A "linker" or "linker moiety" is any chemical moiety capable of covalently connecting a compound, usually a drug moiety such as a chemotherapeutic drug, to another moiety, such as an antibody moiety. The linker may be sensitive or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and / or disulfide bond cleavage, provided that the compound or antibody remains active.

[0055] 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 material. The terms "therapeutic agent," "drug," or "drug moiety" refer to an agent that can modulate a biological process and / or has biological activity.

[0056] The term "chemotherapeutic agent" or "anticancer agent" is used herein to refer to any chemical compound that is effective in treating cancer, regardless of mechanism of action. Inhibition of metastasis or angiogenesis is often a characteristic of chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustards, ethylenimine compounds, and alkylsulfonates; antimetabolites, such as folic acid, purine, or pyrimidine antagonists; antimitotic agents, such as antitubulin agents, vinca alkaloids, and auristatins, such as eribulin or eribulin mesylate (Halaven™) or its derivatives, cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, such as DNA minor groove binding agents; and growth factor receptor antagonists. In addition, chemotherapeutic agents include antibodies, biological molecules, and small molecules. Chemotherapeutic agents may be cytotoxic or cytostatic agents. The term "cytostatic agent" refers to an agent that inhibits or suppresses cell growth and / or proliferation.

[0057] The term "cytotoxic drug" refers to a substance that causes cell death primarily by interfering with the expression, activity, and / or function of a cell. Examples of cytotoxic drugs include, but are not limited to, antimitotic drugs 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, taxol, and colchicine; anthracyclines (e.g., daunorubicin, doxorubicin, diazepam, thiazolinone); hydroxyanthracinediones); cytotoxic antibiotics (e.g., mitomycin, actinomycin, duocarmycin (e.g., CC-1065), auromycin, duomycin, calicheamicin, endomycin, phenomycin); alkylating agents (e.g., cisplatin); intercalating agents (e.g., ethidium bromide); topoisomerase inhibitors (e.g., etoposide, tenoposide); radioactive isotopes, e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 or 213 , P 32 , 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), chrycin, crotin, Sapaonaria officinalis inhibitor, glucocorticoids).

[0058] The term "eribulin," as used herein, refers to a synthetic analog of halichondrin B, a macrocyclic compound originally isolated from the marine sponge Halichondria okadais. The term "eribulin drug moiety" refers to the component of an ADC having the structure of eribulin and attached to the linker of the ADC via its C-35 amine. Eribulin is a microtubule dynamics inhibitor that is thought to induce cell cycle arrest at the G2 / M phase by binding to tubulin and inhibiting mitotic spindle assembly. The term "eribulin mesylate" refers to the mesylate salt of eribulin, which is sold under the trade name Halaven™.

[0059] The term "homologue" refers to a molecule that exhibits homology to another molecule, for example, by having the same or similar sequences of chemical residues at corresponding positions.

[0060] The terms "inhibit" or "inhibition of," as used herein, mean to reduce by a measurable amount and may include, but does not require, complete prevention or inhibition.

[0061] The terms "target negative" or "target antigen negative" refer to the absence of expression of a target antigen by a cell or tissue. The terms "target positive" or "target antigen positive" refer to the presence of expression of a target antigen. For example, a cell or cell line that does not express a target antigen may be described as target negative, while a cell or cell line that expresses the target antigen may be described as target positive.

[0062] The term "bystander killing" or "bystander effect" refers to the killing of target-negative cells in the presence of target-positive cells, where 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, allows for bystander killing. This type of killing is distinguished from "off-target killing," which refers to the indiscriminate killing of target-negative cells. "Off-target killing" can be observed even in the absence of target-positive cells.

[0063] The term "cancer" refers to a physiological condition in mammals in which a cell population is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer (e.g., triple-negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. Triple negative breast cancer refers to breast cancer that is negative for expression of the genes for estrogen receptor (ER), progesterone receptor (PR), or Her2 / neu.

[0064] The terms "tumor" and "neoplasm" refer to any mass of tissue resulting from excessive cell growth or proliferation, either benign or malignant, including precancerous lesions.

[0065] The terms "cancer cell" and "tumor cell" refer to individual cells or the entire population of cells derived from a tumor, including both non-tumorigenic cells and cancer stem cells. As used herein, the term "tumor cell" is modified by the term "non-tumorigenic" when referring only to tumor cells that lack the ability to reproduce and differentiate, to distinguish them from cancer stem cells.

[0066] 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, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0067] The terms "co-administration" or administration "in combination with" one or more therapeutic agents includes simultaneous and consecutive administration in any order.

[0068] A "pharmaceutical composition" refers to a preparation that is in a form that allows for administration to subsequently bring about the intended biological activity of the active ingredient(s) and / or achieve a therapeutic effect, and that does not contain additional components that are unacceptably toxic to the subject to whom the formulation will be administered. The pharmaceutical composition may be sterile.

[0069] "Pharmaceutical excipients" include substances such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like.

[0070] "Pharmaceutically acceptable" means approved or capable of being approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.

[0071] An "effective amount" of an ADC as disclosed herein is an amount sufficient to produce a therapeutic effect following administration to achieve a specifically stated purpose, such as, for example, a reduction in tumor growth rate or tumor volume, a reduction in symptoms of cancer, or some other sign of treatment effectiveness. An effective amount can be determined by routine methods 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 an 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 alleviate 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 a subject before or at an early stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0072] As used herein, "to treat" or "therapeutic" and grammatically related terms refer to any improvement in any prognosis of a disease, such as sustained survival, lower morbidity, and / or alleviation of side effects that are a by-product of alternative treatment modalities. As will be readily appreciated in the art, complete eradication of the disease is preferred, but is not a requirement for therapeutic action. "Treatment" or "treating," as used herein, refers to administration of a described ADC to a subject, e.g., a patient. Treatment may be to cure, cure, alleviate, relieve, alter, correct, improve, palliate, ameliorate, or affect a disorder, symptoms of a disorder, or a predisposition to a disorder, e.g., cancer.

[0073] In some embodiments, a labeled ADC is used. Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.

[0074] As used herein, "protein" refers to at least two covalently attached amino acids. This term includes polypeptides, oligopeptides, and peptides. In some embodiments, the two or more covalently attached amino acids are linked by a peptide bond. For example, if the protein is recombinantly produced using an expression system and host cell, the protein may be composed of naturally occurring amino acids and peptide bonds. Alternatively, the protein may include synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine) or peptidomimetic structures, i.e., "peptide or protein analogs," such as peptoids. Peptoids are an exemplary group of peptidomimetics whose side chains are attached to nitrogen atoms of the peptide backbone rather than the α-carbon (as in amino acids) and have different hydrogen bonding and conformational characteristics compared to peptides (see, e.g., Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367). Thus, peptoids can be resistant to proteolysis or other physiological or storage conditions, and can be effective in permeating cell membranes. Such synthetic amino acids can be incorporated, particularly when antibodies are synthesized in vitro by conventional methods known in the art. Additionally, peptidomimetics, any combination of synthetic and naturally occurring residues / structures, can be used. "Amino acid" also includes imino acid residues, e.g., proline and hydroxyproline. An amino acid "R group" or "side chain" can be in either the (L)- or (S)-configuration. In a specific embodiment, the amino acid is in the (L)- or (S)-configuration.

[0075] A "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid, using any techniques and methods known in the art. Methods and techniques for producing recombinant proteins are well known in the art.

[0076] An "isolated" protein is free from at least some of the materials normally associated with it in its native state, e.g., constituting at least about 5% by weight, or at least about 50% by weight, of the total protein in a given sample. It is understood that an isolated protein may constitute 5-99.9% by weight of the total protein content, depending on the circumstances. For example, the protein may be produced at significantly higher concentrations through the use of inducible or high-expression promoters such that the protein is produced at high concentration levels. This definition includes the production of antibodies in a wide variety of organisms and / or host cells known in the art.

[0077] For amino acid sequences, sequence identity and / or similarity can be determined using methods such as, 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 method of similarity searching 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 algorithm described by Devereux et al. (1984) Nucl. Acid Res. 12:387-95, and the sequence identity and similarity algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the method of similarity searching 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.)), and the Best Fit algorithm described by Devereux et al. (1984) Nucl. Acid Res. 12:387-95. It can be determined using standard techniques known in the art, including the Fit sequence program, preferably using default settings or by inspection. Preferably, percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and connection penalty of 30 ("Current Methods in Sequence Comparison and Analysis", Macromolecules Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc.).

[0078] An example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignment to create a multiple sequence alignment from a group of related sequences. It can also plot a phylogenetic 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; this method is similar to the method 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.

[0079] 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, derived from Altschul et al. (1996) Methods in Enzymology 266:460-80. WU-BLAST-2 uses several search parameters, many of which are set to default values. Adjustable parameters are set with the following values: overlap span = 1, overlap fraction = 0.125, and word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values ​​established by the program that are themselves dependent on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is being searched; however, the values ​​may be adjusted to increase sensitivity.

[0080] An additional useful algorithm is Gapped BLAST as reported by Altschul et al. (1993) Nucl. Acids Res. 25:3389-402. Gapped BLAST uses a BLOSUM-62 substitution score; a threshold T parameter set to 9; a two-hit method for initiating ungapped extensions, imposing a cost of 10+k on gap length k; Xu set to 16, and Xg set to 40 in the database search phase and 67 in the output phase of the algorithm. Gapped alignments are initiated by scores corresponding to approximately 22 bits.

[0081] Generally, the amino acid homology, similarity, or identity between the proteins disclosed herein and variants thereof, including FRA variants, her2 variants, tubulin sequence variants, and antibody variable domain variants (including individual variant CDRs), is at least 80% relative to the sequences set forth herein, with more typically increasing homology or identity of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and near or 100% preferred.

[0082] Similarly, "percent (%) nucleic acid sequence identity" with respect to nucleic acid sequences of 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 of the coding sequence in the antigen-binding protein. A particular method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

[0083] Although the site or region for introducing amino acid sequence changes is predetermined, the mutation itself does not need to be predetermined.For example, to optimize the performance of mutations at a given site, random mutagenesis can be performed at the target codon or region, and the expressed antigen-binding protein CDR variants can be screened for the optimal combination of desired activity.Techniques for making substitution mutations at a predetermined site of DNA with a known sequence are well known, such as MI3 primer mutagenesis and PCR mutagenesis.

[0084] Antibody-drug conjugates Compounds of the present disclosure include those with anticancer activity. In particular, the compounds comprise an antibody moiety (including an antigen-binding fragment thereof) conjugated (i.e., covalently attached via a linker) to a drug moiety, where the drug moiety has a cytotoxic or cytostatic effect when not conjugated to the antibody moiety. In various embodiments, the drug moiety exhibits reduced or no cytotoxicity when attached within the conjugate, but regains cytotoxicity after cleavage from the linker and antibody moiety. In various embodiments, the drug moiety exhibits reduced or no bystander killing when attached within the conjugate (e.g., using a non-cleavable linker), but exhibits increased bystander killing after cleavage from the conjugate (e.g., a conjugate having a cleavable Val-Cit cleavable moiety).

[0085] The development and production of ADCs for use as human therapeutics, e.g., as oncologic agents, can require more than identifying an antibody that binds to a desired target or targets and is capable of conjugating to a drug that will be used alone to treat cancer. Conjugation of an antibody to a drug can have significant and unpredictable effects on the activity of either or both the antibody and the drug, effects that vary depending on the type of linker and / or drug selected. Thus, in some embodiments, the components of the ADC are selected to (i) retain one or more therapeutic properties exhibited by the antibody and drug moiety alone, (ii) maintain the specific binding properties of the antibody moiety; (iii) optimize drug loading and drug-to-antibody ratio; (iv) enable delivery of the drug moiety, e.g., intracellular delivery, by stable attachment to the antibody moiety; (v) maintain ADC stability as an intact conjugate until transport or delivery to the target site; (vi) minimize aggregation of the ADC before or after administration; (vii) enable the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage within the cellular environment; (viii) exhibit in vivo anti-cancer efficacy comparable to or superior to that of the antibody and drug moiety alone; (ix) minimize off-target killing by the drug moiety; and / or (x) exhibit desirable pharmacokinetic and pharmacodynamic properties, formulatability, and toxicological / immunological profile. Screening for each of these properties may be required to identify improved ADCs for therapeutic use (Ab et al. (2015) Mol. Cancer Ther. 14:1605-13).

[0086] In various embodiments, the ADCs disclosed herein exhibit unexpectedly favorable properties in some or each of the above-listed categories. For example, in some embodiments, ADC constructs comprising a Mal linkage to the antibody, a PEG spacer unit (preferably a short PEG spacer unit), and / or a peptide cleavable linker (e.g., a Val-Cit linker) exhibit surprisingly favorable drug loading, aggregation, and / or stability profiles, and / or maintain improved antibody binding function, drug activity, and / or bystander killing, while reducing off-target killing, when compared to ADCs using other cleavable or non-cleavable linker structures.

[0087] In some embodiments, ADCs comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to an antibody (e.g., an anti-FRA antibody such as MORAb-003) exhibit particularly favorable properties across the listed categories when compared to other cleavable or non-cleavable linkers connecting eribulin to the antibody moiety. In some embodiments, ADCs comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to an antibody (e.g., an anti-FRA antibody such as MORAb-003) exhibit particularly favorable bystander killing properties when compared to non-cleavable ADCs. In some embodiments, ADCs comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to an antibody (e.g., an anti-FRA antibody such as MORAb-003) exhibit particularly favorable bystander killing properties when compared to ADCs using alternative cleavable linker structures.

[0088] In some embodiments, ADCs comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to MORAb-003 exhibit a high and desirable drug:antibody ratio (i.e., about 3-4), for example, relative to ADCs comprising a linker that is attached to the antibody by an alternative moiety (e.g., a succinimide moiety). In some embodiments, ADCs comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to MORAb-003 exhibit a high and desirable drug:antibody ratio and / or a low level of aggregation, for example, relative to ADCs comprising a longer spacer unit (e.g., (PEG)8). In some embodiments, ADCs comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to MORAb-003 demonstrate high and desirable drug:antibody ratios, lower aggregation levels, increased on-target killing, and / or decreased off-target killing, for example, relative to ADCs comprising an alternative cleavable moiety (i.e., a non-peptide cleavable moiety, e.g., a cleavable disulfide or sulfonamide). In some embodiments, ADCs comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to MORAb-003 demonstrate increased stability, increased on-target killing, decreased off-target killing, lower aggregation levels, and / or high and desirable drug:antibody ratios, for example, relative to ADCs comprising an alternative amino acid unit (e.g., Ala-Ala-Asn) or an alternative cleavable moiety (e.g., a cleavable disulfide or sulfonamide).

[0089] In some embodiments, some or all of the desirable characteristics described above for ADCs comprising a Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to MORAb-003 may be observed in ADCs comprising a Mal-(PEG)2-Val-Cit-pAB-eribulin linker-toxin conjugated to trastuzumab, or an anti-her2 antibody, such as an anti-mesothelin antibody.

[0090] The ADC compounds of the present disclosure can selectively deliver effective doses of cytotoxic or cytostatic drugs to cancer cells or tumor tissues. It has been discovered that the ADCs of the present disclosure have potent cytotoxic and / or cytostatic activity against cells expressing their respective target antigens (e.g., FRA or Her2). In some embodiments, the cytotoxic and / or cytostatic activity of the ADC depends on the level of target antigen expression in the cells. In some embodiments, the ADCs of the present disclosure are particularly effective at killing cancer cells expressing high levels of the target antigen compared to cancer cells expressing the same antigen at low levels. In some embodiments, the ADCs of the present disclosure are particularly effective at killing cancer cells expressing a moderate level of the target antigen compared to cancer cells expressing the same antigen at low levels. Exemplary high FRA-expressing cancers include, but are not limited to, ovarian cancer (e.g., serous ovarian cancer, clear cell ovarian cancer), lung carcinoid, triple-negative breast cancer, endometrial cancer, and non-small cell lung cancer (e.g., adenocarcinoma). Exemplary cancers with moderate FRA expression include, but are not limited to, gastric cancer and colorectal cancer. Exemplary cancers with low FRA expression include, but are not limited to, melanoma and lymphoma. Exemplary cancers with high HER2 expression include, but are not limited to, breast cancer, gastric cancer, esophageal cancer, ovarian cancer, and endometrial cancer. Exemplary cancers with moderate HER2 expression include, but are not limited to, lung cancer and bladder cancer.

[0091] In some embodiments, cleavage of the ADC releases eribulin from the antibody moiety and linker. In some embodiments, cleavage and release of eribulin improves the cytotoxicity of the ADC. In some embodiments, ADCs comprising cleavable linkers are particularly effective at killing cancer cells, including bystander killing, compared to comparable treatment with ADCs comprising non-cleavable linkers. In some embodiments, ADCs comprising cleavable linkers (e.g., Val-Cit linkers) demonstrate increased on-target cell killing and / or decreased off-target cell killing relative to ADCs comprising non-cleavable linkers (e.g., non-cleavable (PEG)2 or (PEG)4 linkers), particularly when the cells and / or cancers treated with the ADC do not express high levels of the target antigen.

[0092] In some embodiments, the ADCs of the present disclosure also demonstrate bystander killing activity, but with low off-target cytotoxicity. Without being bound by theory, the bystander killing activity of ADCs may be particularly advantageous when their penetration into solid tumors is limited and / or when target antigen expression within tumor cells is heterogeneous. In some embodiments, ADCs comprising cleavable linkers are particularly effective at bystander killing and / or demonstrate improved bystander killing activity compared to comparable treatment with ADCs comprising non-cleavable linkers.

[0093] Provided herein are ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab) that targets tumor cells, a drug moiety (D), and a linker moiety (L) that covalently links the Ab to D. In certain aspects, the antibody or antigen-binding fragment can bind to a tumor-associated antigen (e.g., FRA or her2) with high specificity and high affinity. In certain embodiments, upon binding, the antibody or antigen-binding fragment is internalized into the target cell, e.g., into an intracellular degradation compartment. Thus, preferred ADCs are those that, upon binding to the target cell, are internalized, undergo degradation, and release a drug moiety that kills the cancer cell. The drug moiety can be released from the antibody and / or linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

[0094] An exemplary ADC has Formula I: Ab-(LD) p (I) where Ab = antibody moiety (i.e., antibody or antigen-binding fragment), L = linker moiety, D = drug moiety, and p = number of drug moieties per antibody moiety.

[0095] antibody The antibody moiety (Ab) of Formula I includes within its scope any antibody or antigen-binding fragment that specifically binds to a target antigen on a cancer cell. The antibody or antigen-binding fragment may have a dissociation constant (K) of ≦1 mM, ≦100 nM, or ≦10 nM, as measured, for example, by BIAcore® analysis. D ), or any amount therebetween. D In some embodiments, K D is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

[0096] In some embodiments, the antibody portion is a four-chain antibody (also called an immunoglobulin) comprising two heavy chains and two light chains, hi some embodiments, the antibody portion is a two-chain immunoglobulin half body (one light chain and one heavy chain), or an antigen-binding fragment.

[0097] In some embodiments, the antibody portion is an internalizing antibody or an internalizing antigen-binding fragment thereof. In some embodiments, the internalizing antibody binds to a target cancer antigen expressed on the surface of a cell and, upon binding, enters the cell. In some embodiments, the drug portion of the ADC is released from the antibody portion of the ADC after the ADC enters and resides in a cell expressing the target cancer antigen (i.e., after the ADC is internalized).

[0098] The amino acid and nucleic acid sequences of exemplary antibodies of the disclosure are set forth in Tables 1-9.

[0099] [Table 1]

[0100] [Table 2] TIFF2024174991000003.tif209149

[0101] [Table 3] TIFF2024174991000005.tif226149 TIFF2024174991000006.tif226149 TIFF2024174991000007.tif226149 TIFF2024174991000008.tif226149 TIFF2024174991000009.tif58149

[0102] [Table 4] TIFF2024174991000011.tif226149 TIFF2024174991000012.tif175149

[0103]

Table 5

[0104]

Table 6

[0105]

Table 7

[0106]

Table 8

[0107]

Table 9

[0108] In various embodiments, the ADCs disclosed herein may comprise any set of heavy and light chain variable domains listed in the table above (e.g., the MORAb-003 heavy and light chain variable domains, or the trastuzumab heavy and light chain variable domains), or a set of six CDR sequences from a heavy and light chain set. In some embodiments, the ADC further comprises human heavy and light chain constant domains, or fragments thereof. For example, the ADC may comprise a human IgG heavy chain constant domain (such as an IgG1) and a human kappa or lambda light chain constant domain. In various embodiments, the antibody portion of the described ADC comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain together with a human Ig kappa light chain constant domain.

[0109] In various embodiments, the target cancer antigen for the ADC is folate receptor alpha (“FRA”).

[0110] In various embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as defined by the Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987 and 1991)) as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:2, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:3, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:4; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:7, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:8, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:9.

[0111] In some embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as defined by the IMGT numbering system (International ImMunoGeneTics Information System (IMGT®)): heavy chain CDR1 consisting of SEQ ID NO: 13, heavy chain CDR2 consisting of SEQ ID NO: 14, heavy chain CDR3 consisting of SEQ ID NO: 15; light chain CDR1 consisting of SEQ ID NO: 16, light chain CDR2 consisting of SEQ ID NO: 17, and light chain CDR3 consisting of SEQ ID NO: 18.

[0112] In various embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 23 and the light chain variable region amino acid sequence of SEQ ID NO: 24, or sequences at least 95% identical to the foregoing sequences. In some embodiments, the anti-FRA antibody or antigen-binding fragment thereof has 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: 23 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: 24.

[0113] In various embodiments, the anti-FRA antibody comprises a human IgG1 heavy chain constant domain together with a human Ig kappa light chain constant domain.

[0114] In various embodiments, the anti-FRA antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 1, or a sequence at least 95% identical to SEQ ID NO: 1, and a light chain amino acid sequence of SEQ ID NO: 6, or a sequence at least 95% identical to SEQ ID NO: 6. In specific embodiments, the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence of SEQ ID NO: 6, or a sequence at least 95% identical to the foregoing sequences. In some embodiments, the anti-FRA 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: 1 and / or 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: 6. In some embodiments, the anti-FRA antibody comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 11 (including nucleotides encoding the leader sequence) or SEQ ID NO: 345 (excluding nucleotides encoding the leader sequence); and a light chain encoded by the nucleotides of SEQ ID NO: 12 (including nucleotides encoding the leader sequence) or SEQ ID NO: 346 (excluding nucleotides encoding the leader sequence). In some embodiments, the heavy chain amino acid sequence lacks the C-terminal lysine. In various embodiments, the anti-FRA antibody has the amino acid sequence of the antibody produced by the cell line deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209) under accession number PTA-7552 on April 24, 2006, or such a sequence lacking the heavy chain C-terminal lysine. In various embodiments, the anti-FRA antibody is MORAb-003 (USAN name: farletuzumab) (Ebel et al. (2007) Cancer Immunity 7:6), or an antigen-binding fragment thereof.

[0115] In various other embodiments, the target cancer antigen for the ADC is human epidermal growth factor receptor 2 ("her2").

[0116] In various embodiments, the anti-her2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as defined by the Kabat numbering system: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 71, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 72, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 73; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 74, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 75, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 76.

[0117] In some embodiments, the anti-her2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as defined by the IMGT numbering system: heavy chain CDR1 consisting of SEQ ID NO: 191, heavy chain CDR2 consisting of SEQ ID NO: 192, heavy chain CDR3 consisting of SEQ ID NO: 193; light chain CDR1 consisting of SEQ ID NO: 194, light chain CDR2 consisting of SEQ ID NO: 195, and light chain CDR3 consisting of SEQ ID NO: 196.

[0118] In various embodiments, the anti-her2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-her2 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 27 and the light chain variable region amino acid sequence of SEQ ID NO: 28, or sequences at least 95% identical to the foregoing sequences. In some embodiments, the anti-her2 antibody or antigen-binding fragment thereof has 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: 27 and / or 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: 28.

[0119] In various embodiments, the anti-her2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

[0120] In various embodiments, the anti-her2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 327, or a sequence at least 95% identical to SEQ ID NO: 327, and a light chain amino acid sequence of SEQ ID NO: 328, or a sequence at least 95% identical to SEQ ID NO: 328. In certain embodiments, the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 327 and a light chain amino acid sequence of SEQ ID NO: 328, or a sequence at least 95% identical to the foregoing sequences. In some embodiments, the anti-her2 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: 327, 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: 328. In various embodiments, the anti-her2 antibody is trastuzumab, or an antigen-binding fragment thereof.

[0121] In various embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of MORAb-003, or the CDRs comprise one, two, three, four, five, or no more than six amino acid additions, deletions, or substitutions in HCDR1 (SEQ ID NO: 2 according to Kabat, or SEQ ID NO: 13 according to IMGT), HCDR2 (SEQ ID NO: 3 according to Kabat, or SEQ ID NO: 14 according to IMGT), HCDR3 (SEQ ID NO: 4 according to Kabat, or SEQ ID NO: 15 according to IMGT); LCDR1 (SEQ ID NO: 7 according to Kabat, or SEQ ID NO: 16 according to IMGT), LCDR2 (SEQ ID NO: 8 according to Kabat, or SEQ ID NO: 17 according to IMGT), and LCDR3 (SEQ ID NO: 9 according to Kabat, or SEQ ID NO: 18 according to IMGT).

[0122] In various other embodiments, the anti-her2 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of trastuzumab, or the CDRs comprise one, two, three, four, five, or no more than six amino acid additions, deletions, or substitutions in HCDR1 (SEQ ID NO: 71 according to Kabat, or SEQ ID NO: 191 according to IMGT), HCDR2 (SEQ ID NO: 72 according to Kabat, or SEQ ID NO: 192 according to IMGT), HCDR3 (SEQ ID NO: 73 according to Kabat, or SEQ ID NO: 193 according to IMGT); LCDR1 (SEQ ID NO: 74 according to Kabat, or SEQ ID NO: 194 according to IMGT), LCDR2 (SEQ ID NO: 75 according to Kabat, or SEQ ID NO: 195 according to IMGT), and LCDR3 (SEQ ID NO: 76 according to Kabat, or SEQ ID NO: 196 according to IMGT).

[0123] In various embodiments, amino acid substitutions are single residue substitutions. Insertions typically range from about 1 to about 20 amino acid residues, although much larger insertions can be tolerated as long as biological function (e.g., binding to FRA or her2) is maintained. Deletions typically range from about 1 to about 20 amino acid residues, although in some cases deletions can be much larger. Substitutions, deletions, insertions, or any combination thereof, can be used to arrive at the final derivative or variant. Generally, these changes are made on a few amino acids to minimize alteration of the molecule, particularly the immunogenicity and specificity of the antigen-binding protein. However, larger changes can be tolerated in certain circumstances. Conservative substitutions are generally made according to the following chart, as shown in Table 10:

[0124] [Table 10]

[0125] Substantial changes in function or immunological identity are achieved by selecting substitutions that are less conservative than those shown in Table 10. For example, substitutions may be made that more significantly affect the structure of the polypeptide backbone in the region of the change, e.g., alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. In general, the substitutions predicted to produce the greatest change in the properties of a polypeptide are those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted with (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline is substituted with (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted with (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted with (or by) one having no side chain, e.g., glycine.

[0126] In various embodiments, when variant antibody sequences are used in ADCs, the variants typically exhibit the same qualitative biological activity and induce the same immune response, although, if necessary, variants can be selected to alter the characteristics of the antigen-binding protein. Alternatively, variants can be designed to alter the biological activity of the antigen-binding protein. For example, glycosylation sites can be altered or removed, as discussed herein.

[0127] A variety of antibodies may be used in the ADCs used herein to target cancer cells. As shown below, the linker-toxin in the ADCs disclosed herein is unexpectedly effective with various tumor antigen-targeting antibodies. Suitable antigens against which antibodies are directed that are expressed on tumor cells but not on healthy cells, or that are expressed at higher levels on tumor cells than on healthy cells, are known in the art. These antibodies can be used with the linkers and toxins (e.g., eribulin) disclosed herein. In some embodiments, the antibody moiety targets FRA. In some embodiments, the FRA-targeting antibody moiety is MORAb-003. In some embodiments, the linkers and toxins (eribulin) of the present disclosure were unexpectedly effective with several different tumor-targeting antibodies, with FRA-targeting antibody moieties such as MORAb-003 providing specific improvements in drug:antibody ratio, tumor targeting, bystander killing, treatment efficacy, and reduced off-target killing. Improved treatment efficacy can be measured in vitro or in vivo and can include a decrease in tumor growth rate and / or a decrease in tumor volume.

[0128] In certain embodiments, antibodies against other antigen targets are used, which provide at least some of the favorable functional properties of ADCs comprising an FRA-targeting antibody moiety such as MORAb-003 (e.g., improved drug:antibody ratio, improved treatment efficacy, reduced off-target killing, etc.). In some embodiments, some or all of these favorable functional properties are observed when the linkers and toxins (eribulin) of the present disclosure are conjugated to a her2-targeting antibody moiety such as trastuzumab. In some embodiments, the antibody moiety targets her2. In some embodiments, the her2-targeting antibody moiety is trastuzumab. In some embodiments, some or all of these favorable functional properties are observed when the linkers and toxins (eribulin) of the present disclosure are conjugated to an MSLN-targeting antibody moiety such as MORAb-009. In some embodiments, the antibody moiety targets MSLN. In some embodiments, the MSLN-targeting antibody moiety is MORAb-009.

[0129] Linker In various embodiments, the linker in the ADC is stable extracellularly so as to be sufficiently therapeutically effective. In some embodiments, the linker is stable extracellularly so that the ADC remains intact when present in extracellular conditions (e.g., before transport or delivery to a cell). The term "intact" as used in the context of an ADC means that the antibody moiety remains attached to the drug moiety. As used herein, "stable" in the context of a linker or an ADC comprising a linker means that no more than about 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 in a sample of the ADC (or any percentage therebetween) are cleaved when the ADC is present under extracellular conditions (or in cases where the entire ADC is not otherwise intact).

[0130] Whether a linker is stable extracellularly can be determined, for example, by placing the ADC in plasma for a predetermined period of time (e.g., 2, 4, 6, 8, 16, or 24 hours) and then quantifying the amount of free drug moiety present in the plasma. Stability can allow the ADC to localize to target tumor cells and prevent premature release of the drug, which would reduce the therapeutic index of the ADC by indiscriminately damaging both normal and tumor tissue. In some embodiments, the linker is stable outside the target cell but releases the drug moiety from the ADC inside the cell so that the drug moiety can bind to its target (e.g., to microtubules). Thus, an effective linker: (i) maintains the specific binding properties of the antibody moiety; (ii) enables delivery of the drug moiety, e.g., intracellular delivery, via stable attachment to the antibody moiety; (iii) remains stable and intact until the ADC is transported or delivered to its target site; and (iv) enables the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage.

[0131] Linkers can affect the physicochemical properties of ADCs. Because many cytotoxic drugs are hydrophobic in nature, conjugating them to antibodies with additional hydrophobic moieties can result in aggregation. ADC aggregates are insoluble and often limit the drug loading achievable on the antibody, which can negatively affect the efficacy of the ADC. Protein aggregation of biologics is also commonly linked to increased immunogenicity. As shown below, the linkers disclosed herein result in ADCs with low aggregation levels and desirable levels of drug loading.

[0132] Linkers may be "cleavable" or "non-cleavable" (Ducry and Stump, Bioconjugate Chem. (2010) 21:5-13). Cleavable linkers are designed to release the drug upon exposure to specific environmental factors, for example, upon internalization within target cells, while non-cleavable linkers generally rely on degradation of the antibody moiety itself.

[0133] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the drug moiety of the ADC is released by degradation of the antibody moiety. Non-cleavable linkers tend to remain covalently associated with at least one amino acid of the antibody and the drug upon internalization by and degradation within the target cell. Non-cleavable linkers generally comprise a thioether bond prepared by conjugation of a thiol group on the drug or antibody with a maleimide or haloacetamide group on the antibody or drug, respectively (Goldmacher et al., In Cancer Drug Discovery and Development: Antibody-Drug Conjugates and Immunotoxins (GLP Hillips ed., Springer, 2013)). Exemplary non-cleavable linkers include thioether, cyclohexyl, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-hydroxysuccinimide (NHS), or one or more polyethylene glycol (PEG) moieties, e.g., 1, 2, 3, 4, 5, or 6 PEG moieties. In some embodiments, the non-cleavable linker comprises (PEG). In other embodiments, the non-cleavable linker comprises (PEG).

[0134] In some embodiments, the linker is a cleavable linker. A cleavable linker refers to any linker that includes 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 that include a cleavable moiety may allow for release of the drug moiety from the ADC upon cleavage at a specific site within the linker. In various embodiments, cleavage of the antibody from the attached toxin activates or increases the activity of the toxin. In some embodiments, ADCs that include a cleavable linker (e.g., a Val-Cit linker) demonstrate increased on-target cell killing and / or decreased off-target cell killing compared to ADCs that include a non-cleavable linker (e.g., a non-cleavable (PEG)2 or (PEG)4 linker). In some embodiments, ADCs containing cleavable linkers exhibit improved therapeutic efficacy relative to ADCs containing non-cleavable linkers when cells and / or cancers treated with the ADCs do not express high levels of the target antigen (e.g., FRA or her2). In some embodiments, cleavage of the antibody from the attached toxin is necessary to achieve improved therapeutic efficacy of the ADC, as measured in vitro and / or in vivo.

[0135] 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 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 entry into the cell. In some embodiments, the linker comprises a cleavable moiety that is positioned such that, upon cleavage, no portion of the linker or antibody portion remains attached to the drug moiety. Exemplary cleavable linkers include acid-labile linkers, protease / peptidase-sensitive linkers, photolabile linkers, dimethyl-, disulfide-, or sulfonamide-containing linkers.

[0136] In some embodiments, the linker is a pH-sensitive linker, which is sensitive to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are cleavable under acidic conditions. This cleavage strategy generally takes advantage of the lower pH in endosomal (pH about 5-6) and lysosomal (pH about 4.8) intracellular compartments compared to the cytosol (pH about 7.4) to initiate hydrolysis of an acid-labile group in the linker, e.g., a hydrazone (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid-labile and / or hydrolytically unstable linker. For example, an acid-labile linker that is hydrolyzable in the lysosome and contains an acid-labile group (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) can be used. See, for example, U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 5,622,929; Dubowchik and Walker (1999) Pharm. Therapeutics 83:67-123; Neville et al. (1989) Biol. Chem. 264:14653-61. Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to a therapeutic agent via an acylhydrazone bond). See, for example, U.S. Patent No. 5,622,929.

[0137] In some embodiments, the linker is cleavable under reducing conditions. In some embodiments, the linker is cleavable in the presence of a reducing agent such as glutathione or dithiothreitol. In some embodiments, the linker is a cleavable disulfide linker or a cleavable sulfonamide linker.

[0138] In some embodiments, the linker is a cleavable disulfide linker. A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB, and SMPT. See, e.g., Thorpe et al. (1987) Cancer Res. 47:5924-31; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987). See also U.S. Patent No. 4,880,935. Disulfide linkers are typically used to take advantage of abundant intracellular thiols that can facilitate cleavage of these disulfide bonds. The intracellular concentration of reduced glutathione, the most abundant intracellular thiol, generally ranges from 1 to 10 nM, approximately 1,000-fold higher than the concentration of the most abundant small thiol in blood (i.e., cysteine), which is approximately 5 μM (Goldmacher et al., In Cancer Drug Discovery and Development: Antibody-Drug Conjugates and Immunotoxins (GLP Hillips ed., Springer, 2013)). Intracellular enzymes of the protein disulfide isomerase family can also contribute to intracellular cleavage of disulfide linkers. As used herein, a cleavable disulfide linker refers to any linker containing a cleavable disulfide moiety. The term "cleavable disulfide moiety" refers to a disulfide bond that can be cleaved and / or reduced, for example, by a thiol or enzyme. In some embodiments, the cleavable disulfide moiety is disulfidyl-dimethyl.

[0139] In some embodiments, the linker is a cleavable sulfonamide linker. As used herein, a cleavable sulfonamide linker refers to any linker that contains a cleavable sulfonamide moiety. The term "cleavable sulfonamide moiety" refers to a sulfonamide group, i.e., a sulfonyl group attached to an amine group, in which case the sulfur-nitrogen bond can be cleaved.

[0140] In some embodiments, the linker may be a dendritic-type linker for covalently linking more than one drug moiety to an antibody moiety via a branched polyfunctional linker moiety. See, e.g., Sun et al. (2002) Bioorg. Med. Chem. Lett. 12:2213-5; Sun et al. (2003) Bioorg. Med. Chem. 11:1761-8. Dendritic linkers can increase the molar ratio of drug to antibody, i.e., drug loading, which correlates with the efficacy of an ADC. Thus, for example, multiple drug moieties can be attached via a dendritic linker when the antibody moiety has only one reactive cysteine ​​thiol group. In some embodiments, the linker moiety or linker-drug moiety can be attached to the antibody via reduced disulfide crosslinking chemistry or limited lysine utilization techniques. See, e.g., International Publication Nos. WO2013173391 and WO2013173393.

[0141] In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker may be, for example, 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 includes a cleavable peptide moiety. The term "cleavable peptide moiety" refers to any chemically bonded crosslinking amino acid (natural or synthetic amino acid derivative) that can be cleaved by an agent present in the intracellular environment. For example, the linker may include an alanine-alanine-asparagine (Ala-Ala-Asn) sequence or a valine-citrulline (Val-Cit) sequence that is cleavable by a peptidase such as a cathepsin, e.g., cathepsin B.

[0142] In some embodiments, the linker is an enzyme-cleavable linker, and the cleavable peptide moiety in the linker is cleavable by an enzyme. In some embodiments, the cleavable peptide moiety is cleavable by a lysosomal enzyme, for example, a cathepsin. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the cleavable peptide moiety in the linker is cleavable by a lysosomal cysteine ​​cathepsin, for example, 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 can be cleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69). In some embodiments, ADCs comprising a cleavable peptide moiety demonstrate lower aggregation levels and / or higher drug loading (p) relative to ADCs comprising an alternative cleavable moiety (e.g., a cleavable disulfide moiety or a cleavable sulfonamide moiety).

[0143] In some embodiments, the linker or cleavable peptide moiety within the linker comprises an amino acid unit. In some embodiments, the amino acid unit, upon exposure to one or more intracellular proteases, e.g., one or more lysosomal enzymes, allows the protease to cleave the linker, thereby facilitating release of the drug moiety from the ADC (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, dipeptides, tripeptides, tetrapeptides, and pentapeptides. 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-Val), valine-alanine (Val-Ala), valine-lysine (Val-Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucine-citrulline (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), glycine-glycine-glycine (Gly-Gly-Gly), phenylalanine-phenylalanine-lysine (Phe-Phe-Lys), and glycine-phenylalanine-lysine (Gly-Phe-Lys). Other exemplary amino acid units include, but are not limited to, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-N, as described, for example, in U.S. Patent No. 6,214,345. 9 -tosyl-Arg, and Phe-N 9-nitro-Arg. In some embodiments, the amino acid unit in the linker comprises Val-Cit. In some embodiments, the amino acid unit in the linker comprises Ala-Ala-Asn. In some embodiments, ADCs comprising Val-Cit demonstrate reduced off-target cell killing, increased on-target cell killing, lower aggregation levels, and / or higher drug loading (p) relative to ADCs comprising alternative amino acid units or alternative cleavable moieties. The amino acid unit may comprise naturally occurring amino acid residues and / or minor amino acids and / or non-naturally occurring amino acid analogs, e.g., citrulline. The amino acid unit may be designed and optimized for enzymatic cleavage by specific enzymes, e.g., tumor-associated proteases, lysosomal proteases, e.g., cathepsin B, C, D, or S, or plasmin proteases.

[0144] In some embodiments, the linker in any of the ADCs disclosed herein may comprise at least one spacer unit connecting the antibody moiety to the drug moiety. In some embodiments, the spacer unit connects a cleavage site (e.g., a cleavable peptide moiety) in the linker to the antibody moiety. In some embodiments, the linker, and / or the spacer unit in the linker, is substantially hydrophilic. The use of a hydrophilic linker can reduce the extent to which the drug is effluxed from resistant cancer cells via multidrug resistance (MDR) or functionally similar transporters. In some aspects, the linker comprises one or more polyethylene glycol (PEG) moieties, e.g., 1, 2, 3, 4, 5, or 6 PEG moieties. In some embodiments, the linker is a short PEG linker, which provides improved stability and reduced aggregation over longer PEG linkers.

[0145] In some embodiments, the spacer unit in the linker comprises one or more PEG moieties. In some embodiments, the spacer unit is -(PEG) mand m is an integer between 1 and 10. In some embodiments, m is in the range of 1 to 10; 2 to 8; 2 to 6; 2 to 5; 2 to 4; or 2 to 3. In some embodiments, m is 8. In some embodiments, m is 4. In some embodiments, m is 3. In some embodiments, m is 2. In some embodiments, the spacer unit comprises (PEG)2, (PEG)4, (PEG)8, (PEG)9, (PEG)3-triazole-(PEG)3, (PEG)4-triazole-(PEG)3, or dibenzylcyclooctene-triazole-(PEG)3. In some preferred embodiments, the spacer unit comprises (PEG)2. In some embodiments, ADCs comprising short spacer units (e.g., (PEG)2) demonstrate lower aggregation levels and / or higher drug loading (p) relative to ADCs comprising long spacer units (e.g., (PEG)8).

[0146] In some embodiments, the spacer unit in the linker comprises an alkyl moiety. In some embodiments, the spacer unit comprises -(CH) n -, where n is an integer between 1 and 10 (i.e., n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n is 5. In some embodiments, ADCs comprising short spacer units (e.g., (CH2)5) demonstrate lower aggregation levels and / or higher drug loading (p) relative to ADCs comprising long spacer units (e.g., (PEG)8).

[0147] The spacer unit can be used, for example, to directly or indirectly link the antibody moiety to the drug moiety. In some embodiments, the spacer unit directly links the antibody moiety to the drug moiety. In some embodiments, the antibody moiety and drug moiety are linked via a spacer unit comprising one or more PEG moieties (e.g., (PEG)2 or (PEG)4). In some embodiments, the spacer unit indirectly links the antibody moiety to the drug moiety. In some embodiments, the spacer unit indirectly links the antibody moiety to the drug moiety via a cleavable moiety (e.g., a cleavable peptide, cleavable disulfide, or cleavable sulfonamide) and / or a linking moiety, e.g., a maleimide moiety, that connects the spacer unit to the antibody moiety.

[0148] The spacer unit, in various embodiments, is attached to the antibody moiety (i.e., antibody or antigen-binding fragment) via a maleimide moiety (Mal). In some embodiments, ADCs comprising linkers attached to the antibody moiety via a maleimide moiety demonstrate higher drug loading (p) relative to ADCs comprising linkers attached to the antibody moiety via an alternative linking moiety, such as a succinimide moiety.

[0149] A spacer unit attached to an antibody or antigen-binding fragment via a Mal is referred to herein as a "Mal-spacer unit." The term "maleimide moiety," as used herein, refers to a compound that contains a maleimide group and is reactive with sulfhydryl groups, such as the sulfhydryl groups of cysteine ​​residues on an antibody moiety. Other functional groups reactive with sulfhydryl groups (thiols) include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate. In some embodiments, a Mal-spacer unit is reactive with a cysteine ​​residue on an antibody or antigen-binding fragment. In some embodiments, a Mal-spacer unit connects to an antibody or antigen-binding fragment via a cysteine ​​residue. In some embodiments, a Mal-spacer unit comprises a PEG moiety. In some embodiments, a Mal-spacer unit comprises an alkyl moiety.

[0150] In certain embodiments, the linker comprises a 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 Ala-Ala-Asn. In some embodiments, the linker comprises a Mal-spacer unit and Val-Cit. In some embodiments, the linker comprises Mal-(PEG)2 and Val-Cit. In some embodiments, the linker comprises Mal-(PEG) m and Val-Cit, where m is 2 to 8, or 2 to 5, or 2, 3, 4, or 5. In some embodiments, the linker comprises Mal-(PEG)8 and Val-Cit. In certain embodiments, the linker comprises Mal-(CH2)5 and Val-Cit. In some embodiments, the linker comprises a Mal-spacer unit and Ala-Ala-Asn. In some embodiments, the linker comprises Mal-(PEG)2 and Ala-Ala-Asn.

[0151] In some embodiments, the linker comprises a Mal-spacer unit and a cleavable disulfide moiety. In some embodiments, the cleavable disulfide moiety is disulfidyl-dimethyl. In some embodiments, the linker comprises a Mal-spacer unit and disulfidyl-dimethyl. In some embodiments, the linker comprises Mal-(PEG)4-triazole-(PEG)3 and disulfidyl-dimethyl.

[0152] In some embodiments, the linker comprises a Mal-spacer unit and a cleavable sulfonamide moiety, hi some embodiments, the linker comprises Mal-(PEG)4-triazole-(PEG)3 and a sulfonamide.

[0153] In various embodiments, the spacer unit is attached to the antibody or antigen-binding fragment via a succinimide moiety (OSu). A spacer unit that is attached to the antibody or antigen-binding fragment via OSu is referred to herein as an "OSu-spacer unit." The term "succinimide moiety," as used herein, refers to a compound containing a succinimide compound that is reactive with amine groups, for example, amine groups of lysine residues on an antibody moiety. An exemplary succinimide moiety is N-hydroxysuccinimide (NHS). In some embodiments, the OSu-spacer unit is reactive with lysine residues on an antibody or antigen-binding fragment. In some embodiments, the OSu-spacer unit connects to the antibody or antigen-binding fragment via a lysine residue. In some embodiments, the OSu-spacer unit comprises a PEG moiety. In some embodiments, the OSu-spacer unit comprises an alkyl moiety.

[0154] In certain embodiments, the linker comprises an OSu-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 Ala-Ala-Asn. In some embodiments, the linker comprises an OSu-spacer unit and Val-Cit. In some embodiments, the linker comprises OSu-(PEG)2 and Val-Cit. In other embodiments, the linker comprises OSu-(PEG)9 and Val-Cit. In other embodiments, the linker comprises OSu-(CH2)5 and Val-Cit. In certain embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3 and Val-Cit. In some embodiments, the linker comprises an OSu-spacer unit and Ala-Ala-Asn. In some embodiments, the linker comprises OSu-(PEG)2 and Ala-Ala-Asn.

[0155] In some embodiments, the linker comprises an OSu-spacer unit and a cleavable disulfide moiety. In some embodiments, the cleavable disulfide moiety is disulfidyl-dimethyl. In some embodiments, the linker comprises an OSu-spacer unit and disulfidyl-dimethyl. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3 and disulfidyl-dimethyl. In other embodiments, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3 and disulfidyl-dimethyl.

[0156] In some embodiments, the linker comprises an OSu-spacer unit and a cleavable sulfonamide moiety. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3 and a sulfonamide. In other embodiments, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3 and a sulfonamide.

[0157] In some embodiments, a Mal-spacer unit or an OSu-spacer unit links an antibody moiety (i.e., an antibody or antigen-binding fragment) to a cleavable moiety within the linker. In some embodiments, a Mal-spacer unit or an OSu-spacer unit links an antibody or antigen-binding fragment to a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises a Mal-spacer unit-amino acid unit or an OSu-spacer unit-amino acid unit. In some embodiments, the Mal-spacer unit or the OSu-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit or the OSu-spacer unit comprises an alkyl moiety. In some embodiments, the amino acid unit comprises Val-Cit. In other embodiments, the amino acid unit comprises Ala-Ala-Asn.

[0158] In some embodiments, the linker comprises the structure: Mal-spacer unit-Val-Cit. In some embodiments, the linker comprises the structure: Mal-(PEG)2-Val-Cit. In some embodiments, the linker comprises the structure: Mal-(PEG)2-Val-Cit-pAB. In some embodiments, the linker comprises Mal-(PEG)8-Val-Cit. In certain embodiments, the linker comprises Mal-(CH2)5-Val-Cit. In some embodiments, the linker comprises Mal-spacer unit-Ala-Ala-Asn. In some embodiments, the linker comprises Mal-(PEG)2-Ala-Ala-Asn.

[0159] In some embodiments, the linker comprises OSu-spacer unit-Val-Cit. In some embodiments, the linker comprises OSu-(PEG)2-Val-Cit. In other embodiments, the linker comprises OSu-(PEG)9-Val-Cit. In other embodiments, the linker comprises OSu-(CH2)5-Val-Cit. In other embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3-Val-Cit. In some embodiments, the linker comprises OSu-spacer unit-Ala-Ala-Asn. In some embodiments, the linker comprises OSu-(PEG)2-Ala-Ala-Asn.

[0160] In various embodiments, a Mal-spacer unit or an OSu-spacer unit links the antibody or antigen-binding fragment to the cleavable disulfide moiety. In some embodiments, the linker comprises a Mal-spacer unit-disulfide or an OSu-spacer unit-disulfide. In some embodiments, the disulfide is disulfidyl-dimethyl. In some embodiments, the linker comprises a Mal-spacer unit-disulfidyl-dimethyl. In some embodiments, the linker comprises a Mal-(PEG)4-triazole-(PEG) 3- In other embodiments, the linker comprises OSu-spacer unit-disulfidyl-dimethyl. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG) 3- In another embodiment, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3-disulfidyl-dimethyl.

[0161] In certain embodiments, a Mal-spacer unit or an OSu-spacer unit links the antibody or antigen-binding fragment to a cleavable sulfonamide moiety. In some embodiments, the linker comprises a Mal-spacer unit-sulfonamide or an OSu-spacer unit-sulfonamide. In some embodiments, the linker comprises Mal-(PEG)4-triazole-(PEG)3-sulfonamide. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3-sulfonamide. In other embodiments, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3-sulfonamide.

[0162] In various embodiments, the cleavable moiety in the linker is directly connected to the drug moiety. In other embodiments, a separate spacer unit is used to connect the cleavable moiety in the linker to the drug moiety. In various embodiments, the drug moiety is eribulin. In various embodiments, eribulin is connected to the cleavable moiety in the linker by a spacer unit. In some embodiments, eribulin is connected to the cleavable moiety in the linker by a self-immolative spacer unit. In certain embodiments, eribulin is connected to the cleavable moiety in the linker by a self-immolative spacer unit, the cleavable moiety comprises Val-Cit, and an additional spacer unit comprising PEG connects the cleavable moiety to the antibody moiety. In certain embodiments, eribulin is connected to the anti-FRA antibody via a Mal-spacer unit in the linker connected to the Val-Cit cleavable moiety and the pAB self-immolative spacer unit. In certain other embodiments, eribulin is connected to the anti-her2 antibody via a Mal-spacer unit in the linker connected to the Val-Cit cleavable moiety and the pAB self-immolative spacer unit.

[0163] A spacer unit may be "self-immolative" or "non-self-immolative." A "non-self-immolative" spacer unit is one in which some or all of the spacer unit remains attached to the drug moiety when the linker is cleaved. Examples of non-self-immolative spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. A non-self-immolative spacer unit may eventually degrade over time, but does not readily release the original drug attached thereto completely under cellular conditions. A "self-immolative" spacer unit allows the original drug moiety to be released under intracellular conditions. A "original drug" is one in which no part of the spacer unit or other chemically modified moiety remains after cleavage / degradation of the spacer unit.

[0164] Self-immolative chemistries are known in the art and can be readily selected for the ADCs of the present disclosure. In various embodiments, the spacer unit connecting the cleavable moiety in the linker to the drug moiety (e.g., eribulin) is self-immolative and self-destructs simultaneously with, or shortly before / after, cleavage of the cleavable moiety under intracellular conditions.

[0165] In certain embodiments, the self-immolative spacer unit in the linker comprises a p-aminobenzyl unit. In some embodiments, p-aminobenzyl alcohol (pABOH) 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 created between pABOH 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-aminobenzyloxycarbonyl (pAB). Without being bound by theory, the self-immolation of pAB is thought to involve a spontaneous 1,6-elimination reaction (Jain et al. (2015) Pharm Res 32:3526-40).

[0166] In various embodiments, the structure of p-aminobenzyloxycarbonyl (pAB) used in the ADCs of the disclosure is shown below: [ka]

[0167] In various embodiments, a self-immolative spacer unit connects a cleavable moiety in the linker to the C-35 amine on eribulin. In some embodiments, the self-immolative spacer unit is pAB. In some embodiments, pAB connects a cleavable moiety in the linker to the C-35 amine on eribulin. In some embodiments, pAB self-immolates upon cleavage of the cleavable moiety, releasing eribulin from the ADC in its native, active form. In some embodiments, an anti-FRA antibody (e.g., MORAb-003) is connected to the C-35 amine of eribulin by a linker comprising Mal-(PEG)2-Val-Cit-pAB. In other embodiments, an anti-her2 antibody (e.g., trastuzumab) is connected to the C-35 amine of eribulin by a linker comprising Mal-(PEG)2-Val-Cit-pAB.

[0168] In some embodiments, pAB self-destructs upon cleavage of the cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises the amino acid unit -pAB. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pAB(VCP). In certain embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pAB.

[0169] In some embodiments, the pAB self-destructs upon cleavage of a cleavable disulfide moiety in the linker. In some embodiments, the linker comprises disulfide-pAB. In some embodiments, the linker comprises disulfidyl-dimethyl-pAB.

[0170] In some embodiments, the pAB self-destructs upon cleavage of the cleavable sulfonamide moiety in the linker, hi some embodiments, the linker comprises a sulfonamide-pAB.

[0171] In various aspects, the antibody portion of the ADC is conjugated to the drug portion via a linker, wherein the linker comprises a Mal-spacer unit, a cleavable amino acid unit, and pAB. In some embodiments, the spacer unit comprises a PEG moiety. In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the linker comprises Mal-(PEG)2-amino acid unit-pAB. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit-pAB. In other embodiments, the linker comprises Mal-(PEG)2-Ala-Ala-Asn-pAB. In some embodiments, the linker comprises Mal-(PEG)8-amino acid unit-pAB. In some embodiments, the linker comprises Mal-(PEG)8-Val-Cit-pAB. In some embodiments, the linker comprises Mal-(CH2)5-amino acid unit-pAB. In some embodiments, the linker comprises Mal-(CH2)5-Val-Cit-pAB.

[0172] In various embodiments, the antibody portion of the ADC is conjugated to the drug portion via a linker, wherein the linker comprises Mal-spacer unit-disulfide-pAB. In some embodiments, the spacer unit comprises a PEG moiety. In some embodiments, the linker comprises Mal-(PEG)4-triazole-(PEG)3-disulfide-pAB. In some embodiments, the linker comprises Mal-(PEG)4-triazole-(PEG)3-disulfidyl-dimethyl-pAB.

[0173] In some embodiments, the antibody portion of the ADC is conjugated to the drug portion via a linker, wherein the linker comprises Mal-spacer unit-sulfonamide-pAB. In some embodiments, the spacer unit comprises a PEG moiety. In some embodiments, the linker comprises Mal-(PEG)4-triazole-(PEG)3-sulfonamide-pAB.

[0174] In some aspects, the antibody portion of the ADC is conjugated to the drug portion via a linker, wherein the linker comprises OSu-spacer unit-amino acid unit-pAB. In some embodiments, the spacer unit comprises a PEG moiety. In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the linker comprises OSu-(PEG)2-amino acid unit-pAB. In some embodiments, the linker comprises OSu-(PEG)2-Val-Cit-pAB. In other embodiments, the linker comprises OSu-(PEG)2-Ala-Ala-Asn-pAB. In some embodiments, the linker comprises OSu-(PEG)9-amino acid unit-pAB. In some embodiments, the linker comprises OSu-(PEG)9-Val-Cit-pAB. In some embodiments, the linker comprises OSu-(CH2)5-amino acid unit-pAB. In some embodiments, the linker comprises OSu-(CH2)5-Val-Cit-pAB. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3-amino acid unit-pAB, hi some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3-Val-Cit-pAB.

[0175] In some embodiments, the antibody portion of the ADC is conjugated to the drug portion via a linker, wherein the linker comprises OSu-spacer unit-disulfide-pAB. In some embodiments, the spacer unit comprises a PEG moiety. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3-disulfide-pAB. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3-disulfidyl-dimethyl-pAB. In some embodiments, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3-disulfide-pAB. In some embodiments, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3-disulfidyl-dimethyl-pAB.

[0176] In some embodiments, the antibody portion of the ADC is conjugated to the drug portion via a linker, wherein the linker comprises OSu-spacer unit-sulfonamide-pAB. In some embodiments, the spacer unit comprises a PEG moiety. In some embodiments, the linker comprises OSu-(PEG)3-triazole-(PEG)3-sulfonamide-pAB. In some embodiments, the linker comprises OSu-dibenzylcyclooctene-triazole-(PEG)3-sulfonamide-pAB.

[0177] In various embodiments, the linker is designed to promote bystander killing (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, thereby reducing toxicity to off-target tissues (e.g., non-cancerous tissues), while maintaining ADC binding to the target tissue and bystander killing of cancerous tissues that do not express the antigen targeted by the antibody portion of the ADC but that surround the target cancer tissue that does express the antigen. In some embodiments, linkers comprising a maleimide moiety (Mal), a polyethylene glycol (PEG) moiety, a valine-citrulline (Val-Cit or "vc"), and pAB provide these functional characteristics. In some embodiments, a linker comprising Mal-(PEG)2-Val-Cit-pAB is particularly effective in achieving these functional characteristics, e.g., when connecting an anti-FRA antibody moiety, such as MORAb-003, and a drug moiety, such as eribulin. In some embodiments, at least some of these functional characteristics may also be observed without the anti-FRA antibody moiety and / or without MORAb-003. For example, in some embodiments, a linker comprising Mal-(PEG)2-Val-Cit-pAB is effective in achieving some or all of these functional characteristics, e.g., when connecting an anti-her2 antibody moiety, such as trastuzumab, and a drug moiety, such as eribulin.

[0178] In some embodiments, the antibody moiety is conjugated to the drug moiety via a linker comprising a maleimide moiety (Mal), a polyethylene glycol (PEG) moiety, valine citrulline (Val-Cit or "vc"), and pAB. In these embodiments, the maleimide moiety covalently attaches the linker-drug moiety to the antibody moiety, and pAB acts as a self-immolative spacer unit. Such linkers may be referred to as "m-vc-pAB" linkers, "Mal-VCP" linkers, "Mal-(PEG)2-VCP" linkers, or "Mal-(PEG)2-Val-Cit-pAB" linkers. In some embodiments, the drug moiety is eribulin. The structure of Mal-(PEG)2-Val-Cit-pAB-eribulin is shown in Table 46. pAB of the Mal-(PEG)2-Val-Cit-pAB linker is attached to the C-35 amine on eribulin.

[0179] ADCs comprising Mal-(PEG)2-Val-Cit-pAB-eribulin, particularly when paired with an anti-FRA antibody such as MORAb-003 or an antigen-binding fragment thereof, have been discovered to demonstrate a particular combination of desirable properties, including, but not limited to, effective levels of drug loading (p ≈4), low levels of aggregation, stability under storage conditions or in the systemic circulation (e.g., serum stability), maintenance of affinity for target-expressing cells comparable to unconjugated antibodies, potent cytotoxicity against target-expressing cells, low levels of off-target cell killing, high levels of bystander killing, and / or effective in vivo anti-cancer activity, all compared to ADCs using other linker-toxin and / or antibody moieties. While numerous linker options and spacer and cleavage site combinations are known in the art and may offer particular benefits in one or more of these functional categories, the particular combination of Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to an antibody moiety, such as an anti-FRA antibody (e.g., MORAb-003), may offer good or excellent properties across a wide range of functional properties desirable for therapeutic ADCs. In some embodiments, the good or excellent functional properties offered by the particular combination of Mal-(PEG)2-Val-Cit-pAB linker connecting eribulin to an antibody moiety may be observed with this linker-toxin conjugated to, for example, an anti-her2 antibody, such as trastuzumab.

[0180] In some embodiments, an ADC comprises Mal-(PEG)2-Val-Cit-pAB-eribulin and an antibody portion comprising an internalizing antibody or antigen-binding fragment thereof that retains the ability to target and be internalized into tumor cells. In some embodiments, an ADC comprises Mal-(PEG)2-Val-Cit-pAB-eribulin and an internalizing antibody or internalizing antigen-binding fragment thereof that targets FRA-expressing tumor cells. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof that targets FRA-expressing tumor cells comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:2 (HCDR1), SEQ ID NO:3 (HCDR2), and SEQ ID NO:4 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:7 (LCDR1), SEQ ID NO:8 (LCDR2), and SEQ ID NO:9 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3). In some embodiments, an internalizing antibody or internalizing antigen-binding fragment thereof that targets FRA-expressing tumor cells comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, an internalizing antibody or internalizing antigen-binding fragment thereof that targets FRA-expressing tumor cells comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain.

[0181] In some embodiments, the ADC has Formula I: Ab-(LD) p (I) [In the formula, (i) the Ab is an internalizing anti-folate receptor alpha (FRA) antibody or an internalizing antigen-binding fragment thereof, which comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:2 (HCDR1), SEQ ID NO:3 (HCDR2), and SEQ ID NO:4 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:7 (LCDR1), SEQ ID NO:8 (LCDR2), and SEQ ID NO:9 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3); (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; (iv) p is an integer from 1 to 20.

[0182] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the internalizing antibody is MORAb-003. In some embodiments, p is 1 to 8, or 1 to 6. In some embodiments, p is 2 to 8, or 2 to 5. In some embodiments, p is 3 to 4. In some embodiments, p is 4.

[0183] In another embodiment, the ADC comprises Mal-(PEG)2-Val-Cit-pAB-eribulin and an internalizing antibody or internalizing antigen-binding fragment thereof that targets her2-expressing tumor cells. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof that targets her2-expressing tumor cells comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 71 (HCDR1), SEQ ID NO: 72 (HCDR2), and SEQ ID NO: 73 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 74 (LCDR1), SEQ ID NO: 75 (LCDR2), and SEQ ID NO: 76 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 191 (HCDR1), SEQ ID NO: 192 (HCDR2), and SEQ ID NO: 193 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 194 (LCDR1), SEQ ID NO: 195 (LCDR2), and SEQ ID NO: 196 (LCDR3). In some embodiments, an internalizing antibody or internalizing antigen-binding fragment thereof that targets her2-expressing tumor cells comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, an internalizing antibody or internalizing antigen-binding fragment thereof that targets her2-expressing tumor cells comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain.

[0184] In some embodiments, the ADC has Formula I: Ab-(LD) p (I) [In the formula, (i) the Ab is an internalizing anti-human epidermal growth factor receptor 2 (her2) antibody or an internalizing antigen-binding fragment thereof, which comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:71 (HCDR1), SEQ ID NO:72 (HCDR2), and SEQ ID NO:73 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:74 (LCDR1), SEQ ID NO:75 (LCDR2), and SEQ ID NO:76 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:191 (HCDR1), SEQ ID NO:192 (HCDR2), and SEQ ID NO:193 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:194 (LCDR1), SEQ ID NO:195 (LCDR2), and SEQ ID NO:196 (LCDR3); (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; (iv) p is an integer from 1 to 20.

[0185] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the internalizing antibody is trastuzumab. In some embodiments, p is 1 to 8, or 1 to 6. In some embodiments, p is 2 to 8, or 2 to 5. In some embodiments, p is 3 to 4. In some embodiments, p is 4.

[0186] In another embodiment, the ADC comprises Mal-(PEG)2-Val-Cit-pAB-eribulin and an internalizing antibody or internalizing antigen-binding fragment thereof that targets mesothelin (MSLN)-expressing tumor cells. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof that targets MSLN-expressing tumor cells comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 65 (HCDR1), SEQ ID NO: 66 (HCDR2), and SEQ ID NO: 67 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 68 (LCDR1), SEQ ID NO: 69 (LCDR2), and SEQ ID NO: 70 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 185 (HCDR1), SEQ ID NO: 186 (HCDR2), and SEQ ID NO: 187 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 188 (LCDR1), SEQ ID NO: 189 (LCDR2), and SEQ ID NO: 190 (LCDR3). In some embodiments, an internalizing antibody or internalizing antigen-binding fragment thereof that targets MSLN-expressing tumor cells comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an internalizing antibody or internalizing antigen-binding fragment thereof that targets MSLN-expressing tumor cells comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain.

[0187] In some embodiments, the ADC has Formula I: Ab-(LD) p (I) [In the formula, (i) the Ab is an internalizing anti-mesothelin antibody or an internalizing antigen-binding fragment thereof, which comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 65 (HCDR1), SEQ ID NO: 66 (HCDR2), and SEQ ID NO: 67 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 68 (LCDR1), SEQ ID NO: 69 (LCDR2), and SEQ ID NO: 70 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 185 (HCDR1), SEQ ID NO: 186 (HCDR2), and SEQ ID NO: 187 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 188 (LCDR1), SEQ ID NO: 189 (LCDR2), and SEQ ID NO: 190 (LCDR3); (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; (iv) p is an integer from 1 to 20.

[0188] In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the internalizing antibody is MORAb-003, MORAb-009, or trastuzumab. In some embodiments, p is 1 to 8, or 1 to 6. In some embodiments, p is 2 to 8, or 2 to 5. In some embodiments, p is 3 to 4. In some embodiments, p is 4.

[0189] Drug portion The drug moiety (D) of the ADCs described herein can be any chemotherapeutic agent. A useful group of chemotherapeutic agents includes, for example, antitubulin agents. In certain embodiments, the drug moiety is an antitubulin agent. Examples of antitubulin agents include cryptophycin and eribulin. A preferred drug moiety for use in the described ADCs is eribulin.

[0190] In various embodiments, the drug moiety is eribulin. In these embodiments, the linker of the ADC is attached through the C-35 amine on eribulin.

[0191] In various embodiments, the native form of eribulin used to connect the linker and antibody moiety is shown below: [ka]

[0192] In certain embodiments, an intermediate that is a precursor to the linker is reacted with a drug moiety under appropriate conditions. In certain embodiments, a reactive group is used on the drug and / or the intermediate or linker. The product of the reaction between the drug and the intermediate, the derivatized drug, is then reacted with an antibody or antigen-binding fragment under appropriate conditions. Alternatively, the linker or intermediate may be first reacted with the antibody or derivatized antibody, and then reacted with the drug or derivatized drug.

[0193] Several different reactions are available for covalently linking drugs and / or linkers to antibody moieties. This is often achieved by reaction of one or more amino acid residues on 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 various moieties of aromatic amino acids. For example, nonspecific covalent linkages may be achieved using the carbodiimide reaction to couple carboxy (or amino) groups on a compound to amino (or carboxy) groups on the antibody moiety. In addition, bifunctional agents such as dialdehydes or imidoesters may be used to couple amino groups on a compound to amino groups on the antibody moiety. The Schiff base reaction can also be used to couple drugs to binding agents. This method involves periodate oxidation of a drug containing a glycol or hydroxy group to form an aldehyde, which is then reacted with the binding agent. Linkage occurs through the formation of a Schiff base with the amino group of the binding agent. Isothiocyanates can also be used as coupling agents to covalently link drugs to binding agents. Other techniques are known to those skilled in the art and are within the scope of this disclosure.

[0194] Drug burden Drug loading is represented by p, also referred to herein as the drug-to-antibody ratio (DAR). Drug loading can range from 1 to 20 drug moieties per antibody moiety. In some embodiments, p is an integer from 1 to 20. In some embodiments, p is an integer from 1 to 10, 1 to 9, 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 10, 2 to 9, 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 3 to 4. In other embodiments, p is 1, 2, 3, 4, 5, or 6, preferably 3 or 4.

[0195] 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 is attached 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 through a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., at the N- or C-terminus, to the epsilon-amino group of one or more lysine residues, to the free carboxylic acid groups of one or more glutamic or aspartic acid residues, or to the sulfhydryl groups of one or more cysteine ​​residues). The linker attachment site may be a native residue in the amino acid sequence of the antibody moiety or may have been introduced into the antibody moiety by, for example, recombinant DNA techniques (e.g., by introducing a cysteine ​​residue into the amino acid sequence) or protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

[0196] 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, if the linkage is a cysteine ​​thiol group, the antibody may have only one or a few cysteine ​​thiol groups, or only one or a few sufficiently reactive thiol groups through which a linker can be attached. Generally, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be conjugated to a drug moiety. In fact, many cysteine ​​thiol residues in antibodies exist as disulfide bridges. Over-conjugation of linker-toxins to antibodies can destabilize the antibody by reducing the number of cysteine ​​residues available to form disulfide bridges. Therefore, the optimal drug:antibody ratio should increase the potency of the ADC (by increasing the number of conjugated drug moieties per antibody) without destabilizing the antibody moiety. In some embodiments, the optimal ratio may be about 3-4.

[0197] In some embodiments, linkers attached to the antibody moiety via a Mal moiety exhibit a ratio of about 3 to 4. In some embodiments, linkers attached to the antibody moiety via an alternative moiety (e.g., an OSu moiety) may exhibit a lower optimal ratio (e.g., a lower ratio, such as about 0 to 3). In some embodiments, linkers comprising a short spacer unit (e.g., a short PEG spacer unit such as (PEG)2 or (PEG)4, or a short alkyl spacer unit such as (CH2)5) exhibit a ratio of about 3 to 4. In some embodiments, linkers comprising a long spacer unit (e.g., (PEG)8) may exhibit a lower optimal ratio (e.g., a lower ratio, such as about 0 to 3). In some embodiments, linkers comprising a peptide cleavable moiety exhibit a ratio of about 3 to 4. In some embodiments, linkers comprising an alternative cleavable moiety (e.g., a cleavable disulfide or cleavable sulfonamide) may exhibit a lower optimal ratio (e.g., a lower ratio, such as about 0 to 3). In some embodiments, ADCs comprising Mal-(PEG)2-Val-Cit-pAB-eribulin linked to an antibody, such as an anti-FRA antibody (e.g., MORAb-003), have a ratio of about 3 to 4. In some embodiments, a ratio of about 3 to 4 is observed for ADCs comprising Mal-(PEG)2-Val-Cit-pAB-eribulin linked to a different antibody, such as an anti-her2 antibody (e.g., trastuzumab). In some embodiments, the optimal ratio observed for ADCs comprising Mal-(PEG)2-Val-Cit-pAB-eribulin linker-toxin is antibody-independent.

[0198] In some embodiments, the antibody moiety is exposed to reducing conditions prior to conjugation to generate one or more free cysteine ​​residues. In some embodiments, the antibody can 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 can be generated by partial reduction with a limited molar equivalent of TCEP, which preferentially reduces the interchain disulfide bonds connecting the light and heavy chains (one pair per HL pairing) and the two heavy chains in the hinge region (two pairs per HH pairing in the case of human IgG1) while leaving the intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol. Biol. 1045:145-71). In embodiments, disulfide bonds within the antibody are reduced electrochemically, for example, by using a working electrode that applies alternating reduction and oxidation voltages. This approach allows for on-line coupling of disulfide bond reduction to an analytical device (e.g., an electrochemical detection device, NMR spectrometer, or mass spectrometer) or chemical separation device (e.g., a liquid chromatograph (e.g., HPLC) or an electrophoresis device (see, e.g., U.S. Patent Application Publication No. 20140069822)). In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups on amino acid residues, e.g., lysine or cysteine.

[0199] The drug loading of an ADC can be controlled in various ways, for example, (i) by limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody; (ii) by limiting the conjugation reaction time or temperature; (iii) by partially or limiting the reducing conditions for cysteine ​​thiol modification; and / or (iv) by recombinantly manipulating the amino acid sequence of the antibody so that the number and position of cysteine ​​residues are modified to control the number and / or position of linker-drug bonds.

[0200] In some embodiments, a free cysteine ​​residue is introduced into the amino acid sequence of the antibody moiety. For example, cysteine-engineered antibodies can be prepared in which one or more amino acids of a parent antibody are replaced with cysteine ​​amino acids. Any form of antibody can be engineered, i.e., mutated, in this manner. For example, a parent Fab antibody fragment can be engineered to form a cysteine-engineered Fab, referred to as a "ThioFab." Similarly, a parent monoclonal antibody can be engineered to form a "ThioMab." Single-site mutation results in a single engineered cysteine ​​residue in a ThioFab, and single-site mutation results in two engineered cysteine ​​residues in a ThioMab due to the dimeric nature of IgG antibodies. DNA encoding amino acid sequence variants of a parent polypeptide can be prepared by various methods known in the art (see, for example, the methods described in WO 2006 / 034488). These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of initially prepared DNA encoding the polypeptide. Recombinant antibody variants can also be constructed by restriction fragment engineering or overlap extension PCR with synthetic oligonucleotides. ADCs of Formula I include, but are not limited to, antibodies with one, two, three, or four engineered cysteine ​​amino acids (Lyon et al. (2012) Methods Enzymol. 502:123-38). In some embodiments, without engineering, one or more free cysteine ​​residues are already present in the antibody moiety, in which case the existing free cysteine ​​residues can be used to conjugate the antibody moiety to the drug moiety.

[0201] In some embodiments, high drug loading (e.g., p>5) may result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. High drug loading may also negatively impact the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, low drug loading (e.g., p<3) may reduce the potency of certain ADCs against target-expressing cells and / or bystander cells. In some embodiments, drug loading in ADCs of the present disclosure ranges from 1 to about 8; from about 2 to about 6; from about 2 to about 5; from about 3 to about 5; or from about 3 to about 4.

[0202] When more than one nucleophilic group reacts with a drug-linker intermediate or linker moiety reagent, followed by a drug moiety reagent, in a reaction mixture containing multiple copies of an antibody moiety and a linker moiety, 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 some embodiments, the drug loading in the mixture of ADCs resulting from the conjugation reaction ranges from 1 to 20 attached drug moieties per antibody moiety. The average number of drug moieties per antibody moiety (i.e., average drug loading, or average p) can be calculated by any conventional method known in the art, for example, 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 about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or about 3.3 to about 3.7. In some embodiments, the average number of drug moieties per antibody moiety is about 3.2 to about 3.8. In some embodiments, the average number of drug moieties per antibody moiety is about 3.8. In some embodiments, the average number of drug moieties per antibody moiety is 3 to 4; 3.1 to 3.9; 3.2 to 3.8; 3.2 to 3.7; 3.2 to 3.6; 3.3 to 3.8; or 3.3 to 3.7. In some embodiments, the average number of drug moieties per antibody moiety is 3.2 to 3.8. In some embodiments, the average number of drug moieties per antibody moiety is 3.8.

[0203] In some embodiments, the average number of drug moieties per antibody moiety is about 3.5 to about 4.5; about 3.6 to about 4.4; about 3.7 to about 4.3; about 3.7 to about 4.2; or about 3.8 to about 4.2. In some embodiments, the average number of drug moieties per antibody moiety is about 3.6 to about 4.4. In some embodiments, the average number of drug moieties per antibody moiety is about 4.0. In some embodiments, the average number of drug moieties per antibody moiety is 3.5 to 4.5; 3.6 to 4.4; 3.7 to 4.3; 3.7 to 4.2; or 3.8 to 4.2. In some embodiments, the average number of drug moieties per antibody moiety is 3.6 to 4.4. In some embodiments, the average number of drug moieties per antibody moiety is 4.0.

[0204] In various embodiments, the term "about" when used in reference to the average number of drug moieties per antibody moiety means + / - 10%.

[0205] Individual ADC compounds, or "species," can be identified in a mixture by mass spectrometry and separated by UPLC or HPLC, e.g., hydrophobic interaction chromatography (HIC-HPLC). In certain embodiments, homogeneous or nearly homogeneous ADCs having a single loading value can be isolated from a conjugation mixture, e.g., by electrophoresis or chromatography.

[0206] In some embodiments, a drug loading and / or average drug loading of about 4 results in advantageous properties. In some embodiments, a drug loading and / or average drug loading of less than about 4 may result in unacceptably high levels of unconjugated antibody species, which may compete with the ADC for binding to the target antigen and / or may result in reduced treatment efficacy. In some embodiments, a drug loading and / or average drug loading greater than about 4 may result in unacceptably high levels of product heterogeneity and / or ADC aggregation. A drug loading and / or average drug loading greater than about 4 may also affect the stability of the ADC due to loss of one or more chemical bonds necessary to stabilize the antibody moiety.

[0207] In some embodiments, the ADC has Formula I: Ab-(LD) p (I) [In the formula, (i) the Ab is an internalizing anti-folate receptor alpha antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; (iv) p is an integer of 3 to 4.

[0208] In other embodiments, the ADC has Formula I: Ab-(LD) p (I) [In the formula, (i) the Ab is an internalizing anti-human epidermal growth factor receptor 2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28; (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; (iv) p is an integer of 3 to 4.

[0209] In some embodiments, p is 4.

[0210] The present disclosure includes methods for producing the described ADCs. Briefly, an ADC comprises an antibody or antigen-binding fragment as the antibody portion, a drug moiety, and a linker connecting the drug moiety and the antibody portion. In some embodiments, the ADC can be prepared using a linker with a reactive functional group for covalently linking the drug moiety and the antibody portion. For example, in some embodiments, a cysteine ​​thiol of the antibody portion can form a bond with a reactive functional group (e.g., a maleimide moiety) of a linker or a drug-linker intermediate to produce an ADC. Generation of an ADC can be achieved by any technique known to those of skill in the art.

[0211] In some embodiments, ADCs are produced by sequentially contacting the antibody moiety with a linker and a drug moiety, such that the antibody moiety is first covalently bound to the linker and then the preformed antibody-linker intermediate is reacted with the drug moiety. The antibody-linker intermediate may or may not be subjected to a purification step before contacting the drug moiety. In other embodiments, ADCs are produced by contacting the antibody moiety with a linker-drug compound that is preformed by reacting the linker with the drug moiety. The preformed linker-drug compound may or may not be subjected to a purification step before contacting the antibody moiety. In other embodiments, the antibody moiety is contacted with the linker and drug moiety in one reaction mixture, allowing for simultaneous formation of covalent bonds between the antibody moiety and the linker and between the linker and the drug moiety. This method of producing ADCs may involve contacting the antibody moiety with the antibody moiety before adding the linker to the reaction mixture, or vice versa. In certain embodiments, ADCs are produced by reacting an antibody moiety with a linker attached to a drug moiety, such as Mal-(PEG)2-Val-Cit-pAB-Eribulin, under conditions that allow conjugation.

[0212] The ADC prepared according to the above method may be subjected to a purification step. The purification step may include any biochemical method known in the art for purifying proteins, or any combination of such methods. These include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge- or isoelectric-based chromatography, mixed-mode chromatography, e.g., CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size-exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

[0213] Therapeutic Uses and Compositions Disclosed herein are methods of using the ADCs of the present disclosure in treating a subject for a disorder, e.g., a tumor disorder. The ADCs may be administered alone or in combination with a second therapeutic agent, and may be administered in any pharmaceutically acceptable formulation, dosage, and administration regimen. ADC treatment efficacy may be assessed for toxicity and for efficacy indicators and adjusted accordingly. Measures of efficacy include, but are not limited to, cytostatic and / or cytotoxic effects observed in vitro or in vivo, reduction in tumor volume, inhibition of tumor growth, and / or sustained survival.

[0214] Methods for determining whether an ADC exerts a cytostatic and / or cytotoxic effect on cells are known. For example, the cytotoxic or cytostatic activity of an ADC can be measured by exposing it to mammalian cells expressing the target protein of the ADC in cell culture medium, culturing the cells for 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, and induction of apoptosis (caspase activation) by the ADC.

[0215] A thymidine incorporation assay may be used to determine whether an antibody-drug conjugate exerts a cytostatic effect. For example, cancer cells expressing a target antigen are cultured in a 96-well plate at a density of 5,000 cells / well for 72 hours, and then 0.5 μCi of thymidine in the last 8 hours of the 72-hour period. 3 H-thymidine can be exposed to cultured cells. 3 H-thymidine incorporation is measured in the presence and absence of ADC.

[0216] To determine cytotoxicity, necrosis or apoptosis (programmed cell death) may be measured. Necrosis is typically achieved by increased plasma membrane permeability; cellular swelling; and plasma membrane disruption. Apoptosis is typically characterized by cell membrane blebbing, cytoplasmic condensation, and activation of endogenous endonucleases. Determining either of these effects on cancer cells indicates that the ADC is useful in treating cancer.

[0217] Cell viability may be measured, for example, by determining the uptake of a dye such as neutral red, trypan blue, crystal violet, or ALAMAR™ blue into the cells (see, e.g., Page et al. (1993) Intl. J. Oncology 3:473-6). In such assays, cells are incubated in medium containing the dye, the cells are washed, and the remaining dye, which reflects cellular uptake of the dye, is measured spectrophotometrically. In certain embodiments, the in vitro potency of the prepared ADC is assessed using a crystal violet assay. Crystal violet is a triarylmethane dye that accumulates in the nuclei of viable cells. In this assay, cells are exposed to an ADC or a control agent for a specified period of time, after which the cells are stained with crystal violet, washed extensively with water, and then solubilized with 1% SDS and read spectrophotometrically. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al. (1990) J. Natl. Cancer Inst. 82:1107-12).

[0218] Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercially available photometric methods for quantitatively determining DNA fragmentation in vitro are available. Examples of such assays, including TUNEL (detecting the incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals).

[0219] Apoptosis can also be determined by measuring morphological changes in cells. For example, similar to necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells are described in Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992) pp. 3.17.1-3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide), and the cells can be observed for chromatin condensation and margination along the inner nuclear membrane. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased cell membrane blebbing, and cell shrinkage.

[0220] The ADCs of the present disclosure may also be evaluated for bystander killing activity. Bystander killing activity can be determined, for example, by an assay using two cell lines, one positive for the target antigen and one negative for the target antigen. The cell lines are preferably labeled to distinguish them. For example, IGROV1 cells (FRA+) labeled with Nuclight™ Green (NLG) and HL-60 cells (FRA-) labeled with Nuclight™ Red (NLR) can be co-cultured and treated with an anti-FRA ADC, followed by monitoring cytotoxicity. Killing of target antigen-negative cells when mixed with target antigen-positive cells indicates bystander killing, while killing of target antigen-negative cells in the absence of target antigen-positive cells indicates off-target killing.

[0221] In some aspects, the disclosure features methods for killing, inhibiting or modulating the growth of, or interfering with the metabolism of cancer cells or tissues by disrupting tubulin, which can be used in any subject where disruption of tubulin provides a therapeutic effect. Subjects who may benefit from disruption of tubulin include, but are not limited to, subjects having or at risk of having gastric cancer, ovarian cancer (e.g., serous ovarian cancer), lung cancer (e.g., non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), endometrial cancer (e.g., serous endometrial carcinoma), osteosarcoma, Kaposi's sarcoma, testicular germ cell cancer, leukemia, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's lymphoma), myeloma, head and neck cancer, esophageal cancer, pancreatic cancer, prostate cancer, brain cancer (e.g., glioblastoma), thyroid cancer, colorectal cancer, and / or skin cancer (e.g., melanoma), or any metastasis thereof (Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9:790-803). In various embodiments, the ADCs of the present disclosure can be administered to any cell or tissue that expresses FRA, e.g., FRA-expressing cancer cells or tissues. Exemplary embodiments include methods of inhibiting FRA-mediated cell signaling or killing cells. This method can be used on any cell or tissue that expresses FRA, e.g., cancerous cells or metastatic lesions. Non-limiting examples of FRA-expressing cancers include gastric cancer, serous ovarian cancer, clear cell ovarian cancer, non-small cell lung cancer, colorectal cancer, triple-negative breast cancer, endometrial cancer, serous endometrial cancer, lung carcinoid, and osteosarcoma. Non-limiting examples of FRA-expressing cells include IGROV1 and OVCAR3 human ovarian cancer cells, NCI-H2110 human non-small cell lung cancer cells, and cells containing a recombinant nucleic acid encoding FRA, or a portion thereof.

[0222] In various other embodiments, the ADCs of the present disclosure can be administered to any cell or tissue that expresses her2, e.g., her2-expressing cancer cells or tissues. Exemplary embodiments include methods of inhibiting her2-mediated cell signaling or killing cells. This method can be used on any cell or tissue that expresses her2, e.g., cancerous cells or metastatic lesions. Non-limiting examples of her2-expressing cancers include breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol. Diagn. Ther. 17:85-99). Non-limiting examples of her2-expressing cells include NCI-N87-luc human gastric cancer cells, ZR75 and BT-474 human breast ductal carcinoma cells, and cells containing a recombinant nucleic acid encoding her2, or a portion thereof.

[0223] In various other embodiments, the ADCs of the present disclosure can be administered to any cells or tissues that express mesothelin (MSLN), e.g., MSLN-expressing cancer cells or tissues. Exemplary embodiments include methods of inhibiting MSLN-mediated cell signaling or killing cells. This method can be used on any cells or tissues that express MSLN, e.g., cancerous cells or metastatic lesions. Non-limiting examples of MSLN-expressing cancers include mesothelioma, pancreatic cancer (e.g., pancreatic adenocarcinoma), ovarian cancer, and lung cancer (e.g., lung adenocarcinoma) (Wang et al. (2012) PLoS ONE 7:e33214). Non-limiting examples of MSLN-expressing cells include OVCAR3 human ovarian cancer cells, HEC-251 human endometrioid cells, H226 human lung squamous cell mesothelioma cells, and cells containing a recombinant nucleic acid encoding MSLN, or a portion thereof.

[0224] An exemplary method includes contacting cells with an effective amount, i.e., an amount sufficient to kill the cells, of an ADC as described herein. This method can be used on cells in culture, e.g., in vitro, in vivo, ex vivo, or in situ. For example, cells expressing FRA, her2, and / or MSLN (e.g., cells collected from a 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 performed by adding the ADC to the culture medium. This method results in the death of cells expressing FRA, her2, and / or MSLN, including, in particular, tumor cells expressing FRA, her2, and / or MSLN. Alternatively, the ADC can be administered to a subject by any suitable route of administration (e.g., intravenously, subcutaneously, or by direct contact with tumor tissue) so as to be effective in vivo.

[0225] The in vivo efficacy of the disclosed ADC therapeutic compositions can be evaluated using appropriate animal models. For example, xenogeneic cancer models can be used, in which cancer xenografts or serial xenograft tissues are introduced into immunocompromised animals, such as nude or SCID mice (Klein et al. (1997) Nature Med. 3:402-8). Efficacy can be predicted using assays that measure tumor formation inhibition, tumor regression, or metastasis.

[0226] In vivo assays that assess the promotion of apoptosis can also be used. In one embodiment, xenografts from tumor-bearing mice treated with a therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

[0227] Further provided herein are methods for treating cancer. The ADCs disclosed herein can be administered to non-human mammals or human subjects for therapeutic purposes. The treatment method involves administering to a tumor-bearing mammal a biologically effective amount of an ADC comprising a selected chemotherapeutic agent (e.g., eribulin) linked to a targeting antibody that binds to an expressed antigen available for binding or localized on the surface of cancer cells. An exemplary embodiment is a method of delivering a chemotherapeutic agent to cells expressing FRA, comprising conjugating the chemotherapeutic agent to an antibody that immunospecifically binds to an FRA epitope and exposing the cells to the ADC. Exemplary FRA-expressing tumor cells for which the ADCs of the present disclosure are applicable include cells from gastric cancer, serous ovarian cancer, non-small cell lung cancer, colorectal cancer, breast cancer (e.g., triple-negative breast cancer), lung carcinoid tumor, osteosarcoma, endometrial cancer, and endometrial cancer containing serous tissue.

[0228] Another exemplary embodiment is a method of delivering a chemotherapeutic agent to a cell that expresses her2, comprising conjugating the chemotherapeutic agent to an antibody that immunospecifically binds to a her2 epitope and exposing the cell to an ADC. Exemplary tumor cells that express her2 for which the ADCs of the present disclosure are indicated include cells from breast, gastric, bladder, urothelial, esophageal, lung, cervical, endometrial, and ovarian cancers.

[0229] Another exemplary embodiment is a method of delivering a chemotherapeutic agent to a cell that expresses MSLN, comprising conjugating the chemotherapeutic agent to an antibody that immunospecifically binds to an MSLN epitope and exposing the cell to an ADC. Exemplary MSLN-expressing tumor cells for which the ADCs of the present disclosure are indicated include cells from mesothelioma, pancreatic cancer (e.g., pancreatic adenocarcinoma), ovarian cancer, and lung cancer (e.g., lung adenocarcinoma).

[0230] Another exemplary embodiment is a method of treating a patient having or at risk of having a cancer that expresses a target antigen for the antibody portion of the ADC, e.g., FRA, her2, or MSLN, comprising administering to the patient a therapeutically effective amount of an ADC of the present disclosure. In some embodiments, the patient is non-responsive or poorly responsive to treatment with an anti-FRA antibody when administered alone and / or to treatment with a drug moiety (e.g., eribulin) when administered alone. In other embodiments, the patient is non-responsive or poorly responsive to treatment with an anti-her2 antibody when administered alone and / or to treatment with a drug moiety (e.g., eribulin) when administered alone. In other embodiments, the patient is non-responsive or poorly responsive to treatment with an anti-MSLN antibody when administered alone and / or to treatment with a drug moiety (e.g., eribulin) when administered alone. In other embodiments, the patient is intolerant to treatment with a drug moiety (e.g., eribulin) when administered alone. For example, patients may require doses of eribulin to treat cancer that result in systemic toxicity, which is overcome by targeted delivery to cancers that express the target antigen for the antibody portion of the ADC, e.g., FRA, her2, or MSLN, thereby reducing off-target killing.

[0231] Another exemplary embodiment is a method of reducing or inhibiting the growth of a target antigen-expressing tumor (e.g., an FRA-expressing tumor, a HER2-expressing tumor, or an MSLN-expressing tumor), comprising administering a therapeutically effective amount of an ADC. In some embodiments, the treatment is sufficient to reduce or inhibit tumor growth, reduce the number or size of metastatic lesions, reduce tumor burden, reduce primary tumor burden, reduce invasiveness, prolong survival, and / or maintain or improve quality of life in a patient. In some embodiments, the tumor is resistant or refractory to treatment with an anti-FRA antibody when administered alone and / or to treatment with a drug moiety (e.g., eribulin) when administered alone. In other embodiments, the tumor is resistant or refractory to treatment with an anti-her2 antibody when administered alone and / or to treatment with a drug moiety (e.g., eribulin) when administered alone. In some embodiments, the tumor is resistant or refractory to treatment with an anti-MSLN antibody when administered alone and / or to treatment with a drug moiety (e.g., eribulin) when administered alone.

[0232] Additionally, antibodies of the present disclosure can be administered to non-human mammals expressing an antigen to which the ADC can bind, for veterinary purposes or as animal models of human disease. For the latter, such animal models can be useful for assessing the therapeutic efficacy of the ADCs of the present disclosure (e.g., for testing dosages and time courses of administration).

[0233] Further, therapeutic uses of the ADCs of the present disclosure are provided herein. An exemplary embodiment is the use of the ADCs in the treatment of target antigen-expressing cancers (e.g., FRA-expressing, her2-expressing, or MSLN-expressing cancers). ADCs for use in the treatment of target antigen-expressing cancers (e.g., FRA-expressing, her2-expressing, or MSLN-expressing cancers) are also disclosed. Methods for identifying subjects with cancers that express FRA, her2, and / or MSLN are known in the art and can be used to identify patients suitable for treatment with the disclosed ADCs.

[0234] Another exemplary embodiment is the use of the ADC in a method for manufacturing a medicament for treating a target antigen-expressing cancer (e.g., an FRA-expressing, her2-expressing, or MSLN-expressing cancer).

[0235] The therapeutic compositions used in carrying out the above-described methods can be formulated into pharmaceutical compositions containing a pharmaceutically acceptable carrier appropriate for the desired delivery method. An exemplary embodiment is a pharmaceutical composition containing an ADC of the present disclosure and a pharmaceutically acceptable carrier. Suitable carriers include any substance that, when combined with the therapeutic composition, maintains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, mesylate, and the like, as well as combinations thereof. In many cases, it is preferable to include an isotonicity agent, such as a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride, in the composition. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the ADC.

[0236] Therapeutic formulations can be solubilized and administered via any route that can deliver the therapeutic composition to the tumor site. Potentially effective administration routes include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intravisceral, orthotopic, etc. Therapeutic protein formulations can be lyophilized and stored as sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water (e.g., containing benzyl alcohol preservative) or sterile water before injection. Therapeutic formulations can include ADCs or pharmaceutically acceptable salts thereof, such as mesylate salts.

[0237] The ADCs disclosed herein can be administered to a patient in need thereof at dosages ranging from about 0.2 mg / kg to about 10 mg / kg. In some embodiments, the ADC is administered to a patient daily, every other month, or any time period in between. Dosages and administration protocols for treating cancer using the above-described methods vary depending on the method and target cancer, and generally depend on several other factors recognized in the art.

[0238] A variety of delivery systems are known and can be used to administer one or more ADCs of the present disclosure. Methods of administering ADCs include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration can be used, for example, by using an inhaler or nebulizer and a formulation containing an aerosol agent. See, for example, U.S. Patent Application Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and CT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903 for compositions and methods for pulmonary administration. ADCs can be administered by any conventional route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local.

[0239] Therapeutic compositions disclosed herein are sterile and stable under the conditions of manufacture and storage. In some embodiments, one or more of the ADCs or pharmaceutical compositions are supplied as anhydrous, sterile, lyophilized powders or water-free concentrates in airtight, sealed containers, and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. Preferably, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions are supplied as anhydrous, sterile, lyophilized powders in airtight, sealed containers in unit dosage amounts of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg, or any amount therebetween. In some embodiments, the lyophilized ADCs or pharmaceutical compositions are stored in their original containers at 2°C to 8°C. In some embodiments, one or more of the ADCs or pharmaceutical compositions described herein are supplied in liquid form in airtight, sealed containers, e.g., containers indicating the amount and concentration of the agent. In some embodiments, the liquid form of the administered composition is supplied in an airtight sealed container with an ADC at at least 0.25 mg / mL, at least 0.5 mg / mL, at least 1 mg / mL, at least 2.5 mg / mL, at least 5 mg / mL, at least 8 mg / mL, at least 10 mg / mL, at least 15 mg / mL, at least 25 mg / mL, at least 50 mg / mL, at least 75 mg / mL, or at least 100 mg / mL. The liquid form may be stored in the original container at 2°C to 8°C.

[0240] In some embodiments, the ADCs of the present disclosure can be incorporated into pharmaceutical compositions suitable for parenteral administration. Injectable solutions can consist of either liquid or lyophilized dosage forms in flint or amber vials, ampoules, or pre-filled syringes, or other known delivery or storage devices.

[0241] The 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.

[0242] In various embodiments, treatment comprises a single bolus or multiple administrations of the ADC formulation via an acceptable route of administration.

[0243] Patients can be evaluated for the level of target antigen (e.g., the level of target antigen-expressing cells) in a given sample, such as to aid in determining the most effective dosing regimen. An exemplary embodiment is a method for determining whether a patient will respond to treatment with an ADC of the present disclosure, comprising obtaining a biological sample from the patient and contacting the biological sample with the ADC. Exemplary biological samples include tissue or bodily fluids, such as inflammatory exudate, blood, serum, intestinal fluid, a stool sample, or a tumor biopsy (e.g., a tumor biopsy from a patient having or at risk for a target antigen-expressing cancer, e.g., an FRA-expressing cancer, a HER2-expressing cancer, or an MSLN-expressing cancer). In some embodiments, a sample (e.g., tissue and / or bodily fluid) can be obtained from a subject, and protein expression of the target antigen (e.g., FRA, HER2, or MSLN) can be detected and / or measured using an appropriate immunological method. Such evaluations can also be used to monitor objectives throughout treatment and, in combination with evaluation of other parameters, are useful for accurately measuring the success of treatment.

[0244] In some embodiments, the effectiveness of an ADC can be assessed by contacting a tumor sample from a subject with the ADC and assessing tumor growth rate or volume. In some embodiments, if the ADC is determined to be effective, it can be administered to the subject.

[0245] The above therapeutic approaches can be combined with any one of a wide variety of additional surgical, chemotherapy, or radiation therapy regimens.

[0246] Also disclosed herein is the use of one or more ADCs of the present disclosure in the manufacture of a medicament for treating cancer, e.g., according to the methods described above. In some embodiments, the ADCs disclosed herein are used to treat cancer, e.g., according to the methods described above.

[0247] In various embodiments, kits for use in the laboratory and therapeutic applications described herein are within the scope of the present disclosure. Such kits may include a carrier, package, or container compartmentalized to accommodate one or more containers, e.g., vials, tubes, etc., wherein each of the container(s) contains one of the separate elements used in the methods disclosed herein, along with a label or insert containing instructions, such as for use, as described herein. The kit may also include a container containing a drug moiety. The present disclosure also provides one or more of the ADCs, or pharmaceutical compositions thereof, packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of drug.

[0248] Kits may include the above-described container and one or more other containers associated therewith that contain materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; labels for the carrier, package, container, vial and / or tube that list the contents and / or instructions, and package inserts containing instructions for use.

[0249] A label may be present on or associated with the container to indicate that the composition is used for a particular therapeutic or non-therapeutic application, such as prognosis, prevention, diagnosis, or laboratory application. The label may also indicate instructions for in vivo or in vitro use, such as those described herein. Instructions and other information may also be included on package insert(s) or label(s), which accompany or are included with the kit. The label may be on or associated with the container. The label may be on the container if letters, numbers, or other symbols forming the label are molded or etched into the container itself. If present in a receptacle or carrier that also houses the container, the label may be associated with the container, for example, as a package insert. The label may indicate that the composition is used to diagnose or treat a condition, such as cancer, as described herein.

[0250] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present invention described herein will be apparent and that they may be made using appropriate 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 illustrative purposes only and are not intended to be limiting. [Example]

[0251] Example 1 1. Materials and Methods The MORAb-003 used to prepare the ADC was derived from Lot#AA0312. 1.1 Cytotoxins The structures of cytotoxins that can be conjugated are shown in Table 11.

[0252] [Table 11] TIFF2024174991000052.tif226149 TIFF2024174991000053.tif226149 TIFF2024174991000054.tif226149

[0253] 1.2 Antibody-drug conjugation 1.2.1 Partial reduction using TCEP Partial reduction conditions for MORAb-003 were established by varying the concentration of the non-thiol reducing agent tris(2-carboxyethyl)phosphine (TCEP), antibody concentration, and reduction time. MORAb-003 was buffer-exchanged into Dulbecco's phosphate-buffered saline (DPBS) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and then concentrated to 10 mg / mL using a centrifugal concentrator equipped with a 10 kD molecular weight cutoff (MWCO) centrifugal filter. The antibody was diluted to the appropriate concentration, and TCEP was added to the indicated final concentration and gently mixed at room temperature for 1 hour. TCEP was removed by desalting using a 5 or 10 mL Zeba™ spin desalting column with DPBS / 1 mM EDTA (Thermo Fisher, 40 kD MWCO) as the buffer according to the manufacturer's protocol. Samples were analyzed for free thiol content using a thiol fluorescence quantification kit (Abcam) according to the manufacturer's protocol. SDS-PAGE analysis under non-reducing conditions was performed to determine the magnitude and location of disulfide bond cleavage, as described in Section 1.3.3. In some cases, desalted MAbs were diluted in DPBS to 1-2 mg / mL and subjected to biotinylation to determine conjugation potential and drug-to-antibody (DAR) ratios. 10 mM maleimide-PEG2-biotin (Thermo Fisher) in dimethyl sulfoxide (DMSO) was added to the antibody (mAb) at a 10:1 molar ratio and incubated at room temperature for 4 hours with gentle agitation. After conjugation, unreacted compound was removed by desalting using a Zeba™ spin desalting column (Thermo Fisher). Samples were then analyzed by LC-MS to determine the DAR, as detailed in Section 1.3.4.

[0254] 1.2.2 Cytotoxin conjugation The partially reduced antibody was brought to 2.5 mg / mL in 0.5x DPBS, 0.5 mM EDTA and mixed thoroughly. Then, if used, organic cosolvents were added and mixed thoroughly. The cosolvents tested were propylene glycol (final concentrations of 20% and 50%), dimethyl sulfoxide (DMSO) (10%), N,N-dimethylformamide (20%), N,N-dimethylacetamide (20%), and N,N-dimethylpropionamide (20%). Maleimide-modified cytotoxin (6 mM stock solution in DMSO) was added to the antibody at a molar ratio of 1:6 (mAb:compound) and mixed thoroughly. Conjugation proceeded for 3.5 hours at room temperature with gentle mixing. 50% propylene glycol was chosen as the final organic modifier and used in all subsequent conjugation reactions.

[0255] 1.2.3 Purification To remove unreacted maleimide-cytotoxin and propylene glycol, the conjugated antibody was purified using 26 / 10 HiTrap® desalting columns (GE Healthcare) in a chromatography run by fast protein liquid chromatography (FPLC) (GE Healthcare). The MORAb-003 ADC, including MORAb-003-mal-VCP-eribulin (MORAb-202), was formulated in DPBS (the formulation buffer was used as the running buffer during FPLC chromatography).

[0256] 1.3 Biophysical characterization 1.3.1 BCA assay Prepared bicinchoninic acid (BCA) reagent (200 μL) was added to 25 μL of serially diluted ADC or bovine gamma globulin (Thermo Fisher) 2 mg / mL standard, and the sample was mixed thoroughly. Samples were incubated at 37°C for 20 minutes. Plates were read at 595 nm on a SpectraMax® M5 plate reader (Molecular Devices). Data were analyzed using SoftMax® Pro (ver. 3.2) with a 4-parameter fitting model.

[0257] 1.3.2 SEC-HPLC analysis Antibody aggregation was analyzed by size-exclusion high-performance liquid chromatography (SEC-HPLC) using an Agilent 1100. mAbs were diluted to 1 mg / mL in DPBS. Antibodies (20 μL) were injected onto a TSKgel® SuperSW guard column (4.6 mm × 3.5 cm, 4 μm pore size, Tosoh Bioscience) followed by a TSKgel® SuperSW3000 column (4.6 mm × 30 cm, 4 μm pore size). The columns were eluted with 0.1 M sodium phosphate (pH 7.4) containing 0.15 M NaCl and 0.05% NaN3 at a flow rate of 0.3 mL / min for 20 minutes. All data were analyzed using Agilent ChemStation software. Percent aggregation was calculated as [PA 凝集 / PA 合計 ] × 100 (where PA = integrated peak area).

[0258] 1.3.3 SDS-PAGE analysis Protein samples (0.1–10 μg) were diluted to 1x with lithium dodecyl sulfate (LDS) sample buffer. For nonreduced samples, incubation was performed at room temperature for 10 min before electrophoresis. For reduced samples, dithiothreitol (DTT) was added to a final concentration of 20 mM, and the samples were heated to 95°C for 10 min and placed on ice before electrophoresis. Samples were loaded onto 10-, 12-, or 15-well Bis-Tris SDS-PAGE gels (Thermo Fisher) with 1x MOPS or 1x MES as the running buffer. Electrophoresis was performed at 185 V (constant voltage) for 1 h. Gels were stained with InstantBlue stain (Expedeon) and destained in water. Documentation was performed with an UltraLum gel documentation system using a 600 nm orange filter.

[0259] 1.3.4 UPLC / ESI-MS Analysis of Drug-to-Antibody Ratio (DAR) The ADC was deglycosylated using PNGase F (New England BioLabs). G7 buffer (10 μL) and PNGase F (2 μL) were added to mAb (90 μL, 1 mg / mL in DPBS). The reaction was incubated in a Discover microwave (CEM) for two cycles: (1) 10 W microwave power, 37°C, 10 min, followed by a 5 min rest; (2) 2 W microwave power, 37°C, 10 min. A portion of the sample was reduced by adding DTT to a final concentration of 20 mM, followed by incubation at 60°C for 3 min. The sample was then analyzed using a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) and a quadrupole time-of-flight (Q-Tof) Premier mass spectrometer. Samples (0.5–2 μg each) were injected onto a MassPrep™ microdesalting column at 65°C and eluted from the column at 0.05 mL / min with a 5-minute equilibration in 95% mobile phase A, a 10-minute gradient (5–90% B), and a 10-minute re-equilibration in 95% mobile phase A. Mobile phase A was 0.1% formic acid in water. Mobile phase B was 0.1% formic acid in acetonitrile. The Q-Tof mass spectrometer was operated in positive ion mode (V mode) with detection in the m / z range of 500–4000. Source parameters were as follows: capillary voltage, 2.25 kV (intact antibody) to 2.50 kV (reduced antibody); sampling cone voltage, 65.0 V (intact antibody) or 50.0 V (reduced antibody); source temperature, 100°C; desolvation temperature, 250°C; desolvation gas flow, 550 L / h. Protein peaks were deconvoluted using the MassLynx® MaxEnt1 function. The relative intensities of each unconjugated, single-conjugated, and multiply-conjugated heavy and light chain mass were summed to calculate the overall DAR using the following formula: 2[[I LC+1 +2(I LC+2 )+3(I LC+3 )+…n(I LC+n )] / ΣI LC tot]+2[[I HC+1 +2(I HC+2 )+3(I HC+3)+…n(I HC+n )] / ΣI HC tot] [In the formula, I LC+1 is the mass intensity of the light chain conjugated to one cytotoxin, and I LC+2 is the mass intensity of the light chain conjugated to two cytotoxins, etc. HC is the intensity from the corresponding conjugated heavy chain, and ΣI LC tot and ΣI HC tot is the total intensity of all unconjugated and conjugated light and heavy chains, respectively.

[0260] 1.3.5 HIC-HPLC DAR analysis In addition to DAR analysis by UPLC / electrospray ionization (ESI)-MS, MORAb-003-vcp-eribulin DAR and MORAb-003-0285DAR were also analyzed using hydrophobic interaction HPLC (HIC-HPLC). Samples were injected onto a TSKgel® Ether-5PW column (7.5 mm x 7.5 cm, 10 μM pore size) and eluted at 0.7 mL / min with a 3-minute equilibration in 100% mobile phase A, a 15-minute gradient (0-100% B), a 5-minute hold at 100% B, a 1-minute change to 100% A, and a 5-minute re-equilibration in 100% mobile phase A. Mobile phase A was 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0. Mobile phase B was 25 mM sodium phosphate, 25% isopropanol, pH 7.0. Detection was performed at 280 nm (reference 320 nm). DAR was determined by the following formula: [AUC +1 +2(AUC +2 )+3(AUC +3 )+…n(AUC +n )] / ΣAUC tot ] [In the formula, AUC +1 is the area under the curve at the mAb peak corresponding to one cytotoxin-conjugated ADC, and AUC +2 is the area under the curve at the mAb peak corresponding to the ADC conjugated with two cytotoxins, etc. ΣAUCtot is the total area under the curve over all peaks].

[0261] 1.4 Cytotoxicity analysis 1.4.1 Crystal violet assay IGROV1(FR hi ) and SJSA-1(FR neg ) Cells were subcultured and seeded at 10,000 cells / well into 96-well tissue culture plates in complete growth medium and incubated overnight (16 hours) at 37°C, 5% CO2. Typically, test reagents were serially diluted 1:4 in a 2 mL deep-well dilution plate, starting at 1 μM (for a total of 10 dilutions). 100 μL of diluted sample was added to the cell plate (starting at a test sample concentration of 500 nM). The plate was incubated for an additional 48 hours at 37°C, 5% CO2. The medium was discarded, and the plate was washed once with 200 μL of DPBS and stained with 50 μL of 0.2% crystal violet solution for 15 minutes at room temperature, followed by extensive washing with tap water. The plate was air-dried, and the crystal violet was dissolved in 200 μL of 1% SDS solution. The plate was read at 570 nm. Data were analyzed using GraphPad Prism 6. Assays were performed using a seeding density of 1,000 cells per well, and compound exposure was over a total of 5 days. If a shorter exposure was desired, the cytotoxic drug-containing medium was removed after 4 hours and replaced with fresh growth medium before the 5-day incubation. For OVCAR3, CaOV3, and NCI-H2110, cells were seeded at 3,000 cells / well and incubated with the ADC for 5 days. For competition experiments, titrated ADCs were pre-incubated with 2 μM (final) unconjugated MORAb-003 before incubation with the cells.

[0262] 1.4.2 Bystander killing assay The day before the start of the experiment, Nuclight™ Green (NLG) IGROV1 cells were seeded at 5,000 cells / well into a 96-well round-bottom plate, followed by centrifugation at 1,000 rpm for 3 minutes at room temperature to ensure cell pellet formation. The plate was placed in an Incucyte Zoom® (EssenBio science) container and incubated overnight at 37°C / 5% CO2. A program was set to collect images of cell growth and determine the total number of green-stained and red-stained nuclear cells, as well as the cell phase confluency every 2 hours. On the day of the experiment, MORAb-003 ADC or free drug was diluted in complete RPMI medium and serially diluted, starting at 400 nM. 50 μL of cytotoxin solution was added to the NLG-IGROV1 cells and incubated for 30 minutes. During the incubation period, Nuclight™ Red (NLR) HL-60 (FR neg ) cells in fresh medium, 2 × 10 5 , 1×10 5 or 5×10 4 The cells were diluted to 1000 cells / mL. 50 μL of the NLR-HL60 cell suspension or medium alone was added to the NLG-IGROV1 wells, followed by centrifugation at 1,000 rpm for 3 minutes at room temperature to ensure cell pellet re-formation. The plate was placed back into the Incucyte Zoom (EssenBio science) container and incubated at 37°C / 5% CO2 for up to 5 days. Relative cell proliferation of NLG-IGROV1 was determined by comparing the number of green cells with the number of cells when no ADC was added to the sample or when only free drug was added. Relative cell proliferation of HL60 was performed similarly, except that the number of red cells was determined. IC for both NLG-IGROV1 and NLR-HL-60 50 Values ​​were determined using Prism (GraphPad).

[0263] 1.4.3 Serum Stability Assay 20 μL of MORAb-003ADC was thoroughly mixed with 80 μL of DPBS, normal pooled human serum (Bioreclamation, Lot BRH552911), or normal pooled mouse serum (Bioreclamation, Lot MSE152591) and incubated at 37°C for 0, 4, 24, and 48 hours. After incubation, samples were frozen and stored at -20°C until evaluation in cytotoxicity and binding assays. For cytotoxicity analysis, samples were evaluated on IGROV1 and SJSA-1 cells as detailed in Section 1.4.1. For binding evaluation, samples were evaluated using the solution-based MSD ECL assay. Samples were incubated with biotinylated folate receptor alpha and sulfo-tagged anti-MORAb-003, then captured on a streptavidin plate and detected using electrochemiluminescence on an MSD Sector Imager 2400.

[0264] 2. Results 2.1 Preparation of MORAb-003ADC To select the best combination of linker and cytotoxin for conjugation with MORAb-003, ADCs were prepared using three methods. According to the conjugation strategy shown in Figure 1, unpaired cysteines are generated by partial reduction with a limited molar equivalent of the non-thiol reducing agent TCEP. This strategy preferentially reduces the interchain disulfide bonds connecting the light and heavy chains (one pair per HL pairing) and the two heavy chains in the hinge region (two pairs per HH pairing in the case of human IgG1), while leaving the intrachain disulfide bonds intact.

[0265] The second conjugation strategy for preparing the MORAb-003 ADC utilized reduced disulfide cross-linking chemistry, which re-cross-links the free thiols of cysteine ​​residues released during the partial reduction process, mimicking the role of disulfide bonds and thus maintaining the stability and functionality of the ADC.

[0266] A third conjugation strategy for preparing the MORAb-003 ADC employed limited lysine utilization, which results in the conjugation of a very limited number of the estimated 70+ solvent-exposed lysines available on a typical human IgG molecule and can potentially result in a mixture of ADC products with less uniformity relative to strategies involving cysteine ​​modifications.

[0267] 2.1.1 Preparation of VCP-eribulin for MORAb-003 ADC Eribulin (1) (10 mg, 14 μmol) (Figure 2) was dissolved in N,N-dimethylformamide (DMF) (1 mL) and mixed thoroughly. N,N-Diisopropylethylamine (Hunig's base or iPrNEt) (3.6 μL, 21 μmol) and Fmoc-Val-Cit-para-aminobenzyl-para-nitrophenol (Fmoc-VCP-PNP) (2) (16 mg, 21 μmol, Concortis Biosystems, cat#VC1003) were added. The reaction mixture was stirred at room temperature for 4-16 h and monitored using a ninhydrin test kit (Anaspec, cat#25241) until completion. Diethylamine (EtNH) (0.014 mL, 0.14 mmol) was then added to the reaction mixture and stirred at 18-25 °C for 2 h to remove the Fmoc protecting group. The reaction was monitored using a ninhydrin test kit. Upon completion, the solvent was evaporated under vacuum to give crude VCP-eribulin (3) (16 mg). It was purified using a ZOBAX SB-C18 column (5 μm pore size, 9.4 × 150 mm) on a Waters Alliance e2695 HPLC system with a mobile phase of HO-CHCN containing 0.1% formic acid, with a gradient of 15 to 70% B. VCP-eribulin (3) (16 mg) was dissolved in DMF (1 mL). Hunig's base (7.2 μL, 41 μmol) and maleimide-PEG-NHS (4) (9.7 mg, 27 μmol) were added. The reaction mixture was stirred at 18-25 °C for 3 h. The reaction mixture was purified by HPLC (HO-CHCN containing 0.1% formic acid) with a gradient of 15 to 70% B. The solvent was removed by lyophilization to give mal-(PEG)2-Val-Cit-p-aminobenzyloxycarbonyl (pAB)-eribulin (mal-(PEG)2-VCP-eribulin) (5).

[0268] 2.1.2 Optimization of reduction conditions MORAb-003 ADC was prepared by generating unpaired cysteines via partial reduction with a limited molar equivalent of the non-thiol reducing agent tris(2-carboxyethyl)phosphine (TCEP). Initial studies were performed with MORAb-003, whereby antibody concentration, TCEP concentration, and incubation time were varied with the goal of generating an average of four potential conjugation sites per antibody molecule. The number of free thiol sites was determined using a fluorometric thiol quantification assay. The results of this analysis are shown in Table 12. The extent of cleavage of HH and HL bonds after 10, 30, 60, or 120 minutes of incubation was also analyzed by SDS-PAGE (Figure 3). For this analysis, non-reduced and reduced samples were loaded onto an SDS-PAGE gel, and electrophoresis was performed at 185 V for 1 hour. In Figure 3, lane M corresponds to the protein standard. Lane 1 corresponds to untreated, non-reduced MORAb-003. Lane 2 corresponds to MORAb-003 (5.3 mg / mL) reduced in 70.6 μM TCEP. Lane 3 corresponds to MORAb-003 (5.3 mg / mL reduced) at 141.2 μM TCEP. Lane 4 corresponds to MORAb-003 (1.5 mg / mL) reduced in 20 μM TCEP. Lane 5 corresponds to MORAb-003 (1.5 mg / mL) reduced in 40 μM TCEP. The identity of each band is indicated on the lower right gel. "H" indicates heavy chain, and "L" indicates light chain.

[0269] [Table 12]

[0270] Analysis of SDS-PAGE and thiol content suggested that a 60-minute incubation of 5.3 mg / mL of mAb at a 4-fold molar ratio of TCEP to mAb represented a reasonable starting point, since there appeared to be limited reduction of intramolecular disulfides (as determined by free thiol content) and very little non-reduced mAb remained (which would act as a competitive inhibitor in in vitro and in vivo studies using the prepared ADC). Further studies were performed with MORAb-003, starting at a concentration of 5.0 mg / mL, to confirm this optimized molar ratio of TCEP to mAb using SDS-PAGE analysis (Figure 4). In Figure 4, lane 1 corresponds to the protein standard; lane 2 corresponds to untreated, non-reduced MORAb-003; and lane 3 corresponds to MORAb-003 treated at a 1:1 ratio of MORAb-003:TCEP. Lane 4 corresponds to MORAb-003 treated at a ratio of 1:2 MORAb-003:TCEP. Lane 5 corresponds to MORAb-003 treated at a ratio of 1:3 MORAb-003:TCEP. Lane 6 corresponds to MORAb-003 treated at a ratio of 1:4 MORAb-003:TCEP. To simulate the conjugation of a cytotoxin for ADC preparation, conjugation using maleimide-PEG2-biotin after reduction and TCEP removal was also performed. DAR analysis was performed using LC-MS. The results of these studies are shown in Table 13.

[0271] [Table 13]

[0272] After biotin conjugation, free thiol analysis showed that no free thiols were present in MORAb-003-biotin. This indicated that after reduction of the disulfide bond, conjugation typically occurred with both generated thiols, and any unconjugated reduced disulfides were reoxidized to reform disulfide bonds. The final conditions selected for reduction to produce the ADC were an antibody concentration of 5.0 mg / mL, a TCEP concentration of 110 μM, and an incubation time of 60 minutes. This resulted in a mAb with a DAR of 4 after conjugation.

[0273] 2.1.3 Optimization of ADC conjugation Because the first cytotoxin used for ADC preparation was cryptophycin, a hydrophobic compound, initial conjugation optimization experiments were performed with a "surrogate" anti-human mesothelin antibody with two unpaired cysteines (one per light chain) available for conjugation at specific positions. This greatly facilitates analysis of conjugation efficiency by mass spectrometry, since only the light chain needs to be analyzed. Propylene glycol titration during conjugation of maleimide-LL3-cryptophycin to the surrogate antibody was performed, followed by analysis of the conjugation efficiency of the light chain by LC-MS (Table 14).

[0274] [Table 14]

[0275] 50% propylene glycol resulted in complete occupancy of available sites and was selected as the final concentration used. No loss of mAb binding was observed after conjugation (data not shown), indicating that propylene glycol had no adverse effects on the antibody. Therefore, the final conjugation reaction conditions selected were a final 2.5 mg / mL mAb, a 6:1 molar ratio of maleimide-linker-cytotoxin to mAb (final concentration after propylene glycol addition) in 0.5x DPBS, 0.5 mM EDTA, 50% propylene glycol, pH 7.2, and room temperature for 3.5–4 hours. In these reactions, propylene glycol was added prior to the addition of the maleimide-linker-cytotoxin.

[0276] 2.1.4 Preparation and Biophysical Characterization of ADCs The first 10 MORAb-003 ADCs listed in Table 15 were prepared using the established reduction and conjugation conditions described in Section 2.1.2. With the exception of M-MMAE and M-DM1, the remaining ADCs were prepared by either reduced disulfide bridging or limited lysine utilization. M-MMAE and M-DM1 were prepared by Concortis Biosystems, Inc. and administered in conjugated form.

[0277] Reduction disulfide crosslinking chemistry crosslinks the free thiols generated during the partial reduction process, resulting in one cytotoxin per reduced disulfide. Theoretically, an antibody with a DAR of 4 would recrosslink both the HL and hinge disulfides, resulting in an ADC with increased stability and homogeneity over conventional conjugation approaches. Limited lysine utilization results in conjugation of a very limited number of the estimated 70+ solvent-exposed lysines available on a typical human IgG molecule. The MORAb-003 conjugate prepared using this method yielded a DAR of 2.0, suggesting that only one lysine per HL pair was utilized.

[0278] All ADCs were purified by HiPrep 26 / 10 desalting chromatography and formulated in DPBS. DAR analysis was performed on all prepared ADCs by LC-MS, and aggregation levels were determined by SEC-HPLC. The results of these DAR and aggregation analyses are listed in Table 15 next to the individual ADCs.

[0279] [Table 15]

[0280] DAR values ​​for all ADCs were within the predetermined range (DAR between 3 and 4). Although aggregation levels with cryptophycin-based ADCs were significantly higher than desired (>10%), eribulin-based (VCP-eribulin) and maytansine-based maleimide-linker-cytotoxins (ER-001161318, ER-001161319, and M-MMAE) all demonstrated acceptable aggregation levels. Other organic cosolvents were investigated in conjugation reactions to MORAb-003 using VCP-cryptophycin. The cosolvents tested were DMSO (10%), N,N-dimethylformamide (20%), N,N-dimethylacetamide (20%), and N,N-dimethylpropionamide (20%). Aggregation levels after conjugation using these cosolvents were all comparable to or higher than those using 50% propylene glycol.

[0281] Non-reducing SDS-PAGE analysis was performed on a subset of ADCs (Figure 5). Because the DAR for all these ADCs was determined to be 4, it was assumed that these ADCs should migrate as intact IgGs of approximately 160 kD, since both HL and both hinge disulfides should be recrosslinked. This subset of ADCs included M-MMAE (lane 2), M-DM1 (lane 3), M-0026 (lane 4), M-0260 (lane 5), M-0267 (lane 6), M-0272 (lane 7), M-0285 (lane 8), M-292 (lane 9), M-027-0381 (lane 10), and M-0384 (lane 11) (Figure 5). In Figure 5, lane 1 corresponds to the protein standard.

[0282] This analysis reveals that in the reduced disulfide bridge chemistry ADCs (lanes 4-9, 11), there is a significant HL monovalent species (80 kD) in addition to the intact ADC, indicating the presence of significant intrachain hinge disulfide bridges in addition to interchain hinge bridges. SEC-HPLC analysis shows that the ADCs migrate as single, intact IgGs, indicating that in ADCs with intrachain HH bridges, the heavy chains are not covalently linked in the final ADC.

[0283] 2.2 Analysis of In Vitro Potency of MORAb-003 ADC 2.2.1 Cytotoxicity in IGROV1 and SJSA-1 cells The in vitro potency of the prepared ADCs was assessed using the crystal violet assay as detailed in Section 1.4.1.

[0284] Initial screening of all MORAb-003 ADCs was performed using IGROV1 (FR hi(+++) ) and SJSA-1(FR neg(-)The screening of selected ADCs was also performed in CaOV3 (human ovarian carcinoma, FR) cells. IGROV1 cells are derived from a human ovarian epithelial carcinoma and express high levels of folate receptor alpha (FR), the target antigen of MORAb-003. SJSA-1 cells are a human osteosarcoma tumor cell line that is negative for folate receptor alpha. Screening of selected ADCs was also performed in CaOV3 (human ovarian carcinoma, FR) cells. med(++) ), NCI-H2110 (human non-small cell lung cancer, FR med(++) ), and / or OVCAR3 (human ovarian cancer, FR med(++) The results of this screening are shown in Table 16.

[0285] [Table 16]

[0286] The VCP-eribulin ADC was potent against IGROV1 cells (54 pM) and showed little killing in SJSA-1 cells. In these cell lines, the VCP-eribulin ADC demonstrated high potency and specificity relative to ADCs with comparable DAR values, such as M-MMAE and M-DM1. The VCP-eribulin ADC also demonstrated potent cytotoxicity against additional FR-expressing tumor cell lines derived from ovarian (CaOV3 and OVCAR3) and non-small cell lung cancer (NC-H2110).

[0287] The ADCs VCP-eribulin, LL2-cryptophycin, LL3-cryptophycin, VCP-cryptophycin, ER-001161318, ER-001161319, and ER-001159200 were used in combination with CaOV3 (FR med(++)(2012) demonstrated specific cytotoxicity (>2-log specificity) in IGROV1 cells. Many of these ADCs demonstrated subnanomolar potency. The cryptophycin conjugates also demonstrated high levels of potency (23 pM-86 pM) in IGROV1 cells (with the exception of VCP-cryptophycin) and measurable cytotoxicity in SJSA-1 cells. The cleavable maytansine conjugates ER-001161318 and ER-001161319 had intermediate potency in IGROV1 (0.26 nM and 0.49 nM) and little off-target killing of SJSA-1 cells.

[0288] All of the limited lysine-utilizing conjugates did not demonstrate specificity and were not further evaluated. Cleavable conjugates using reduced disulfide crosslinking technology, Duostatin-3 (M-0285), Duostatin-5 (M-0115), and Duostatin-14 (M-292 and M-0026), all demonstrated specific cytotoxicity in the IGROV1 cell line and little cytotoxicity in the SJSA-1 cell line. The auristatin derivatives, Duostatin-3 and Duostatin-5 conjugates, were slightly more potent than the maytansine derivative, Duostatin-14 conjugate. Potency and specificity were comparable to the control M-MMAE conjugate, which uses a Val-Cit-pAB (VCP) linker attached to monomethyl E. All of the noncleavable reduced disulfide chemistry conjugates lacked sufficient potency or specificity and were not further analyzed.

[0289] 2.2.2 Cytotoxicity in the human folate receptor-expressing ovarian cancer cell line CaOV3 The potency of selected MORAb-003 ADCs was also demonstrated in the human ovarian tumor cell lines OVCAR3 and CaOV3, as well as the human NSCLC cell line NCI-H2110 (Table 16). Unlike the potency observed in IGROV1 cells, the cryptophycin conjugate demonstrated measurably higher potency than the VCP-eribulin conjugate in the human ovarian cell line CaOV3. This may be due to lower expression levels of folate receptor alpha in CaOV3 cells compared to IGROV1, or the higher potency of cryptophycin for these cells compared to eribulin. The maytansine-based conjugates ER-001161318, ER-001161319, and ER-001159200 all had potencies similar to or lower than VCP-eribulin.

[0290] 2.3 Bystander Killing of VCP-eribulin, ER-001161318, and M-0285 To assess bystander killing activity, an assay was set up using two labeled cell lines. In this assay, IGROV1 cells (FR hi ) and Nuclight™ Red-labeled HL-60 (FR neg ) were co-cultured at various cell number ratios and treated with dose-ranging MORAb-003 ADCs VCP-eribulin, ER-001161318, or M-0285. VCP-eribulin is an eribulin-based ADC containing a maleimide-PEG-Val-Cit-pAB cleavable linker; ER-001161318 is a maytansine-based ADC containing a maleimide-(CH)-Val-Cit-pAB cleavable linker; and M-0285 is a duostatin-based ADC containing a PEG-pAB cleavable linker. Cytotoxicity was monitored using an Incucyte Zoom® Cell Imager. The results of this bystander cytotoxicity assay are shown in Table 17 and Figures 6A-C.

[0291] [Table 17]

[0292] HL-60(FR neg ) cells with IGROV1(FR hi When cultured at a 2:1 ratio with HL-60 cells, treatment with MORAb003-VCP-eribulin resulted in a 2-log increase in killing of HL-60 cells compared to HL-60 cells alone (Table 17 and Figure 6A). These data suggest that folate receptor alpha (FR) target-negative cells are more effectively killed by MORAb003-VCP-eribulin when cocultured with FR target-positive cells, referred to herein as bystander killing. Bystander killing is distinct from off-target killing, defined as the killing of target-negative cells by themselves, in the absence of target-positive cells, and independent of coculture with them. The observed increase in bystander killing was also nearly identical to that observed after treatment of HL-60 cells with free eribulin, indicating a potential mechanism for bystander effect. Without wishing to be bound by any theory, MORAb003-VCP-eribulin may be cleaved in or near FR-positive IGROV1 cells, which also undergo apoptosis and release free eribulin in culture. The released cytotoxin may kill FR-negative HL-60 cells.

[0293] In contrast, only a slight shift was observed with MORAb003-ER-001161318 (Figure 6B), and no shift was observed with MORAb003-0285 (Figure 6C). When the HL-60:IGROV1 ratio was reduced from 2:1 to 1:2, measurable killing of HL-60 cells was observed with MORAb003-ER-001161318, but the bystander effect of MORAb003-0285, although detectable, remained low. These data suggest that the MORAb-003 ADCs evaluated can be ranked in terms of bystander killing as follows: VCP-eribulin > ER-001161318 > M-0285.

[0294] 2.4 Serum stability analysis Given the long in vivo circulatory half-life of ADCs and the potential for toxicity if cytotoxins are released into the circulation, ADCs should demonstrate stability in serum. The MORAb-003 ADCs VCP-eribulin, ER-001161319, and M-0285 were preincubated in human or mouse serum at 37°C for up to 48 hours and then evaluated in cytotoxicity assays using IGROV1 and SJSA-1 cells. ER-001161319 is a maytansine-based ADC containing the same cleavable linker, maleimide-PEG2-Val-Cit-pAB, as VCP-eribulin. PBS and serum controls were included to correct for any serum effects on assay performance. The results of this study are shown in Table 18.

[0295] [Table 18]

[0296] While VCP-eribulin and M-0285 were stable in both serum samples for at least 48 hours, ER-001161319 demonstrated a significant decrease in potency after 48 hours. This may be due to the aziridino-carbamate linkage to maytansine, which has not been previously described in the literature. Since no increase in cytotoxicity was observed in SJSA-1 cells, the released form of the compound is unlikely to be highly potent.

[0297] 2.5 In vitro studies with MORAb003-VCP-eribulin 2.5.1 HIC-HPLC Analysis of DAR and Product Heterogeneity To assess the DAR by alternative methods and examine the heterogeneity of the product and the content of unconjugated antibody (competitor), MORAb003-VCP-eribulin and MORAb003-0285 were analyzed by HIC-HPLC. MORAb003-VCP-eribulin was shown to have DAR species of 0, 2, 4, and 6, consistent with the method used for reduction and conjugation (Figure 7A). A very small amount of the DAR=0 species was observed. The overall DAR based on AUC calculation was 3.80, consistent with the value determined by LC-MS. MORAb003-0285 migrated as a single peak by HIC-HPLC, indicating a single DAR species (Figure 7B). This was designated a DAR of 4.0.

[0298] 2.5.2 Specificity by competitive assay Using a competitive assay format, the antigen specificity of MORAb-003-VCP-eribulin cytotoxicity was demonstrated for the VCP-eribulin conjugate (Figure 8). In this experiment, a titration of MORAb-003-VCP-eribulin (starting at 100 nM) was co-incubated with 2 μM unconjugated MORAb-003. Unconjugated MORAb-003 demonstrated a 2-log shift in potency in IGROV1 cells, similar to results obtained with IMGN853, an anti-human folate receptor alpha-maytansine ADC from Immunogen currently in Phase II clinical trials, against KB cells (Moore et al., 2015 American Society of Clinical Oncology (ASCO) Annual Meeting, Abstract 5518).

[0299] 2.5.3 Cytotoxicity in NCI-H2110 NSCLC cells Cytotoxicity of both MORAb003-VCP-eribulin and MORAb003-0285 against the human NSCLC cell line NCI-H2110 was performed using a crystal violet assay. The results of this assay are shown in Table 16. MORAb003-VCP-eribulin had an IC50 of 0.73 nM. 50and MORAb003-0285 has an IC of 14 nM 50 had.

[0300] 2.6 In vivo testing 2.6.1 Maximum Tolerated Dose (MTD) of MORAb-003-VCP-Eribulin (MORAb-202) in the CD-1 Mouse Strain Naive CD-1 mice were intravenously injected with 200 μL of MORAb-202 according to the schedule in Table 19. Body weights were measured before dosing on the day of dosing, 24 hours after dosing, and three times weekly thereafter. Animals were observed for clinical health throughout the study. Two weeks after dosing, final body weights were measured and recorded. Mice euthanized at the end of the study (and mice euthanized or found dead during the study, if any) were processed for necropsy. Organs were examined for signs of tissue damage.

[0301] [Table 19]

[0302] Compared to the PBS-treated control group, no significant weight loss or any clinical findings indicating toxicity during treatment were observed in any of the treatment groups. Individual mouse weights are shown in Table 20, and group means and SEM are shown in Table 21. Weight change kinetics (group means and SEM) for each group are shown in Figure 9. MORAb-202 at doses up to 80 mg / kg administered intravenously via bolus did not result in toxicity. Therefore, the MTD is greater than 80 mg / kg.

[0303] [Table 20]

[0304] [Table 21]

[0305] 2.6.2 Maximum tolerated dose of eribulin in CD-1 mice Naive CD-1 mice were intravenously injected with 200 μL of eribulin according to the schedule in Table 22. Body weights were measured three times a week, including before dosing on each dosing day and 24 hours after each dose. Animals were observed for clinical health throughout the study period (2 weeks after the last dose). Final body weights were measured and recorded. Mice euthanized at the end of the study (and mice euthanized or found dead during the study, if any) were processed for necropsy. Organs were examined for signs of tissue damage.

[0306] [Table 22]

[0307] No significant weight loss or clinical findings indicative of toxicity were observed in animals administered eribulin at doses up to 1.6 mg / kg using a q4d x 3 dosing regimen (one dose every four days for a total of three doses). Administration of 3.2 mg / kg on the same schedule induced piloerection in all three mice after the second dose. Severe weight loss (23% loss in one mouse, #552, after the second dose; 17% and 8% in the remaining mice, #551 and #552, after the third dose) was observed compared to PBS-treated controls. No gross changes were observed in mouse organs during necropsy. Individual mouse weights are shown in Table 23, and group means and SEMs are shown in Table 24. The weight change kinetics (group means and SEM) for each group are shown in Figure 10.

[0308] Doses of eribulin up to 1.6 mg / kg using a q4d x 3 regimen did not result in toxicity, and 3.2 mg / kg induced severe weight loss. Therefore, the MTD of eribulin in this study is 1.6 mg / kg q4d x 3.

[0309] [Table 23]

[0310] [Table 24]

[0311] 2.6.3 Evaluation of the Minimum Effective Dose of MORAb003-VCP-Eribulin (MORAb-202) in the hNSCLC NCI-H2110 Model in CB17-SCID Mice Human NSCLC, NCI-H2110 cells, passage 47, were subcutaneously implanted into 30 CB17 SCID mice (female, 5-6 weeks old, weighing 20 grams). 14 days after implantation, the mice were randomized into five groups. The mean tumor volume in each group on the day of treatment (day 0) was 154-175 mm. 3 (Table 27). According to the study design (Table 25), enrolled mice were treated with 1, 2.5, or 5 mg / kg of MORAb003-VCP-eribulin (MORAb-202) (Lot#NB2900-87E 10 / 07 / 15), 5 mg / kg of MORAb-003-0285 (Lot#042-150-002) as a control, or PBS. If any animal in a group had a tumor volume >2000 mm 3 Each group was removed from the study when the age reached 61 days.

[0312] [Table 25]

[0313] Tumor volumes for individual mice are shown in Table 26, and group means and SEM are shown in Table 27. Tumor growth kinetics for each group (group means and standard error of the mean, SEM) are shown in Figure 11, and tumor volumes for individual mice, as well as group means and SEM, are shown in Figure 12. Day 17 (first tumor >2000 mm 3Based on tumor volume (when tumor volume was observed), MORAb-202 produced 47% tumor growth inhibition (TGI) at 1 mg / kg (p=0.002 vs. saline) and 96% TGI at 2.5 mg / kg (p<0.0001 vs. saline). However, regressed tumors regrew 1-2 weeks after the end of treatment. No tumors were detected in mice treated with 5 mg / kg MORAb-202. These mice remained tumor-free for more than 60 days after single-dose treatment. MORAb-003-0285 produced 89.7% TGI at 5 mg / kg (p<0.0001 vs. saline).

[0314] Individual mouse body weights are shown in Table 28, and group means and SEM are shown in Table 29. The kinetics of body weight change (group means and SEM) in each group are shown in Figure 13.

[0315] No significant weight loss was observed in any of the treatment groups compared to controls.

[0316] MORAb-202 showed significant effects on NCI-H2110 tumor growth. Tumor regression was achieved with a bolus treatment of 2.5 mg / kg, with a TGI of 94% (vs. PBS). Therefore, the minimum effective dose of MORAb-202 tested in this model is 2.5 mg / kg. Complete tumor eradication was achieved with a single dose of 5 mg / kg. No tumor growth was observed for more than 60 days.

[0317] [Table 26]

[0318] [Table 27]

[0319] [Table 28]

[0320] [Table 29]

[0321] 2.6.4 Evaluation of the Minimum Effective Dose of Eribulin in the hNSCLC NCI-H2110 Model in CB17-SCID Mice Human NSCLC, H2110 cells, passage 46, were subcutaneously implanted into 30 CB17 SCID mice (female, 5-6 weeks old, weighing 20 grams). Eleven days after implantation, the mice were randomized into five groups. The five animals with the most tumor volume deviation from the mean were excluded. The mean tumor volume in each group on the day of treatment (day 0) was 87.6-89.4 mm. 3 According to the study design (Table 30), enrolled mice were treated with 0.05, 0.2, 0.8, or 1.6 mg / kg of eribulin (Lot#N1201193) or with PBS. 3 Each group was terminated when a tumor volume of 0.05 mg / kg / day was first observed. The study was terminated on day 38 (30 days after the last dose).

[0322] [Table 30]

[0323] Tumor volumes for individual mice are shown in Table 31, and group means and SEM are shown in Table 32. Tumor growth kinetics (group means and SEM) for each group are shown in Figure 14, and tumor volumes for individual mice, as well as tumor volumes at day 24 (>2000 mm in PBS-treated mice), are shown in Table 33. 3Group means and SEM at doses (when tumor volumes of 10 ...

[0324] Individual mouse body weights are shown in Table 33, and group means and SEM are shown in Table 34. The kinetics of body weight change (group means and SEM) in each group are shown in Figure 16.

[0325] No significant weight loss was observed in any of the treatment groups compared to the saline-treated control group. No clinical signs of toxicity were observed during treatment.

[0326] Eribulin at doses of 0.2 mg / kg or higher administered intravenously q4d x 3 demonstrated significant effects on H2110 tumor growth. Tumor regression was achieved. When lower doses were administered (0.05 mg / kg), tumor regression was not achieved. Therefore, the minimum effective dose tested in this study was 0.2 mg / kg.

[0327] [Table 31]

[0328] [Table 32]

[0329] [Table 33]

[0330] [Table 34]

[0331] Example 2 1. Materials and Methods MORAb003-VCP-eribulin (MORAb-202) was synthesized by conjugating MORAb-003 (humanized anti-human folate receptor alpha) to the MAL-PEG2-Val-Cit-PAB-eribulin (ER-001159569) compound described in section 1.1 of Example 3. The conjugation method is described in section 1.4.1 of Example 4.

[0332] 1.1 Tumor model Human tumor cell lines used in further in vitro evaluation of MORAb-202 include IGROV1 (human ovarian carcinoma, FR hi(+++) ), OVCAR3 (human ovarian cancer, FR med(++) ), NCI-H2110 (human non-small cell lung cancer, FR med(++) ), A431-A3 (A431 parent cell line stably transfected with human mesothelin, FR lo(+ / -) ), SJSA-1 (human osteosarcoma, FR neg(-) ), and HL-60 (human leukemia, FR neg(-) All of these cell lines were obtained directly from the American Type Culture Collection (ATCC). For in vitro testing, xenograft mouse models derived from patients with non-small cell lung cancer, triple-negative breast cancer, and endometrial cancer were established and maintained at Oncotest GmbH (Freiburg, Germany), Oncodesign (Dijon, France), and EPO Berlin-Buch GmbH (Berlin, Germany), respectively.

[0333] 1.2 In vitro cytotoxicity assay 1.2.1 Crystal violet assay IGROV1(FR hi(+++) ), A431-A3(FR lo(+ / -) ), and SJSA-1(FRneg(-) ) Cells were subcultured and seeded at 10,000 cells / well into 96-well tissue culture plates in complete growth medium and incubated overnight (16 hours) at 37°C, 5% CO2. Typically, test reagents were serially diluted 1:4 in a 2 mL deep-well dilution plate, starting at 1 μM (for a total of 10 dilutions). 100 μL of diluted sample was added to the cell plate (starting at a test sample concentration of 100 nM). The plate was incubated for an additional 48 hours at 37°C, 5% CO2. The medium was discarded, and the plate was washed once with 200 μL of DPBS and stained with 50 μL of 0.2% crystal violet solution for 15 minutes at room temperature, followed by extensive washing with tap water. The plate was air-dried, and the crystal violet was dissolved in 200 μL of 1% SDS solution. The plate was read at 570 nm. Data were analyzed using GraphPad Prism 6. OVCAR3(FR med(++) ) and NCI-H2110(FR med(++) ), cells were seeded at 3,000 cells / well and incubated with MORAb-202 for 5 days.

[0334] 1.3 In vivo testing 1.3.1 NCI-H2110 xenograft model Animal preparation: CB17 SCID mice (female, 6 weeks old) were housed at 5 mice per ventilated cage. Sterile food pellets and water bottles were available ad libitum to the animals. Animals were allowed to acclimate for 5-7 days before tumor implantation.

[0335] Cell culture: Human NCI-H2110 cells were thawed from cryopreservation (NB2813-65) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in 5% CO at 37°C. After two passages, when they reached approximately 70% confluence, cells were harvested by using cell dissociation solution, washed twice with serum-free medium, and counted.

[0336] Tumor implantation: Cell suspension in serum-free medium was mixed with ice-cold Matrigel at a 1:1 (v:v) ratio of 1.0 × 10 8Each mouse was mixed to a final concentration of 1.0 × 10 cells / mL. 7 100 μL of the mixture was injected subcutaneously at 100 μL per mouse. A 27G needle was used for all injections. Mice were monitored for clinical health and tumors were measured by digital caliper three times a week starting on day 3 post-implantation. Tumor volume (mm 3 ) was calculated using the formula: W (mm) × L (mm) × D (mm) × π / 6. 3 (>70~approx. 130mm 3 When tumor volume reached a mean of 1000 mg / kg, animals were randomized to 4-5 animals per group. The five animals with the greatest deviation in tumor volume from the mean were excluded.

[0337] Study Design: Enrolled experimental mice were intravenously injected with 200 μL of vehicle or 1.0, 2.5, and 5 mg / kg of MORAb-202 on the day of randomization according to the study design (Table 35). Body weights were measured before dosing and twice weekly during the study. At the end of the study, final body weights were measured and recorded. Individual tumor volumes of 2000 mm 3 Animals were euthanized when the tumor volume exceeded 100%. Criteria for early termination before reaching the maximum allowable tumor volume included: (1) tumor ulceration exceeding 50% (v:v); (2) paralysis; (3) >20% weight loss; and (4) 50% of the animals in a group were terminated. Any mice that were euthanized or found dead during the study were handled according to the termination procedure described above.

[0338] [Table 35]

[0339] 1.3.2 Patient-Derived Xenograft (PDx) Models 1.3.2.1 Non-small cell lung cancer (NSCLC) PDx model: LXFA-737 (Oncotest) Tumor Implantation: NSCLC tumor fragments were obtained from serially passaged LXFA-737 tumor xenografts in nude mice. After removal from the donor mice, tumors were cut into fragments (3-4 mm on each side) and placed in phosphate-buffered saline (PBS) containing 10% penicillin / streptomycin. Recipient animals were anesthetized by inhalation of isoflurane and received tumor implants subcutaneously in the flanks, either unilaterally or bilaterally. Tumor xenografts were implanted at one or two tumors per mouse, with a survival rate of <65%. In cases of bilateral survival, one of these tumors was explanted prior to randomization. Animals and tumor implants were monitored daily until solid tumor growth was detectable in a sufficient number of animals. At randomization, the volume of the growing tumors was determined to be approximately 100-120 mm. 3 For the purpose of comparable median and mean group tumor volumes, animals meeting the randomization criteria (i.e., 50–250 mm) were randomized. 3 , preferably 80 to 200 mm 3 The mice (bearing tumors) were distributed into experimental groups consisting of 5-6 animals per group. Animals not used in the experiment were euthanized. The day of randomization was designated day 0 of the experiment.

[0340] Study Design: On the day of randomization, enrolled experimental mice were injected intravenously with vehicle, 5 mg / kg MORAb-003, or 5 mg / kg MORAb-202 according to the study design (Table 36). Body weights were measured pre-dose on each dosing day and twice weekly during the study. At the end of the study, terminal body weights were measured and recorded. Individual tumor volumes of 2000 mm 3 When the body weight exceeded 100g, the animals were euthanized.

[0341] [Table 36]

[0342] 1.3.2.2 Triple-Negative Breast Cancer (TNBC) PDx Model: OD-BRE-0631 (Oncodesign) Tumor implantation: Nine female Swiss nude mice were subcutaneously injected with patient-derived TNBC tumor fragments in the right flank. Tumor volumes ranged from 500 to 1000 mm.3 When tumors reached a mass of 200–300 mm, tumor-bearing mice were euthanized and tumors were surgically excised. 24–72 h after whole-body irradiation with a gamma ray source (2 Gy, 60Co, BioMEP, France), tumor fragments (30–50 mg) were orthotopically implanted into the mammary fat pad region of 34 female Swiss nude mice. 3 When the tumors reached a mean volume of 1000 mg / kg, 24 of the 34 animals were randomized into two groups (n = 12 animals) according to their individual tumor volumes using Vivo Manager® software (Biosystemes, Couternon, France). Statistical tests (analysis of variance) were performed to assess the homogeneity between groups. The day of randomization was designated day 0 of the experiment.

[0343] Study design: On day 1 (1 day after randomization and 2 days before treatment), three mice from each of the two untreated groups were terminated. On day 3, the remaining experimental mice were intravenously injected with vehicle or 5 mg / kg of MORAb-202 according to the study design (Table 37). On day 8 (5 days after treatment), three mice from each of the two treatment groups were terminated. Immediately after termination, tumor tissues were collected and fixed in 4% neutral buffered formalin for 24-48 hours, then embedded in paraffin (Histosec®, Merck, Darmstadt, Germany). Paraffin-embedded samples were stored at room temperature for subsequent immunohistochemical analysis.

[0344] [Table 37]

[0345] Immunohistochemistry (IHC) analysis: To evaluate both MORAb-202 occupancy and expression in cancer-associated fibroblasts, IHC staining of formalin-fixed, paraffin-embedded tumor tissues was performed. Prior to staining, slides were dewaxed, and antigens were retrieved in citrate buffer (pH 6.0) preheated to 85°C in a LabVision™ PT Module (Thermo Scientific) using the following program: warm to 97°C; incubate at 97°C for 30 minutes; cool to 60°C. Slides were then transferred to double-distilled water at room temperature for 5 minutes. Staining was performed using a LabVision™ Autostainer 360 (Thermo Scientific). Briefly, slides were washed twice in 1x Tris-buffered saline / Tween-20 (TBST) for 6 minutes per wash. The tissue sections were then incubated in blocking buffer (300 μL) (10% goat serum (Jackson Immunoresearch Laboratory Inc., Cat. No. 005-000-121), 3% bovine serum albumin (BSA) diluted in phosphate-buffered saline (PBS)) for 1 hour, conjugated antibody (200 μL) (Table 38) for 1 hour, and washed five times in 1x TBST for 6 minutes per wash. Slides were counterstained with DAPI in mounting medium, and slides with coverslips were allowed to harden for 30 minutes. Slides were processed on a Panoramic MIDI scanner (3DHISTECH), and IHC images were analyzed using Halo software (Indica Labs). The antibodies used in this analysis targeted human IgG, which can detect the presence of α-smooth muscle actin (SMA), a specific marker of cancer-associated fibroblasts, and MORAb-202.

[0346] [Table 38]

[0347] 1.3.2.3 Endometrial Cancer PDx Models: Endo-12961 and Endo-10590 (EPO Berlin) Tumor Implantation: Endometrial cancer tumor fragments were obtained from serially passaged Endo-12961 and Endo-10590 tumor xenografts and stored as stocks in liquid nitrogen. Tumor fragments were implanted subcutaneously into the left flank of 40 female NMRI nu / nu mice, and tumor volumes were monitored. The tumor volumes ranged from 100 to 160 mm. 3 Mice with tumor volumes of 0.01% or less were randomized into one of four groups (Groups A-D, Table 39). Satellite mice for randomization were included in a fifth group (Group E, Table 39). Each group consisted of eight animals. The day of randomization was designated day 0 of the experiment.

[0348] Study Design: On the day of randomization, enrolled experimental mice were intravenously injected with PBS, 3.2 mg / kg or 0.1 mg / kg eribulin, or 5 mg / kg MORAb-202 according to the study design (Table 39). Tumor growth was assessed by measuring two perpendicular diameters twice weekly, and tumor volume (TV), relative tumor volume (RTV), and treatment / control (T / C) values ​​were calculated. Body weight was also assessed twice weekly as a parameter for toxicity, along with calculation of body weight change per group and body weight change relative to the start of treatment (BWC). Individual tumor volumes of 1 cm 3 The animals were sacrificed when the stool mass exceeded 100 mg / kg or at the end of the study.

[0349] [Table 39]

[0350] 1.4 Mechanism of action 1.4.1 Three-dimensional (3D) co-culture system in zPredicta All mesenchymal stem cell (MSC)-containing 3D coculture experiments were performed in zPredicta using organ-specific 3D extracellular matrix systems, such as rStomach™. Bone marrow mesenchymal stem cells (BM-MSCs) in rStomach™ were cocultured with the Nuc Red Light MKN-74 gastric cancer cell line in quadruplicate in a 48-well format for 12 days. MKN-74 cells have previously been shown to express sufficient folate receptor alpha (FR) for MORAb-202 treatment to induce cell apoptosis. Prior to culture, BM-MSCs were assessed by flow cytometry for target antigen expression and for markers of MSC differentiation (Table 40).

[0351] [Table 40]

[0352] rStomach™ cultures were treated with either MORAb-202, unconjugated MORAb-003 antibody, eribulin, or controls, as described in Table 41. Controls included untreated and vehicle-treated (PBS and DMSO) cultures. MSC differentiation was monitored by light microscopy. Once visible differentiation was observed, samples were taken for staining and flow cytometry analysis.

[0353] [Table 41]

[0354] 1.4.2 Time course analysis of the effects of MORAb-202 on cancer-associated fibroblasts Subcutaneous H2110 xenograft tumor-bearing mice were prepared as described in Section 1.3.1. Tumor samples were collected on days 0, 3, 5, 7, and 9 after administration of vehicle or 5 mg / kg of MORAb-202. Collected tumor samples were processed onto slides and analyzed by IHC for cancer-associated fibroblast expression as described in Section 1.3.2.2.

[0355] 2. Results 2.1 In vitro cytotoxicity analysis 2.1.1 Cytotoxicity of MORAb-202 The in vitro potency of MORAb-202 was assessed using a crystal violet assay as detailed in Section 1.2.1. Screening was performed using IGROV1 (FR hi(+++) ), OVCAR3(FR med(++) ), NCI-H2110(FR med(++) ), A431-A3(FR lo(+ / -) ), and SJSA-1(FR neg(-) The results of this screening are shown in Figure 17 and Table 42.

[0356] [Table 42]

[0357] MORAb-202 demonstrated folate receptor alpha expression-dependent cytotoxicity and low levels of off-target killing against tumor cell lines. MORAb-202 demonstrated the highest level of potency (0.01 nM) in IGROV1 cells, but little cytotoxicity (>100 nM) in folate receptor alpha-negative SJSA-1 cells. Intermediate potency was observed in OVCAR3 and NCI-H2110 cells (0.16 nM and 0.74 nM, respectively).

[0358] 2.2 In vivo testing 2.2.1 Efficacy of MORAb-202 in the NC1-H2110 xenograft model Mice bearing subcutaneous H2110 tumors were intravenously injected with vehicle or 1, 2.5, and 5 mg / kg of MORAb-202. Significant tumor regression was observed after a single dose of 5 mg / kg of MORAb-202 (Figure 18 and Table 43). Using this xenograft model with high folate receptor alpha expression and single-dose administration, the therapeutic window for MORAb-202 was shown to be 1 mg / kg for tumor growth delay (stable disease) and ≥ 2.5 mg / kg for tumor regression. In this study, MORAb-202 at a dose of 2.5 mg / kg induced a partial response, and MORAb-202 at a dose of 5 mg / kg induced a complete response.

[0359] [Table 43]

[0360] 2.2.2 Efficacy of MORAb-202 in NSCLC PDx Model: LXFA-737 Subcutaneous NSCLC PDx tumor-bearing mice were intravenously injected with vehicle, 5 mg / kg MORAb-003, or 5 mg / kg MORAb-202. In contrast to a single dose of unconjugated MORAb-003 antibody (5 mg / kg), which did not demonstrate significant antitumor activity in this model, a single dose of MORAb-202 (5 mg / kg) resulted in significant tumor regression (Figure 19A). Five of the six mice treated with MORAb-202 were deemed tumor-free on day 32 of the study (Table 44), and four remained tumor-free through day 74 (the end of the study). In addition, no significant weight loss was observed in the treated groups compared with the vehicle-treated control group, indicating a lack of toxicity during treatment (Figure 19B).

[0361] [Table 44]

[0362] 2.2.3 Relative Efficacy of MORAb-202 and Eribulin in Endometrial Cancer PDx Models: Endo-12961 and Endo-10590 Endo-12961 and Endo-10590 xenografts express high levels of folate receptor alpha. Subcutaneous endometrial cancer PDx tumor-bearing mice were intravenously injected with PBS, 3.2 mg / kg or 0.1 mg / kg eribulin, or 5 mg / kg MORAb-202. The maximum tolerated dose (MTD) of eribulin in this model is 3.2 mg / kg, whereas 0.1 mg / kg is equivalent to the eribulin dosage achieved by MORAb-202 administered at 5 mg / kg. Throughout the study, significant antitumor activity was observed in both animal models after treatment with MORAb-202 (5 mg / kg) and the MTD dose of eribulin (3.2 mg / kg), whereas no significant antitumor activity was observed after treatment with 0.1 mg / kg eribulin (Figures 20A and 20C). However, the regressed tumors in mice treated with 3.2 mg / kg eribulin began to regrow during the study period, while no significant tumor regrowth was noted in mice treated with MORAb-202. In this study, MORAb-202 proved to be significantly more effective than eribulin. Eribulin treatment also temporarily affected body weight during the first week after treatment (Figures 20B and 20D). In contrast, no weight loss was observed in animals treated with MORAb-202.

[0363] 2.3 Mechanism of action of MORAb-202 2.3.1 IHC and Efficacy of MORAb-202 in the TNBC PDx Model: OD-BRE-0631 Mice bearing subcutaneous TNBC PDx tumors were intravenously injected with vehicle or 5 mg / kg MORAb-202. Tumor tissues were collected from each group of mice before (day 1) and after (day 8) treatment. IHC analysis of the collected tumor tissues revealed that MORAb-202, administered as a single dose (5 mg / kg) on ​​day 3, occupied folate receptor alpha-expressing tumor cells 5 days after treatment (day 8). Cell occupancy was assessed using an anti-human IgG antibody (Figure 21A). MORAb-202 treatment also reduced the structure of cancer-associated fibroblasts, as shown by IHC staining with anti-α-smooth muscle actin (SMA)-FITC antibody (Figure 21B). In terms of efficacy, MORAb-202 treatment resulted in maximum tumor regression, measured by a relative tumor volume (RTV) of 0.62, 11 days after treatment (Figure 21C).

[0364] 2.3.2 Effects of MORAb-202, MORAb-003, and eribulin in 3D co-culture systems Bone marrow mesenchymal stem cells (BM-MSCs) in rStomach™ were co-cultured with the MKN-74 gastric cancer cell line for 12 days. Prior to culture, BM-MSCs were assessed for folate receptor alpha expression and for markers of MSC differentiation by flow cytometry. rStomach™ cultures were then treated with either MORAb-202, unconjugated MORAb-003 antibody, eribulin, or a control. Once visible MSC differentiation was observed by light microscopy, samples were taken for staining and flow cytometry analysis. The results of these analyses are shown in Figure 22.

[0365] A total treatment period of 7 days (with supplemental treatment during the period) was sufficient to produce a measurable effect on the differentiation of human BM-MSCs in cultures containing MKN-74 cells. Relative to vehicle control, treatment with MORAb-202 (10 nM) resulted in an increase in the MSC and adipocyte populations and a decrease in the pericyte population (Table 45). These data indicate that MORAb-202 can have a significant effect on the tumor microenvironment.

[0366] [Table 45]

[0367] 2.3.3 Time course analysis of the effects of MORAb-202 on cancer-associated fibroblasts Tumor samples were collected from mice bearing subcutaneous H2110 xenograft tumors on days 0, 3, 5, 7, and 9 after administration of vehicle or 5 mg / kg of MORAb-202. Collected tumor samples were processed onto slides, and cancer-associated fibroblast (CAF) expression was analyzed by IHC. CAF network structure, assessed and quantified by staining with anti-α-smooth muscle actin (SMA)-FITC antibody, was prominent on days 3 and 5 after administration of a single dose of 5 mg / kg of MORAb-202 (Figure 23). However, by day 7, the majority of this structure had significantly decreased.

[0368] Example 3 1. Materials and Methods Conjugable eribulin compounds having the structures shown in Table 46 were synthesized according to the following procedure and used in the preparation of ADCs (Example 4).

[0369] All solvents used in synthesis reactions were anhydrous grade (EMD Millipore). All solvents used for workup or purification were high-performance liquid chromatography (HPLC) grade (EMD Millipore). Unless otherwise noted, all chemicals were commercially available. Column chromatography was performed using a Biotage® SP4. Solvent removal was performed using either a rotary evaporator (Buchi Laboratechik AG) or a centrifugal evaporator (Genevac, SP Scientific). Preparative liquid chromatography-mass spectrometry (LC / MS) was performed using a Waters AutoPurification System and an XTerra MS C18 column (5 μm, 19 mm × 100 mm) under acidic mobile phase conditions. Nuclear magnetic resonance (NMR) spectra were obtained using deuterated chloroform (CDCl3) and recorded at 400 or 500 MHz using a Varian instrument (Agilent Technologies) unless otherwise noted. Mass spectra were obtained using a Waters Acquity Ultra Performance LC / MS. As used herein, the term "inerting" refers to replacing the air in a reactor (e.g., a reaction vessel, a flask, a glass reactor) with an essentially moisture-free inert gas, such as nitrogen or argon. Multiplicities are indicated using the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sxt = sextet, m = multiplet, dd = double doublet, ddd = double doublet of doublets, dt = doublet of triplets, br s = broad singlet.

[0370] [Table 46] TIFF2024174991000090.tif175149 TIFF2024174991000091.tif215149 TIFF2024174991000092.tif179149 TIFF2024174991000093.tif187149 TIFF2024174991000094.tif226149

[0371] 1.1 Preparation of MAL-PEG2-Val-Cit-PAB-Eribulin (ER-001159569) [ka]

[0372] Eribulin (ER-000086526) (61.5 mg, 0.074 mmol) was dissolved in N,N-dimethylformamide (DMF) (6.0 mL) and then mixed with Hunig's base (0.027 mL, 0.156 mmol) and Fmoc-Val-Cit-PAB-PNP (86 mg, 0.112 mmol). The reaction was stirred at room temperature for 18 hours until coupling was complete, as determined by high-performance liquid chromatography (HPLC) analysis. Diethylamine (0.078 mL, 0.745 mmol) was added to the mixture, and the mixture was stirred for an additional 2 hours until the reaction was complete. The solvent was removed by evaporation, and the residue was purified by flash chromatography to give Val-Cit-PAB-Eribulin (ER-001228950) as a white solid (60 mg, 71% yield). 1HNMR(400 MHz, CD3OD) δ ppm 7.56 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz,2H), 5.14 (s, 1H), 5.06 (d, J = 12.4 Hz, 1H), 5.03 (s, 1H), 5.01 (d, J = 12.4 Hz,1H), 4.87 (s, 1H), 4.83 (s, 1H), 4.71 (t, J = 4.4 Hz, 1H), 4.62 (t, J = 4.4 Hz,1H), 4.57 (dd, J = 4.8, 8.8 Hz, 1H), 4.47 (d, J = 10.8 Hz, 1H), 4.32-4.27 (m, 2H),4.18 (dd, J = 4.8, 6.4 Hz, 1H), 4.13-4.07 (m, 2H), 3.98 (t, J = 10.4 Hz, 1H), 3.88-3.82(m, 3H), 3.76-3.70 (m, 4H), 3.60 (d, J = 6.0 Hz, 1H), 3.38 (s, 3H), 3.26-3.10 (m,3H), 2.93 (dd, J = 2.0, 11.2 Hz, 1H), 2.91-2.84 (m, 1H), 2.75-2.64 (m, 2H), 2.44-2.29(m, 5H), 2.21-1.97 (m, 8H), 1.93-1.83 (m, 3H), 1.79-1.72 (m, 5H), 1.68-1.29 (m,8H), 1.11 (d, J = 6.8 Hz, 3H), 1.07-1.01 (m, 1H), 1.06 (d, J = 7.2 Hz, 3H), 1.02(d, J = 7.2 Hz, 3H)。LCMS(M+H)=1135.7。

[0373] Val-Cit-PAB-Eribulin (ER-001228950) (16 mg, 14 μmol) was dissolved in DMF (1 mL). N,N-Diisopropylethylamine (7.2 μL, 41 μmol) and Mal-PEG2-NHS (9.7 mg, 27 μmol) were then added to the solution at room temperature, and the reaction mixture was stirred at room temperature for 1 hour. Upon completion of the reaction, the crude mixture was purified by reverse-phase HPLC using an acetonitrile-water mobile phase containing 0.1% formic acid. The collected fractions were concentrated under vacuum at room temperature in an unheated water bath to give Mal-PEG2-Val-Cit-PAB-Eribulin (ER-001159569) (7.1 mg, 5.2 μmol, 38% yield). 1HNMR (400 MHz, CD3OD) δ ppm 7.59 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.81 (s, 2H), 5.13 (s, 1H), 5.06 (d, J = 12.4 Hz, 1H), 5.02 (s, 1H), 5.01 (d, J = 12.4 Hz, 1H), 4.87 (s, 1H), 4.82 (s, 1H), 4.71 (t, J = 4.0 Hz, 1H), 4.61 (t, J = 4.4 Hz, 1H), 4.50 (dd, J = 5.2, 9.2 Hz, 1H), 4.47 (d, J = 10.8 Hz, 1H), 4.32 - 4.27 (m, 2H), 4.19 (dd, J = 6.8, 11.6 Hz, 1H), 4.13 - 4.07 (m, 2H), 3.98 (t, J = 10.4 Hz, 1H), 3.88 - 3.82 (m, 3H), 3.76 - 3.64 (m, 6H), 3.62 - 3.51 (m, 6H), 3.38 (s, 3H), 3.22 - 3.08 (m, 4H), 2.93 (dd, J = 2.4, 9.6 Hz, 1H), 2.92 - 2.84 (m, 1H), 2.76 - 2.63 (m, 2H), 2.52 (t, J = 6.0 Hz, 2H), 2.44 - 2.29 (m, 5H), 2.21 - 1.97 (m, 8H), 1.93 - 1.83 (m, 3H), 1.80 - 1.66 (m, 5H), 1.66 - 1.28 (m, 10H), 1.11 (d, J = 6.4 Hz, 3H), 1.07 - 1.01 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.4 Hz, 3H). LCMS (M + H) = 1374.9.

[0374] 1.2 Preparation of NHS-PEG2-Val-Cit-PAB-Eribulin (ER-001236940) [Chemical formula]

[0375] Val-Cit-PAB-Eribulin (ER-001228950) (45 mg, 0.04 mmol) and bis(2,5-dioxopyrrolidin-1-yl) 3,3'-(ethane-1,2-diylbis(oxy))dipropanoate (79 mg, 0.198 mmol) were mixed in DMF (1.5 mL), and then EtN (44.2 μL, 0.317 mmol) was added. The mixture was stirred for 18 hours until the reaction was complete, as determined by HPLC analysis. The solvent was evaporated, and the residue was purified by flash chromatography to give NHS-PEG2-Val-Cit-PAB-Eribulin (ER-001236940) as a white solid (38 mg, 68% yield). 1HNMR (400 MHz, CD3OD) δ ppm 7.58 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 5.14 (s, 1H), 5.05 (d, J = 12.4 Hz, 1H), 5.03 (s, 1H), 5.01 (d, J = 12.4 Hz, 1H), 4.87 (s, 1H), 4.83 (s, 1H), 4.71 (t, J = 4.4 Hz, 1H), 4.62 (t, J = 4.4 Hz, 1H), 4.51 (dd, J = 4.8, 8.8 Hz, 1H), 4.50 - 4.47 (m, 1H), 4.32 - 4.27 (m, 2H), 4.21 (dd, J = 4.8, 6.4 Hz, 1H), 4.14 - 4.08 (m, 2H), 3.99 (t, J = 10.4 Hz, 1H), 3.88 - 3.82 (m, 3H), 3.78 - 3.70 (m, 4H), 3.62 (s, 2H), 3.62 - 3.58 (m, 1H), 3.50 - 3.46 (m, 2H), 3.39 (s, 4H), 3.36 (s, 3H), 3.22 - 3.08 (m, 3H), 2.93 (dd, J = 2.0, 11.2 Hz, 1H), 2.91 - 2.87 (m, 1H), 2.84 (s, 2H), 2.80 (s, 2H), 2.75 - 2.64 (m, 2H), 2.59 - 2.52 (m, 2H), 2.44 - 2.29 (m, 5H), 2.21 - 1.97 (m, 10H), 1.93 - 1.83 (m, 3H), 1.79 - 1.72 (m, 5H), 1.68 - 1.29 (m, 8H), 1.11 (d, J = 6.8 Hz, 3H), 1.08 - 0.98 (m, 1H), 1.00 (d, J = 7.2 Hz, 3H), 0.98 (d, J = 7.2 Hz, 3H). LCMS (M + H) = 1421.0.

[0376] 1.3 Preparation of NHS-(CH2)5-Val-Cit-PAB-Eribulin (ER-001236941)

Chemical Structure

[0377] Heptanedioic acid (1.6 g, 9.99 mmol) was dissolved in tetrahydrofuran (THF) (100 mL), and then 1-hydroxypyrrolidine-2,5-dione (2.299 g, 19.98 mmol) was added, followed by DCC (4.12 g, 19.98 mmol). The mixture was stirred at room temperature for 18 hours until HPLC analysis showed the reaction was complete. The solid was removed by filtration through a Celite pad and washed with THF (3 × 2 mL). The combined filtrate was concentrated and purified by flash chromatography to give bis(2,5-dioxopyrrolidin-1-yl)heptanedioate (ER-001236140) as a white solid (2.5 g, 71% yield). 1 HNMR(400 MHz) δ ppm 2.83 (s, 8H), 2.64 (t, J = 7.6 Hz, 4H), 1.80 (dt, J = 7.6 Hz, 4H),1.59-1.51 (m, 2H). LCMS(M+H)=355.2.

[0378] Using the same procedure described above for the preparation of NHS-PEG-Val-Cit-PAB-eribulin (ER-001236940), NHS-(CH)-Val-Cit-PAB-eribulin (ER-001236941) was prepared from VCP-eribulin (ER-001228950) and bis(2,5-dioxopyrrolidin-1-yl)heptanedioate (ER-001236140) (8.5 mg, 47% yield). 1HNMR (400 MHz, CD3OD) δ ppm 7.56 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 5.13 (s, 1H), 5.04 (d, J = 12.0 Hz, 1H), 5.01 (s, 1H), 5.00 (d, J = 12.4 Hz, 1H), 4.86 (s, 1H), 4.82 (s, 1H), 4.70 (t, J = 4.4 Hz, 1H), 4.60 (t, J = 4.4 Hz, 1H), 4.50 (dd, J = 4.8, 8.8 Hz, 1H), 4.46 (d, J = 10.8 Hz, 1H), 4.36 - 4.25 (m, 2H), 4.17 (dd, J = 4.8, 6.4 Hz, 1H), 4.13 - 4.06 (m, 2H), 3.97 (t, J = 10.4 Hz, 1H), 3.87 - 3.80 (m, 3H), 3.74 - 3.68 (m, 2H), 3.37 (s, 3H), 3.20 - 3.06 (m, 4H), 2.94 (dd, J = 2.0, 11.2 Hz, 1H), 2.90 - 2.82 (m, 1H), 2.82 (s, 4H), 2.74 - 2.65 (m, 2H), 2.61 (t, J = 8.0 Hz, 2H), 2.46 - 2.26 (m, 7H), 2.24 - 1.81 (m, 13H), 1.78 - 1.28 (m, 19H), 1.10 (d, J = 6.8 Hz, 3H), 1.06 - 0.96 (m, 1H), 0.97 (d, J = 7.2 Hz, 3H), 0.95 (d, J = 7.2 Hz, 3H). LCMS (M + H) = 1375.1.

[0379] 1.4 Preparation of Mal-(CH2)5-Val-Cit-PAB-Eribulin (ER-001235638)

Chemical formula

[0380] Eribulin (ER-000086526) (10 mg, 0.012 mmol) was dissolved in DMF (1 mL) and mixed with MC-Val-Cit-PAB-PNP (9.02 mg, 0.012 mmol) and Hunig's base (4.44 μL, 0.025 mmol). The mixture was then stirred at room temperature for 12 hours until HPLC analysis showed the reaction was complete. The reaction mixture was concentrated and purified by flash chromatography to give Mal-(CH)-Val-Cit-PAB-Eribulin (ER-001235638) as a white solid (11.3 mg, 63% yield). 1 HNMR(400 MHz, CD3OD) δ ppm 7.57 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz,2H), 6.79 (s, 2H), 5.13 (s, 1H), 5.05 (d, J = 12.4 Hz, 1H), 5.02 (s, 1H), 5.00 (d,J = 12.4 Hz, 1H), 4.87 (s, 1H), 4.83 (s, 1H), 4.71 (t, J = 4.4 Hz, 1H), 4.61 (t,J = 4.4 Hz, 1H), 4.56-4.46 (m, 3H), 4.35-4.27 (m, 2H), 4.20-4.07 (m, 4H), 3.98 (t,J = 10.8 Hz, 1H), 3.87-3.83 (m, 3H), 3.73-3.70 (m, 2H), 3.48 (t, J = 7.6 Hz, 2H),3.38 (s, 3H), 3.20-3.08 (m, 4H), 2.93 (dd, J = 1.6, 9.6 Hz, 1H), 2.89-2.85 (m, 1H),2.69 (dt, J = 11.2, 16.8 Hz, 2H), 2.44-2.33 (m, 5H), 2.27-1.83 (m, 13H), 1.78-1.68(m, 5H), 1.66-1.27 (m, 14H), 1.11 (d, J = 7.2 Hz, 3H), 1.07-0.98 (m, 1H), 0.98 (d,J = 7.2 Hz, 3H), 0.96 (d, J = 7.2 Hz, 3H). LCMS(M+H)=1328.9.

[0381] 1.5 Preparation of Mal-PEG8-Val-Cit-PAB-Eribulin (ER-001242287) [ka]

[0382] VCP-eribulin (ER-001228950) (10 mg, 8.808 μmol) and 2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oate (6.07 mg, 8.808 μmol) were mixed in DMF (1 mL), followed by the addition of EtN (9.82 μL, 0.07 mmol). The reaction mixture was stirred at room temperature for 18 hours until HPLC analysis showed the reaction was complete. The solvent was removed by evaporation, and the residue was purified by flash chromatography to give Mal-PEG8-Val-Cit-PAB-Eribulin (ER-001242287) as a white solid (3.0 mg, 20% yield). 1HNMR(400 MHz, CD3OD)δ ppm 7.58 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 6.80 (s, 2H), 5.12 (s, 1H), 5.04 (d, J = 12.4 Hz, 1H), 5.01 (s, 1H), 4.99 (d,J = 12.4 Hz, 1H), 4.85 (s, 1H), 4.80 (s, 1H), 4.69 (t, J = 4.4 Hz, 1H), 4.59 (t,J = 4.4 Hz, 1H), 4.50-4.42 (m, 2H), 4.32-4.24 (m, 2H), 4.20-4.14 (m, 2H), 4.12-4.04(m, 3H), 3.96 (t, J = 10.4 Hz, 1H), 3.86-3.80 (m, 3H), 3.76-3.57 (m, 4H), 3.48 (t,J = 6.0 Hz, 1H), 3.36 (s, 3H), 3.20-3.08 (m, 3H), 2.91 (dd, J = 2.0, 11.2 Hz, 1H), 2.90-2.82 (m, 1H), 2.74-2.60 (m, 2H), 2.44-2.29 (m, 5H), 2.21-1.97 (m, 10H), 1.93-1.83(m, 3H), 1.79-1.20 (m, 19H), 1.09 (d, J = 6.8 Hz, 3H), 1.04-0.98 (m, 1H), 0.99 (d,J = 7.2 Hz, 3H), 0.97 (d, J = 7.2 Hz, 3H). LCMS(M+H)=1711.6.

[0383] 1.6 Modulation of NHS-PEG9-Val-Cit-PAB-エリブリン(ER-001242288)

change

[0384] Using the same procedure described above for the preparation of NHS-PEG2-Val-Cit-PAB-eribulin (ER-001236940), NHS-PEG9-Val-Cit-PAB-eribulin (ER-001242288) was prepared from VCP-eribulin (ER-001228950) and bis-NHS-PEG9 (13 mg, 85% yield). 1 HNMR(400 MHz, CD3OD) δ ppm 7.61 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz,2H), 5.16 (s, 1H), 5.06 (d, J = 12.4 Hz, 1H), 5.01 (s, 1H), 5.00 (d, J = 12.4 Hz,1H), 4.87 (s, 1H), 4.82 (s, 1H), 4.71 (t, J = 4.4 Hz, 1H), 4.61 (t, J = 4.4 Hz,1H), 4.52-4.45 (m, 2H), 4.34-4.26 (m, 2H), 4.20-4.19 (m, 1H), 4.14-4.06 (m, 2H),3.98 (t, J = 10.4 Hz, 1H), 3.88-3.80 (m, 3H), 3.76-3.70 (m, 4H), 3.66-3.58 (m, 37H),3.38 (s, 3H), 3.24-3.10 (m, 3H), 2.93 (dd, J = 2.0, 11.2 Hz, 1H), 2.91-2.84 (m,1H), 2.84 (s, 4H), 2.76-2.64 (m, 2H), 2.58-2.50 (m, 4H), 2.46-2.28 (m, 5H), 2.22-1.96(m, 8H), 1.91-1.82 (m, 3H), 1.79-1.68 (m, 5H), 1.64-1.24 (m, 8H), 1.11 (d, J = 6.8Hz, 3H), 1.08-0.96 (m, 1H), 0.99 (d, J = 7.2 Hz, 3H), 0.97 (d, J = 7.2 Hz, 3H). LCMS(M+H)=1729.7.

[0385] 1.7 Preparation of NHS-PEG3-triazole-PEG3-Val-Cit-PAB-Eribulin (ER-001243700) [ka]

[0386] VCP-eribulin (ER-001228950) (25 mg, 0.022 mmol) was dissolved in DMF (2.5 mL) and then mixed with EtN (24.55 μL, 0.176 mmol) and azido-PEG-NHS (8.34 mg, 0.024 mmol). The mixture was stirred at room temperature for 18 hours until HPLC analysis showed the reaction was complete. The mixture was concentrated in vacuo, and the residue was purified by preparative HPLC (MeCN and water containing 0.1% formic acid). The fraction containing azido-PEG-Val-Cit-PAB-eribulin was extracted with dichloromethane (CHCl) (3 × 20 mL), and the CHCl was evaporated to give azido-PEG-Val-Cit-PAB-eribulin (ER-001243116) as a white solid (18.9 mg, 63% yield). 1HNMR(400 MHz, CD3OD) δ ppm 7.58 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz,2H), 5.14 (s, 1H), 5.04 (d, J = 12.4 Hz, 1H), 5.03 (s, 1H), 5.01 (d, J = 12.4 Hz,1H), 4.85 (s, 1H), 4.81 (s, 1H), 4.70 (t, J = 4.4 Hz, 1H), 4.61 (t, J = 4.4 Hz,1H), 4.52-4.48 (m, 2H), 4.31-4.25 (m, 2H), 4.20-4.15 (m, 1H), 4.13-4.07 (m, 2H),3.99 (t, J = 10.4 Hz, 1H), 3.84-3.79 (m, 3H), 3.77-3.65 (m, 4H), 3.64-3.56 (m, 13H),3.38 (s, 3H), 3.20-3.05 (m, 3H), 2.95-2.80 (m, 2H), 2.75-2.60 (m, 2H), 2.55-2.50(m, 2H), 2.43-2.25 (m, 5H), 2.21-1.97 (m, 8H), 1.93-1.83 (m, 3H), 1.79-1.72 (m,5H), 1.68-1.29 (m, 10H), 1.08 (d, J = 6.8 Hz, 3H), 1.05-0.95 (m, 1H), 0.98 (d, J= 7.2 Hz, 3H), 0.95 (d, J = 7.2 Hz, 3H)。LCMS(M+H)=1365.1。

[0387] Azido-PEG3-VCP-eribulin (ER-001243116) (9.6 mg, 7.035 μmol) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)propanoate (6.61 mg, 0.021 mmol) were mixed in water (0.6 mL) and t-butanol (1.8 mL). N was bubbled through the mixture for 45 minutes. Copper iodide on amberlyst-21 (1.23 mmol / g, 10 mg) was added to the mixture, and N was bubbled through the mixture for another 30 minutes. The reaction mixture was then stirred at room temperature for 72 hours until the starting material was completely consumed. LCMS analysis showed that the desired NHS ester product was not observed, only the hydrolyzed carboxylic acid. The mixture was filtered through a short Celite pad to remove the CuI resin. The filtrate was concentrated in vacuo, and the resulting residue was purified by preparative thin-layer chromatography (prep-TLC) (20% MeOH / CH2Cl2) to give acid-PEG3-triazole-PEG3-Val-Cit-PAB-eribulin (ER-001243701) as a white solid (3.7 mg, 33% yield). LCMS (ES) (M+H) = 1581.2.

[0388] Acid-PEG-triazole-PEG-Val-Cit-PAB-eribulin (ER-001243701) (3.0 mg, 1.898 μmol) was dissolved in DMF (200 μL) and 1-hydroxypyrrolidine-2,5-dione (0.437 mg, 3.796 μmol) was added, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.728 mg, 3.796 μmol). After stirring at room temperature for 18 hours, the reaction was approximately 50% complete. EDC (1.46 mg, 7.8 μmol) was added, and the mixture was stirred for an additional 18 hours until HPLC analysis showed >95% conversion to NHS-PEG-triazole-PEG-Val-Cit-PAB-eribulin. The mixture was concentrated in vacuo, and the residue was purified by preparative-TLC (15% MeOH / CH2Cl2) to give NHS-PEG3-triazole-PEG3-Val-Cit-PAB-Eribulin (ER-001243700) as a white solid (2.2 mg, 69% yield). 1HNMR(400 MHz, CD3OD) δ ppm 8.00 (s, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.31 (d,J = 8.4 Hz, 2H), 5.13 (s, 1H), 5.04 (d, J = 12.4 Hz, 1H), 5.02 (s, 1H), 5.00 (d,J = 12.4 Hz, 1H), 4.87 (s, 1H), 4.83 (s, 1H), 4.71 (t, J = 4.0 Hz, 1H), 4.63 (s,2H), 4.61 (t, J = 4.4 Hz, 1H), 4.57-4.55 (m, 2H), 4.51-4.45 (m, 1H), 4.32-4.28 (m,2H), 4.21-4.17 (m, 2H), 4.13-4.10 (m, 2H), 3.98 (t, J = 10.8 Hz, 1H), 3.88-3.80(m, 5H), 3.75-3.70 (m, 4H), 3.68-3.55 (m, 18H), 3.45-3.40 (m, 2H), 3.38 (s, 3H), 3.20-3.08 (m, 4H), 2.93-2.80 (m, 2H), 2.75-2.50 (m, 2H), 2.68 (s, 4H), 2.48-2.30(m, 7H), 2.28-1.92 (m, 10H), 1.90-1.68 (m, 8H), 1.65-1.27 (m, 8H), 1.11 (d, J =6.8 Hz, 3H), 1.05-0.95 (m, 1H), 0.99 (d, J = 7.2 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H). LCMS(M+H)=1678.3.

[0389] 1.8 Modulation of Mal-PEG2-Ala-Ala-Asn-PAB-エリブリン(ER-001231679) and びMal-PEG2-(Ala-Ala-Asn-PAB)2-エリブリン(ER-001231690)

change

[0390] Eribulin (ER-000086526) (10 mg, 0.014 mmol) was dissolved in DMF (0.5 mL) and mixed with Hunig's base (3.59 μL, 0.021 mmol). (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-4-amino-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (15.76 mg, 0.021 mmol) was then added. The resulting yellow solution was stirred at room temperature for 3 days until HPLC analysis showed complete consumption of the starting material. Diethylamine (14.23 μL, 0.137 mmol) was added to the reaction mixture, which was then stirred at room temperature for an additional 2 hours until 100% cleavage of the Fmoc protection was observed. The reaction mixture was concentrated to remove diethylamine, and the residue was redissolved in DMF (1.5 mL). EtN (0.015 mL, 0.11 mmol) was added at room temperature, followed by 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)propanoate (9.71 mg, 0.027 mmol). The reaction mixture was stirred at room temperature for 16 hours until the reaction was complete, as determined by LCMS analysis. The mixture was concentrated under high vacuum and purified by flash chromatography to give Mal-PEG-Ala-Ala-Asn-PAB-eribulin (ER-001231679) (9.2 mg, 49% yield) and Mal-PEG-(Ala-Ala-Asn-PAB)-eribulin (ER-001231690) (6.0 mg, 18% yield) as colorless oils.

[0391] Mal-PEG2-Ala-Ala-Asn-PAB-Eribulin (ER-001231679): 1HNMR(400 MHz) δ ppm 9.23 (s, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H),7.38 (d, J = 6.8 Hz, 1H), 7.24 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 7.2 Hz, 1H), 6.68(s, 2H), 6.30 (br s, 1H), 6.04-6.00 (m, 1H), 5.77 (br s, 1H), 5.42 (br s, 1H), 5.07(s, 1H), 5.06-4.98 (m, 2H), 4.93 (s, 1H), 4.88 (s, 1H), 4.90-4.82 (m, 1H), 4.80(s, 1H), 4.69 (t, J = 4.0 Hz, 1H), 4.60 (t, J = 4.0 Hz, 1H), 4.49-4.42 (m, 1H),4.38-4.25 (m, 4H), 4.19 (t, J = 4.8 Hz, 1H), 4.15-4.08 (m, 1H), 4.03 (t, J = 4.8Hz, 1H), 3.97-3.85 (m, 3H), 3.83-3.50 (m, 12H), 3.41 (s, 3H), 3.50-3.10 (m, 3H),3.02-2.64 (m, 6H), 2.52-2.30 (m, 7H), 2.30-1.65 (m, 14H), 1.65-1.20 (m, 12H), 1.10(d, J = 6.8 Hz, 3H), 1.13-1.05 (m, 1H)。LCMS(M+Na)=1396.6。

[0392] Mal-PEG2-(Ala-Ala-Asn-PAB)2-エリブリン(ER-001231690): 1HNMR(400 MHz, CD3OD) δ ppm 7.65 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz,2H), 7.28 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 6.79 (s, 2H), 5.13 (s,1H), 5.02 (s, 1H), 5.06-4.98 (m, 4H), 4.87 (s, 1H), 4.82 (s, 1H), 4.85-4.72 (m,2H), 4.71 (t, J = 4.8 Hz, 1H), 4.61 (t, J = 4.4 Hz, 1H), 4.47 (d, J = 11.2 Hz, 1H),4.30-4.06 (m, 9H), 3.97 (t, J = 4.8 Hz, 1H), 3.89-3.80 (m, 3H), 3.75-3.48 (m, 12H),3.38 (s, 3H), 3.17 (d, J = 6.8 Hz, 2H), 2.94-2.62 (m, 8H), 2.50-2.28 (m, 7H), 2.22-1.65(m, 14H), 1.58-1.30 (m, 18H), 1.10 (d, J = 6.8 Hz, 3H), 1.06-0.97 (m, 1H). LCMS(M+Na)=1802.8.

[0393] 1.9 Preparation of NHS-PEG2-Ala-Ala-Asn-PAB-Eribulin (ER-001231691) [ka]

[0394] Using the same procedure as described above for the preparation of Val-Cit-PAB-eribulin (ER-001228950), Ala-Ala-Asn-PAB-eribulin (ER-001231678) was prepared from eribulin (ER-000086526) and Fmoc-Ala-Ala-Asn-PAB-PNP (15 mg, quantitative yield). LCMS (M+H) = 1135.5.

[0395] Using the same procedure described above for the preparation of NHS-PEG2-Val-Cit-PAB-eribulin (ER-001236940), NHS-PEG2-Ala-Ala-Asn-PAB-eribulin (ER-001231691) was prepared from Ala-Ala-Asn-PAB-eribulin (ER-001231678) and bisNHS-PEG2 (12.4 mg, 64% yield). 1 HNMR(400 MHz) δ ppm 9.21 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H),7.62 (d, J = 8.8 Hz, 2H), 7.58-7.52 (m, 1H), 7.28 (br s, 1H), 7.24 (d, J = 8.4 Hz,2H), 7.10 (br s, 1H), 6.29 (d, J = 12.4 Hz, 1H), 5.83 (br s, 1H), 5.38 (br s, 1H),5.07 (s, 1H), 5.05-4.95 (m, 2H), 4.93 (s, 1H), 4.88 (s, 1H), 4.90-4.83 (m, 1H),4.81 (s, 1H), 4.69 (t, J = 4.4 Hz, 1H), 4.60 (t, J = 4.4 Hz, 1H), 4.46-4.41 (m,1H), 4.36-4.25 (m, 4H), 4.19 (dd, J = 4.8, 6.0 Hz, 1H), 4.15-4.09 (m, 1H), 4.03(dd, J = 4.8, 6.0 Hz, 1H), 3.99-3.89 (m, 3H), 3.85-3.50 (m, 10H), 3.41 (s, 3H),3.40-3.10 (m, 3H), 3.01-2.60 (m, 10H), 2.60-2.35 (m, 7H), 2.35-1.65 (m, 14H), 1.65-1.20(m, 14H), 1.10 (d, J = 6.8 Hz, 3H), 1.15-1.03 (m, 1H). LCMS(ES)(M+H)=1442.7.

[0396] 1.10 Preparation of Azido-PEG3-disulfide-PAB-Eribulin (ER-001237508) [ka]

[0397] 4-(((tert-Butyldimethylsilyl)oxy)methyl)benzoic acid (1.0 g, 3.754 mmol) was dissolved in dichloromethane (DCM) (25 mL) cooled to 0 °C. Triethylamine (0.549 mL, 3.941 mmol) was then added, followed by diphenylphosphorazidate (1.085 mg, 3.941 mmol). The reaction mixture was allowed to warm slowly to room temperature and stirred for 14 h. The crude mixture was diluted with ethyl acetate (EtOAc) / Hep (1:1, 100 mL) and passed through a short silica plug eluting with EtOAc / Hep (50%). The solvent was removed in vacuo to give 1.10 g of 4-(((tert-butyldimethylsilyl)oxy)methyl)benzoyl azide (ER-001131970). 1 HNMR (400 MHz) δ ppm 7.98 (d, 2 H, J = 8.0 Hz), 7.40 (d, 2 H, J = 8.0 Hz), 4.79 (s,2 H), 0.94 (s, 9 H), 0.10 (s, 6 H).

[0398] 4-(((tert-Butyldimethylsilyl)oxy)methyl)benzoyl azide (ER-001131970) (1.1 g, 3.775 mmol) dissolved in toluene (20 mL) was heated at 110° C. for 3 h. The product did not show a single spot, but thin layer chromatography (TLC) analysis indicated that the starting material had been consumed. The reaction mixture was then cooled to room temperature, transferred under nitrogen to a sealed vial, and stored at −20° C. as a solution in toluene (1 mL = 32.6 mg).

[0399] Triethylamine (0.099 mL, 0.709 mmol) was added to a solution of tert-butyl((4-isocyanatobenzyl)oxy)dimethylsilane (165 mg, 0.626 mmol) in toluene (5 mL), followed by the addition of alcohol (90.0 mg, 0.591 mmol), and the reaction mixture was stirred at 36 °C for 6 h. The reaction progress was monitored by UPLC / MS. A saturated solution of sodium bicarbonate (NaHCO) (10 mL) was then added, and the mixture was extracted with EtOAc / Hep (1:1, 60 mL), washed with brine, dried over sodium sulfate, and concentrated. The crude material was purified by flash chromatography (EtOAc / Hep 10% to 40%) to give 215 mg of 2-methyl-2-(methyldisulfanyl)propyl(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)carbamate (ER-001131973). 1 HNMR (400 MHz) δ ppm 7.34 (d, 2 H, J = 8.4 Hz), 7.26 (d, 2 H, J = 7.6 Hz), 6.63 (brs, 1 H), 4.69 (s, 2 H), 4.17 (s, 2 H), 2.42 (s, 3 H), 1.35 (s, 6H), 0.93 (s, 9H), 0.08 (s, 6H).

[0400] 2-Methyl-2-(methyldisulfanyl)propyl(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)carbamate (ER-001131973) (198 mg, 0.476 mmol) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (325 mg, 0.87 mmol) were dissolved in DMF (6.6 mL). Cesium carbonate (621 mg, 1.905 mmol) was then added, followed by tetrabutylammonium iodide (45 mg, 0.122 mmol), and the reaction mixture was stirred at 36° C. for 15 hours. The progress of the reaction was monitored by UPLC / MS. A saturated solution of NH4Cl (30 mL) was then added, extracted with EtOAc / Hep (2:1, 150 mL), washed with brine (10 mL), dried over sodium sulfate, and concentrated in vacuo. The crude material was purified by flash chromatography (20% to 50% EtOAc / Hep) to give 248 mg of 2-methyl-2-(methyldisulfanyl)propyl(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)carbamate (ER-001140141). 1 HNMR (400 MHz) δ ppm 7.28 (d, 2 H, J = 8.4 Hz), 7.20 (d, 2 H, J = 8.0 Hz), 4.73 (s,2 H), 4.06 (br s, 2 H), 3.83 (dd, 2 H, J = 6.4, 5.6 Hz), 3.68- 3.56 (m, 12 H), 3.37(dd, 2 H, J = 5.6, 5.2 Hz), 2.33 (s, 3 H), 1.14 (br s, 6 H), 0.93 (s, 9 H), 0.09(s, 6 H).

[0401] 2-Methyl-2-(methyldisulfanyl)propyl(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)carbamate (ER-001140141) (81 mg, 0.131 mmol) was dissolved in a mixture of methanol (5 mL) and water (0.5 mL). Acetic acid (0.5 mL, 8.734 mmol) was then added to the reaction mixture and stirred at 38° C. for 14 hours. The reaction mixture was cooled to room temperature and the solvent was removed in vacuo. The residue was diluted with EtOAc (30 mL), washed with water (2×5 mL), NaHCO, and brine (3 mL), dried over sodium sulfate, and concentrated in vacuo. The crude material was purified by flash chromatography (EtOAc / Hep 30% to 90%) to afford 61.0 mg of 2-methyl-2-(methyldisulfanyl)propyl(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)(4-(hydroxymethyl)phenyl)carbamate (ER-001140549). 1 HNMR (400 MHz) δ ppm 7.34 (d, 2 H, J = 8.8 Hz), 7.26 (d, 2 H, J = 8.0 Hz), 4.69 (d,2 H, J = 4.4 Hz), 4.06 (br s, 2 H), 3.84 (dd, 2 H, J = 6.2, 6.2 Hz), 3.66-3.56 (m,12 H), 3.37 (dd, 2 H, J = 5.2, 5.2 Hz), 2.33 (s, 3 H), 1.74 (br s, 1 H), 1.14 (brs, 6 H).

[0402] 2-Methyl-2-(methyldisulfanyl)propyl(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)(4-(hydroxymethyl)phenyl)carbamate (ER-001140549) (60 mg, 0.119 mmol) was dissolved in DCM (2 mL) and Py (0.019 mL, 0.239 mmol) cooled to 0° C. Then, 4-nitrophenyl carbonochloridate (38.5 mg, 0.191 mmol) and dimethylaminopyridine (DMAP) (2.9 mg, 0.024 mmol) in DCM (2 mL) were added, and the reaction mixture was stirred for 30 min at 0° C. The reaction mixture was slowly warmed to room temperature and stirred until the starting material was consumed (approximately 2.5 h). The solvent was then removed under vacuum and the residue was purified by flash chromatography (EtOAc / Hep 10% to 35%) to give 78 mg of 2-methyl-2-(methyldisulfanyl)propyl(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (ER-001140550). 1 HNMR (400 MHz) δ ppm 8.27 (dd, 2 H, J = 6.8, 2.4 Hz), 7.41 (d, 2 H, J = 8.8 Hz),7.37 (dd, 2 H, J = 7.2, 2.4 Hz), 7.33(d, 2 H, J = 8.8 Hz), 5.27 (s, 2 H), 4.08 (brs, 2 H), 3.85 (dd, 2 H, J = 5.8, 5.8 Hz), 3.66- 3.57 (m, 12 H), 3.36 (dd, 2 H, J= 5.2, 5.2 Hz), 2.33 (br s, 3 H), 1.19 (br s, 6 H).

[0403] 2-Methyl-2-(methyldisulfanyl)propyl(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamate (ER-001140550) (30 mg, 0.045 mmol) in DCM (3 mL, 46.625 mmol) was placed in a 25 mL flask under nitrogen and cooled to 0° C. The amine (40.8 mg, 0.049 mmol) in DCM (2 mL) and Hunig's base (0.024 mL, 0.135 mmol) was added, followed by DMAP (1.4 mg, 0.011 mmol). The reaction mixture was then slowly warmed to room temperature, stirred for 3 hours, concentrated in vacuo, and purified by flash chromatography (EtOAc / Hep 50% to 100%, followed by MeOH / EtOAc 3% to 8%) to give 45.0 mg of pure azido-PEG-disulfide-PAB-eribulin (ER-001237508). 1HNMR (400 MHz) δ ppm 7.32 (d, 2 H, J = 8.0 Hz), 7.25 (d, 2 H, J = 7.2 Hz), 5.28 (dd,1 H, J = 5.6, 5.6 Hz), 5.11-5.04 (m, 3 H), 4.93 (s, 1 H), 4.88 (s, 1 H), 4.81 (s,1 H), 4.69 (dd, 1 H, J = 4.4, 4.4 Hz), 4.60 (dd, 1 H, J = 4.2, 4.2 Hz), 4.36 (brs, 1 H), 4.33(dd, 1 H, J = 4.0, 2.0), 4.29 (ddd, 1 H, J = 9.6, 4.4, 4.4 Hz), 4.18(dd, 1 H, J = 6.4, 4.4 Hz), 4.14-4.04 (m, 3 H), 4.03 (dd, 1 H, J = 6.4, 4.4 Hz),3.97-3.89 (m, 3 H), 3.84-3.78 (m, 3 H), 3.67-3.56 (m, 14 H), 3.42 (s, 3 H), 3.40-3.35(m, 1 H), 3.37 (dd, 2 H, J = 5.2, 5.2 Hz), 3.27 (d, 1 H, J = 3.2 Hz), 3.20 (ddd,1 H, J = 12.8, 6.0, 6.0Hz), 2.91-2.83 (m, 2 H), 2.70 (dd, 1 H, J = 16.0, 10.0 Hz),2.52-2.40 (m, 3 H), 2.35-2.13 (m, 9 H), 2.10-2.06 (m, 1 H), 2.01-1.89 (m, 4 H),1.78-1.64 (m, 4 H), 1.60-1.52 (m, 4 H), 1.49-1.28 (m, 5 H), 1.22-1.07 (m, 6 H),1.09 (d, 3 H, J = 6.0 Hz).

[0404] 1.11 Preparation of Mal-PEG4-triazole-PEG3-disulfide-PAB-eribulin (ER-001237504) [ka]

[0405] A mixture of azide (9.0 mg, 7.151 μmol) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3,6,9,12-tetraoxapentadeca-14-yn-1-yl)propanamide (6.8 mg, 0.018 mmol) in tert-butanol (1.5 mL) and water (0.5 mL) was degassed for 45 min. Copper iodide on amberlyst-21 (1.23 mmol / g, 10 mg) was then added and degassed for an additional 30 min. The reaction mixture was stirred at room temperature for 18 h and monitored by UPLC / MS. Most of the starting material was consumed, and the desired product was shown as the major peak. The mixture was then separated from the resin and purified by HPLC (acetonitrile / water containing 0.05% formic acid) to give 1.5 mg of Mal-PEG4-triazole-PEG3-disulfide-PAB-eribulin (ER-001237504). 1HNMR (400 MHz) δ ppm 7.74 (s, 1 H), 7.32 (d, 2 H, J = 8.4 Hz), 7.27-7.25 (m, 2 H),6.69 (br s, 2 H), 5.43 (dd, 1 H, J = 5.6, 5.6 Hz), 5.14-5.06 (m, 3 H), 4.95 (s,1 H), 4.89 (s, 1 H), 4.82 (s, 1 H), 4.70 (dd, 1 H, J = 4.4, 4.4 Hz), 4.66 (s, 2H), 4.62 (dd, 1 H, J = 4.4, 4.4 Hz), 4.52(dd, 1 H, J = 5.2, 5.2 Hz), 4.38-4.31 (m,2 H), 4.30 (ddd, 1 H, J = 10.4, 4.0, 4.0 Hz), 4.20 (dd, 1 H, J = 6.4, 4.4 Hz), 4.16-4.05(m, 3 H), 4.04 (dd, 1 H, J = 6.4, 4.4 Hz), 3.99-3.91 (m, 3 H), 3.87-3.80 (m, 6 H),3.70-3.59 (m, 22 H), 3.53 (dd, 2 H, J = 5.2, 5.2 Hz), 3.44 (s, 3 H), 3.43-3.36 (m,3 H), 3.29 (d, 1 H, J = 2.8 Hz), 3.18 (ddd, 1 H, J = 12.9, 6.2, 6.2 Hz), 2.92-2.84(m, 2 H), 2.72 (dd, 1 H, J = 16.0, 10.0 Hz), 2.54-2.42 (m, 5 H), 2.37-1.90 (m, 19H), 178-1.52 (m, 3H), 1.50-1.14 (m, 16H), 1.10 (d, 3H, J = 6.0 Hz). LCMS(M+H)=1642.1.

[0406] 1.12 Preparation of NHS-PEG3-triazole-PEG3-disulfide-PAB-eribulin (ER-001244129) [ka]

[0407] A mixture of azide (9 mg, 7.151 μmol) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)propanoate (4.5 mg, 14.30 μmol) in tert-butanol (1 mL) and water (0.5 mL) was degassed for 45 min. Copper iodide on amberlyst-21 (1.23 mmol / g, 10 mg, 7.151 μmol) was then added and degassed for an additional 30 min. The reaction mixture was stirred at room temperature for 18 h and monitored by UPLC / MS. Most of the starting material was consumed, and the desired product was shown as the major peak. The mixture was then separated from the resin by filtration, extracted with DCM (15 mL), washed with brine (3 × 3 mL), dried over sodium sulfate, and concentrated in vacuo. The residue (5 mg, 3.39 μmol) was azeotroped with toluene, dissolved in THF (1 mL), and cooled to 0° C. DCC (4.2 mg, 0.02 mmol) was added, followed by 1-hydroxypyrrolidine-2,5-dione (2.2 mg, 0.019 mmol), and the reaction mixture was stirred at room temperature for 18 hours. Most of the starting material was consumed, and the desired product appeared as the major peak as determined by UPLC / MS. The reaction mixture was then concentrated and purified by preparative TLC (DCM / i-propanol, 8%) to give 2.5 mg of NHS-PEG3-triazole-PEG3-disulfide-PAB-eribulin (ER-001244129) as a colorless oil. 1HNMR (400 MHz, CD2Cl2) δ ppm 7.72 (s, 1 H), 7.32 (d, 2 H, J= 8.8 Hz), 7.25 (d, 2 H, J = 8.8 Hz), 5.08-5.04 (m, 3 H), 4.93 (s, 1 H), 4.85 (s,1 H), 4.78 (s, 1 H), 4.64 (dd, 1 H, J = 4.4, 4.4 Hz), 4.58 (s, 2 H), 4.55 (dd, 1H, J = 4.4, 4.4 Hz), 4.48 (dd, 2 H, J = 5.0, 5.0 Hz), 4.32 (d, 1 H, J = 6.6 Hz),4.27-4.22 (m, 2 H), 4.14 (dd, 1 H, J = 6.6, 4.8 Hz), 4.10-4.01 (m, 3 H), 4.00 (dd,1 H, J = 6.8, 4.4 Hz), 3.92-3.78 (m, 9 H), 3.65-3.53 (m, 19 H), 3.44-3.39 (m, 4H), 3.37 (s, 3 H), 3.26 (d, 1 H, J = 3.2 Hz), 3.13 (ddd, 1 H, J = 12.4, 6.0, 6.0Hz), 2.91-2.73 (m, 11 H), 2.70-2.64 (m, 2 H), 2.54-2.41 (m, 3 H), 2.38-1.80 (m,16 H), 1.74-1.52 (m, 3 H), 1.41-1.13 (m, 10 H), 1.07 (d, 3 H, J = 6.4 Hz). LCMS(M+H)=1572.3.

[0408] 1.13 Preparation of Azido-PEG3-Sulfonamide-PAB-Eribulin (ER-001138856) [ka]

[0409] 4-(((tert-Butyldimethylsilyl)oxy)methyl)aniline (315 mg, 1.327 mmol) was dissolved in DCM (10 mL) cooled to 0 °C. Pyridine (0.268 mL, 3.317 mmol) was then added over 15 min, followed by 5-cyanopyridine-2-sulfonyl chloride (365 mg, 1.801 mmol) in DCM (10 mL). The reaction mixture was allowed to warm slowly to room temperature over 1 h and stirred for 2 h. The reaction mixture was diluted with EtOAc (50 mL), washed with brine, dried over sodium sulfate, and concentrated in vacuo to afford 610 mg (103%) of N-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-5-cyanopyridine-2-sulfonamide (ER-001137670). The crude product was reasonably pure but colored. 1 HNMR (400 MHz) δ ppm 8.94 (dd, 1 H, J = 1.8, 0.6 Hz), 8.10 (dd, 1 H, J = 8.4, 2.0Hz), 7.99 (dd, 1 H, J = 8.0, 0.8 Hz), 7.18 (d, 2 H, J = 8.2 Hz), 7.15 (br s, 1 H),7.11 (dd, 2 H, J = 6.8, 0.8 Hz), 4.64 (s, 2 H), 0.90 (s, 9 H), 0.05 (s, 6 H).

[0410] N-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-5-cyanopyridine-2-sulfonamide (ER-001137670) (105.0 mg, 0.26 mmol) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (143 mg, 0.383 mmol) were dissolved in DMF (4 mL). Potassium carbonate (K2CO3) (144 mg, 1.041 mmol) was then added, followed by tetrabutylammonium iodide (19.2 mg, 0.052 mmol), and the reaction mixture was stirred at 50 °C for 36 h. The progress of the reaction was monitored by UPLC / MS. A saturated solution of NH4Cl (10 mL) was added, extracted with EtOAc / Hep (2:1, 30 mL), washed with brine, dried over sodium sulfate, and concentrated. The crude material was purified by flash chromatography (EtOAc / Hep 25% to 80%) to afford 118.0 mg of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-5-cyanopyridine-2-sulfonamide (ER-001138452) (75%). 1 HNMR (400 MHz) δ ppm 8.99 (dd, 1 H, J = 1.8, 0.6 Hz), 8.08 (dd, 1 H, J = 8.2, 2.2Hz), 7.86 (dd, 1 H, J = 8.0, 0.8 Hz), 7.24 (d, 2 H, J = 10 Hz), 7.09 (d, 2 H, J= 8.8 Hz), 4.69 (s, 2 H), 4.06 (dd, 2 H, J = 6.0, 6.0 Hz), 3.67 (dd, 2 H, J = 5.2,5.2 Hz), 3.65 - 3.62 (m, 4 H), 3.58 (dd, 2 H, J = 6.2, 6.2 Hz), 3.56 - 3.53 (m,4 H), 3.38 (dd, 2 H, J = 5.2, 5.2 Hz), 0.93 (s, 9 H), 0.08 (s, 6 H).

[0411] N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-5-cyanopyridine-2-sulfonamide (ER-001138452) (150 mg, 0.248 mmol) was dissolved in methanol (6 mL). Water (0.60 mL) was then added, followed by acetic acid (AcOH) (0.60 mL, 10.481 mmol). The reaction mixture was slowly warmed to 38° C. and stirred for 14 hours. Most of the solvent was removed under vacuum. The residue was diluted with EtOAc (30 mL), washed with water (2×5 mL), NaHCO3, and brine, dried over sodium sulfate, and concentrated under vacuum. The crude material was purified by flash chromatography (EtOAc / Hep 35% to 90%) to afford 105.0 mg (84%) of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-cyano-N-(4-(hydroxymethyl)phenyl)pyridine-2-sulfonamide (ER-001138455). 1 HNMR (400 MHz) δ ppm 8.99 (d, 1 H, J = 1.2 Hz), 8.09 (dd, 1 H, J = 8.4, 2.0 Hz),7.88 (dd, 1 H, J = 8.4, 0.8 Hz), 7.30 (d, 2 H, J = 8.8 Hz), 7.15 (d, 2 H, J = 8.4Hz), 4.67 (s, 2 H), 4.06 (dd, 2 H, J = 6.2, 6.2 Hz), 3.66 (dd, 2 H, J = 5.0, 5.0Hz), 3.65 - 3.58 (m, 6 H), 3.55 - 3.51 (m, 4 H), 3.38 (dd, 2 H, J = 5.2, 5.2 Hz.

[0412] N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-cyano-N-(4-(hydroxymethyl)phenyl)pyridine-2-sulfonamide (ER-001138455) (45 mg, 0.092 mmol) was dissolved in DCM (3 mL) and cooled to 0° C. after the addition of pyridine (0.015 mL, 0.183 mmol). 4-Nitrophenyl carbonochloridate (20.3 mg, 0.101 mmol) and DMAP (2.3 mg, 0.018 mmol) in DCM (2 mL) were then added. The reaction mixture was allowed to warm slowly to room temperature and stirred for 2 h. UPLC / MS indicated that some starting material remained. The reaction mixture was then concentrated in vacuo and purified by flash chromatography (EtOAc / Hep 12% to 40%) to afford 35 mg (58%) of 4-((N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-cyanopyridine)-2-sulfonamido)benzyl (4-nitrophenyl)carbonate (ER-001235286) and 20 mg of starting material. 1 HNMR (400 MHz) δ ppm 8.99 (d, 1 H, J = 0.8 Hz), 8.27 (dd, 2 H, J = 9.2, 2.0 Hz),8.12 (dd, 1 H, J = 7.6, 2.0 Hz), 7.92 (d, 1 H, J = 8.4 Hz), 7.38 (d, 4 H, J = 9.6Hz), 7.26 (d, 2 H, J = 8.8 Hz), 5.45 (s, 2 H), 4.06 (dd, 2 H, J = 5.8, 5.8 Hz),3.67 - 3.58 (m, 8 H), 3.58 - 3.50 (m, 4 H), 3.38 (dd, 2H, J = 6.1, 6.1 Hz).

[0413] 4-(N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-cyanopyridine-2-sulfonamido)benzyl(4-nitrophenyl)carbonate (ER-001235286) (35.0 mg, 0.053 mmol) was placed in a 25 mL flask under nitrogen and cooled to 0° C. Then, the amine (48.5 mg, 0.059 mmol) in DCM (3 mL, 46.625 mmol) and Hunig's base (0.037 mL, 0.214 mmol) was added, followed by DMAP (2.61 mg, 0.021 mmol). The reaction mixture was stirred at 0° C. for 30 minutes and then at room temperature for an additional 6 hours. The reaction mixture was concentrated in vacuo and purified by flash chromatography (EtOAc / Hep 50% to 100%, followed by MeOH / EtOAc 3% to 8%) to give 61.0 mg of pure azido-PEG3-sulfonamide-PAB-eribulin (ER-001138856). 1HNMR (400 MHz) δ ppm 8.98 (d, 1 H, J = 1.2 Hz), 8.10 (dd, 1 H, J = 8.2, 1.8 Hz), 7.87 (d, 1 H, J = 8.0 Hz), 7.26 (d, 2 H, J = 6.8 Hz), 7.13 (d, 2 H, J = 8.4 Hz), 5.29 (dd, 1 H, J = 5.6, 5.6 Hz), 5.08 - 5.00 (m, 3 H), 4.92 (s, 1 H), 4.87 (s, 1 H), 4.80 (s, 1 H), 4.68 (dd, 1 H, J = 4.6, 4.6 Hz), 4.59 (dd, 1 H, J = 4.6, 4.6 Hz), 4.38 - 4.30 (m, 2 H), 4.28 (ddd, 1 H, J = 10.4, 4.0, 4.0, Hz), 4.17 (dd, 1 H, J = 6.2, 4.6 Hz), 4.13 - 4.01 (m, 4 H), 3.97 - 3.88 (m, 3 H), 3.82 - 3.78 (m, 1 H), 3.67 - 3.50 (m, 15 H), 3.41 (s, 3 H), 3.40 - 3.33 (m, 1 H), 3.37 (dd, 2 H, J = 4.8, 4.8 Hz), 3.27 (d, 1 H, J = 3.2 Hz), 3.15 (ddd, 1 H, J = 12.8, 6.4, 6.4 Hz), 2.90 - 2.82 (m, 2 H), 2.70 (dd, 1 H, J = 16.0, 10.0 Hz), 2.51 - 2.40 (m, 3 H), 2.34 - 2.13 (m, 7 H), 2.10 - 2.05 (m, 1 H), 1.99 - 1.88 (m, 4 H), 1.78 - 1.64 (m, 5 H), 1.62 - 1.52 (m, 2 H), 1.50 - 1.29 (m, 4 H), 1.08 (d, 3 H, J = 6.8 Hz).

[0414] 1.14 Preparation of Mal-PEG4-triazole-PEG3-sulfonamide-PAB-erythrin (ER-001237505) [Chemical Structure]

[0415] A mixture of azide (10 mg, 8.023 μmol) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3,6,9,12-tetraoxapentadeca-14-yn-1-yl)propanamide (9.20 mg, 0.024 mmol) in tert-butanol (2.1 mL) and water (0.7 mL) was degassed for 45 min. Copper iodide on amberlyst-21 (1.23 mmol / g, 15 mg) was then added and degassed for an additional 30 min. The reaction mixture was stirred at room temperature for 18 h and monitored by UPLC / MS. Most of the starting material was consumed, and the desired product was shown as the major peak. The reaction mixture was then cleaved from the resin and purified by preparative TLC (DCM / methanol, 7%) to give 5.5 mg of Mal-PEG4-triazole-PEG3-sulfonamide-PAB-eribulin (ER-001237505). 1HNMR (400 MHz, CD2Cl2) δ ppm 9.01 (s, 1 H), 8.15 (dd, 1 H,J = 8.0, 1.8 Hz), 7.87 (d, 1 H, J = 8.0 Hz), 7.75 (s, 1 H), 7.28 (d, 2 H, J = 8.0Hz), 7.14 (d, 2 H, J = 8.4 Hz), 6.68 (s, 2 H), 6.47 (br s, 1 H), 5.44 (br s, 1 H),5.10-5.02 (m, 3 H), 4.94 (s, 1 H), 4.86 (s, 1 H), 4.80 (s, 1 H), 4.68 (dd, 1 H,J = 4.4, 4.4 Hz), 4.59 (s, 2 H), 4.56 (dd, 1 H, J = 4.4, 4.4 Hz), 4.51(dd, 2 H,J = 5.2, 5.2, Hz), 4.34(d, 1 H, J = 7.6, Hz), 4.30-4.23 (m, 2 H), 4.19 - 4.14 (m,2 H), 4.08 (dd, 1 H, J = 4.0, 4.0 Hz), 4.03 - 3.98 (m, 2 H), 3.94 - 3.72 (m, 8 H),3.68 - 3.46 (m, 28 H), 3.38 (s, 3 H), 3.38 - 3.33 (m, 3 H), 3.27 (d, 1 H, J = 3.2Hz), 3.16 - 3.02 (m, 2 H), 2.90 - 2.81 (m, 2 H), 2.68 (dd, 1 H, J = 16.2, 9.8 Hz),2.54-2.40 (m, 7H), 2.40-1.8 (m, 11 H), 1.80-1.50 (m, 3H), 1.48-1.25 (m, 3H), 1.09(d, 3H, J = 6.4 Hz). LCMS(M+H)=1630.0.

[0416] 1.15 Preparation of NHS-PEG3-triazole-PEG3-sulfonamide-PAB-eribulin (ER-001244623) [ka]

[0417] A mixture of azide (14 mg, 0.011 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)propanoate (8.80 mg, 0.028 mmol) in tert-butanol (2 mL) and water (1 mL) was degassed for 45 min. Copper iodide on amberlyst-21 (1.23 mmol / g, 20 mg) was then added and degassed for another 30 min. The reaction mixture was stirred at room temperature for 18 h and monitored by UPLC / MS. Most of the starting material was consumed, and the desired product was shown as the major peak. The reaction mixture was then separated from the resin by extraction with DCM (2 × 10 mL). The DCM layer was washed with brine (4 × 5 mL), dried over sodium sulfate, and concentrated to the desired product, which was used in the next step without any further purification.

[0418] The crude acid (15.0 mg, 10.255 μmol) was dissolved in THF (1.5 mL) and cooled to 0° C. DCC (15.2 mg, 0.074 mmol) was then added, followed by 1-hydroxypyrrolidine-2,5-dione (8.3 mg, 0.072 mmol). The reaction mixture was stirred at room temperature for 18 hours. UPLC / MS showed that most of the starting material had been consumed, with the desired product appearing as the major peak. The reaction mixture was concentrated and purified by preparative TLC (DCM / i-propanol, 8%) to give 2.5 mg of NHS-PEG3-triazole-PEG3-sulfonamide-PAB-eribulin (ER-001244623). 1HNMR (400 MHz, CD2Cl2) δ ppm 9.00 (s, 1 H), 8.12 (d, 1 H, J= 8.4 Hz), 8.00 (d, 1 H, J = 8.0 Hz), 7.72 (s, 1 H), 7.26 (d, 2 H, J = 8.0 Hz),7.12 (d, 2 H, J = 8.0 Hz), 5.37 (br s, 1 H), 5.08-5.02 (m, 3 H), 4.93 (s, 1 H),4.85 (s, 1 H), 4.78 (s, 1 H), 4.66-4.62 (m, 1 H), 4.58-4.56 (m, 4 H), 4.33 (d, 1H, J = 10.8 Hz), 4.29-4.21 (m, 2 H), 4.10-3.96 (m, 4 H), 3.93-3.76 (m, 6 H), 3.74-3.44(m, 27 H), 3.36 (s, 3 H), 3.34-3.24 (m, 2 H), 3.15-3.06 (m, 1 H), 2.97 (br s, 1H), 2.90-2.78 (m, 8 H), 2.74-2.08 (m, 13 H), 2.05 -1.78 (m, 5 H), 1.73-1.50 (m,2 H), 1.41-1.25 (m, 4 H), 1.07 (d, 3 H, J = 6.0 Hz). LCMS (M+H) = 1560.0.

[0419] 1.16 Preparation of Mal-PEG2-Eribulin Eribulin (5 mg, 7 μmol) was dissolved in DMF (0.5 mL) and mixed with maleimide-PEG2-NHS (5 mg, 14 μmol; Broadpharm, Cat. No. BP-21680) and Hunig's base (2.4 μL, 14 μmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then purified by HPLC (30-70% water-acetonitrile gradient containing 0.1% formic acid). The eluent was collected by mass and lyophilized to dryness. The final yield was 3.7 mg (3.8 μmol, 54%). The predicted accurate mass was 968.5 Da. The measured mass was 969.6 Da [M+H].

[0420] 1.17 Preparation of Mal-PEG4-Eribulin Eribulin (5 mg, 7 μmol) was dissolved in DMF (0.5 mL) and mixed with maleimide-PEG4-NHS (6.2 mg, 14 μmol; Broadpharm, Cat. No. BP-20554) and Hunig's base (2.4 μL, 14 μmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then purified by HPLC (30-70% water-acetonitrile gradient containing 0.1% formic acid). The eluent was collected by mass and lyophilized to dryness. The final yield was 3.7 mg (3.5 μmol, 50%). The predicted accurate mass was 1056.5 Da. The measured mass was 1057.7 Da [M+H].

[0421] 1.18 Preparation of azido-PEG2-eribulin Eribulin (5 mg, 7 μmol) was dissolved in DMF (0.5 mL) and mixed with azido-PEG2-NHS (4.2 mg, 14 μmol; Broadpharm, Cat. No. BP-20524) and Hunig's base (2.4 μL, 14 μmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then purified by HPLC (30-70% water-acetonitrile gradient containing 0.1% formic acid). The eluent was collected by mass and lyophilized to dryness. The final yield was 2.2 mg (2.4 μmol, 34%). The predicted accurate mass was 914.5 Da. The measured mass was 915.7 Da [M+H].

[0422] 1.19 Preparation of azido-PEG4-eribulin Eribulin (5 mg, 7 μmol) was dissolved in DMF (0.5 mL) and mixed with azido-PEG4-NHS (5.5 mg, 14 μmol; Broadpharm, Cat. No. BP-20518) and Hunig's base (2.4 μL, 14 μmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then purified by HPLC (30-70% water-acetonitrile gradient containing 0.1% formic acid). The eluent was collected by mass and lyophilized to dryness. The final yield was 3.0 mg (3.0 μmol, 43%). The predicted accurate mass was 1002.5 Da. The measured mass was 1003.7 Da [M+H].

[0423] 1.20 Preparation of Azido-PEG4-Val-Cit-PAB-Eribulin Eribulin (15 mg, 21 μmol) was dissolved in DMF (1.5 mL) and mixed thoroughly. Hunig's base (5.5 μL, 32 μmol) and Fmoc-VCP-PNP (24 mg, 22 μmol; Levena Biopharma, Cat. No. VC1003) were then added. The reaction mixture was stirred overnight (16 hours) at room temperature. Upon completion of the reaction, diethylamine (20 μL, 0.21 mmol) was added to the reaction mixture and stirred at room temperature for 2 hours to remove the Fmoc protecting group. The deprotection reaction was monitored using a Waters SQD mass spectrometer. Upon completion of the reaction, the reaction mixture was transferred to a pre-weighed 1.5 mL microcentrifuge tube. The solvent was evaporated under vacuum using a refrigerated Centrivap concentrator set at 30°C. The yield was 16 mg (14 μmol) of crude NH 2 -Val-Cit-pAB-Eribulin (accurate mass 1134.6 Da, yield 67%).

[0424] NH2-Val-Cit-PAB-Eribulin (16 mg, 14.1 μmol) was dissolved in DMF (1.5 mL). Hunig's base (7.2 μL, 41 μmol) and azido-PEG4-NHS (11 mg, 28.2 μmol) were then added. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was then purified by HPLC (48-72% water-acetonitrile gradient containing 0.1% formic acid). The eluent at m / z 1409 was collected and lyophilized to give azido-PEG4-Val-Cit-PAB-Eribulin (exact mass 1407.7 Da). 13 mg (9.2 μmol) of azido-PEG4-Val-Cit-PAB-Eribulin was obtained (step yield 65%, overall yield 44%).

[0425] Example 4 1. Materials and Methods All reagents used were of research grade or better unless otherwise indicated and were obtained from commercial suppliers.

[0426] 1.1 Antibodies MORAb-003 (humanized anti-human folate receptor alpha, 25 mg / mL) and MORAb-009 (mouse-human chimeric anti-human mesothelin, 25 mg / mL) used in the following studies were from Lot #NB02962-19 and Lot #030A14, respectively. Trastuzumab was obtained commercially (Clingen) and was from Lot #503345.

[0427] Rabbit-human chimeric and humanized anti-human mesothelin antibodies (Table 1) bearing an unpaired cysteine ​​at LCcys80 were expressed transiently or as stable selected pools in 293F cells. Conditioned media was purified and decysteinylated as described in section 1.4.1.2.1.

[0428] 1.2 Cytotoxins Conjugable eribulin compounds were synthesized (Table 46) as described in Example 3. Stocks (10 mM) were prepared in DMSO and stored at -20°C until use.

[0429] 1.3 Tumor cell lines Human tumor cell lines used in the analysis of MORAb-003, MORAb-009, and trastuzumab ADCs p...

Claims

1. Antibody-drug conjugates of formula (I): Ab-(L-D) ｐ (I) [wherein Ab is an internalizing antibody or an internalizing antigen-binding fragment thereof that targets a tumor cell; D is eribulin; L is a cleavable linker covalently linking Ab to D; and p is an integer from 1 to 20.

2. The antibody-drug conjugate of claim 1, wherein p is 1 to 8, or 1 to 6.

3. The antibody-drug conjugate of claim 1 or claim 2, wherein p is 2 to 8, or 2 to 5.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein p is 3 to 4.

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein p is 4.

6. The antibody-drug conjugate of any one of claims 1 to 5, wherein the cleavable linker comprises a cleavable moiety that is arranged such that, upon cleavage, no portion of the linker or the antibody or antigen-binding fragment remains attached to eribulin.

7. The antibody-drug conjugate of any one of claims 1 to 6, wherein the cleavable linker comprises a cleavable peptide moiety.

8. The antibody-drug conjugate of claim 7 , wherein the cleavable peptide moiety is enzymatically cleavable.

9. The antibody-drug conjugate of claim 7 or claim 8, wherein the cleavable peptide moiety is cleavable by cathepsin.

10. The antibody-drug conjugate of any one of claims 7 to 9, wherein the cleavable peptide moiety is cleavable by cathepsin B.

11. The antibody-drug conjugate of any one of claims 1 to 10, wherein the cleavable peptide moiety or cleavable linker comprises an amino acid unit.

12. The antibody-drug conjugate of claim 11, wherein the amino acid unit comprises valine-citrulline (Val-Cit).

13. The antibody-drug conjugate of claim 11, wherein the amino acid unit comprises alanine-alanine-asparagine (Ala-Ala-Asn).

14. The antibody-drug conjugate of any one of claims 1 to 6, wherein the cleavable linker comprises a cleavable sulfonamide moiety.

15. The antibody-drug conjugate of any one of claims 1 to 6, wherein the cleavable linker comprises a cleavable disulfide moiety.

16. The antibody-drug conjugate of claim 14 or claim 15, wherein the cleavable linker is cleavable under reducing conditions.

17. The antibody-drug conjugate of any one of claims 1 to 16, wherein the cleavable linker comprises at least one spacer unit.

18. The antibody-drug conjugate of any one of claims 1 to 17, wherein the spacer unit or cleavable linker comprises a polyethylene glycol (PEG) moiety.

19. The PEG moiety is -(PEG) ｍ -, and m is an integer from 1 to 10.

20. The antibody-drug conjugate of claim 19, wherein m is 2 to 8.

21. The antibody-drug conjugate of claim 19 or 20, wherein m is 2 to 5.

22. The antibody-drug conjugate of any one of claims 19 to 21, wherein m is 2.

23. The antibody-drug conjugate of any one of claims 1 to 17, wherein the spacer unit or cleavable linker comprises an alkyl moiety.

24. The alkyl portion is —(CH ２ ) ｎ -, wherein n is an integer from 1 to 10.

25. The antibody-drug conjugate of claim 24, wherein n is 5.

26. The antibody-drug conjugate of any one of claims 17 to 25, wherein the spacer unit is attached to the antibody or antigen-binding fragment via a maleimide moiety (Mal).

27. The antibody-drug conjugate of claim 26, wherein the Mal-spacer unit is reactive with a cysteine ​​residue on the antibody or antigen-binding fragment.

28. The antibody-drug conjugate of claim 26 or claim 27, wherein the Mal-spacer unit is connected to the antibody or antigen-binding fragment via a cysteine ​​residue on the antibody or antigen-binding fragment.

29. The antibody-drug conjugate of any one of claims 26 to 28, wherein the cleavable linker comprises the Mal-spacer unit and a cleavable peptide moiety.

30. The antibody-drug conjugate of claim 29 , wherein the cleavable peptide moiety comprises an amino acid unit.

31. The antibody-drug conjugate of claim 29 or claim 30, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit.

32. The antibody-drug conjugate of claim 29 or claim 30, wherein the cleavable peptide moiety or amino acid unit comprises Ala-Ala-Asn.

33. The antibody-drug conjugate of any one of claims 26 to 28, wherein the cleavable linker comprises the Mal-spacer unit and a cleavable sulfonamide moiety.

34. The antibody-drug conjugate of any one of claims 26 to 28, wherein the cleavable linker comprises the Mal-spacer unit and a cleavable disulfide moiety.

35. The antibody-drug conjugate of any one of claims 26 to 34, wherein the Mal-spacer unit comprises a PEG moiety.

36. The antibody-drug conjugate of any one of claims 26 to 34, wherein the Mal-spacer unit comprises an alkyl moiety.

37. The antibody-drug conjugate of any one of claims 26 to 36, wherein the Mal-spacer unit connects the antibody or antigen-binding fragment to the cleavable moiety within the linker.

38. The antibody-drug conjugate of claim 37, wherein the cleavable moiety in the linker comprises a cleavable peptide moiety.

39. The antibody-drug conjugate of claim 38, wherein the cleavable peptide moiety comprises an amino acid unit.

40. The antibody-drug conjugate of claim 38 or claim 39, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit or Ala-Ala-Asn.

41. The cleavable linker is Mal-(PEG). ２ The antibody-drug conjugate of any one of claims 37 to 40, comprising -Val-Cit.

42. The cleavable linker is Mal-(PEG). ８ The antibody-drug conjugate of any one of claims 37 to 40, comprising -Val-Cit.

43. The cleavable linker is Mal-(CH ２ ) ５ The antibody-drug conjugate of any one of claims 37 to 40, comprising -Val-Cit.

44. The cleavable linker is Mal-(PEG). ２ The antibody-drug conjugate of any one of claims 37 to 40, comprising -Ala-Ala-Asn.

45. The antibody-drug conjugate of any one of claims 37 to 40, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit.

46. The antibody-drug conjugate of claim 37, wherein the cleavable moiety in the linker comprises a cleavable sulfonamide moiety.

47. The cleavable linker is Mal-(PEG). ４ -triazole-(PEG) ３ 47. The antibody-drug conjugate of claim 46, comprising a sulfonamide.

48. The antibody-drug conjugate of claim 37, wherein the cleavable moiety in the linker comprises a cleavable disulfide moiety.

49. The cleavable linker is Mal-(PEG). ４ -triazole-(PEG) ３ - The antibody-drug conjugate of claim 48, comprising a disulfide.

50. The antibody-drug conjugate of any one of claims 37 to 49, wherein the Mal-spacer unit comprises a PEG moiety.

51. The antibody-drug conjugate of any one of claims 37 to 49, wherein the Mal-spacer unit comprises an alkyl moiety.

52. The antibody-drug conjugate of any one of claims 17 to 25, wherein the spacer unit is attached to the antibody or antigen-binding fragment via a succinimide moiety (OSu).

53. The antibody-drug conjugate of claim 52, wherein the OSu-spacer unit is reactive with a lysine residue on the antibody or antigen-binding fragment.

54. The antibody-drug conjugate of claim 52 or claim 53, wherein the OSu-spacer unit is connected to the antibody or antigen-binding fragment via a lysine residue.

55. The antibody-drug conjugate of any one of claims 52 to 54, wherein the cleavable linker comprises the OSu-spacer unit and a cleavable peptide moiety.

56. The antibody-drug conjugate of claim 55, wherein the cleavable peptide moiety comprises an amino acid unit.

57. The antibody-drug conjugate of claim 55 or claim 56, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit.

58. The antibody-drug conjugate of claim 55 or claim 56, wherein the cleavable peptide moiety or amino acid unit comprises Ala-Ala-Asn.

59. The antibody-drug conjugate of any one of claims 52 to 54, wherein the cleavable linker comprises the OSu-spacer unit and a cleavable sulfonamide moiety.

60. The antibody-drug conjugate of any one of claims 52 to 54, wherein the cleavable linker comprises the OSu-spacer unit and a cleavable disulfide moiety.

61. The antibody-drug conjugate of any one of claims 52 to 60, wherein the OSu-spacer unit comprises a PEG moiety.

62. The antibody-drug conjugate of any one of claims 52 to 60, wherein the OSu-spacer unit comprises an alkyl moiety.

63. The antibody-drug conjugate of any one of claims 52 to 62, wherein the OSu-spacer unit connects the antibody or antigen-binding fragment to the cleavable moiety in the linker.

64. The antibody-drug conjugate of claim 63, wherein the cleavable moiety in the linker comprises a cleavable peptide moiety.

65. The antibody-drug conjugate of claim 64, wherein the cleavable peptide moiety comprises an amino acid unit.

66. The antibody-drug conjugate of claim 64 or claim 65, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit or Ala-Ala-Asn.

67. The cleavable linker is OSu-(PEG). ２The antibody-drug conjugate of any one of claims 63 to 66, comprising -Val-Cit.

68. The cleavable linker is OSu-(PEG). ９ The antibody-drug conjugate of any one of claims 63 to 66, comprising -Val-Cit.

69. The cleavable linker is OSu-(PEG). ３ -triazole-(PEG) ３ The antibody-drug conjugate of any one of claims 63 to 66, comprising -Val-Cit.

70. The cleavable linker is OSu-(CH ２ ) ５ The antibody-drug conjugate of any one of claims 63 to 66, comprising -Val-Cit.

71. The cleavable linker is OSu-(PEG). ２ 67. The antibody-drug conjugate of any one of claims 63 to 66, comprising -Ala-Ala-Asn.

72. The antibody-drug conjugate of any one of claims 63 to 66, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit.

73. The antibody-drug conjugate of claim 63, wherein the cleavable moiety in the linker comprises a cleavable sulfonamide moiety.

74. The cleavable linker is OSu-(PEG). ３ -triazole-(PEG) ３ The antibody-drug conjugate of claim 73, comprising a sulfonamide.

75. The antibody-drug conjugate of claim 63, wherein the cleavable moiety in the linker comprises a cleavable disulfide moiety.

76. The cleavable linker is OSu-(PEG). ３ -triazole-(PEG) ３ - The antibody-drug conjugate of claim 75, comprising a disulfide.

77. The antibody-drug conjugate of any one of claims 63 to 76, wherein the OSu-spacer unit comprises a PEG moiety.

78. The antibody-drug conjugate of any one of claims 63 to 76, wherein the OSu-spacer unit comprises an alkyl moiety.

79. The antibody-drug conjugate of any one of claims 17 to 78, wherein the cleavable moiety in the linker is directly connected to eribulin or a spacer unit connects the cleavable moiety in the linker to eribulin.

80. The antibody-drug conjugate of claim 79, wherein cleavage of the conjugate releases eribulin from the antibody and linker.

81. The antibody-drug conjugate of claim 79 or claim 80, wherein the spacer unit that connects the cleavable moiety in the linker to eribulin is self-immolative.

82. The antibody-drug conjugate of any one of claims 79 to 81, wherein the spacer unit that connects the cleavable moiety in the linker to eribulin comprises p-aminobenzyloxycarbonyl (pAB).

83. The antibody-drug conjugate of claim 82, wherein the pAB binds the cleavable moiety in the linker to eribulin.

84. The antibody-drug conjugate of claim 82 or claim 83, wherein the pAB is covalently attached to eribulin via the C-35 amine.

85. The antibody-drug conjugate of any one of claims 79 to 84, wherein the cleavable moiety in the linker comprises a cleavable peptide moiety.

86. The antibody-drug conjugate of claim 85, wherein the cleavable peptide moiety comprises an amino acid unit.

87. The antibody-drug conjugate of claim 85 or claim 86, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit or Ala-Ala-Asn.

88. The antibody-drug conjugate of any one of claims 85 to 87, wherein the cleavable linker comprises Val-Cit-pAB.

89. The antibody-drug conjugate of any one of claims 85 to 87, wherein the cleavable linker comprises Ala-Ala-Asn-pAB.

90. The antibody-drug conjugate of any one of claims 85 to 87, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit.

91. The antibody-drug conjugate of any one of claims 79 to 84, wherein the cleavable moiety in the linker comprises a cleavable sulfonamide moiety.

92. The antibody-drug conjugate of claim 91, wherein the cleavable linker comprises a sulfonamide-pAB.

93. The antibody-drug conjugate of any one of claims 79 to 84, wherein the cleavable moiety in the linker comprises a cleavable disulfide moiety.

94. The antibody-drug conjugate of claim 93, wherein the cleavable linker comprises a disulfide-pAB.

95. The antibody-drug conjugate of any one of claims 1 to 94, wherein the antibody or antigen-binding fragment is an anti-folate receptor alpha antibody.

96. The antibody-drug conjugate of claim 95, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:2 (HCDR1), SEQ ID NO:3 (HCDR2), and SEQ ID NO:4 (HCDR3) as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:7 (LCDR1), SEQ ID NO:8 (LCDR2), and SEQ ID NO:9 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3) as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3).

97. The antibody-drug conjugate of claim 95 or claim 96, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

24.

98. The antibody-drug conjugate of any one of claims 1 to 94, wherein the antibody or antigen-binding fragment is an anti-human epidermal growth factor receptor 2 (HER2) antibody.

99. The antibody-drug conjugate of claim 98, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:71 (HCDR1), SEQ ID NO:72 (HCDR2), and SEQ ID NO:73 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:74 (LCDR1), SEQ ID NO:75 (LCDR2), and SEQ ID NO:76 (LCDR3), as defined by the IMGT numbering system; or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:191 (HCDR1), SEQ ID NO:192 (HCDR2), and SEQ ID NO:193 (HCDR3), as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:194 (LCDR1), SEQ ID NO:195 (LCDR2), and SEQ ID NO:196 (LCDR3).

100. The antibody-drug conjugate of claim 98 or claim 99, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:27, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

28.

101. The antibody-drug conjugate of any one of claims 1 to 94, wherein the antibody or antigen-binding fragment is an anti-mesothelin antibody.

102. The antibody-drug conjugate of claim 101, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:65 (HCDR1), SEQ ID NO:66 (HCDR2), and SEQ ID NO:67 (HCDR3) as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:68 (LCDR1), SEQ ID NO:69 (LCDR2), and SEQ ID NO:70 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:185 (HCDR1), SEQ ID NO:186 (HCDR2), and SEQ ID NO:187 (HCDR3) as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:188 (LCDR1), SEQ ID NO:189 (LCDR2), and SEQ ID NO:190 (LCDR3).

103. The antibody-drug conjugate of claim 101 or claim 102, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

26.

104. Antibody-drug conjugates of formula (I): Ab-(L-D) ｐ (I) [In the formula, (i) the Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:2 (HCDR1), SEQ ID NO:3 (HCDR2), and SEQ ID NO:4 (HCDR3) as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:7 (LCDR1), SEQ ID NO:8 (LCDR2), and SEQ ID NO:9 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3) as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3); (ii) D is eribulin; (iii) L is Mal-(PEG) ２ - a cleavable linker comprising Val-Cit-pAB; (iv) p is an integer from 1 to 8.

105. The antibody-drug conjugate of claim 104, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

24.

106. Antibody-drug conjugates of formula (I): Ab-(L-D) ｐ (I) [In the formula, (i) the Ab has three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:71 (HCDR1), SEQ ID NO:72 (HCDR2), and SEQ ID NO:73 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:74 (LCDR1), SEQ ID NO:75 (LCDR2), and SEQ ID NO:76 (LCDR3), as defined by the IMGT numbering system; and an internalizing anti-HER2 antibody or an internalizing antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 191 (HCDR1), SEQ ID NO: 192 (HCDR2), and SEQ ID NO: 193 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 194 (LCDR1), SEQ ID NO: 195 (LCDR2), and SEQ ID NO: 196 (LCDR3); (ii) D is eribulin; (iii) L is Mal-(PEG) ２ - a cleavable linker comprising Val-Cit-pAB; (iv) p is an integer from 1 to 20.

107. The antibody-drug conjugate of claim 106, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

28.

108. Antibody-drug conjugates of formula (I): Ab-(L-D) ｐ (I) [In the formula, (i) the Ab has three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:65 (HCDR1), SEQ ID NO:66 (HCDR2), and SEQ ID NO:67 (HCDR3), as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:68 (LCDR1), SEQ ID NO:69 (LCDR2), and SEQ ID NO:70 (LCDR3), as defined by the IMGT numbering system; and an internalizing anti-mesothelin antibody or an internalizing antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 185 (HCDR1), SEQ ID NO: 186 (HCDR2), and SEQ ID NO: 187 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 188 (LCDR1), SEQ ID NO: 189 (LCDR2), and SEQ ID NO: 190 (LCDR3); (ii) D is eribulin; (iii) L is Mal-(PEG) ２ - a cleavable linker comprising Val-Cit-pAB; (iv) p is an integer from 1 to 20.

109. The antibody-drug conjugate of claim 108, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

26.

110. The antibody-drug conjugate of any one of claims 104 to 109, wherein p is 1 to 8, or 1 to 6.

111. The antibody-drug conjugate of any one of claims 104 to 110, wherein p is 2 to 8, or 2 to 5.

112. The antibody-drug conjugate of any one of claims 104 to 111, wherein p is 3 to 4.

113. The antibody-drug conjugate of any one of claims 104 to 112, wherein p is 4.

114. Antibody-drug conjugates of formula (I): Ab-(L-D) ｐ (I) [In the formula, (i) Ab is an internalizing anti-folate receptor alpha antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; (ii) D is eribulin; (iii) L is Mal-(PEG) ２ - a cleavable linker comprising Val-Cit-pAB; (iv) p is an integer from 3 to 4.

115. The antibody-drug conjugate of claim 114, wherein p is 4.

116. The antibody-drug conjugate of claim 114 or claim 115, wherein the antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

117. The antibody-drug conjugate of any one of claims 1 to 116, wherein p is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC).

118. The antibody-drug conjugate of any one of claims 1 to 116, wherein p is determined by reverse phase liquid chromatography-mass spectrometry (LC-MS).

119. The antibody-drug conjugate of any one of claims 1 to 118, wherein the cleavable linker is covalently attached to eribulin via the C-35 amine.

120. 120. A composition comprising multiple copies of the antibody-drug conjugate of any one of claims 1 to 119, wherein the average p of said antibody-drug conjugate in said composition is from about 3.2 to about 3.

8.

121. 98. A composition comprising multiple copies of the antibody-drug conjugate of any one of claims 95 to 97, wherein the average p of said antibody-drug conjugate in said composition is from about 3.2 to about 3.

8.

122. 120. A composition comprising multiple copies of the antibody-drug conjugate of any one of claims 1 to 119, wherein the average p of said antibody-drug conjugate in said composition is from about 3.6 to about 4.

4.

123. 98. A composition comprising multiple copies of the antibody-drug conjugate of any one of claims 95 to 97, wherein the average p of said antibody-drug conjugate in said composition is from about 3.6 to about 4.

4.

124. 123. A method of treating a patient having or at risk of having a cancer expressing a target antigen, comprising administering to said patient a therapeutically effective amount of an antibody-drug conjugate of any one of claims 1 to 119 or a composition of any one of claims 120 to 123.

125. 125. The method of claim 124, wherein the target antigen is the folate receptor alpha.

126. 126. The method of claim 125, wherein the cancer expresses high levels of folate receptor alpha.

127. 126. The method of claim 125, wherein the cancer expresses moderate levels of folate receptor alpha.

128. 126. The method of claim 125, wherein the cancer expresses low levels of folate receptor alpha.

129. The method of any one of claims 125 to 128, wherein the folate receptor alpha expressing cancer is gastric cancer, serous ovarian cancer, clear cell ovarian cancer, non-small cell lung cancer, colorectal cancer, triple negative breast cancer, endometrial cancer, serous endometrial cancer, lung carcinoid, or osteosarcoma.

130. The method of any one of claims 125 to 129, wherein the patient is non-responsive or poorly responsive to treatment with (a) an anti-folate receptor alpha antibody, when administered alone, and / or (b) eribulin, when administered alone.

131. The method of any one of claims 125-130, wherein the patient is intolerant, non-responsive or poorly responsive to treatment with eribulin when administered alone.

132. 125. The method of claim 124, wherein the target antigen is human epidermal growth factor receptor 2.

133. 133. The method of claim 132, wherein the cancer expresses high levels of human epidermal growth factor receptor 2.

134. 133. The method of claim 132, wherein the cancer expresses moderate levels of human epidermal growth factor receptor 2.

135. 133. The method of claim 132, wherein the cancer expresses low levels of human epidermal growth factor receptor 2.

136. The method of any one of claims 132 to 135, wherein the human epidermal growth factor receptor 2-expressing cancer is breast cancer, gastric cancer, bladder cancer, or urothelial cell cancer.

137. The method of any one of claims 132-136, wherein the patient is non-responsive or poorly responsive to treatment with (a) an anti-human epidermal growth factor receptor 2 antibody when administered alone, and / or (b) eribulin when administered alone.

138. The method of any one of claims 132-137, wherein the patient is intolerant, non-responsive, or poorly responsive to treatment with eribulin when administered alone.

139. 125. The method of claim 124, wherein the target antigen is mesothelin.

140. The method of claim 139, wherein the patient is non-responsive or poorly responsive to treatment with (a) an anti-mesothelin antibody when administered alone, and / or (b) eribulin when administered alone.

141. The method of claim 139 or claim 140, wherein the patient is intolerant, non-responsive, or poorly responsive to treatment with eribulin when administered alone.

142. A method of reducing or inhibiting the growth of a target antigen-expressing tumor comprising administering a therapeutically effective amount of an antibody-drug conjugate of any one of claims 1 to 119 or a composition of any one of claims 120 to 123.

143. 143. The method of claim 142, wherein the target antigen is the folate receptor alpha.

144. The method of claim 143, wherein the tumor is a folate receptor alpha-expressing gastric cancer, serous ovarian cancer, clear cell ovarian cancer, non-small cell lung cancer, colorectal cancer, triple negative breast cancer, endometrial cancer, serous endometrial carcinoma, lung carcinoid, or osteosarcoma.

145. The method of claim 143 or claim 144, wherein the tumor is resistant or refractory to treatment with an anti-folate receptor alpha antibody when administered alone and / or to treatment with eribulin when administered alone.

146. 143. The method of claim 142, wherein the target antigen is human epidermal growth factor receptor 2.

147. The method of claim 146, wherein the tumor is a human epidermal growth factor receptor 2-expressing breast cancer, gastric cancer, bladder cancer, or urothelial cell carcinoma.

148. The method of claim 146 or claim 147, wherein the tumor is resistant or refractory to treatment with an anti-human epidermal growth factor receptor 2 antibody when administered alone and / or to treatment with eribulin when administered alone.

149. 143. The method of claim 142, wherein the target antigen is mesothelin.

150. The method of claim 149, wherein the tumor is resistant or refractory to treatment with an anti-mesothelin antibody when administered alone and / or to treatment with eribulin when administered alone.

151. An antibody-drug conjugate according to any one of claims 1 to 119 or a composition according to any one of claims 120 to 123 for use in the treatment of a target antigen-expressing cancer.

152. The antibody-drug conjugate for use according to claim 151, wherein the target antigen is the folate receptor alpha.

153. The antibody-drug conjugate for use according to claim 152, wherein the folate receptor alpha expressing cancer is gastric cancer, serous ovarian cancer, clear cell ovarian cancer, non-small cell lung cancer, colorectal cancer, triple negative breast cancer, endometrial cancer, serous endometrial cancer, lung carcinoid, or osteosarcoma.

154. The antibody-drug conjugate for use according to claim 151, wherein the target antigen is human epidermal growth factor receptor 2.

155. The antibody-drug conjugate for use according to claim 154, wherein the human epidermal growth factor receptor 2 expressing cancer is breast cancer, gastric cancer, bladder cancer, or urothelial cell cancer.

156. The antibody-drug conjugate for use according to claim 151, wherein the target antigen is mesothelin.

157. Use of an antibody-drug conjugate according to any one of claims 1 to 119 or a composition according to any one of claims 120 to 123 in the treatment of a target antigen-expressing cancer.

158. The use of claim 157, wherein the target antigen is the folate receptor alpha.

159. The use of claim 158, wherein the folate receptor alpha expressing cancer is gastric cancer, serous ovarian cancer, clear cell ovarian cancer, non-small cell lung cancer, colorectal cancer, triple negative breast cancer, endometrial cancer, serous endometrial cancer, lung carcinoid, or osteosarcoma.

160. The use according to claim 157, wherein the target antigen is human epidermal growth factor receptor 2.

161. The use of claim 160, wherein the human epidermal growth factor receptor 2-expressing cancer is breast cancer, gastric cancer, bladder cancer, or urothelial cell carcinoma.

162. The use of claim 157, wherein the target antigen is mesothelin.

163. Use of an antibody-drug conjugate according to any one of claims 1 to 119 or a composition according to any one of claims 120 to 123 in a method for the manufacture of a medicament for the treatment of a target antigen-expressing cancer.

164. The use of claim 163, wherein the target antigen is the folate receptor alpha.

165. The use of claim 164, wherein the folate receptor alpha expressing cancer is gastric cancer, serous ovarian cancer, clear cell ovarian cancer, non-small cell lung cancer, colorectal cancer, triple negative breast cancer, endometrial cancer, serous endometrial cancer, lung carcinoid, or osteosarcoma.

166. The use according to claim 163, wherein the target antigen is human epidermal growth factor receptor 2.

167. The use of claim 166, wherein the human epidermal growth factor receptor 2-expressing cancer is breast cancer, gastric cancer, bladder cancer, or urothelial cell carcinoma.

168. The use of claim 163, wherein the target antigen is mesothelin.

169. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 119 or the composition of any one of claims 120 to 123, and a pharma- ceutically acceptable carrier.

170. A method of producing the antibody-drug conjugate of any one of claims 1 to 119 or the composition of any one of claims 120 to 123, comprising reacting an antibody or antigen-binding fragment with a cleavable linker attached to eribulin under conditions that allow conjugation.

171. A method for determining whether a patient will be responsive to treatment with an antibody-drug conjugate of any one of claims 1 to 119 or a composition of any one of claims 120 to 123, comprising obtaining a biological sample from the patient and contacting said biological sample with the antibody-drug conjugate of any one of claims 1 to 119 or the composition of any one of claims 120 to 123.

172. The method of claim 171, wherein the biological sample is a tumor biopsy from a patient having or at risk of having a folate receptor alpha-expressing cancer, and the cancer is gastric cancer, serous ovarian cancer, clear cell ovarian cancer, non-small cell lung cancer, colorectal cancer, triple negative breast cancer, endometrial cancer, serous endometrial cancer, lung carcinoid, or osteosarcoma.

173. The method of claim 171, wherein the biological sample is a tumor biopsy from a patient having or at risk of having a human epidermal growth factor receptor 2-expressing cancer, and the cancer is breast cancer, gastric cancer, bladder cancer, or urothelial cell carcinoma.

174. The method of claim 171, wherein the biological sample is a tumor biopsy from a patient having or at risk of having a mesothelin-expressing cancer.

175. -LD, wherein D is eribulin; and L is a cleavable linker covalently attached to D.

176. The composition of claim 175, wherein the cleavable linker is covalently attached to eribulin via the C-35 amine.

177. The composition of claim 175 or claim 176, wherein the cleavable linker comprises valine-citrulline (Val-Cit).

178. The composition of any one of claims 175 to 177, wherein the cleavable linker comprises a PEG spacer unit.

179. The cleavable linker is Mal-(PEG). ２ -Val-Cit-pAB.

180. A composition comprising multiple copies of an antibody-drug conjugate of formula (I): Ab-(L-D) ｐ (I) [In the formula, (i) the Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:2 (HCDR1), SEQ ID NO:3 (HCDR2), and SEQ ID NO:4 (HCDR3) as defined by the Kabat numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:7 (LCDR1), SEQ ID NO:8 (LCDR2), and SEQ ID NO:9 (LCDR3); or three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3) as defined by the IMGT numbering system; and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3); (ii) D is eribulin; (iii) L is Mal-(PEG) ２ - a cleavable linker comprising Val-Cit-pAB; (iv) p is the average number of -L-D moieties per Ab, and the average p of the antibody-drug conjugates in the composition is from about 3.6 to about 4.4; said average p is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC).

181. The composition of claim 180, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

24.

182. The composition of any one of claims 120 to 123, wherein the average p of the antibody-drug conjugates in the composition is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC).

183. The composition of any one of claims 120 to 123, wherein the average p of the antibody-drug conjugates in the composition is determined by reverse phase liquid chromatography-mass spectrometry (LC-MS).

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